Annual Report 2015

Annual Report
2015

IOM-NCGM
Research Collaboration Oﬃce

March 2016
Kathmandu, Nepal
Tokyo, Japan

Annual Report
2015

IOM-NCGM
Research Collaboration Office

March 2016
Kathmandu, Nepal
Tokyo, Japan
ISBN 978-4-909675-31-6

Preface

The Medical Education Project, which was implemented from 1980 to 1996, contributed
to the establishment and enhancement of basic and the clinical medicine of the Institute ofMedicine (IOM) at Tribhuvan University along with its attached teaching hospital (TUTH). During
the project period experts dispatched from NCGM worked together with IOM staff and good
relationship was built between the two institutions.
Nineteen years have passed since completion of the project. During this period IOM
achieved further development and currently it is greatly contributing to medical education,
medical care, researches and human resource development in Nepal. Besides, reliable
relationship between NCGM and IOM has been maintained until today. This is one of thevaluable outcomes and legacies of the project.
In January 2013, Memorandum of Understanding (MOU) was concluded between
NCGM and IOM to start a unique cooperation focusing on research and related human resource
development. Three years have passed since the start of the new cooperation between NCGM
and IOM. Collaborations initiated based on the MOU are on track, and a number of fruitful
outcomes have been obtained.
It is a great pleasure for us to summarize the outline report of our new collaboration as
Annual Report 2015 following the publications of Annual Report 2013 and 2014. We also would
like to thank all those who worked hard for the success of the project and toward the realizationof new collaborations between NCGM and IOM. I sincerely hope that the relationship of mutual
trust between the two institutions will be further strengthened.

Hiroshi Ohara, M.D.,Ph.D.
Director, Division of Global Networking,Bureau of International Health Cooperation
National Center for Global Health and Medicine
Tokyo, Japan

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IOM-NCGM Research Collaboration Office Annual Report 2015

IOM-NCGM Research Collaboration Office Annual Report 20153

Abbreviations

4

COPD		

Chronic Obstructive Pulmonary DiseaseCT		

Computed TomographyDM		

Diabetes Mellitus

ESBL		

Extended Stratum beta Lactamase

GFATM		

Global Fund against AIDS, Tuberculosis and Malaria

IOM		

Institute of Medicine

IT		Information Technology

JICA		

Japan International Cooperation Agency

JOCVs		

Japan Overseas Cooperation Volunteers

MBBS		

Medicine Bachelor Bachelor Surgery

MOHP		

Ministry of Health and Population

MOU		

Memorandum of UnderstandingNCD		

Non Communicable Diseases

NCGM		

National Center for Global Health and Medicine

NDM

New Delhi Methalo-β-Lactamase

ODA		

Official Development Assistance

TUTH		

Tribhuvan University Teaching Hospital

WHO		

World Health Organization

WPRO		

Western Pacific Regional Office

IOM-NCGM Research Collaboration Office Annual Report 2015

Contents

Preface
1. NCGM............................................................................................................................. 02
2. IOM................................................................................................................................. 03
Abbreviations...................................................................................................................... 04
Contents.............................................................................................................................. 05
I.

Outline of Collaboration between NCGM and IOM
1. Introduction.................................................................................................................... 06
2. Before conclusion of MOU.............................................................................................. 06
3. Conclusion of MOU and start of new cooperation.......................................................... 09
4. Purpose of the Collaboration between NCGM and IOM ............................................... 10

II. Current IOM
1. General information, Clinical medicine, Basic medicine, Prospects................................ 12
Table 1: Situation and activities of IOM/TUTH : Comparison between 1985 and 2015......... 15
III. Nepal earthquake in April 2015 and contribution of IOM/TUTH						to medical care for casualties.......................... 16
IV. Research activities
1. General information........................................................................................................ 18
2. Progress of research activities up to FY 2015.................................................................. 20
3. Outline of each research................................................................................................. 26
Table 2: Collaborative researches in NCGM-IOM collaboration............................................ 27
Researches No.1- No.7.......................................................................................................... 28
Attachment:

Publications on Scientific Papers.................................................................. 42

Table 3: List of papers published on international journals................................................... 42
Scientific papers.................................................................................................................. 44

IOM-NCGM Research Collaboration Office Annual Report 2015

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I. Outline of Collaboration between NCGM and IOM

1.

Introduction
The Institute of Medicine (IOM) at Tribhuvan University was established as the first medical school

in Nepal with the support of Japan’s Grant Aid in 1980 and then a technical cooperation project (Medical
Education Project) was implemented from 1980 to 1996 supported by Japan International Cooperation
Agency (JICA). During this period, the National Center for Global Health and Medicine (NCGM) dispatched a
project team leader, doctors and other medical professionals. Currently, IOM functions as a core in medical
education, medical care and medical human resource development in Nepal, and its attached Tribhuvan
University Teaching Hospital (TUTH) has gained the trust and popularity of the people in Nepal.
Recently, NCGM has begun some unique activities focused mainly on research and human resource
development based on the relationship of mutual trust built between NCGM and IOM during the JICA
project. At the beginning of these activities, a memorandum of understanding (MOU) was concluded
between NCGM and IOM, and the IOM-NCGM Research Collaboration Office was established.
2.

Before the conclusion of MOU(1)Grant-aid project and Technical cooperation project (1980-1996)
Since most of the medical education in Nepal was dependent on foreign countries and there werelimited number of medical workers in the past, increasing the number of doctors was crucial in order towidespread modern medicine and improve the medical and hygienic standards. Hence, in 1978, the Nepalese
government requested assistance from the Japanese government in order to establish a medical university.
After several surveys, a grant-aid project by
JICA was initiated for the purpose of establishing a
medical faculty as the 10th faculty of the Tribhuvan
University located in the capital, Kathmandu. The
project aimed to widely provide medical services to
the general public by strengthening and developing
the medical faculty as a core institution for medical
education and healthcare in Nepal. The project
was conducted by a combination of grant aid andtechnical cooperation.
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IOM-NCGM Research Collaboration Office Annual Report 2015

Tribuvan University Institute of Medicine (TUTH) in 1995

For the grant aid project, main facilities such as hospital buildings, academic building for basic
science and nursing school were constructed, and essential equipment was provided.
<Grant-aid project>•

1981 Construction of outpatient building, etc.

•

1982 Construction of patients’ ward, etc.

•

1990 Construction of academic building, etc.

•

1991 Extension of patient’s ward, etc.

•

1992 Repair of operation theater, etc.
The Medical Education Project was conducted

as a technical cooperation project in two phases. In thefirst phase (1980-1989), strengthening education, clinicalpractice, and the research infrastructure for basic medicine
and clinical medicine were mainly implemented. In thesecond phase (1989-1996), a technical cooperation project
was implemented, focusing mainly on improving medical
education to have international accreditation, enhancing thefunction of basic medicine, clinical medicine, and research,
as well as improving hospital management function.
Technical guidance in nephrology (1996)<Technical cooperation project: Medical Education Project>
Medical Education Project Phase 1 (1980-1989)•

Dispatch of Japanese Experts

12 (14JOCVs are included)•

Invitation for Nepalese Trainees

29•

Equipment Provision

Medical Education project Phase 2 (1989-1996)•

Dispatch of Japanese Experts

86•

Invitation for Nepalese Trainees

28•

Equipment ProvisionInternational Symposium on Infectious and Tropical diseases (1996)

IOM-NCGM Research Collaboration Office Annual Report 20157

Local and International Seminar/ Symposium held by the Project•

Nepal-JICA Joint Symposium on Cholelithiasis			

6-8 January 1991•

Congress on Altitude Medicine and Physiology			

8 April 1992•

Nursing Research Conference					2-4 November 1993•

International Symposium on Obstructive Jaundice			

17-18 February 1994•

International Symposium on Diabetes Mellitus			

23-24 March 1995•

International Symposium on Infectious and Tropical Diseases

20-21 march, 1996

During the 16 year technical cooperation period almost all the basic and advanced techniques
regarding diagnosis, examination, treatment and clinical record, which were indispensable to the daily
medical care and medical education in IOM/TUTH, were guided (However, nosocomial infection control
was not included as awareness on it was still low in those days even in advanced countries). IOM/TUTH
had grown to be a medical institution that carried out a kernel function of Nepalese medical care, and had
gained extremely high reliance from Nepalese people.
IOM/TUTH developed to be an internationally acknowledged medical institute; the MBBS (medicine
bachelor bachelor surgery) degree awarded by IOM, Tribhuvan University was recognized by dental andmedical councils of Bangladesh and Pakistan, and medical councils of India and Sri Lanka. Graduates of
IOM had already pursued post-graduates education in Japan, Thailand, United Kingdom, U.S.A., and othercountries.
As a result of the technical cooperation project over 16 years, the foundation for a medical
education system, hospital management, clinical medicine, research, and nursing education was constructed
at IOM.
(2)

After Completion of the Technical Cooperation Project (1996- )The Medical Education Project was completed in July 1996. Since then, IOM has greatly contributed

to healthcare in Nepal, gaining the trust of the people as the most important medical institution in Nepal.
Human resources trained by the technical cooperation project play leading roles not only in the capital
Kathmandu but throughout the nation, furthermore globally, greatly contributing to the medical andhealthcare field in Nepal.
After completion of the project, based on the good relationship developed during the project,
NCGM and IOM have conducted small scale collaborative research activities on hepatitis, helicobacter
infection, diarrhea, multi-drug resistant bacteria in respiratory tract infections, etc.
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IOM-NCGM Research Collaboration Office Annual Report 2015

Nepal suffered political instability from 1996 to 2006 followed by a transition from the Kingdomof Nepal to the Federal Democratic Republic of Nepal in 2008. The political instability affected economic
growth and compromised the delivery of social and public health interventions in the country. During thisperiod, cooperative relations between IOM and NCGM were temporarily suspended, but with the stabilizingof political conditions in recent years, the good relationship has been recovering.
In September 2009, a joint symposium on nosocomial infection control was held at IOM jointly
organized by NCGM and IOM, resulting in greater awareness of the importance of infection control andresearch collaboration. At the conference, successful technical cooperation in nosocomial infection control
in Vietnam was introduced inviting director of Bach Mai Hospital in Hanoi. In 2013, “the 1st Joint Conferenceon Infectious Diseases with Growing Concern in Recent Years in Nepal” was held at IOM.

3.

Conclusion of MOU and start of new cooperation (2013-2015 )On Januar y 18, 2013, NCGM concluded anagreement of cooperation with IOM in Nepal for researchand related human resource development, and IOM became
the fifth overseas platform for NCGM. The Memorandumof Understanding (MOU) was signed by the President of
NCGM in Tokyo firstly, and then, was signed by the Deanof IOM at IOM in the capital Kathmandu on January 18,
2013. Based on this MOU, unique activities of NCGM,
such as collaborative research on infectious diseases, dual
burden of infectious diseases and non-communicable

Joint Symposium on nosocomicao infection Control(September 2009)diseases, and development of human resources related tothese researches came to be actively promoted, seeking
to improve healthcare in Nepal. Collaboration between
NCGM and IOM including related institutions (Kathmandu
University School of Medicine, Clinics in Kathmandu,etc.) mainly on research field will be continued hereafter.
Conclusion of MOU is considered to be highly effective forthe smooth and efficient implementation of collaborations
including related institutions.

Signing of MOU at NCGM and IOM (January 2013)

IOM-NCGM Research Collaboration Office Annual Report 2015

9

In September 2013, the IOM-NCGM Research Collaboration Office was established in the academic
building of IOM, and the necessary equipment (desk, chair, computer, projector and scanner) was installed.
One assistant was employed and given an orientation. Currently, activities utilizing grants from theInternational Health Cooperation Research (24-5, 24-5, 25-7), a grant from the Ministry of Health, Labor andWelfare of Japan, are progressing utilizing the office as a platform. IOM’s Department of Microbiology and
NCGM’s Bureau of International Medical Cooperation manage the office operation.
The persons in charge of divisions of basic
and clinical medicines are the professors for the
department of Microbiology and Internal Medicine,respectively. In addition, there is a Research
Department to manage research, which serves as acounterpart.

IOM-NCGM Research Collaboration Office4.

Purpose of the Collaboration between NCGM and IOM and expected outcomes
The purpose of this collaboration is to contribute to the healthcare in both countries by collaborative

research and related human resource development, and to strengthen a reliable relationship.
Expected Outcomes
1. To strengthen research activities by the publication and presentation at academic conferences, etc. of
the collaborative research by IOM and NCGM.
2. To obtain research results effectively through collaboration with IOM that is the main medical care andmedical education institution in Nepal.
3. To provide benefits to younger staff members of NCGM by learning about the actual conditions of
medical care in developing countries and how international collaborations are conducted.
4. To promote a relationship by utilizing a good relationship built on the results of the technicalcooperation project.
5. To contribute to the quality improvement of medical care in Nepal by conducting researches focused onhigh-priority infectious diseases in Nepal and providing related technical assistance. These results arealso beneficial for medical care in Japan.

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IOM-NCGM Research Collaboration Office Annual Report 2015

6. To enable wider range of cooperation in the future, because the inclusion of other medical institutions
other than IOM allows providing other possible collaboration fields.
7. To contribute to expansion of the benefit of ODA projects.

Main gate of Trubhuvan University Teaching Hospital (TUTH)

Nursing School

Academic building of Institute of Medicine (IOM)

IOM-NCGM Research Collaboration Office Annual Report 201511

II. Current IOM

1.

General information
IOM was established as the first medical school in Nepal in 1980. Thereafter the number of medical

schools (faculty of medicine, medical university) increased and at the time of project termination in 1986
there were 4 medical schools in Nepal. As of January 2015, the number of medical schools in Nepal is 22,
in which 7 are affiliated with IOM. Post-graduate curriculums including diploma and degree programs havebeen improved so as to provide better medical personnel who can contribute efficiently to medical service.
Since its inception, IOM has produced more than 1,800 medical doctors (as of 1996 it was 307).
These doctors are working, not only in Kathmandu Valley, but also in rural and remote areas of Nepal,
and rendering valuable services. Beside, increasing number of doctors are studying or working in foreigncountries. It is noteworthy that not a few graduates of IOM are working in medical schools across thecountry as professors. Similarly, nursing and co-medical staff trained in IOM/TUTH are rendering medicalcare to the Nepalese people. IOM is carrying out quite important role in medical care as well as medical
education in Nepal.
Table 1 shows comparison of hospital general information between 1985 and 2015
2.

Clinical Medicine
TUTH is a general hospital which has 740 beds (as of December 2015) under 28 Clinical

Departments. The equipment which was provided during the project period has been gradually replacedwith new ones, but not a few old equipment are still used. Advanced equipment such as CT scan, MRI,
hemodialyzers are also equipped purchased by Nepalese government. TUTH can provide advanced
medical care as well as essential medical care and examinations. TUTH has been conferred duties androles as the most important top referral medical institution in Nepal. Since 1990, Cardiovascular Center,
Obstetrics & Gynecology Center and Emergency Center have been constructed by Nepalese Government.
The number of out-patients, in-patients,and surgical operation has been ever increasing
since its establishment, which means that theimportance of TUTH in Nepalese medical care has
been increasing year by year.

Patient waiting for doctor’s consultation (TUTH)

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IOM-NCGM Research Collaboration Office Annual Report 2015

3.

Basic Medicine
Basic Medicine consists of 8 departments, which are responsible for giving lectures and practicalstudies to MBBS students and a variety of post-graduate courses to MBBS graduates. These departments
are integrated in the academic building which was completed in 1992.
Research activities are another important role of the Basic Medicine departments in the university.
The efforts to establish the basis for conducting research were made during the project period, andcurrently active researches are being done (currently more than 200 researches are conducted in IOM/
TUTH). The results of research activities which were conducted in IOM/TUTH have been presented invarious domestic and international medical congresses and journals. The Journal of Institute of Medicine,which was founded by IOM in 1984 as the first medical
journal in Nepal developed to be a high level journal
carrying a lot of valuable medical papers and it has
become internationally acknowledged journal. Besides
the 8 basic medicine departments, Research department,
Department of Information Technology and National
Center for Health Professional Education were set up after
the project termination.

4.

Bacteriological laboratory

Prospects
As a result of the 16-year technical cooperation, IOM/TUTH has developed to be one of thebest medical institutions which receives the deep reliance of the nation and makes many contributions
to medical care in Nepal. In addition, the friendly relationship between Nepalese staff and Japanese
staff, and what is more, the Nepalese nation and the Japanese nation, has been firmly established. It isquite important to make the utmost effort to maintain these good results. It is true that the nationwide
expectation for IOM/TUTH as well as duties and roles has been increasing. However, IOM/TUTH has
problems such as small out-patient clinic space, obsolete equipment along with increasing number of noncommunicable diseases cases. Besides, due to the catastrophic earthquake in 2015, a large number of
equipment was damaged.
Hereafter, more and more importance in the quality of medical care will be attached. We expect
IOM/TUTH will make every possible effort in order to live up to these expectations, addressing the existing
challenges.
IOM-NCGM Research Collaboration Office Annual Report 201513

Patient’s ward (TUTH)

Residents of Pulmonary Dept. (TUTH)

Clinical conference(TUTH)

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IOM-NCGM Research Collaboration Office Annual Report 2015

Table 1		

Situation and activities of IOM/TUTH : Comparison between 1985 and 2015
1985

2015

1

Total No. of beds

401

740

2

No. of charity beds

40

63

3

No. of outpatients served (per year)

229,516

381,848

4

No. of in-patients served (per year)

11,973

23,107

5

Average length of hospitalization (days)

NA

6.73

6

No. of operations (per year)

5,237

12,531

7

Average bed occupancy rate (%)

NA

83%

8

Number of Clinical Departments

13

28

9

Number of Basic Science Departments(including forensic medicine and public health)

8

8

10

No. of students (per school year grade)

40

76

11

Research department

-

1

12

Department of Information Technology

-

1

13

National Center for Health Professional Education-

1

IOM-NCGM Research Collaboration Office Annual Report 201515

III. Nepal earthquake in April 2015 and contribution of IOM/
TUTH to medical care for casualties

The recent Nepal earthquake of April 25, 2015 of magnitude 7.8 on the Richter scale had its
epicenter in the area near Barpak, a mountain village between the capital, Kathmandu, and the tourist
town of Pokhara. The earthquake was followed by many powerful after shocks on the same day and a very
powerful one (6.7 on the Richter scale) hit Nepal on the very next day, Sunday April 26. The earthquakes,
which caused extensive damage to buildings and thousands of deaths and injuries were even felt inPakistan, India and Bangladesh.
Since immediately after the earthquake, the TUTH could start to offer medical services to a numberof victims. This was because, even though some of the buildings in TUTH had cracks during the earthquake,all buildings in TUTH were not collapsed following the earthquake.
Furthermore, TUTH contributed to people in Kathmandu not only for medical services. TUTH wasdesignated one of the two mortuaries in Kathmandu city, where many victims' dead bodies were placed.
The dead bodies were kept cold during two months at the morgue. During that time, they tried their best tofind their families, e.g. showing dead people’s belongings.
As of December 2015, there were still many of tents (shelters), tentative houses in skirt ofKathmandu city. Rubbles are still remained in these areas. Even though the difficult situation, medical staff
actively offered medical services with compassion. They called for a long lasting supports.
(Cf. P. Earthquake disaster-associated health effects and the need for improved preventive measures)

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IOM-NCGM Research Collaboration Office Annual Report 201517

IV. Research Activities

1.

General information
To implement collaborative activities and strengthen the IOM-NCGM Research Collaboration Office,the following grants were utilized in 2015.
XX Grants from the International Health Cooperation Research, grants from the Ministry of Health, Laborand Welfare of Japan.
1 ) 24-5: Studies on factors and trends of infectious diseases with growing concern in recent years in
Nepal and Vietnam
2 ) 25-5: Studies on enhancement of human resource development utilizing the IOM-NCGM Research
Collaboration Office ‐ taking into consideration of the dissemination of the benefits of ODA project3 ) 25-7: Fact-finding survey of nosocomial infection control in major hospitals in Nepal and planning ofeffective improvement4 ) 27-4: Study on effective use of the surveillance results for drug resistant pathogens in nosocomial
infection control
XX Grant of Japan Society for the Promotion of Science: Scientific Research (B) (Overseas Academic
Research) to NM: The survey of the present status of viral hepatitis treatment in Nepal²² The main theme of the ongoing researches is “Studies on Factors and Trends of Infectious Diseases with
Growing Concern in Recent Years in Nepal.” Infectious diseases change over time, as can be clearly seenby the appearance of emerging or re-emerging infectious diseases and multi-drug resistant bacteria,
and the control history of various infectious diseases by the appropriate measures. These changes
in infectious diseases are considered to be associated with influential factors such as development,
population movement, change in climate, nutrition, changes in the health system and disease structure,
implementation of disease specific control programs, and support conditions of foreign counties. In thisresearch, we summarized the overview of trends of infectious diseases chronologically and analyzed thefactors that caused change in infectious diseases. Such overview and analysis are crucial to implement
effective control measures.
As the results of preliminary study, we recognized some infectious diseases that fit this main theme.
Among them we selected the following diseases and studies focusing on them were started. (Research
grant 24-5)

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IOM-NCGM Research Collaboration Office Annual Report 2015

① Malaria control and health system② Diarrhea caused by emerging pathogens③ Multi-drug resistant bacteria④ Healthcare associated opportunistic infections⑤ Dual burden of infectious disease and non-communicable diseases²² Recently in Nepal, although there has been increased awareness regarding nosocomial infection control,
implementation of control measures is rather slow. In the Medical Education Project, technical guidance
was provided in almost all fields of medical care, but only control measures against nosocomial infection
were not included in the technical cooperation subjects. This was partly because of the poor awarenessfor nosocomial infection control even in developed countries including Japan at that time. Nosocomial
infection control is also an important research theme for NCGM-IOM collaborative research.
Effective nosocomial infection control is crucial in the healthcare facilities of developing countries,
but in actual fact, attention to it is still limited and control measures are not functioning well in many
countries. This study has been conducted with the purpose to investigate the actual conditions of
nosocomial infection control in Kathmandu City, Nepal as a basis for the possible contribution to its
improvement. (Research grants 25-7, 27-4)²² Collaborative researches are being conducted in Nepal utilizing the IOM-NCGM Research Collaboration
Office as a base (platform). Improvement of the base functions is essential to conduct researchsmoothly. We are trying to strengthen the function of the IOM-NCGM Research Collaboration Officeaiming at smooth implementation of researches and human resource development. 1n 2014 thefollowing activities were conducted. (Research grant 25-5)① Strengthening of management capacity of IOM-NCGM Collaboration Office② Comparative study on outcomes of Medical Education Project and current IOM/TUTH③ Preparation for enhancement of human resource development④ Making the Annual Report 2014, NCGM-IOM Research Collaboration Office²² In 2015 a new research “The survey of the present status of viral hepatitis treatment in Nepal” started
with the Grant of Japan Society for the Promotion of Science.

IOM-NCGM Research Collaboration Office Annual Report 201519

Discussion on collaborative research Microbiology Dept. (IOM)

2.

Progress of research activities up to 2015

2-1 Summary of the activities of Research Grant 24-5
Collaborative research based on the Research Grant 24-5 “Studies on factors and trends ofinfectious diseases with growing concern in recent years in Nepal and Vietnam” had been conducted forthree years from April 2012 to March 2015, and fruitful results were obtained. The summary of the research
is described below.
(1) Purpose:
The purpose of this study is to analyze the latest situation of the infectious diseases which have growing
issues in recent years but appropriate studies along with control measures have not been done (namely,“Emerging health priorities in infectious diseases” as a new issue in the field of infectious diseases), and to
discuss the cause of the spread of such diseases. This study has been carried out mainly in Nepal and partly
in Vietnam with the aim of contribution to effective control of these diseases.
(2) Subjects and their significance
After the discussions with health authorities in each country, following subjects were selected andcollaborative studies are going on: antibiotic resistance, health care-associated and opportunistic infections,malaria, diarrheal diseases caused by emerging pathogens (Rotavirus, Cryptosporisium, etc.), and dual
burden of infectious diseases and non-communicable diseases. Necessities of conducting studies from theviewpoint of emerging health priorities for each subject are as follows:•

Antibiotic resistance: Bacterial resistance to antibiotics is increasing however studies about the spreadof multi-drug resistant bacteria in developing countries along with measures to address such situationis still limited. To provide effective guidance for control, identification of the fact and analysis of thecausative factors are needed.

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IOM-NCGM Research Collaboration Office Annual Report 2015

•

Antibiotic resistance: Bacterial resistance to antibiotics is increasing however studies about the spreadof multi-drug resistant bacteria in developing countries along with measures to address such situationis still limited. To provide effective guidance for control, identification of the fact and analysis of thecausative factors are needed.

•

Health care-associated and opportunistic infections: These types of infections have been increasing evenin developing countries alongside poor control measures, increase of compromised hosts, antibiotics
abuse, application of modern technology (respirator, hemodializer, endoscope, etc.). However ingeneral, these countries have a low awareness and control measures are not effectively done. To
provide effective guidance for improvement, identification of the fact and analysis of the causative
factors are needed.

•

Malaria: Progress has been made in developing countries regarding this disease under the support of
the Global Fund against AIDS, Tuberculosis and Malaria (GFATM). However, in Nepal and Vietnam (alsoin many other endemic countries) newly developed malaria-endemic areas have been reported, and theenvironment and social factors have been indicated as the leading cause. It is necessary to analyze thesefactors in order to take effective control measures against malaria.

•

Diarrheal diseases caused by emerging pathogens: Diarrheal diseases are frequent in developing
countries. In Nepal, diarrheal diseases caused by emerging pathogens such as Rotavirus,
Cryptosporisium, Cyclospora are also frequently experienced at medical care settings. In order to
conduct effective control, analysis of these pathogens is needed. (Basic research on the fact of these
diseases and pathogens was carried out by this study group.)•

Dual burden of communicable diseases and non-communicable diseases: These have been raised as
new problems in developing countries, and their detailed analysis is needed. To address this problem
it is necessary to investigate the actual conditions of the issues and identify causative factors throughbasic studies.

(3) Major achievements
Major achievements of collaborative studies on these subjects up to now are shown below. These havebeen published in conference presentations, scientific papers and reports. Furthermore, through joint
conferences (held in Kathmandu in January 2013, Nagasaki in October 2013, Kathmandu in December2014) and a focal group discussions (held in Kathmandu in April 2014), study results have been shared withrepresentatives of each country concerned (hospital personnel, WHO, Ministries of Health and Population,
Embassy of Japan and JICA). Discussions have been held over the issues, and recommendations have been
given based on study results.
IOM-NCGM Research Collaboration Office Annual Report 201521

ØØ Factors related to successful measures against malaria and current issues were analyzed. The results
are expected to serve as useful information that contributes to measures against malaria. Besides,
our in-depth study revealed that in malaria endemic-areas, the distribution rate of bed-nets was low
among poverty groups which did not receive aid from the government or other countries. Also, medical
facilities were not properly utilized. These are considered to be important information in order toachieve universal health coverage.
ØØ A high frequency of diarrheal diseases caused by emerging pathogens such as Rotavirus,
Cryptosporisium, and Cyclospora in Nepal was indicated.
ØØ In medical settings in Nepal, a significant growth in drug resistance of gram negative bacilli, which cause
nosocomial infections, was clearly observed. (Multiple drug resistant bacteria with strong resistance toCarbapenem and Amynoglycoside were isolated.)ØØ The following new findings were obtained by analyzing nosocomial pathogens:•

The new variant of New Delhi methalo-β-lactamase producers were identified from Escherichia coli
and named NDM-8 and NDM-12 respectively (the first case in the world).

•“AAC (6')-Ial” gene was identified for the first time in Serratia marcescens,•“AAC (6’)-Iak” gene in Strenotrophomonas martophilia was identified (the first case in Nepal).

•

Providencia rettgeri producing NDM-1 Metallo-β-Lactamase and ArmA 16S rRNA•

Methylase was detected (the second case in the world).

•

The new variant of New Delhi methalo-β-lactamase producers were identified from Escherichia coli
in 2015 and named NDM-13.

✓✓ The following new findings regarding the epidemiology of extended-spectrum β-lactamase (ESBL)producing E. coli were obtained: Patients infected with ESBL-producing E. coli were significantly younger
than patients in Japan. Among them, infection rate was high in pregnant women. Moreover, E. coli 025bST131 clone, which is of growing global concern, was shown to have a high frequency in Nepal.
✓✓ A high rate of double burden of diabetes and tuberculosis was discovered. Furthermore, a trend toward
an especially high dual burden rate of both diabetes and tuberculosis was seen among patients over theage of 40. From the above, the possibility of clinics for non-communicable diseases (NCD) and those fortuberculosis may be able to collaborate in early diagnosis of their counterpart illness.

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IOM-NCGM Research Collaboration Office Annual Report 2015

Discovery of NDM variants
NDM variants

Organism and country

References

NDM-1

E. coli and K. pneumoniae (Sweden)

Yong D. et. al., 2009

NDM-2

Acinetobactor baumanii (Egypt)

Kaase M. et all., 2011

NDM-3

E. coli (Australia)

Rogers B.A. et. al., 2013

NDM-4

E. coli (India)

Nordmann P. et. al., 2012

NDM-5

E. coli (United Kingdom)Hornsey M. et. al., 2011

NDM-6

E. coli (New Zealand)

Williamson D.A. et. al. 2012

NDM-7

E. coli (Canada)

Accession no. JX262694

NDM-8

E. coli (Nepal)

Tada T. et. al., 2013

NDM-9

K. pneumoniae (China)

Accession no. KC999080

NDM-10K. pneumoniae (India)

Accession no. KF361506

NDM-11

Assigned (not known)

www.lahey.org/studies

NDM-12E. coli (Nepal)

Tada T. et. al., 2014

NDM-13

E. coli (Nepal)

Shrestha B.,Tada T. et. al., 2015

Joint conferences and information sharing
carried out in this study have contributed toincreased awareness of medical personnel in eachcountry concerned (especially in Nepal) regarding
emerging health priorities of infectious diseases(multi-drug resistant bacteria, equity in the
distribution of bed-nets for malaria prevention,and double burden of infectious diseases and non-

Opening ceremony of the 2nd Joint Conference (IOM, December 2014)

communicable diseases).
Especially in Nepal, the use of antibiotics, poor countermeasures for nosocomial infections,health knowledge of local residents, environmental change caused by development processes, population
movement, and lifestyle habits including diet are considered to contribute to the increase and transitionof infectious diseases (studied by this research group). It is important to seek measures that address theseemerging priority issues taking account of such potential factors.
IOM-NCGM Research Collaboration Office Annual Report 201523

Our studies revealed the spread of multi-drug resistant bacteria in medical settings in Nepal.
Measures must be urgently taken to address this situation, in which not only medical care but alsocountermeasures for nosocomial infections have great influence. To implement measures effectively, active
intervention not only by medical facilities but also by governmental agencies, furthermore inter sectoralcollaboration, is needed.
The significance of these diseases studied have not been fully recognized and active control
measures have not been actively carried out. Our contribution to a heightened awareness by medical
personnel of each country concerned can be considered as great progress. We aim to contribute to proper
measures by further analyzing factors of growing issues regarding these infectious diseases (those studied
by this study group).

Collaborative research on multi-drugresistant bacteria (IOM)

Collaborative research on multi-drugresistant bacteria (NCGM)

2-2 Outline of other researches
Research Grant: 25-5
The purpose of this study is to strengthen human resource development capacity at IOM, Tribhuvan
University, by taking advantage of the overseas platform of the NCGM located at IOM (IOM-NCGM Research
Collaboration Office). In the past, IOM received grant aid and technical cooperation projects from Japan’s
Official Development Assistance (ODA), and currently plays a leading and core role in Nepal’s medicalcare. At IOM, studies have been carried out with the aim of sharing the benefits of medical care throughhuman resource development, and to contribute to the improvement of medical standards in Nepal andthe extension of ODA's achievements. In particular IOM focuses on improving management capacity at the
IOM-NCGM Research Collaboration Office and human resource development in main areas of specialization(internal medicine and public health). In FY 2014, the following studies and activities were carried out:
24

IOM-NCGM Research Collaboration Office Annual Report 20151 ) Improvement of Management Capacity at the IOM-NCGM Research Collaboration Office:The IOM-NCGM Research Collaboration Office was established in January, 2013. This well-maintained
office contributes to the smooth implementation of study group activities. Management of theoffice and liaison with other study groups have been carried out in collaboration with Department ofMicrobiology, Institute of Medicine, Tribhuvan University. Of particular note is the well-establishedframework for cooperation and the continuing good relationship with departments at the Institute ofMedicine, Tribhuvan University.
2 ) Improvements in Human Resource Development:
Planning, implementation and analysis (all or in part) of the following studies were carried out with thecounterpart departments, contributing to human resource development by improving study and training
capacity.
ØØ Regarding “Knowledge, Attitude and Hand washing Practices among Health Care Professionals
Working in Teaching Hospital, Kathmandu, Nepal”, the main study carried out by the Department of
Study and Training, instructions were provided on data analysis and report preparation.
ØØ Data analysis and report preparation of the “Study on malaria control and the equity in bed-net
distribution” by the Department of Public Health were carried out with the counterpart department.
ØØ In the “Study on pulmonary fibrosis using diagnostic imaging” carried out by Pulmonary Medicine,
support was given regarding radiologic interpretation and data input, contributing to humanresource development.
ØØ Planning of “Training for Newly Employed Nurses on Measures for Nosocomial Infections” scheduled
to be conducted at Nursing Department was carried out.
ØØ It is noteworthy that IOM/TUTH contributed greatly to conduct medical care for the woundedpeople after the catastrophic earthquake which hit Nepal in April 2015
Research Grant: 25-7
Although important for the enhancement of quality in medical care, effective measures fornosocomial infection control have been limited in developing countries. The main purpose of this study
group is to follow up JICA's “Training Course for Specialists of Nosocomial Infection Control and Prevention
in Developing Countries” held at NCGM. This study includes Nepal, where such training has not been given
up to now. In the study in Nepal, examination of actual conditions of measures for nosocomial infections,and analysis of problems was carried out, followed by important advice regarding improvement based on
the obtained results and monitoring of the progress of improvement. In other words, the study in Nepal has
IOM-NCGM Research Collaboration Office Annual Report 201525

a meaning of a negative control in contrast to the countries where training has already been provided by
the above JICA’s training course.
In 2015, the following two surveys were carried out, followed by some advice for improvement. KAP
Study on Medical Personnel regarding Nosocomial Infection Control.
This study was carried out with an aim to assess the level of awareness and actual practice of “handwashing", which is a basic prevention method of nosocomial infection control among medical personnel,
and then use the obtained results for improvement.
The study indicated that doctors at TUTH have a relatively good knowledge regarding hand-washing,but do not follow it in actual practice.
A large number of nurses and laboratory technicians lack knowledge, and have a low degree ofhand-washing in actual practice prior to contact with patients. It is important to provide improved training
for medical staff regarding nosocomial infection control and prevention, which are mainly based on standard
precautions.
3.

Outline of each research
Table 2 shows overview of the collaborative researches between NCGM and IOM. Progress statusof each research (No. 1-7) is described on pages 28-42. We have already obtained some results, and theseresults were published at medical conferences and on scientific papers as shown on pages 43-46 (papers
published in 2015 were put in full paper).
Among these researches it is particularly outstanding that new strains of New Delhi metalo-βlactactamase producer were identified among the nosocomial infection cases and named NDM-8, 12 and
13.

26

IOM-NCGM Research Collaboration Office Annual Report 2015

Table 2		
No.

Collaborative researches in NCGM-IOM Collaboration Center, Nepal
Chief Researchers in Japan and Nepal

Affiliation in Nepal

Subject

Sourceof fund1• Hiroshi Ohara (Bureau of International • I n s t i t u t e o f M e d i c i n e , Assessment of the interface
Medical Cooperation, NCGM)
Tribhuvan University
between malaria control• Jeevan B Sherchand (Dept. of Public Health, • F a c u l t y o f M e d i c i n e , program and health system
Insitutie of Meidicne, Tribhuvan University)
Kathandu University
strengthening

24-5*

2

• Teruo Kirikae (Research Institute,NCGM)
Molecular epidemiology of• Institute of Medicine,• Bharat M. Pokhrel (Dept. of Microbiology,
nosocomial pathogens in
Tribhuvan University
Institute of Medicine, Tribhuvan University)
developing countries

24-5*

3

Evaluation of changing• Norio Ohmagari, Kayoko Hayakawa (Disease
epidemiology of infectious
Control and Prevention Center, NCGM)• S c h o o l o f M e d i c i n e , diseases in a developing• Jatan Sherchan (School of Medicine,
Kathmandu University
country: The role of healthcare
Kathmandu University)
associated opportunistic
infections

24-5*

4

• Naohiko Masaki (The Research Center for
Hepatitis and Immunology, NCGM)
The survey of the present status• Institute of Medicine,• Pradeep K. Shrestha(Dept. of Internalof viral hepatitis treatment in
Tribhuvan University
Medicine, Teaching Hospital, Tribhuvan
Nepal
University)§5• Hiroshi Ohara (Bureau of International
Fact-finding survey ofMedical Cooperation, NCGM)
nosocomial infection control• Institute of Medicine,• Bharat M. Pokhrel (Dept. of Microbiology,
in major hospitals in Nepal
Tribhuvan University
Institute of Medicine, Tribhuvan University,and discussion on effective
Nepal)
improvement plans

25-7*

6

Studies on enhancement of
human resource development• Hiroshi Ohara (Bureau of International, • I n s t i t u t e o f M e d i c i n e ,utilizing the IOM-NCGM
Medical Cooperation, NCGM)
Tribhuvan University,
Collaboration Center - taking• Bharat M. Pokhrel (Dept. of Microbiology, • S c h o o l o f M e i c i n e ,into consideration of theInstitute of Medicine, Tribhuvan University)
Kathmandu University
dissemination of the benefits of
ODA project

25-5*

7

• Chieko Matsubara(Bureau of International • I n s t i t u t e o f M e d i c i n e ,Fact-finding study of antibiotic
Medical Cooperration, NCGM)
Tribhuvan University,
stewardship programs in Nepali• Bharat M. Pokhrel (Dept. of Microbiology, • S c h o o l o f M e i c i n e ,Hospitals
Institute of Medicine, Tribhuvan University)
Kathmandu University

27-4** grants from the International Health Cooperation Research, a grant from the Ministry of Health, Labor and Welfare of Japan§ Grant of Japan Society for the Promotion of Science : Scientific Research (B) (Overseas Academic Research) to NM

IOM-NCGM Research Collaboration Office Annual Report 201527

Research No.1
1.

Title(in English)

Assessment of the interface between malaria control program and health systemstrengthening

2.

Title(in Japanese)

マラリア対策とヘルスシステム強化に関する研究

3.

Main researcher

Hiroshi Ohara (Bureau of International Medical Cooperation, National Center for Global
Health and Medicine)4.

Co-Researcher(s)Jeevan B. Sherchand (Dept. of Public Health, Institute of Medicine, Tribhuvan University,
Nepal)
Jatan B. Sherchan (Dept. of Microbiology, Faculty of Medicine, Kathmandu University)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (24-5)

6.

Affiliation(s) in Nepal

Department of Public Health, Institute of Medicine, Tribhuvan University,

7.

Period of the research

January 2012- March 20168.

Publications in FY
2015

Ohara H, Sherchand JB, Pokhrel BM, et al. Assessment of health systems in relation
to interface between malaria control programs and health system strengthening:
comparative study between Nepal and Viet Nam. J Inst Med, 37(1): 11-20, 2015.
Ohara H, Sherchand JB, Vu HN et al. Assessment of malaria control programs in relation
to general health systems with special reference to equity in bed net use. NCGM
report, available at:
http://ncgmimcj.ecnet.jp/HP/library/research-_doc/ncgm_report_oct 2013.pdf

9.

Summary:
Malaria has been a high priority issue in many tropical and sub-tropical countries. In order to implement malaria
control program effectively, it is crucial to utilize health system effectively. In this study, interactions between
malaria control program and health system strengthening was assessed.
The studies were conducted in Nepal and Vietnam with the methods of key informant interviews, investigationin malaria endemic areas and document review. As retrospective study, encountered challenges in malaria control
and interventions for them were analyzed from the viewpoint of interactions between disease specific programand general health system using the 6 Building Blocks of Health System Strengthening of WHO (Leadership andGovernance, Service delivery, Workforce, Information system, Medical products and technologies, and Financing).
In addition, current challenges in malaria control were identified and possible interventions were discussed.
In Nepal, malaria was showing high morbidity and mortality rate until the middle of 1990s, however thereafter
it decreased remarkably due to the effective control program. Leading factors contributed to the successful control
were identified as the best practices.
The followings were recognized as leading current challenges in malaria control in Nepal: 1) Increase of malaria
in some areas associated with population movement, 2) Shortage of health manpower in remote areas, 3) Poorly
developed reporting system from the private health sector, 4) Difficulty in treatment due to increasing resistanceof P. falciparum to anti- malaria drugs,
distribution.

285) Low incentive for health workers, 6) existence of inequity of bednets

IOM-NCGM Research Collaboration Office Annual Report 2015

In Vietnam, leading good practices included: 1)Strong government commitment for malaria control, 2)National
strategy for rural development and intensified education for residents, 3)Effective vertical system from national tovillage level for malaria surveillance and service delivery, 4)Domestic antimalarial production and high coverage ofcontrol measures, 5)Strengthening the capacity of health workers along with mobilization of mass organizations,and 6)Support from international organizations.
Effective implementation under the strong leadership of the governments utilizing the existing health system
was outstanding in both countries. Besides, strengthening of the vertical health program appeared to have a good
impact on the general health system, particularly at the primary level.
We made an in-depth study in 4 districts in Terai areas in Nepal with the aim to examine variation in utilizationof bednets by socioecomomic groups and inequities in access to malaria control services. This study revealed thewider disparity and pro-rich inequities in ownership of bednets. In area without bednet intervention, ownershipwas significantly higher in the rich households. There was significant variation in bednet ownership across caste/ethnic groups. Disparity in ownership between the poorest and richest group appeared to be smaller in areawith bednet intervention and people equally use bednets irrespective of caste and ethnic background. Free
mass distribution of bednets allowed equitable ownership and reduce the inequality in usage of bednets acrosssocioecomomic groups.
The results suggested that if provided freely, bednet distribution program will be an important opportunity to
reduce socioecomomic inequity in usage by allowing equitable ownership among the households of malaria risk
area.

IOM-NCGM Research Collaboration Office Annual Report 201529

Research No.2
1.

Title (in English)

Molecular epidemiology of nosocomial pathogens in developing countries

2.

Title (in Japanese)

開発途上国の医療機関で分離される多剤耐性菌の推移に関する研究

3.

Main researcher

Teruo Kirikae (Department of Infectious Diseases, Research Institute, National Center for
Global Health and Medicine)4.

Co-Researcher(s)Bharat M. Pokhrel (Dept. of Microbiology, Institute of Medicine, Tribhuvan University,
Nepal)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (24-5)

6.

Affiliation(s) in Nepal

Department of Microbiology, Institute of Medicine, Tribhuban University,

7.

Period of the research

April 2012- March 20158.

Publications
In FY 2015

1.
2.
3.

9.

Summary:
Emergenece of multidrug-resistant pathogens has become one of the most serious problems in medical settings
worldwide. There are serious concerns about dissemination of multidrug-resistant nosocomial pathogens in Nepal.
We started a study project of drug resistant pathogens isolated from inpatients hospitalized in Tribuvan
University Teaching Hospital, Kathomandu, Nepal, in collaboration between Department of Microbiology, Instituteof Medicine, Tribhuvan University and National Center for Global Health and Medicine from April 2012. Professor
Dr. Bharat M. Pokhrel, his colleagues and his students obtained a total of 308 Gram-negative isolates by November
2014, including Acinetobactor baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Providencia rettgeri, Serratia marcesces and Stenotrophomonas maltophilia isolates.
Of E. coli clinical isolates, we detected a novel NDM-type metallo-β-lactamase variant, NDM-13. A novel New

Int J Antimicrob Agents. 2015, 46(5):526-531
Microb Drug Resist. 2015, in press
Antimicrob Agents Chemother. 2015, 59(9):5847-5850

Delhi metallo-β-lactamase, NDM-13, was identified in a carbapenem-resistant Escherichia coli clinical isolate
obtained from the urine of a patient in Nepal. The enzymatic activity of NDM-13 against β-lactams was similar
to that of NDM-1. However, NDM-13 displayed significantly higher k cat/Km ratios for cefotaxime. The genetic
environment of bla NDM-13 was determined to be tnpA-IS30-blaNDM-13-bleMBL-trpF-dsbC-cutA-groES-groL, with
blaNDM-13 located within the chromosome.
Of S. marcescens clinical isolates, we detected a novel 6′-N-aminoglycoside acetyltransferase-encoding gene,aac(6′)-Ial. The encoded protein AAC(6′)-Ial has 146 amino acids, with 91.8% identity to the amino acid sequenceof AAC(6′)-Ic in S. marcescens SM16 and 97.3% identity to the amino acid sequence of AAC(6′)-Ian in S. marcescens
WW4. The minimum inhibitory concentrations of aminoglycosides for E. coli expressing AAC(6′)-Ial were similar to
those for E. coli expressing AAC(6′)-Ic or AAC(6′)-Ian. Thin-layer chromatography showed that AAC(6′)-Ial, AAC(6′)Ic, or AAC(6′)-Ian acetylated all the aminoglycosides tested, except for apramycin, gentamicin, and lividomycin.
Kinetic assays revealed that AAC(6′)-Ial is a functional acetyltransferase against aminoglycosides. The aac(6′)-Ialgene was located on a chromosomal DNA.
To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting
in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii
by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by
30

IOM-NCGM Research Collaboration Office Annual Report 2015

MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II.
Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequencetypes were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%)
were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones
but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as blaNDM-1,
blaOXA-23 or blaOXA-58. MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel
clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes
encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA).

IOM-NCGM Research Collaboration Office Annual Report 201531

Research No.3
1.

Title(in English)

Evaluation of changing epidemiology of infectious diseases in a developing country: The
role of healthcare associated opportunistic infections

2.

Title(in Japanese)

途上国における感染症の変貌と要因に関する研究 - 特に医療に関連した日和見感染に関する検討 -

3.

Main researcher

Norio Ohmagari (Disease Control and Prevention Center, National Center for Global
Health and Medicine)4.

Co-Researcher(s)Jatan Sherchan (Department of Medical Microbiology,
Kathmandu University, School of Medical Sciences, Nepal)
Kayoko Hayakawa (Disease Control and Prevention Center, National Center for Global
Health and Medicine)
Maki Nagamatsu (Disease Control and Prevention Center, National Center for Global
Health and Medicine)
Tohru Miyoshi-Akiyama (Pathogenic Microbe Laboratory, Research Institute, National
Center for Global Health and Medicine)5.

Resource of fund

6.

Affiliation(s) in Nepal

Department of Medical Microbiology,
Kathmandu University, School of Medical Sciences

7.

Period of the research

September 2012- March 20158.

Publications in FY
2015

Clinical epidemiology and molecular analysis of extended spectrum-β-lactamaseproducing Escherichia coli in Nepal: characteristics of sequence types 131 and 648.
Sherchan JB, Hayakawa K, Miyoshi-Akiyama T, Ohmagari N, Kirikae T, Nagamatsu
M, Tojo M, Ohara H, Sherchand JB, Tandukar S. Antimicrob Agents Chemother.
2015;59(6):3424-32.

9.

Summary:
Recently, CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains have emerged
worldwide. In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often
associated with the CTX-M-15 ESBL, has been increasingly reported globally; however, epidemiology reports on
ESBL-producing E. coli in Asia are limited.
Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in
Kathmandu, Nepal, were included in this study. Whole-genome sequencing of the isolates was conducted toanalyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of
acquired drug resistance genes. During the study period, 105 patients with ESBL-producing E. coli isolation wereidentified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n =54; 51.4%), followed by ST648 (n = 15; 14.3%). All ST131 isolates were identified as O25b-ST131 clones, subclone
H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses. ST64isolates
had a proportionally higher resistance to non-β-lactam antibiotics and featured drug-resistant genes more
frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648.
The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were

32

IOM-NCGM Research Collaboration Office Annual Report 2015

from the outpatient clinic, and pregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed
that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by
CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern.

IOM-NCGM Research Collaboration Office Annual Report 201533

Research No.4
1.

Title(in English)

The survey of the present status of viral hepatitis treatment in Nepal

2.

Title(in Japanese)

ネパールにおけるウイルス肝炎治療に関する実態調査

3.

Main researcher

Naohiko Masaki, MD, PhD (The Research Center for Hepatitis and Immunology, National
Center for Global Health and Medicine)4.

Co-Researcher(s)Pradeep K. Shrestha (Dept. of Internal Medicine, Teaching Hospital, Tribhuvan University)

5.

Resource of fund

Grant of Japan Society for the Promotion of Science : Scientific Research (B) (Overseas
Academic Research) to NM

6.

Affiliation(s) in Nepal

Department of Internal Medicine, Teaching Hospital, Tribhuvan University

7.

Period of the research

April 2015-March 20188.

P u b l i c at i o n s i n F Y
2015Masaki N, Shrestha PK, Nishimura S, Ito K, Sugiyama M, Mizokami M.
Use of nucleoside analogs in patients with chronic hepatitis B in Nepal: A prospective
cohort study in a single hospital.
Hepatol Res 45(12):1163-1169, 2015.

9.

Summary:
According to WHO estimate, there are globally 240 millions and 150 millions of hepatitis B virus (HBV) andhepatitis C virus (HCV) carriers, respectively. Especially, most of the carriers are residents of Asian and African
developing countries suffering from poor socio-economical problems and unstable political affairs, where theincrease in numbers of death due to liver cirrhosis and hepatocellular carcinoma has been a serious burden on
public health. Of note is that the people in those areas have been obliged to be far from the benefit of recent
progress in diagnostic and therapeutic modalities, which, on the other hand, are regarded as universal in thedeveloped countries.
We had performed epidemiological survey of HBV and HCV from 2007 to 2012 in Asia (Vietnum, Thailand,
Nepal, Philippins, Taiwan, China, Parkistan, Uzbekistan, Indonesia, Bangladesh) and in Africa (Egypt, Kenya), andreported that 1) relatively cheaper generic drugs of nucleoside analogs for HBV were produced and distributedin those Asian countries, 2) use of expensive interferons for HCV was confined to only wealthy people, except for
Egypt where interferon therapy was introduced as national policy, 3) there were many countries where domestic
determinations of genotypes as well as viral load of HBV or HCV, which are essential to diagnosis and treatment ofchronic liver diseases, were not available, and finally 4) doctors’ prescriptions were not always mandatory for thepatients to buy the anti-viral agents at pharmacies in those developing countries, where health insurance systems
are not established, resulting in low drug adherence.
Our new research project will attempt to clarify the present status of public health administration in charge ofviral hepatitis as well as of use of recently introduced anti-viral agents in clinical settings, in Nepal.
The objectives are as follows:
1. Epidemiological studies of HBV or HCV carriers, including HIV co-infection,among general populations
2. Prevalence of viral genotypes and their chronological changes in patients with chronic hepatitis B and C
3. Use of recently introduced anti-viral agents (nucleoside analogs such as lamivudine, adefovir, entecavir,
tenofovir for HBV; directly-acting anti-virals [inhibitors of NS3/4A, NS5A and NS5B] for HCV) and their
virological effects, including domestic coverage of virological monitoring systems, quality and commercial
cost of anti-viral agents, medical expenses for standard regimens

34

IOM-NCGM Research Collaboration Office Annual Report 2015

4.
5.

Emerging rates of drug-resistant HBV or HCV in real clinical settings
Coverage of universal hepatitis B vaccination, and evaluation of its effect

6.

Present status of HBV reactivation and the preventive strategies against it: prevalence of anti-HBs and antiHBc in blood donors
Serial determinations of whole genome sequence of HBV and HCV by next generation sequencer (deep7.
8.

sequencing), if applicable
A questionnaire survey of public health administration in charge of viral hepatitis and of the Nepalese Societyof Hepatology and Gastroenterology, would be scheduled in the fiscal year 2016.

Our research project was already approved by the Ethical Committees, TUTH, Tribhuvan University, Nepal aswell as National Center for Global Health and Medicine, Japan. Collection of patients’ serum samples and clinicalinformation have just started since December, 2015.

IOM-NCGM Research Collaboration Office Annual Report 201535

Research No.5
1.

Title(in English)

Fact-finding survey of nosocomial infection control in major hospitals in Nepal andplanning of effective improvement

2.

Title(in Japanese)

ネパールの主要病院における院内感染対策の実情分析と効果的な改善策に関する検討

3.

Main researcher

Hiroshi Ohara, Chieko Matsubara (Bureau of International Medical Cooperation, National
Center for Global Health and Medicine)4.

Co-Researcher(s)Pokhrel BM, Shrestha RK, Dahal RK, Mishra SK, Kattel HP, Rijal BP (Dept. of Microbiology,
Institute of Medicine, Tribhuvan University, Nepal)
Jeevan B. Sherchand (Dept. of Public Health, Institute of Medicine, Tribhuvan University,
Nepal)
Shreshta DL (Dept. of Nursing Management, Tribhuvan University, Nepal)
Teruo Kirikae, Yumiko Haneishi (Bureau of International Medical Cooperation, National
Center for Global Health and Medicine)5.

Resource of fund

Grants of National Center for Global Health and Medicine (25-7, 27-4)

6.

Affiliation(s) in Nepal

Department of Microbiology, Institute of Medicine, Tribhuban University,

7.

Period of the research

September 2012- March 20168.

P u b l i c at i o n s i n F Y
2015Oral presentation
88th General Assembly of the Japanese Society of Infectious Diseases, June 2014,
Fukuoka, Japan
Scientific Paper
J Inst Med 2014, 36(3): 38-48.

9.

Summary:
In developing countries, where the incidence of infectious diseases is high and environmental conditions ofhealthcare facilities are poor, nosocomial infections may frequently occur. Effective nosocomial infection control
is crucial in the healthcare facilities of developing countries, but in actual fact, attention to it is still limited andcontrol measures are not functioning well in many countries. This study has been conducted with the purpose
to investigate the actual conditions of nosocomial infection control in Kathmandu City, Nepal as a basis for the
possible contribution to its improvement.
1.

Fact-finding survey of nosocomial infection control in hospitals in Kathmandu, Nepal- a basis for

improvement
The actual condition of nosocomial infection control were examined at the TUTH and five other referral hospitals
in Kathmandu City with the method of questionnaire survey and interview. The obtained results were comparedwith the results of the last survey (2012-2013).
Both the frequency of the meetings of nosocomial infection control committee and the frequency of ICT
rounds by infection control teams increased. Also, some improvement in the monitoring of causative agents andin the information provision system for clinical settings were recognized. Training for newly employed nurses
on nosocomial infections control was enhanced and provided to all newly hired nurses in TUTH. Issues to be
36

IOM-NCGM Research Collaboration Office Annual Report 2015

addressed included improving the quality of the control system and training program, providing training to awider range of staff members, improving waste disposal system, updating manuals, etc.
These findings clearly reflect that there is a need of further improvement of nosocomial infection control
program in the hospitals of capital. Moreover, nosocomial infection control program should also have aim toestimate mortality, morbidity, additional financial burden and length of stay in the hospital due to nosocomial
infection for public awareness.
1)

KAP Study on Medical Personnel regarding Nosocomial Infection Control

This study was carried out with the aim to assess the level of awareness and actual practice of “hand-washing",
which is a basic prevention method of nosocomial infection control among medical personnel, and then use theobtained results for improvement. 163 medical personnel (doctors, nurses and laboratory technicians) at Tribhuvan
University Teaching Hospital (TUTH) were subjects of this study. A questionnaire, direct observation at important
departments, and discussions were used. The following results were obtained
Knowledge: 74.1% of doctors had an accurate knowledge of hand-washing (significance, timing, methods andeffects), while only 19.7% of nurses and 50.0% of laboratory technicians had such knowledge.
Attitude: 83.3% of laboratory technicians, 59.3% of nurses and 29.6% of doctors considered that they had an
accurate knowledge. Regarding motive of hand-washing, fear for infections during medical practices accounted fora relatively high proportion (55.5% in doctors). Reasons given for not practicing hand-washing as instructed were:“I was busy” (46.0%), “I thought it wasn’t necessary because I wore gloves” (33.1%), “Something urgent came up”(11.7%). 38.7% answered that hand-washing is important. Poor levels of hand-washing practice among newly hired
personnel and the importance of education for newly hired staff members were indicated.
Practice: The following results were obtained regarding the degree of hand-washing in actual practice: “Prior tocontact with patients” (55.8%), “After contact with patients” (97.5%), and “At the end of work" (96.1%). Practicelevels were higher in nurses than in doctors. Drying and wiping methods were: hand dryer (52.7%), shared-use
towels (22.7%), personal towel or handkerchief (12.3%), and natural drying (3.7%).
The study indicated that doctors at TUTH have a relatively good knowledge regarding hand-washing, but do
not follow it in actual practice. A large number of nurses and laboratory technicians lack knowledge, and have a
low degree of hand-washing in actual practice prior to contact with patients. It is important to provide improved
training for medical staff regarding nosocomial infection control and prevention, which are mainly based onstandard precautions.

IOM-NCGM Research Collaboration Office Annual Report 201537

Research No.6
1.

Title(in English)

Studies on enhancement of human resource development utilizing the IOM-NCGM
Collaboration Center — taking into consideration of the dissemination of the benefits of
ODA project2.

Title(in Japanese)

ネパール拠点を活用した人材育成能力強化に関する研究 - ODA プロジェクトの成果拡大を視野に入れて3.

Main researcher

Hiroshi Ohara (Bureau of International Medical Cooperation, National Center for Global
Health and Medicine)4.

Co-Researcher(s)Bharat M. Pokhrel
Jeevan B. Sherchand (Dept. of Public Health, Institute of Medicine, Tribhuvan University,
Nepal)
Karbir N. Yogi (Dept. of Pulmonology, TUTH)
Mitsuhiro Kamimura (Dept. of Pulmonology, National Disaster Medical Center)
Pradeep Shrestha (Dept. of Internal Medicine, TUTH)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (25-5)

6.

Affiliation(s) in Nepal

Department of Public Health, Institute of Medicine, Tribhuvan University,

7.

Period of the research

April 2013- March 20158.

Publications in FY
2015

Annual Report of the IOM-NCGM Collaboration Office in 2015 was made.

9.

Summary:
Collaborative researches are being conducted in Nepal utilizing the IOM-NCGM Collaboration Office as abase. Strengthening of the base functions is essential to conduct researches smoothly. This study was started tostrengthen the function of the IOM-NCGM Collaboration Office aiming at smooth implementation of researches
and human resource development.
In FY 2014 the following activities were conducted.
1.

Strengthening of management capacity of IOM-NCGM Collaboration Office:Management system to conduct researches/activities was clarified and some instructions were conducted2.

to local staff. Management of the office and liaison with other study groups have been carried out.
Comparative study on outcomes of Medical Education Project and current IOM/TUTH:

3.

The overview of the Medical Education Project, which was implemented as Official Development Assistanceby JICA from 1980 to 1996, was summarized and the situations of IOM/TUTH at the end of the project andthe present time were compared.
Improvements in Human Resource Development:
Planning, implementation and analysis (all or in part) of the following studies were carried out with thecounterpart departments, contributing to human resource development by improving study and training
capacity.

38

IOM-NCGM Research Collaboration Office Annual Report 2015ØØ Regarding “Knowledge, Attitude and Hand washing Practices among Health Care Professionals Working in
Teaching Hospital, Kathmandu, Nepal”, the main study carried out by the Department of Study and Training,
instructions were provided on data analysis and report preparation.
ØØ Data analysis and report preparation of the “Study on Malaria Control and the Equity in Bed-Net Distribution”
by the Department of Public Health were carried out with the counterpart departments.
ØØ In the “Study on Pulmonary Fibrosis using Diagnostic Imaging” carried out by Pulmonary Medicine, support
was given regarding radiologic interpretation and data input, contributing to human resource development.
ØØ Planning of “Training for Newly Employed Nurses on Measures for Nosocomial Infections” scheduled to be
conducted at Nursing Department was carried out.

IOM-NCGM Research Collaboration Office Annual Report 201539

Research No.7
1.

Title(in English)

Fact-finding study of antibiotic stewardship programs in Nepali Hospitals

2.

Title(in Japanese)

ネパールの病院における抗生剤管理の現状調査

3.

Main researcher

Chieko Matsubara, Hiroshi Ohara (Bureau of International Medical Cooperation, National
Center for Global Health and Medicine)4.

Co-Researcher(s)Jeevan Bahadur Sherchand (Dept. of Public Health, Institute of Medicine, Tribhuvan
University, Nepal)
Bharat Mani Pokhrel (Dept. of Public Health, Institute of Medicine, Tribhuvan University,
Nepal)
Jatan Sherchan (Dept. of Microbiology, Faculty of Medicine, Kathmandu University)

5.

Resource of fund

Grants of National Center for Global Health and Medicine (25-5, 27-4)

6.

Affiliation(s) in Nepal

Department of Public Health, Institute of Medicine, Tribhuvan University,

7.

Period of the research

December 20158.

Publications in FY
2015

In preparation for FY 2016

9.

Summary:
Antibiotic resistance is a global emerging threat in public health issue. New multiple drug-resistant pathogens werereported in Nepal, however, few studies is publicly available to evaluate antibiotic stewardship programs in Nepali
hospitals. The objective of this study is to investigate the actual conditions of antibiotic stewardship programs in
Kathmandu City, Nepal.
We are going to conduct a questionnaire survey and site visits at hospitals in Kathmandu City. Preferably, datacollection will cover various levels of hospitals (for example, academic hospitals and non-academic hospitals).
Potential interviewees are a director of antibiotic team and a responsible person of antibiotics in the hospital, e.g.
a medical doctor, a pharmacist, and a microbiologist. We hope to conduct this study for 1-2 weeks in the middle ofDecember, 2015.
Major expected outcomes are to clarify the type of interventions implemented in hospitals for antibiotic
stewardship programs. The outcomes would reveal the actual conditions of antibiotic stewardship programs andwould contribute as a basis to decide/consider future plans for improvement in antibiotic stewardship at Nepali
hospitals.
In total 27 health facilities participated in this survey (Primary level: 6 facilities, Secondary level: 2 facilities, andTertiary level: 19 facilities) and two of them skipped some questions. Among 21 secondary and tertiary hospitals,
6 facilities provided Infectious Control Team (ICT) and 2 facilities provided antibiotics committee. The 19 among
25 health facilities agreed that antibiotic resistance became an important issue. Among 17 secondary and tertiary
hospitals, 15 facilities offered 24-hours laboratory services and conducted at least one blood-culture test everyday. Further among 17 secondary and tertiary hospitals, 14 facilities offered 24-hours pharmacy services.

40

IOM-NCGM Research Collaboration Office Annual Report 2015

The results suggested that intervention for antibiotic stewardship is necessary to be introduced as soon as
possible. Most of hospitals started 24-hours service at microbiology department and pharmacy. Therefore, if thehospitals developed their hospital system to connect ICT, clinical departments, microbiology department, andpharmacy, they will ensure a successful use of antibiotic resistance information as a shared information to properuse of antibiotic resistance, which will contribute to reduce antibiotic resistance. Further intervention is called
for antibiotic stewardship in Primary Health Centers and Health Posts, where there are no microbiologists andpharmacitis.

IOM-NCGM Research Collaboration Office Annual Report 201541

Attachment

Publication of scientific papers
Scientific papers which were published on international journals as results of collaborative studies during
the period from 2012 to 2015 are shown below.
Full papers published in 2015 are put in this attachment.
Table 3				

List of papers published on international journals (papers in press are included)
2012-2015: Achievement in NCGM-IOM Collaboration Office◎ Full papers are put in this annual report○ Only abstract is put in this annual report

Papers421

Sherchan JB, Ohara H, Sakurada S, Basnet A, Tandukar S, Sherchand JB, Bam DS. Enteric
opportunistic parasitic infections among HIV-seropositive patients in Kathmandu, Nepal.
Kathmandu Univ Med J 2012; 38(2):14-17.

○

2

Sherchan JB, Ohara H, Sherchand JB, Tandukar S, Sakurada S, Gurung B, Ansari S, Rijal BP, Pokhrel BM.
Molecular evidence based hospital acquired rotavirus gastroenteritis in Nepal. Prime J Mirobobiol Res
2011; 1(2): 16-21.

○

3

Shrestha S, Chaudhari R, Karmacharya S, Kattel HP, Mishra SK, Dahal RK, Bam N, Banjade N, Rijal BP,
Sherchand JB, Ohara H, Koirala J, Pokhrel BM. Prevalence of nosocomial lower respiratory tract infections
caused by multi drug resistant pathogens. J Inst Med 2012; 33(2): 7-14.

○

4

Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Shimada K, Kirikae T, Pokhrel BM. NDM-8 metalloβ-lactamase in a multidrug-resistant Escherichia coli strain isolated in Nepal. Antimicrob Agents Chemother2013; 57(5): 2394-2396.

○

5

Shrestha RK, Dahal RK, Mishra SK, Parajuli K, Rijal BP, Sherchand JB, Kirikae T, Ohara H, Pokhrel BM.
Ventilator associaed pneumonia in tertiary care hospital, Maharajgunj, Kathmandu, Nepal. J Inst Med 2013;35(3): 21-28.

〇

6

Ohara H, PokhrelBM, DahalRK, Mishra SK, Kattel HP, Shrestha DL, Haneishi Y, Sherchand JB. Fact-finding
survey of nosocomial infection control in hospitals in Kathmandu, Nepal and trial to improvement. Tropical
Med Health 2013; 41:113-119.

〇

7

Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Shimada K,  Kirikae T, Pokhrel BM. NDM-1 metalloβlactamase and ArmA 16S rRNA methylase producing Providencia rettgeri clinical isolates in Nepal. BMC
Infect Dis 2014; 14:56-60〇

8

Tada T, Miyoshi-Akiyama T, Dahal RK, Mishra SK, Ohara H, Shimada K, Kirikae T, Pokhrel BM. Disseminationof multidrug-resistant Klebsiella pneumoniae clinical isolates with various combinations of carbapenemases(NDM-1 and OXA-72) and 16S rRNA methylases (ArmA, RmtC and RmtF) in Nepal. Int J Antimicrob Agents2014; 42(4):372-374.

〇

9

Tada T, Shrestha B, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T, Pokhrel BM. NDN-12, a Novel
New Delhi Metallo-β-Lactamase Variant from a Carbapenem-Resistant Escherichia coli Clinical
Isolate in Nepal. Antimicrob Agents Chemother 2014; 58(10):6302-6305.

〇

IOM-NCGM Research Collaboration Office Annual Report 2015

Attachment

Papers10

Tada T, Miyoshi-Akiyama T, Dahal RK, Shyam MK, Shimada K, Ohara H, Kirikae T, Pokhrel BM. Identificationof a Novel 6’-N-Aminoglycoside Aetyltransferase, AAC(6’)-Iak from a Multidrug-resistant Clinical Isolates of
Stenotrophomonas maltophilia. Antimicrob Agents Chemother 2014; 58(10):6324-6327.

〇

11

Sah MK, Mishra SK, Ohara H, Kirikae T, Sherchand JB, Rijal BP, Pokhrel BM. Nosocomial bacterial infection
and Antimicrobial resistant Pattern in a tertiary Care hospital in Nepal. J Inst Med 2014; 36(3): 38-48.

〇

12

Sherchan JB, Hayakawa K, Miyoshi-Akiyama T, Ohmagari N, Kirikae T, Nagamatsu M, Tojo M, Ohara
H, Sherchand JB, Tandukar S. Clinical epidemiology and molecular analysis of extended-spectrum
β-lactamase (ESBL)-producing Escherichia coli in Nepal: Characteristics of sequence types 131 and648. Antimicrob Agents Chemother 2015; 59(6): 3424-3432.

◎

13

Ohara H, Sherchand JB, Pokhrel BM, Hirayama T, Nam VH, Sherchan JB. Assessment of health systems in
relation to interface between malaria control programs and health system strengthening: Comparative
study between Nepal and Viet Nam. J Inst Med 2015; 37(1): 11-20.

◎

14

Shrestha S, Tada T, Miyoshi-Akiyama T, Ohara H, Shimada K, Satou K, Teruya K, Nakano K, Shiroma A,
Sherchand JB, Rijal BP, Hirano T, Kirikae T, Pokhrel BM. Molecular epidemiology of multidrug-resistantAcinetobacter baumannii isolate in a university hospital in Nepal reveals the emergence of a novel
epidemic clonal lineage. Int J Antimicrob Agents 2015; 46: 526-531.

◎

15

Shrestha B, Tada T, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T, Pokhrel BM. Identification of a novel
NDM variant, NDM-13, from a multidrug-resistant Escherichia coli clinical isolate in Nepal. Antimicrob
Agents Chemother 2015; 59(9): 5847-5850.

◎

16

Tada T, Miyoshi-Akiyama T, Shimada K, Dahal RK, Mishra SK, Ohara H, Kirikae T, Pokhrel BM. A novel 6’-N
aminoglycoside acetyltransferase, AA(6’)-lai, from a clinical isolate of Serratia marcescens. Microbial Drug
Resist 2015. (in press)◎

17

Masaki N, Shrestha PK, Nishimura S, Ito K, Sugiyama M, Mizokami M. Use of nucleoside analogs in patientswith chronic hepatitis B in Nepal: a prospective cohort study in a single hospital. Hepatology Research
2015; 45: 1163-1169.

◎

18

Sherchand JB. Earthquake disaster-associated health effects and the need for improved preventive
measures. J Inst Med 2015; 37(1): 1-3.

◎

IOM-NCGM Research Collaboration Office Annual Report 201543

Attachment

KATHMANDU UNIVERSITY MEDICAL JOURNAL

Enteric Opportunistic Parasitic Infections Among HIVSeropositive Patients in Kathmandu, Nepal
Sherchan JB,1 Ohara H,2 Sakurada S,2 Basnet A,3 Tandukar S,3 Sherchand JB,3 Bam
D S4Kathmandu University School of Medical Sciences,
Dhulikhel, Nepal

1

National Center for Global Health and Medicine,Tokyo, Japan2

Tribhuvan University Teaching Hospital, Public
Health Research Laboratory, Kathmandu, Nepal

3

Dirgh-Jeevan Health Care Research Center,
Tripureswar, Kathmandu, Nepal

4

ABSTRACT
Background
Enteric opportunistic parasitic infections are the major source of diarrheal disease
in developing countries mainly in Human Immunodeficiency virus (HIV) infectedpatients.
Objective
The study was to detect enteric parasites causing diarrhea and their association
with immune status in HIV-seropositive patients.
Methods

Corresponding Author
Jatan B. Sherchan
Department of Microbiology
Kathmandu University School of Medical Sciences

The present study was conducted in Dirgh-Jeevan Health Care Research Center
and Tribhuvan University Teaching Hospital, Public Health Research Laboratory,
Kathmandu, Nepal between June 2010 and May 2011 involving 146 Human
Immunodeficiency virus (HIV) positive patients. Serostatus from these patientswere detected by Enzyme Linked Immunosorbent assay. CD4+ T cell counts weredone by flow cytometry. Stool was examined for enteric parasites by microscopy
with special staining methods.

Dhulikhel, Nepal

Results

Email: jatansherchan@gmail.com

A total of 146 HIV sero-positive patients with and without diarrhea age between 20
to 45 years were included in the study. Of the 146 patients, the protozoan parasitic
infection was found in 30.13% (44/146). Out of 146 patients, 78 had diarrhea
in which parasitic infection was 39 (50%) and 7.35% (5/68) protozoal parasites
positive cases did not have diarrhea. A significant difference (p<0.05) was observedin the level of infection of intestinal protozoan between the HIV seropositive withdiarrhea and HIV-seropositive without diarrhea. Out of 43 patients whose CD4+ T
cells were <200/µl, 29 (67.4%) had opportunistic parasitic infection whereas out of
103 patients whose CD4+ T cells were ≥200/µl, only 15 (14.56%) had opportunistic
parasitic infection (P < 0.05).

Citation
Sherchan JB, Ohara H, Sakurada S, Basnet A3,
Tandukar S, Sherchand JB, et al. Enteric Opportunistic Parasitic Infections Among HIV-seropositive
Patients in Kathmandu, Nepal. Kathmandu Univ Med
J 2012;38(2):14-17.

Conclusion
Enteric opportunistic parasitic infections were detected in 30.1% among HIVseropositive patients and low CD4+ T count indicated high enteric opportunistic
infection. Early detection of enteric parasitic infections will help in the management
and to improve the quality of life for HIV-infected individuals.

KEYWORDS
Diarrhea, HIV, Opportunistic parasites

INTRODUCTIONEnteric opportunistic parasitic infections are major sourceof diarrheal disease in developing countries mainly in HIV
infected patients. The progressive decline and ultimate
destruction of immune system functions, which arecharacteristic for AIDS, usually result in morbidity andultimately death due to opportunistic bacterial, viral, fungiand parasitic infections.1 Gastrointestinal infections are very
common in patients with HIV infection or AIDS.2 Diarrhea
is a common clinical presentation of these infections.
Reports indicate that diarrhea occurs in 30-60 % of AIDS
patients in developed countries and in about 90 per centof AIDS patients in developing countries.3 The presence ofPage 1444

IOM-NCGM Research Collaboration Office Annual Report 2015

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IOM-NCGM Research Collaboration Office Annual Report 201545

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46

IOM-NCGM Research Collaboration Office Annual Report 2015

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IOM-NCGM Research Collaboration Office Annual Report 201547

Attachment

NDM-8 Metallo--Lactamase in a Multidrug-Resistant Escherichia coli
Strain Isolated in Nepal
Tatsuya Tada,a Tohru Miyoshi-Akiyama,a Rajan K. Dahal,c Manoj K. Sah,c Hiroshi Ohara,b Teruo Kirikae,a Bharat M. Pokhrelc
Department of Infectious Diseases, Research Institute,a and Department of International Medical Cooperation,b National Center for Global Health and Medicine, Toyama,
Shinjuku, Tokyo, Japan; Department of Microbiology, Institute of Medicine, Tribhuvan University, Maharajgunj, Kathmandu, Nepalc

A novel metallo--lactamase, NDM-8, was identified in a multidrug-resistant Escherichia coli isolate, IOMTU11 (NCGM37),
obtained from the respiratory tract of a patient in Nepal. The amino acid sequence of NDM-8 has substitutions at positions 130(Asp to Gly) and 154 (Met to Leu) compared with NDM-1. NDM-8 showed enzymatic activities against -lactams similar to
those of NDM-1.

M

etallo--lactamases (MBLs) produced by Gram-negative
bacteria confer resistance to all -lactams except monobactams (1). New Delhi metallo--lactamase-1 (NDM-1), a recently
discovered MBL, was initially isolated from Klebsiella pneumoniae
and Escherichia coli in 2008 in Sweden (2). Since then, NDM-1producing members of the Enterobacteriaceae have been isolated
in various parts of the world, including Australia, Bangladesh,
Belgium, Canada, France, India, Japan, Kenya, the Netherlands,
New Zealand, Pakistan, Singapore, Taiwan, and the United States(3, 4). In addition, isolates producing six NDM variants have beenreported, including NDM-2-producing Acinetobacter baumannii
strains from Egypt (5, 6), Israel (5), Germany (7), and the United
Arab Emirates (8); an NDM-3-producing E. coli strain from Australia (accession no. JQ734687); an NDM-4-producing E. coli
strain from India (9); an NDM-5-producing E. coli strain from theUnited Kingdom (10); an NDM-6-producing E. coli strain fromNew Zealand (11); and an NDM-7-producing E. coli strain from
Canada (accession no. JX262694).
E. coli IOMTU11 (NCGM37) and Pseudomonas aeruginosa
IOMTU9 (NCGM1841) were isolated from pus from a surgicalsite and from sputum of patients, respectively, in 2012 at Tribhuvan University Teaching Hospital in Kathmandu, Nepal. The isolates were phenotypically identified, and species identification was
confirmed by 16S rRNA sequencing (12). MICs were determinedusing the microdilution method recommended by the Clinicaland Laboratory Standards Institute (13). E. coli IOMTU11 wasresistant to all antibiotics tested excepted fosfomycin (MIC, 4
g/ml). The MICs of -lactams are shown in Table 1, and thoseof other antibiotics were as follows: arbekacin, 1,024 g/ml;
amikacin, 1,024 g/ml; colistin, 0.25 g/ml; gentamicin,
1,024 g/ml; and tigecycline, 0.5 g/ml. MBL production
was examined with an MBL Etest (Sysmex; bioMérieux Co.,
Marcy l’Etoile, France), with MICs of 256 g/ml of imipenem
and 2 g/ml of imipenem-EDTA. PCR analysis for MBL genes(14, 15, 16) and 16S rRNA methylase genes (17) was performed. The isolates were positive for blaNDM and rmtB. Sequence analysis showed that the blaNDM was a novel variant,and it was designated blaNDM-8. Multilocus sequence typing(MLST) of IOMTU11 showed that it was ST101 (Escherichia coli
MLST database [http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html]). P. aeruginosa IOMTU9 had blaNDM-1, which
was used as a reference gene.
The sequence of the blaNDM-8 gene showed mutations corre2394

48

aac.asm.org

sponding to two amino acid substitutions compared with
blaNDM-1 (accession number JF798502). Analysis of the predicted
amino acid sequence revealed two substitutions (D130G and
M154L) compared with NDM-1, one substitution (D130G) compared with NDM-4, and one substitution (L88V) compared with
NDM-5.
The blaNDM-8 and blaNDM-1 genes were cloned into the corresponding sites of pHSG398 (TaKaRa Bio, Shiga, Japan) with theprimer set EcoRI-NDM-F (5=-GGGAATTCATGGAATTGCCCA
ATATTATG-3=) and PstI-NDM-R (5=-AACTGCAGTCAGCGC
AGCTTGTCGGCCAT-3=). E. coli DH5 was transformed with
pHSG398-NDM-8 or pHSG398-NDM-1 to determine the MICsof -lactams.
The open reading frames of NDM-1 and NDM-8 without signal
peptide regions were cloned into the expression vector pQE2
(Qiagen, Tokyo, Japan) with the primer set SacI-NDM-F (5=-CCCC
TCGAGCAGCAAATGGAAACTGGCGACCAACGGT-3=) andSalI-NDM-R (5=-CCCGAGCTCTCAGCGCAGCTTGTCGGCC
ATGCGGGCC-3=). The plasmids were transformed into E. coli
BL21-CodonPlus (DE3)-RIP (Agilent Technologies, Santa Clara,
CA). The recombinant NDM proteins were purified using nickelnitrilotriacetic acid (Ni-NTA) agarose according to the manufacturer’s instruction (Qiagen). His tags were removed by digestion
with DAPase (Qiagen), and untagged proteins were purified by anadditional passage over Ni-NTA agarose. The purities of NDM-1
and NDM-8 were over 90%, as estimated by SDS-PAGE. During
the purification procedure, the presence of -lactamase activity
was monitored with nitrocefin (Oxoid Ltd., Basingstoke, United
Kingdom). Initial hydrolysis rates were determined in 50 mM
phosphate buffer (pH 7.0) at 25°C with a UV-visible spectrophotometer (V-530; Jasco, Tokyo, Japan). The Km and kcat values andthe kcat/Km ratio were determined by analyzing -lactam hydrolysis by use of the Lineweaver-Burk plot. Wavelengths and extinc-

Antimicrobial Agents and Chemotherapy

Received 20 December 2012 Returned for modification 13 January 2013
Accepted 23 February 2013
Published ahead of print 4 March 2013
Address correspondence to Teruo Kirikae, tkirikae@ri.ncgm.go.jp.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02553-12

p. 2394 –2396

IOM-NCGM Research Collaboration Office Annual Report 2015

May 2013 Volume 57 Number 5Attachment

21

Original Article

Ventilator Associated Pneumonia in Tertiary Care
Hospital, Maharajgunj, Kathmandu, Nepal
Shrestha RK*, Dahal RK*, Mishra SK*,Parajuli K*, Rijal BP*, Sherchand JB* , Kirikae T, Ohara H**, Pokhrel BM**Department of Microbiology, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal;     

Abstract
Introduction: Ventilator Associated Pneumonia (VAP) is the most common nosocomial
infection among intensive care unit (ICU) patients and lack of much information in Nepal.
                 ­with special reference to multi-drug resistant (MDR), Methicillin-resistant Staphylococcus            
producing bacterial strains.
Methods:                
Department of Microbiology, TUTH as described by American Society for Microbiology(ASM). Combination disk method was done for the detection of ESBL and MBL producing
isolates.
Results: ­  ­       
(44%) was the commonest isolate, followed by Klebsiellapneumoniae (22%), Pseudomonas
aeruginosa (16%) and Staphylococcus aureus (12%). Among MDR Gram negative bacteria(GNB), 39% were MBL and 33% were ESBL-producers. All GNB (61) were sensitive to­          Prevalence of MRSA was 75%, which were all sensitive to Vancomycin.
Conclusion: High prevalence of VAP, MDR along with MRSA or ESBL or MBL producing
strains was found in the study. Thus, suitable control measures must be adopted to cope up   
Key words: VAP, ICU, MDR, MRSA, ESBL, MBL.

Introduction
People with life-threatening injuries and illnesses needcritical care and mechanical ventilation is must. It is often
a life-saving intervention, but carries many potential      
alveolar damage, collapsed lung and ventilator-associatedpneumonia.1

ventilation.3 This is associated with increases in morbidity
and mortality, hospital length of stay, and costs.

           have resulted in emergence and rapid disseminationof Multi drug resistant (MDR), Methicillin Resistant          ­             
episode of pneumonia in a patient who requires a device producing bacteria. Thus, their detection is crucial for theto assist or control respiration through a tracheostomy optimal treatment of patients and to control the spread ofor endotracheal tube at the time of or within 48 hours resistance.So this study is intended to address the issues
before the onset of the infection.2     regarding the prevalence of VAP, MDR, ESBL-, MBLnosocomial pneumoni as are associated with mechanical producing bacterial isolates, and MRSA.

www.jiom.com.np

Journal of Institute of Medicine, December, 2013, 35:3 21-28

IOM-NCGM Research Collaboration Office Annual Report 201549

Attachment

Tropical Medicine and Health Vol. 41 No. 3, 2013, pp. 113-119
doi:10.2149/tmh.2013-03
Copyright 2013 by The Japanese Society of Tropical Medicine

113

Original article
TMHFact-finding Survey of Nosocomial Infection Control in Hospitals

in Kathmandu, Nepal—A Basis for Improvement
Hiroshi Ohara1*, Bharat M. Pokhrel2, Rajan K. Dahal2, Shyam K. Mishra2, Hari P. Kattel2, Dharma L. Shrestha3,
Yumiko Haneishi1 and Jeevan B. Sherchand2??

Received 10 January, 2013

Accepted 21 March, 2013

Published online 29 June, 2013

Abstract:
TheSociety
purposeofofTropicalthis study
was to investigate the actual conditions of nosocomial infection control in© 2013
Japanese
Medicine
Kathmandu City, Nepal as a basis for the possible contribution to its improvement. The survey was conducted at
17 hospitals and the methods included a questionnaire, site visits and interviews. Nine hospitals had manuals on
nosocomial infection control, and seven had an infection control committee (ICC). The number of hospitals that
met the required amount of personal protective equipment preparation was as follows: gowns (13), gloves (13),surgical masks (12). Six hospitals had carried out in-service training over the past one year, but seven hospitals
responded that no staff had been trained. Eight hospitals were conducting surveillance based on the results of bacteriological testing. The major problems included inadequate management of ICC, insufficient training opportunities for hospital staff, and lack of essential equipment. Moreover, increasing bacterial resistance to antibiotics wasrecognized as a growing issue. In comparison with the results conducted in 2003 targeting five governmental hospitals, a steady improvement was observed, but further improvements are needed in terms of the provision of highquality medical care. Particularly, dissemination of appropriate manuals, enhancement of basic techniques, andstrengthening of the infection control system should be given priority.
Key words: Fact finding survey, nosocomial infection control, Kathmandu, Nepal

INTRODUCTION
Recently, nosocomial infections have become a global
concern recognized as a major patient safety issue. They not
only cause a significant burden on patients but also lower
the quality of medical care. In addition, prolonged hospitalization due to nosocomial infections increases costs and unnecessary expenses for the hospital [1, 2]. In the healthcare
setting, particularly in developed countries, various measures including the organization of infection control teams(ICTs), preparation of manuals, strengthening of surveillance systems, and training of staff have been taken to assure effective control. However, it is only some decades agothat importance was attached to nosocomial infection control and effective measures were employed, even in developed countries [3].
In developing countries, where the incidence of infectious diseases is high and environmental conditions ofhealthcare facilities are poor, nosocomial infections may
frequently occur, and some studies have reported a high incidence at healthcare facilities in these countries [4–6]. Effective nosocomial infection control is crucial in the healthcare facilities of developing countries, but in actual fact, attention to it is still limited and control measures are notfunctioning well in many facilities. Furthermore, as implementation of control measures seems to be costly and to
consume resources, nosocomial infection control is often
given a low priority.
Severe acute respiratory syndrome (SARS), which
originated in Guangdong Province, China in November
2002, spread to more than 30 countries. In many hospitals
where SARS cases were encountered, nosocomial infections also broke out, causing many casualties along witheconomic havoc [7, 8]. It is not overstatement to say thatsuch outbreaks have heightened awareness regarding nosocomial infection control even in developing countries. In

1

Bureau of International Medical Cooperation, National Center for Global Health and Medicine, Japan
Department of Microbiology, Institute of Medicine, Tribhuvan University, Nepal
3
Tribhuvan University Teaching Hospital, Institute of Medicine, Nepal*Corresponding author:
Bureau of International Cooperation, International Medical Center of Japan, 1-21-1, Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
Tel: +81-3-3202-7181
Fax: +81-3-3205-7860
E-mail: oharah@it.ncgm.go.jp
250

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Attachment

Tada et al. BMC Infectious Diseases 2014, 14:56
http://www.biomedcentral.com/1471-2334/14/56

RESEARCH ARTICLE

Open Access

NDM-1 Metallo-β-Lactamase and ArmA 16S rRNA
methylase producing Providencia rettgeri clinicalisolates in Nepal
Tatsuya Tada1, Tohru Miyoshi-Akiyama1, Rajan K Dahal3, Manoj K Sah3, Hiroshi Ohara2, Kayo Shimada1,
Teruo Kirikae1* and Bharat M Pokhrel3
Abstract
Background: Drug-resistant Providencia rettgeri producing metallo-β-lactamase and 16S rRNA methylase has
been reported in several countries. We analyzed P. rettgeri clinical isolates with resistance to carbapenems andaminoglycosides in a hospital in Nepal.
Methods: Five clinical isolates of multidrug-resistant P. rettgeri were obtained in a hospital in Nepal. Antimicrobial
susceptibilities were determined using the microdilution method and entire genomes were sequenced todetermine drug-resistant genes. Epidemiological analysis was performed by pulsed-field gel electrophoresis.
Results: Four of the 5 isolates were resistant to carbapenems (imipenem and meropenem), with MICs ≥16 mg/L,
with the remaining isolate showing intermediate resistance to imipenem, with an MIC of 2 mg/L and susceptibility
to meropenem with an MIC ≤1 mg/L. All 5 isolates had blaVEB-1. Of the 4 carbapenem-resistant strains, 3 had
blaNDM-1 and 1 had blaOXA-72. All isolates were highly resistant to aminoglycosides (MICs ≥1,024 mg/L) and harboredarmA. As the result of pulsed-field gel electrophoresis pattern analysis in the 5 P. rettgeri isolates, 4 had identical
PFGE patterns and the fifth showed 95.7% similarity.
Conclusions: This is the first report describing multidrug-resistant P. rettgeri strains harboring blaNDM-1 or blaOXA-72
and armA isolated from patients in Nepal.
Keywords: NDM-1, OXA-72, 16S rRNA methylase, Providencia rettgeri, Molecular epidemiology

Background
Providencia rettgeri has been associated with hospitalacquired infections, including catheter-related urinary
tract infections, bacteremia, skin infections, diarrhea, andgastroenteritis [1,2]. To date, there have been 5 reports of
P. rettgeri isolates harboring metallo-β-lactamase (MBL)
encoding genes, including IMP-type MBL producers inJapan [3,4]; VIM-type MBL, PER-1 extended-spectrum βlactamase (ESBL) and 16S rRNA methylase ArmA in
Korea [5]; and NDM-type MBL in Israel [6] and Brazil [7].
NDM-type MBL was initially identified in Klebsiellapneumoniae and Escherichia coli in 2009 in Sweden [8].

Since then, NDM-1-producing Enterobacteriaceae havebeen isolated in various parts of the world [9,10].
Exogenously acquired 16S rRNA methylase genes responsible for very high levels of resistance to various
aminoglycosides are widely distributed among Enterobacteriaceae and glucose-nonfermentative microbes [11].
Gram-negative pathogens producing 16S rRNA methylase
ArmA have been isolated in various countries [11].
Although co-production of several resistance determinants is not rare in Enterobacteriaceae [12-16], it is lesscommon in P. rettgeri [5]. We describe here P. rettgeri
clinical isolates from Nepal that produce carbapenemase(NDM-1 or OXA-72) and 16S rRNA methylase (ArmA).

* Correspondence: tkirikae@ri.ncgm.go.jp
1
Department of Infectious Diseases, Research Institute, National Center for
Global Health and Medicine, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan
Full list of author information is available at the end of the article© 2014 Tada et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

IOM-NCGM Research Collaboration Office Annual Report 201551

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372

Letters to the Editor / International Journal of Antimicrobial Agents 42 (2013) 367–378

Table 1
Description of the 23 cefotaxime-resistant Salmonella enterica isolates.
Year ofisolation

Isolate ID

Serovar

Phage
type

Genes encoding
cephalosporin
resistance

Sex

Age
(years)TravelabroadResistance proﬁle2008

0812M7303
0811R10895
0809W37247
0809F35063
0808S63221
0807F21428
0806H16365
0806R9615
0805R9530
0911W58164
0910W56953
0910F48822

Typhimurium
Typhimurium
Stanley
Stanley
Typhimurium
Stanley
Stanley
Typhimurium
Typhimurium
Heidelberg
subsp. enterica (I)Isangi

193
RDNC

M
M
F
F
M
F
M
M
M
M
M
M

50
1
37
6
20
22
2
12
47
40
55
<1

ThailandUnknown
No
Unknown
ThailandThailandUnknown
No
GreeceEgypt
ThailandSouth Africa

CHL, CIP, FFN, GEN, SUL, STR, TET
SUL,TET
AMC, CHL, FFN, SUL, STR, TET
AMC, CHL, FFN, GEN, SUL, STR, TET
AMC, CHL, FFN, SUL, STR, TET
AMC, CHL, FFN, GEN, SUL, STR, TET
AMC, CHL, FFN, GEN, SUL, STR, TET
None
AMC, CHL, GEN, SUL, STR, TMP
GEN, SUL, STR
AMC, CHL, CIP, FFN, GEN, NAL, SUL, STR, TET
AMC, CHL, CIP, FFN, GEN, NAL, SUL, STR, TET, TMP

0909F36769
0905W18230
0904R11448
0904W9384
0903T66197
1003F13978
1001M23541
1010H59657
1008R13307
1002H3270
1002W11208

O:6,8; H:e,h:O:4,5,12; H:i:Enteritidis
Typhimurium
O:4,5,12; H:i:O:4,12; H:i: Infantis
Senftenberg
Typhimurium
Stanley
O:4,5,12; H:i:-blaCTX-M-55
blaCTX-M-1
blaCMY-2-likeblaCMY-2-likeblaCMY-2-likeblaCMY-2-likeblaCMY-2-likeblaCTX-M-3
blaCTX-M-14
blaCTX-M-14
blaCMY-2-likeblaCMY-2-like ,blaOXA-10
blaCMY-2-likeblaCTX-M-15
blaCTX-M-15
blaCTX-M-15
blaCTX-M-55
blaCTX-M-55
blaCTX-M-55
blaCTX-M-15
blaCTX-M-55
blaCMY-2-likeblaCTX-M-55

M
M
F
F
F
M
F
M
F
F
F

49
48
44
54
46
16
56
36
21
58
58

No
Unknown
Egypt
No
Unknown
ThailandThailand
Egypt
ThailandThailandUnknown

AMC, CHL, FFN, SUL, STR, TET, TMP
CHL, CIP, GEN, SUL, STR, TET, TMP
GEN
CHL, CIP, FFN, GEN, SUL, STR, TET
GEN, SUL, STR, TET, TMP
CHL, CIP, FFN, SUL, STR, TET
CHL, CIP, FFN, GEN, NAL, SUL, STR, TET, TMP
SUL, TMP
CHL, CIP, FFN, GEN, SUL, STR, TET
AMC, CHL, FFN, SUL, STR, TET
CHL, CIP, FFN, GEN, SUL, STR, TET

2009

2010

NT

U292
NT

U302
1
193
193
193

193
193

CHL, chloramphenicol; CIP, ciproﬂoxacin, FFN, ﬂorfenicol; GEN, gentamicin; SUL, sulfamethoxazole; STR, streptomycin; TET, tetracycline; AMC, amoxicillin/clavulanic acid;
TMP, trimethoprim; NAL, nalidixic acid.

Acknowledgments

Frank Hansen
Steen Ethelberg
Anette M. Hammerum ∗Statens Serum Institut, Copenhagen, Denmark

Pia Møller Hansen and Tune Øst-Jacobsen are thanked for excellent technical assistance.
Funding: Part of this study was supported by the Danish Ministry of Health and Prevention as part of The Danish Integrated
Antimicrobial Resistance Monitoring and Research Programme(DANMAP).
Competing interests: None declared.
Ethical approval: Not required.

∗ Corresponding author. Present address: Department ofMicrobiology and Infection Control, Statens Serum Institut,
Artillerivej 5, DK-2300 Copenhagen S, Denmark.
Tel.: +45 32 68 33 99; fax: +45 32 68 32 31.
E-mail address: ama@ssi.dk (A.M. Hammerum)

References

8 April 2013
http://dx.doi.org/10.1016/j.ijantimicag.2013.06.010[1] Hendriksen RS, Mikoleit M, Kornschober C, Rickert RL, Duyne SV, Kjelsø C, et al.
Emergence of multidrug-resistant Salmonella Concord infections in Europe andthe United States in children adopted from Ethiopia, 2003–2007. Pediatr Infect
Dis J 2009;28:814–18.
[2] Morosini MI, Valverde A, García-Castillo M, Nordmann P, Cantón R. Persistent isolation of Salmonella Concord harbouring CTX-M-15, SHV-12 and QnrA1
in an asymptomatic adopted Ethiopian child in Spain also colonized with
CTX-M-14- and QnrB-producing Enterobacteriaceae. J Antimicrob Chemother
2010;65:1545–6.
[3] Vanhoof R, Gillis P, Stévart O, Boland C, Vandenberg O, Fux F, et al. Transmissionof multiple resistant Salmonella Concord from internationally adopted children
to their adoptive families and social environment: proposition of guidelines. Eur
J Clin Microbiol Infect Dis 2012;31:491–7.
[4] Hansen DS, Schumacher H, Hansen F, Stegger M, Hertz FB, Schønning K, et al.
DANRES Study Group. Extended-spectrum -lactamase (ESBL) in Danish clinicalisolates of Escherichia coli and Klebsiella pneumoniae: prevalence, -lactamase
distribution, phylogroups, and co-resistance. Scand J Infect Dis 2012;44:174–81.
[5] Haldorsen B, Aasnaes B, Dahl KH, Hanssen AM, Simonsen GS, Walsh TR, et al.
The AmpC phenotype in Norwegian clinical isolates of Escherichia coli isassociated with an acquired ISEcp1-like ampC element or hyperproduction of
the endogenous AmpC. J Antimicrob Chemother 2008;62:694–702.
[6] Clinical Laboratory Standards Institute. Performance standards for antimicrobial
susceptibility testing; twenty-ﬁrst informational supplement. Document M100S21. Wayne, PA: CLSI; 2011.
[7] Hendriksen RS, Le Hello S, Bortolaia V, Pulsrikarn C, Nielsen EM, Pornruangmong S, et al. Characterization of isolates of Salmonella enterica serovar
Stanley, a serovar endemic to Asia and associated with travel. J Clin Microbiol
2012;50:709–20.
[8] Yu F, Chen Q, Yu X, Li Q, Ding B, Yang L, et al. High prevalence of extendedspectrum  lactamases among Salmonella enterica Typhimurium isolates frompediatric patients with diarrhea in China. PLoS One 2011;6:e16801.

Mia Torpdahl
Eva Møller Nielsen

52Dissemination of multidrug-resistant Klebsiellapneumoniae clinical isolates with various combinations of carbapenemases (NDM-1 and OXA-72)
and 16S rRNA methylases (ArmA, RmtC andRmtF) in Nepal
Sir,
The carbapenemases NDM-1 and OXA-72 hydrolyse almost
all -lactams. NDM-1-producing Enterobacteriaceae and OXA72-producing Acinetobacter spp. have been reported in various
countries [1,2]. To date, OXA-72-producing isolates of bacterial
species other than Acinetobacter spp. have not been reported.
Acquired 16S rRNA methylase genes responsible for highlevel resistance to various aminoglycosides have been widelydistributed among Enterobacteriaceae, including Klebsiella pneumoniae and glucose-non-fermentative bacteria [3]. 16S rRNA
methylase-producing Gram-negative pathogens have been isolated
in various countries [3], including Nepal [4]. The 16S rRNA methylases ArmA and RmtC are widely spread among various bacterial
species, including Enterobacteriaceae and Acinetobacter spp.
In this study, 25 K. pneumoniae isolates were obtained from
25 inpatients during the period May–October 2012 at Tribhuvan
University Teaching Hospital (Kathmandu, Nepal), of which 13 isolates were obtained from sputa and 12 were from pus samples.
Isolates were identiﬁed phenotypically and species identiﬁcation

IOM-NCGM Research Collaboration Office Annual Report 2015

>1024
>1024
>1024
>1024

512
>1024

>1024
1024

>1024
>1024
>1024
>1024
>1024

>1024
>1024
>1024
>1024
>1024

>1024
>1024

>1024
>1024

>1024

>1024

IOMTU 25IOMTU 40IOMTU 46IOMTU 53IOMTU 67IOMTU 74IOMTU 76IOMTU 83IOMTU 89
IOMTU 100IOMTU 102IOMTU 103IOMTU 111IOMTU 116.1
IOMTU 116.2
IOMTU 117IOMTU 120IOMTU 122IOMTU 125IOMTU 138IOMTU 139IOMTU 145IOMTU 154IOMTU 164

8

1024

128
8

4
128

512
512
512
16
512

512
1024
512
8
1024

1024
512

8
512

1024
256
512
512

512

TZP

64

>1024

128
32

32
256

>1024
>1024
>1024
512
>1024

>1024
>1024
>1024
64
>1024

>1024
128

64
32

>1024
>1024
>1024
>1024

>1024

CAZ

512

1024

512
1024

512
>1024

1024
>1024
1024
>1024
>1024

>1024
>1024
1024
>1024
>1024

512
1024

512
256

512
1024
>1024
>1024

1024

CTX

256

256

256
256

256
>1024

512
>1024
128
>1024
512

256
128
256
64
512

128
128

128
128

128
256
128
512

256

FEP

<0.5

16

2
<0.5

<0.5
<0.5

8
8
4
<0.5
16

32
32
32
<0.5
16

32
32

<0.5
4

32
16
16
32

16

IPM

<0.5

32

2
<0.5

<0.5
2

8
16
4
<0.5
16

32
64
32
<0.5
32

64
32

<0.5
8

64
32
32
32

32

MEM

64

64

512
64

64
512

256
256
128
1024
256

1024
512
256
128
512

512
128

32
128

512
128
512
512

512

ATM

4

>1024

32
4

>1024
>1024

>1024
>1024
1024
>1024
>1024

>1024
>1024
512
1
>1024

>1024
>1024

1
>1024

1024
1024
>1024
>1024

>1024

ABK

4

>1024

32
4

>1024
>1024

>1024
>1024
>1024
>1024
>1024

>1024
>1024
>1024
8
>1024

>1024
>1024

4
>1024

1024
>1024
>1024
>1024

>1024

AMK

<0.5

1024

64
<0.5

>1024
>1024

512
1024
1024
>1024
>1024

>1024
>1024
>1024
32
1024

>1024
>1024

64
>1024

>1024
1024
>1024
>1024

>1024

GEN

<0.5

16

256
2

8
>1024

64
64
64
128
128

128
64
128
128
64

64
128

32
2

128
128
128
64

128

CIP

NDM-1,OXA-72–––

NDM-1,OXA-72
NDM-1
NDM-1
NDM-1
NDM-1,OXA-72–
NDM-1,OXA-72
NDM-1
NDM-1,OXA-72
NDM-1
NDM-1
NDM-1
–
NDM-1,OXA-72
NDM-1
NDM-1
NDM-1
–
NDM-1,OXA-72OXA-72OXA-72

Carbapenemases

CTX-M-15, SHV-83, TEM-1

CTX-M-15, SHV-28, TEM-1

CTX-M-15, SHV-28, TEM-1
CTX-M-15, TEM-1

CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-11, TEM-1

CTX-M-15, SHV-28
CTX-M-15, SHV-28
CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-28,TEM-1
CTX-M-15, SHV-28

CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-28
CTX-M-15, SHV-28, TEM-1
CTX-M-15, TEM-1

CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-28

CTX-M-15, SHV-28, TEM-1
CTX-M-15, SHV-1

CTX-M-15, SHV-28, TEM-1
CTXM-15, SHV-28
CTX-M-15, SHV-11, TEM-1
CTX-M-15, SHV-11, TEM-1

CTX-M-15, SHV-158, TEM-1

ESBL

–

RmtC

–
–

ArmA
RmtF

ArmA
ArmA
ArmA
RmtF
ArmA

RmtF
RmtF
ArmA
–
RmtC

RmtF
ArmA

–
ArmA

RmtF
ArmA
RmtC, RmtF
RmtC, RmtF

RmtC, RmtF

16S rRNA
methylases

No mutation

S83F, D87A

S83Y, D87G
No mutation

S83F, D87A
S83F, D87N

S83F, D87A
S83F, D87A
S83F, D87A
S83Y, D87N
S83F, D87A

S83Y, D87F
S83F, D87A
S83F, D87A
S83F, D87A
S83I

S83I
S83F, D87A

S83F, D87A
S83F, D87A

S83I
S83F, D87A
S83I
S83I

S83I

gyrA

No
mutation

S80I
No
mutation
S80I
No
mutation
S80I

S80I
S80I
S80I
S80I
S80I

S80I
S80I
S80I
S80I
S80I

S80I
S80I

S80I
S80I

S80I
S80I
S80I
S80I

S80I

parC

Mutations in DNA gyrase

MIC, minimum inhibitory concentration; PIP, piperacillin; TZP, piperacillin/tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IPM, imipenem; MEM, meropenem; ATM, aztreonam; ABK, arbekacin; AMK, amikacin;GEN, gentamicin; CIP, ciproﬂoxacin; ESBL, extended-spectrum -lactamase.

>1024

PIP

MIC (g/mL)

IOMTU 23

StrainTable 1
Summary of the characteristics of the 25 Klebsiella pneumoniae strains, including antimicrobial resistance proﬁles and resistance genes.

Attachment

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was conﬁrmed by 16S rRNA sequencing. Minimum inhibitory concentrations of antibiotics were determined by the microdilution
method according to the guidelines of the Clinical and Laboratory
Standards Institute (CLSI) M07-A9.
Entire genomes of the isolates were sequenced by MiSeqTM(Illumina, San Diego, CA). CLC Genomics Workbench v.5.5 (CLC
bio, Tokyo, Japan) was used to search 923 drug resistance genes,
including genes encoding -lactamases, 16S rRNA methylases
and aminoglycoside-acetyl/adenyltransferases, as well as point
mutations in gyrA and parC associated with quinolone resistance. The genetic environments surrounding blaNDM-1 , blaOXA-72
and 16S rRNA methylase-encoding genes were determined.
Multilocus sequence typing (MLST) and clonal complexes (CCs)were determined according to the K. pneumoniae MLST databasewebsite(http://www.pasteur.fr/recherche/genopole/PF8/mlst/
Kpneumoniae.html) and eBURST v.3 (http://eburst.mlst.net),respectively.
Pulsed-ﬁeld gel electrophoresis (PFGE) analysis was performed
and ﬁngerprinting patterns were analysed by the unweighted pairgroup method.
All isolates were resistant to piperacillin, of which 19 isolates
were resistant to piperacillin/tazobactam. All isolates were resistant to ceftazidime, cefotaxime and cefepime. Seventeen isolates
were resistant to carbapenems (imipenem and meropenem). All
isolates are resistant to aztreonam. Twenty isolates were resistantto all aminoglycosides tested (arbekacin, amikacin and gentamicin). Twenty-two isolates were resistant to ciproﬂoxacin (Table 1).
The majority of isolates had various combinations of genes
encoding carbapenemases (blaNDM-1 and blaOXA-72 ) and 16S
rRNA methylases (armA, rmtC and rmtF) (Table 1). These isolates also had extended-spectrum -lactamase-encoding genes,
including blaCTX-M-15 , blaTEM-1 and/or blaSHV -type, as well asaminoglycoside-modifying enzymes, including aac(6� )-Ib and/or
aadA2. Twenty-three isolates had two or three point mutations in
the quinolone resistance-determining regions of gyrA and parC.
The genetic environment surrounding blaNDM-1 (GenBank accession no. AB824738) including rmtC was auniquestructure,
which
was
orf1–tniB–orf2–orf3–rmtC–
blaNDM-1 –bleMBL –trpF–dsbC–cutA1–qroL. The genetic environment surrounding armA (GenBank accession no. AB825954)
from nucleotides 19 to 14 138 had >99.9% sequence identity toa nucleotide sequence from nucleotides 65 492 to 79 611 of theplasmid pCTX-M3 (GenBank accession no. AF550415). The genetic
environment surrounding rmtF (GenBank accession no. AB824739)
from nucleotides 268 to 9812 had >99.9% sequence identity
to a nucleotide sequence from nucleotides 49 291 to 58 835 of
the plasmid pKPX-1 (GenBank accession no. AP012055). The
genetic environment surrounding blaOXA-72 (GenBank accession
no. AB825955) from nucleotides 1 to 8970 was identical to that ofpAB-NCGM253 (GenBank accession no. AB823544).
The clinical isolates of K. pneumoniae tested belonged to one of
the following sequence types (STs): ST11; ST14; ST15; ST29; ST43;
ST340; ST378; ST395; ST437; ST1231; and ST1232. Of these isolates, 14 belonged to CC14 and 5 belonged to CC11. These results
mostly corresponded with the results of PFGE pattern analysis,
which revealed two clusters showing >60% similarity (clusters I and
II). Cluster I comprised 12 isolates belonging to CC14 and cluster II
comprised 4 isolates belonging to CC11.
NDM-1-producers have epidemiological links to the Indian subcontinent as of 2011 [5]. There was, nevertheless, no report of
NDM-1-producers in Nepal. We recently found NDM-1-producing
Pseudomonas aeruginosa and a novel variant NDM-8-producing
Escherichia coli isolates in Nepal [4].
This is the ﬁrst report describing OXA-72-producers in SouthAsia, suggesting that OXA-72-producers have disseminated inthis region. This is also the ﬁrst report of OXA-72-producing

54

K. pneumoniae clinical isolates. Up to now, OXA-72-producers werereported to be only Acinetobacter spp.
The present study suggests that aminoglycoside-resistantGram-negative pathogens producing ArmA, RmtC and RmtF disseminated in medical settings in Nepal. These pathogens producing
16S rRNA methylases may also disseminate in neighbouring
countries. Hidalgo et al. [6] recently reported that 14% ofEnterobacteriaceae isolates from an Indian hospital had 16S rRNA
methylases, of which 24% produced RmtF.
Funding: This study was supported by grants from the International
Health Cooperation Research [23-A-301 and 24-S-5], a grant from
the Ministry of Health, Labor and Welfare of Japan [H24-ShinkoIppan-010], and JSPS KAKENHI [grant no. 24790432].
Competing interests: None declared.
Ethical approval: This study protocol was ethically reviewed andapproved by the Institutional Review Board of the Institute ofMedicine, Tribhuvan University (Kathmandu, Nepal) [ref. 6-11-E].
References[1] Cornaglia G, Giamarellou H, Rossolini GM. Metallo--lactamases: a last frontierfor -lactams? Lancet Infect Dis 2011;11:381–93.
[2] Povilonis J, Seputiene V, Krasauskas R, Juskaite R, Miskinyte M, Suziedelis K,
et al. Spread of carbapenem-resistant Acinetobacter baumannii carrying a plasmid with two genes encoding OXA-72 carbapenemase in Lithuanian hospitals. J
Antimicrob Chemother 2013;68:1000–6.
[3] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases
found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an
update. Drug Resist Updat 2012;15:133–48.
[4] Tada T, Miyoshi-Akiyama T, Dahal RK, Sah MK, Ohara H, Kirikae T, et al. NDM-8
metallo--lactamase in a multidrug-resistant Escherichia coli strain isolated in
Nepal. Antimicrob Agents Chemother 2013;57:2394–6.
[5] Nordmann P, Poirel L, Walsh TR, Livermore DM. The emerging NDM carbapenemases. Trends Microbiol 2011;19:588–95.
[6] Hidalgo L, Hopkins KL, Gutierrez B, Ovejero CM, Shukla S, Douthwaite S, et al.
Association of the novel aminoglycoside resistance determinant RmtF with NDM
carbapenemase in Enterobacteriaceae isolated in India and the UK. J Antimicrob
Chemother 2013;68:1543–50.

Tatsuya Tada
Tohru Miyoshi-Akiyama
Department of Infectious Diseases, Research Institute, National
Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku,
Tokyo 162-8655, Japan
Rajan K. Dahal
Shyam K. Mishra
Department of Microbiology, Institute of Medicine, Tribhuvan
University, Maharajgunj, Kathmandu, Nepal
Hiroshi Ohara
Department of International Medical Co-operation, National Center
for Global Health and Medicine, 1-21-1 Toyama, Shinjuku, Tokyo,
Japan
Kayo Shimada
Teruo Kirikae ∗Department of Infectious Diseases, Research Institute, National
Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku,
Tokyo 162-8655, Japan
Bharat M. Pokhrel
Department of Microbiology, Institute of Medicine, Tribhuvan
University, Maharajgunj, Kathmandu, Nepal∗ Corresponding author. Tel.: +81 3 3202 7181x2838;

fax: +81 3 3202 7364.
E-mail address: tkirikae@ri.ncgm.go.jp (T. Kirikae)
17 June 2013

http://dx.doi.org/10.1016/j.ijantimicag.2013.06.014

IOM-NCGM Research Collaboration Office Annual Report 2015

Attachment

NDM-12, a Novel New Delhi Metallo--Lactamase Variant from a
Carbapenem-Resistant Escherichia coli Clinical Isolate in Nepal
Tatsuya Tada,a Basudha Shrestha,c Tohru Miyoshi-Akiyama,a Kayo Shimada,a Hiroshi Ohara,b Teruo Kirikae,a Bharat M. Pokhrelc
Department of Infectious Diseases, Research Institute,a and Department of International Medical-Cooperation,b National Center for Global Health and Medicine, Shinjuku,
Tokyo, Japan; Department of Microbiology, Institute of Medicine, Tribhuvan University, Maharajgunj, Kathmandu, Nepalc

A novel New Delhi metallo--lactamase variant, NDM-12, was identified in a carbapenem-resistant Escherichia coli clinical isolate obtained from a urine sample from a patient in Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions(M154L and G222D). The enzymatic activities of NDM-12 against -lactams were similar to those of NDM-1, although NDM-12
showed lower kcat/Km ratios for all -lactams tested except doripenem. The blaNDM-12 gene was located in a plasmid of 160 kb.

M

etallo--lactamases (MBLs) usually confer reduced susceptibility to carbapenems, cephalosporins, and penicillins but
not monobactams (1). Acquired MBLs are produced by Gramnegative bacteria, including Acinetobacer spp., Pseudomonas
aeruginosa, and several Enterobacteriaceae (1). MBLs are categorized by their amino acid sequences into various types (2–4), including AIM (5), DIM (6), FIM (7), GIM (8), IMPs (9), KHM(10), NDMs (11), SMB (12), SIM (13), SPM (14), TMBs (15), andVIMs (16). The most prevalent types of MBLs are IMP-, VIM-,
and NDM-type enzymes (1, 2, 17). NDM-1 was initially isolated
from Klebsiella pneumoniae and Escherichia coli in 2008 in Sweden(11). Subsequently, at least 11 NDM variants (www.lahey.org/studies) have been reported in several countries (4, 18–29).
This study was ethically reviewed and approved by the Institutional Review Board of the Institute of Medicine at Tribhuvan
University (reference 6-11-E) and the Biosafety Committee at the
National Center for Global Health and Medicine (approval no.
26-D-088 and 26-D-089).
E. coli IOMTU388.1 was isolated from a urine sample obtained
from a patient in 2013 in a university hospital in Nepal. The isolate
was phenotypically identified, and the species identification was
confirmed by 16S rRNA sequencing (30). E. coli DH5 (TaKaRa
Bio, Shiga, Japan) and E. coli BL21-CodonPlus(DE3)-RIP (Agilent Technologies, Santa Clara, CA) were used as hosts for recombinant plasmids and for expression of blaNDM-1 and blaNDM-12,
respectively.
MICs were determined using the broth microdilution method asrecommended by the Clinical and Laboratory Standards Institute(31). The MICs of -lactams for E. coli IOMTU388.1 are shown in
Table 1, and the MICs of other antibiotics were as follows: amikacin, 1,024 g/ml; arbekacin, 1,024 g/ml; ciprofloxacin, 128
g/ml; colistin, 0.125 g/ml; fosfomycin, 8 g/ml; gentamicin,
1,024 g/ml; kanamycin, 1,024 g/ml; levofloxacin, 32 g/
ml; minocycline, 8 g/ml; tigecycline, 0.125 g/ml; and tobramycin, 1,024 g/ml. PCR analysis was performed to detect the
MBL genes blaDIM, blaGIM, blaIMP, blaNDM, blaSIM, blaSPM, andblaVIM (32, 33). The isolate was PCR positive for blaNDM but
negative for the other MBL genes tested. The DNA sequence of
the PCR product revealed that the isolate had blaNDM-12.
Multilocus sequence typing (MLST) of IOMTU388.1 typed it as
ST635 (E. coli MLST Database; http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html). blaNDM-1 obtained from P.
aeruginosa IOMTU9 (29) was used as a reference gene.
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The blaNDM-12 sequence had 2 amino acid substitutions(M154L and G222D) compared with blaNDM-1 (accession no.
JF798502) and one substitution (G222D) compared with NDM-4(accession no. JQ348841).
The blaNDM-1 and blaNDM-12 genes were cloned into the corresponding sites of pHSG398 (TaKaRa, Shiga, Japan) using theprimer set EcoRI-NDM-F (5=-GGGAATTCATGGAATTGCCCA
ATATTATG-3=) and PstI-NDM-R (5=-AACTGCAGTCAGCGC
AGCTTGTCGGCCAT-3=). E. coli DH5 was transformed with
pHSG398-NDM-1 or pHSG398-NDM-12.
The open reading frames of NDM-1 and NDM-12 without signal
peptide regions were cloned into the pET28a expression vector (Novagen, Inc., Madison, WI) using the primer set BamHI-TEV-NDM-F(5=-ATGGATCCGAAAACCTGTATTTCCAAGGCCAGCAAATGGAAACTGGCGAC-3=) and XhoI-NDM-R (5=-ATCTCGAGTCAG
CGCAGCTTGTCGGCCATG-3=). The resulting plasmids weretransformed into E. coli BL21-CodonPlus(DE3)-RIP (Agilent
Technologies, Santa Clara, CA). Both recombinant NDM-1 andNDM-12 were purified simultaneously using Ni-nitrilotriacetic
acid (NTA) agarose according to the manufacturer’s instruction(Qiagen, Hilden, Germany). His tags were removed by digestion
with TurboTEV protease (Accelagen, San Diego, CA) and untagged proteins were purified by an additional passage over the
Ni-NTA agarose. The purities of NDM-1 and NDM-12, whichwere estimated by SDS-PAGE, were greater than 90%. During thepurification procedure, the presence of -lactamase activity wasmonitored using nitrocefin (Oxoid, Ltd., Basingstoke, United
Kingdom). Initial hydrolysis rates were determined in 50 mM
Tris-HCl buffer (pH 7.4) containing 0.3 M NaCl and 5 M
Zn(NO3)2 at 37°C, using a UV-visible spectrophotometer (V-530;
Jasco, Tokyo, Japan). The Km and kcat values and the kcat/Km ratio
were determined by analyzing -lactam hydrolysis with a Lineweaver-Burk plot. Wavelengths and extinction coefficients for

Antimicrobial Agents and Chemotherapy

Received 15 May 2014 Returned for modification 14 June 2014
Accepted 26 July 2014
Published ahead of print 4 August 2014
Address correspondence to Teruo Kirikae, tkirikae@ri.imcj.go.jp.
T.T. and B.S. contributed equally to this article.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.03355-14

p. 6302– 6305

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Attachment

Identification of a Novel 6=-N-Aminoglycoside Acetyltransferase,
AAC(6=)-Iak, from a Multidrug-Resistant Clinical Isolate of
Stenotrophomonas maltophilia
Department of Infectious Diseases, Research Institute,a and Department of International Medical Cooperation,b National Center for Global Health and Medicine, Shinjuku,
Tokyo, Japan; Department of Microbiology, Institute of Medicine, Tribhuvan University, Maharajgunj, Kathmandu, Nepalc

Stenotrophomonas maltophilia IOMTU250 has a novel 6=-N-aminoglycoside acetyltransferase-encoding gene, aac(6=)-Iak. The
encoded protein, AAC(6=)-Iak, consists of 153 amino acids and has 86.3% identity to AAC(6=)-Iz. Escherichia coli transformed
with a plasmid containing aac(6=)-Iak exhibited decreased susceptibility to arbekacin, dibekacin, neomycin, netilmicin, sisomicin, and tobramycin. Thin-layer chromatography showed that AAC(6=)-Iak acetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin but not apramycin, gentamicin, or lividomycin.

Stenotrophomonas maltophilia is a globally emerging multidrug-resistant Gram-negative pathogen that is most commonly associated with respiratory infections in humans (1) andcauses an increasing number of nosocomial respiratory tract andbloodstream infections in immunocompromised patients. S.
maltophilia exhibits resistance to a broad spectrum of antibiotics,
namely, -lactam antibiotics, macrolides, cephalosporins, fluoroquinolones, aminoglycosides, carbapenems, chloramphenicol,
tetracyclines, and polymyxins (1). Several intrinsic antibiotic resistance traits in S. maltophilia are known; an increase in membrane permeability and the presence of chromosomally encoded
multidrug resistance efflux pumps have also been observed (2).
Aminoglycoside-resistant mechanisms involve primarily aminoglycoside-modifying enzymes (3) and 16S rRNA methylases(4). The 6=-N-aminoglycoside acetyltransferases [AAC(6=)s] areof particular interest because they can modify a number of clinically important aminoglycosides. There are two main AAC(6=)
subclasses, which differ in their activities against amikacin andgentamicin. The AAC(6=)-I-type enzymes effectively acetylateamikacin but not gentamicin, whereas the AAC(6=)-II-type enzymes effectively acetylate gentamicin but not amikacin (5). To
date, 45 genes encoding AAC(6=)-I types, designated aac(6=)-Ia to-Iaj, have been cloned, and their bacteriological or biochemical
properties have been characterized (5–8).
S. maltophilia IOMTU250 was isolated from the endotracheal
tube of a patient in a medical ward of a hospital in Nepal in 2012.
Escherichia coli DH5 (TaKaRa Bio, Shiga, Japan) and Escherichia
coli BL21-CodonPlus (DE3)-RIP (Agilent Technologies, Santa

Clara, CA) were used as hosts for recombinant plasmids and protein expression, respectively. MICs were determined using the microdilution method (9). The MICs of tested aminoglycosides for
S. maltophilia IOMTU250 are shown in Table 1. The MICs ofother antibiotics were as follows: ampicillin, 1,024 g/ml; ampicillin-sulbactam, 128 g/ml; aztreonam, 128 g/ml; ceftazidime, 8 g/ml; cephradine, 1,024 g/ml; cefepime, 64 g/ml; cefotaxime, 64 g/ml; cefoxitin, 512 g/ml; chloramphenicol, 8 g/
ml; colistin, 32 g/ml; fosfomycin, 128 g/ml; imipenem, 256
g/ml; levofloxacin, 1 g/ml; meropenem, 64 g/ml; minocycline, 0.25 g/ml; penicillin, 512 g/ml; ticarcillin-clavulanate,
8 g/ml; tigecycline, 0.25 g/ml; and trimethoprim-sulfamethoxazole, 4 g/ml.
Genomic DNA was extracted from S. maltophilia IOMTU250
using DNeasy blood and tissue kits (Qiagen, Tokyo, Japan) andsequenced with a MiSeq system (Illumina, San Diego, CA). Morethan 20-fold coverage was achieved. A new 6=-N-aminoglycosideacetyltransferase variant was designated aac(6=)-Iak.
A synthetic aac(6=)-Iz gene (462 bp) was produced by Funako-

Received 14 May 2014 Returned for modification 27 May 2014
Accepted 1 August 2014
Published ahead of print 4 August 2014
Address correspondence to Teruo Kirikae, tkirikae@ri.imcj.go.jp.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.03354-14

TABLE 1 MICs of various aminoglycosides for S. maltophilia IOMTU250 and E. coli strains transformed with aac(6=)-Iak and aac(6=)-Iz
MICb (g/ml)
Straina

ABK

AMK

APR

DIB

GEN

ISP

KAN

LIV

NEO

NET

SIS

TOB

S. maltophilia IOMTU250
E. coli DH5/pSTV28
E. coli DH5/pSTV28-aac(6=)-Iak
E. coli DH5/pSTV28-aac(6=)-Iz

512
0.5
2
4

64
0.25
1
2

512
1
1
1

512
0.25
16
16

32
0.25
0.25
0.25

64
0.25
0.5
0.5

128
1
2
2

512
2
2
2

512
0.5
4
4

512
0.25
1
8

64
0.25
2
2

64
0.5
4
16

a

The MICs for S. maltophilia and E. coli strains were determined with Mueller-Hinton broth preparations and individual aminoglycosides.
ABK, arbekacin; AMK, amikacin; APR, apramycin; DIB, dibekacin; GEN, gentamicin; ISP, isepamicin; KAN, kanamycin; LIV, lividomycin; NEO, neomycin; NET, netilmicin; SIS,
sisomicin; TOB, tobramycin.
b

6324

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Tatsuya Tada,a Tohru Miyoshi-Akiyama,a Rajan K. Dahal,c Shyam K. Mishra,c Kayo Shimada,a Hiroshi Ohara,b Teruo Kirikae,aBharat M. Pokhrelc

Attachment

IOM-NCGM Research Collaboration Office Annual Report 201557

Attachment

Clinical Epidemiology and Molecular Analysis of Extended-Spectrum-Lactamase-Producing Escherichia coli in Nepal: Characteristics ofSequence Types 131 and 648
Jatan Bahadur Sherchan,a Kayoko Hayakawa,b Tohru Miyoshi-Akiyama,c Norio Ohmagari,b Teruo Kirikae,d Maki Nagamatsu,b,dMasayoshi Tojo,b,d Hiroshi Ohara,e Jeevan B. Sherchand,f Sarmila Tandukarf
Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel, Nepala; Disease Control and Prevention Center, National Center for
Global Health and Medicine, Tokyo, Japanb; Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo, Japanc;
Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japand; Department of International Medical Cooperation,
National Center for Global Health and Medicine, Tokyo, Japane; Public Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital,
Kathmandu, Nepalf

Escherichia coli is a part of the normal human and animal gastrointestinal flora; it is the most common cause of urinary tract
infections and also causes various other infectious conditions,such as intra-abdominal infections, neonatal meningitis, and septicemia (1–3).
Recently, extended-spectrum-beta-lactamase (ESBL) producing E. coli strains, particularly strains producing CTX-M-type
ESBLs, have emerged worldwide (4). In particular, E. coli with O
antigen type 25 (O25) and sequence type 131 (ST131) is often
associated with the CTX-M-15 ESBL and has been increasingly
reported globally. These bacteria are resistant to classes of antibiotics distinct from -lactams, such as fluoroquinolones and trimethoprim-sulfamethoxazole (5, 6). Epidemiology reports on ESBLproducing E. coli in Asia are limited to date. To the best of ourknowledge, there has been no report on the prevalence of pandemic ESBL-producing E. coli ST131, or other potentially dominant ESBL-producing E. coli STs, or clinical and microbiologicalinformation pertaining to their isolation in Nepal. Nepal is locatedin south Asia and adjacent to India, where a high proportion ofresistant Gram-negative bacteria has been reported (7); understanding the epidemiology of ESBL-producing E. coli in this region is therefore particularly important. In addition, the patients
population and their clinical background in developing countries
are different from those in developed countries, where the majority of studies on ESBL-producing E. coli have been conducted. It isthus imperative to reveal the clinical and microbiological charac3424

58

aac.asm.org

teristics of ESBL-producing E. coli in developing countries in order to better understand the global epidemiology of this pathogen.
In this study, we aimed to elucidate the clinical and microbiological characteristics of ESBL-producing E. coli and specifically toreveal the unique aspects of the dominant ESBL-producing E. coli
ST in Nepal.
MATERIALS AND METHODS
Study settings and design. Microbiological investigations and clinical epidemiological analyses of ESBL-producing E. coli were conducted among
patients from whom ESBL-producing E. coli was isolated in the Tribhuvan
University teaching hospital, which serves as a tertiary referral hospital in
Kathmandu, Nepal. Institutional review boards at Tribhuvan University

Received 3 February 2015 Returned for modification 23 February 2015
Accepted 21 March 2015
Accepted manuscript posted online 30 March 2015
Citation Sherchan JB, Hayakawa K, Miyoshi-Akiyama T, Ohmagari N, Kirikae T,
Nagamatsu M, Tojo M, Ohara H, Sherchand JB, Tandukar S. 2015. Clinicalepidemiology and molecular analysis of extended-spectrum--lactamaseproducing Escherichia coli in Nepal: characteristics of sequence types 131 and 648.
Antimicrob Agents Chemother 59:3424 –3432. doi:10.1128/AAC.00270-15.
Address correspondence to Kayoko Hayakawa, kayokohayakawa@gmail.com.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00270-15

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Recently, CTX-M-type extended-spectrum--lactamase (ESBL)-producing Escherichia coli strains have emerged worldwide. In
particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often associated with the CTX-M-15
ESBL, has been increasingly reported globally; however, epidemiology reports on ESBL-producing E. coli in Asia are limited.
Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in Kathmandu, Nepal,
were included in this study. Whole-genome sequencing of the isolates was conducted to analyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of acquired drug resistance genes. During the study period,
105 patients with ESBL-producing E. coli isolation were identified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n  54; 51.4%), followed by ST648 (n  15; 14.3%). All ST131 isolates were identified as
O25b-ST131 clones, subclone H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses.
ST648 isolates had a proportionally higher resistance to non--lactam antibiotics and featured drug-resistant genes more frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648. The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were from the outpatient clinic, andpregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed that the high resistance of ESBL-producing E.
coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern.

Attachment

Epidemiology of ESBL-Producing E. coli in Nepal

RESULTSA total of 105 patients with ESBL-producing E. coli isolation wereidentified during the study period. The STs and phylotypes of theisolated ESBL-producing E. coli are shown in Table 1. The mostdominant ST was ST131, which accounted for 54 (51.4%) of all
ESBL-producing E. coli isolates, followed by ESBL-producing E.
June 2015 Volume 59 Number 6

TABLE 1 Sequence typing and phylotypes of extended-spectrum-lactamase-producing Escherichia coli isolates in Nepal
Sequence
type
ST131
ST648
ST405
ST38
ST167
ST361
ST410
ST10
ST14
ST44
ST315
ST393
ST394
ST421
ST443
ST517
ST617
ST624
ST746
ST1312
ST2562
New ST
Total

No. (%) of isolates (total n  105) in phylotype:
A

B1

D

B2
54 (51.4)

15 (14.3)
5 (4.8)3 (2.9)2 (1.9)2 (1.9)2 (1.9)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)1 (1.0)11 (10.5)

2 (1.9)7 (6.7)

1 (1.0)34 (32.4)

58 (55.2)

coli ST648 (n  15; 14.3%). One isolate of ST131 contained a new
fumC allele (400), but the other 6 MLST locus sequences andphylotypes were consistent with ST131 and were therefore included in the ST131 group for further analysis. All ESBL-producing E. coli ST131 isolates were identified as the O25b-ST131 clone
and the H30-Rx subclone. Among the 15 ST648 isolates, 12 had an
identification rate of 97.4%, and 3 had a 96.0% identification rateof the pabB gene, which is reported to be specific for isolates of theO25b-ST131 clone (18). In order to further understand the characteristics of dominant STs, we conducted analyses comparing thethree groups of ESBL-producing E. coli isolates (ST131, ST648,
and non-ST131/648).
The comparison between the three groups with regard to theirantimicrobial susceptibility is shown in Table 2. ST648 isolates
were the most resistant to multiple antibiotics, including non-lactams such as levofloxacin, gentamicin, trimethoprim-sulfamethoxazole, and minocycline. More than 70% of ST131 (n  38;70%) and ST648 (n  12; 80%) isolates were resistant to bothlevofloxacin and trimethoprim-sulfamethoxazole, compared to
14 (39%) of non-ST131/648 isolates (ST131 versus non-ST131/
648, P  0.004; ST648 versus non-ST131/648, P  0.013).
We next analyzed and compared the profiles of resistancegenes among the three isolate groups (Table 3). Overall, ST131
and ST 648 isolates had resistance genes more frequently than
non-ST131/648. Importantly, aac(3)-IIa was most frequently
identified in ST648 isolates, which would explain the higher rates
of resistance to gentamicin in ST648 isolates. blaCTX-M-15 wascommonly isolated from all 3 groups.
To further elucidate the microbiological characteristics of
ESBL-producing E. coli ST131 and ST648, we investigated the virulence-associated traits (Table 4). Overall, ST131 isolates had a

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approved the study before its initiation. The study period, including chart
reviewing, was from 1 February 2013 to 31 January 2014.
Patients and variables. Patients with clinical isolation of ESBL-producing E. coli between 1 February 2013 and 31 July 2013 were divided into
three groups, i.e., ESBL-producing E. coli ST131, ESBL-producing E. coli
ST648, and ESBL-producing E. coli non-ST131/648 (ESBL-producing E.
coli isolates of ST other than ST131 and ST648), based on multilocus
sequence type (MLST). For patients from whom more than one ESBLproducing E. coli strain was isolated during the study period, only the first
episode was analyzed; this study therefore incorporated only unique patient episodes. Parameters retrieved from the patient records included (i)demographics, (ii) background conditions and clinical diagnosis. (iii) duration of hospital stay, and (iv) antimicrobial treatment during the current hospital stay (or at the outpatient clinic for outpatients).
Isolates. Standard identification and susceptibility testing of E. coli
were performed and interpreted in accordance with the Clinical and Laboratory Standard Institute (CLSI) criteria (8), using an automated brothmicrodilution system (MicroScan; Siemens AG, Germany) unless otherwise stated. To determine the MIC of fosfomycin, an NC6.11J panel (Siemens AG, Germany) was used.
In addition, we tested MICs of flomoxef, cefoperazone-sulbactam,
and fosfomycin, as they are potentially active against ESBL-producing E.
coli (9–11). The breakpoints for susceptibility were 8 g/ml for flomoxef (12), and 16/8 g/ml for cefoperazone-sulbactam (13). ESBL
production was confirmed with disc diffusion tests in accordance with the2009 CLSI criteria (14).
Molecular analysis. Molecular analysis was conducted in the Pathogenic Microbe Laboratory, Research Institute, National Center for Global
Health and Medicine, Tokyo, Japan. Whole-genome sequences of all 105
isolates were obtained using a MiSeq system with Nextera XT library kits
(Illumina, Tokyo, Japan) for the analysis of MLST, phylotypes (15), H30
and H30-Rx subclones of ST131 (16), virulence genotypes (17), O25bST131 clones (18), and distribution of acquired drug resistance genes.
Approximately one million 301-bp by 2 pair-end reads were obtained.
After trimming based on base quality (quality score limit  0.05; removing reads with more than 2 ambiguous nucleotides or of less than 15 bp inlength), the reads were assembled de novo to construct contigs without
annotation using the commercial software CLC genomics workbench(CLC Bio, Tokyo, Japan). The contigs were subjected to further analyses
with the BLAST algorithm (19) and Resfinder (20). An identification rateof more than 98% was considered positive for each targeted gene. The
virulence score was determined as the number of virulence genes detected,
with pap elements counting collectively as a single trait (17).
We selected sequences of contigs that could contain mobile acquiredelements carrying resistance genes in the isolates, based on BLAST (19)and ResFinder (20) analyses. We then conducted BLAST analyses of thesesequences using the National Center for Biotechnology Information andidentified sequences with close to 100% query cover and identity at thenucleotide level. We then identified isolates that contains the aforementioned sequences with 95% identity at the nucleotide level and 80%coverage level.
Statistical analysis. All analyses were performed using IBM-SPSS Statistics 20 (2012). Bivariate analyses were performed using Fisher’s exact
test or the chi-square test for categorical variables and the t test or the
Mann-Whitney U test for continuous variables. All P values were twosided. Throughout, the percentages displayed are the “valid percentage,”which indicates the percentage excluding the missing data from the denominator.

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Sherchan et al.

TABLE 2 Susceptibility profiles among extended-spectrum--lactamase-producing Escherichia coli isolates in Nepal
P valuea

Value for:ST648 vsnon-ST
131/648

0.999
NA

0.001

0.022

12 (33.3)
2, 8

0.001
NA

0.99

0.005

3 (20)
8, 32

3 (8.3)
4, 16

0.358
NA

0.99

0.343

39 (72.2)
2/38, 2/38

12 (80)2/38, 2/38

18 (50)2/38, 2/38

0.743
NA

0.045

0.064

34 (32.4)4, 8

2 (3.7)2, 4

15 (100)
8, 817 (47.2)4, 8<0.001NA<0.001<0.001

Amoxicillin-clavulanic acidNo. (%) of resistant isolates
MIC50, MIC90

76 (72.4)
16/8, 16/8

40 (74.1)
16/8, 16/8

13 (86.7)
16/8, 16/8

23 (63.9)
16/8, 16/8

0.492
NA

0.352

0.177

Cefoperazone-sulbactam
No. (%) of resistant isolates
MIC50, MIC90

32 (30.5)
16/8, 32/16

19 (35.2)
16/8, 32/16

2 (13.3)
16/8, 16/8

11 (30.6)
16/8, 32/16

0.125
NA

0.82

0.297

Cefmetazole
No. (%) of resistant isolates
MIC50, MIC90

5 (4.8)4, 80
4, 40
4, 45 (13.9)
4, 32

NA
NA

0.009

0.305

Flomoxef
No. (%) of resistant isolates
MIC50, MIC90

2 (1.9)8, 80
8, 80
8, 82 (5.6)
8, 8

NA
NA

0.157

0.99

Fosfomycin
No. (%) of resistant isolates
MIC50, MIC900
4, 160
4, 160
4, 160
4, 16

0.999NA

0.532

0.999Both levofloxacin andtrimethoprim-sulfamethoxazole
No. (%) of resistant isolates

64 (61)38 (70.4)

12 (80)14 (38.9)

0.534

0.004

0.013

Antibiotic and parameterE. coli ST131(n  54)

E. coli ST648(n  15)

E. coli non-ST
131/648b(n  36)

ST131 vs
ST648

Levofloxacin
No. (%) of resistant isolatesc
MIC50, MIC90 (g/ml)

92 (87.6)
4, 452 (96.3)
4, 415 (100)
4, 425 (69.4)
4, 4

Gentamicin
No. (%) of resistant isolates
MIC50, MIC90

41 (39)2, 8

17 (31.5)
2, 8

12 (80)8, 8AmikacinNo. (%) of resistant isolates
MIC50, MIC90

11 (10.5)8, 32

5 (9.3)8, 16

Trimethoprim-sulfamethoxazole
No. (%) of resistant isolates
MIC50, MIC90

69 (65.7)
2/38, 2/38

Minocycline
No. (%) of resistant isolates
MIC50, MIC90a

Bold P values represent statistically significant results. NA, data not available.
b
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
cIncluding intermediate and resistant isolates, based on 2013 CLSI criteria (M100-S23) unless otherwise noted.

higher prevalence of multiple virulence genes than ST648 andnon-ST131/648 isolates, except for hlyD, which was more prevalent in ST648 isolates (n  8; 53%) than in ST131 (n  9; 17%)and non-ST131/648 (n  11; 31%) isolates (ST131 versus ST648,
P  0.007; ST131 versus non-ST131/648, P  0.131; ST648 versus
non-ST131/648, P  0.203). The median virulence score washighest in the ST131 group (score  9; interquartile range [IQR],
6 to 10), followed by ST648 (score  7; IQR, 5 to 8) and nonST131/648 (score  5; IQR, 3 to 7).
The clinical characteristics of patients with ESBL-producing E.
coli isolates were also evaluated as a function of ST (Table 5). The
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mean age of the study cohort was 40.7 years (23.2), and 39 patients (37%) were male. With regard to the demographics of thepatients, the underlying conditions, the severity of illness, and theduration of hospitalization, there were no statistically significant
differences among the ST groups. In female patients, the prevalence of pregnancy was high (n  16; 24%), and in male patients,
benign prostatic hyperplasia (n  6; 15%) was common. There
was a tendency that the ST131 group (n  4; 8%) received appropriate empirical antimicrobial therapy less frequently than the
non-ST131/648 group (n  8; 23%); however, this trend did notreach statistical significance (P  0.057).

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ST131 vsnon-ST
131/648

Whole cohort(n  105)

Attachment

Epidemiology of ESBL-Producing E. coli in Nepal

TABLE 3 Resistance genes among extended-spectrum--lactamase-producing Escherichia coli isolates in Nepal
P valuea

No. (%) of isolates
Whole cohort(n  105)

E. coli ST131(n  54)

E. coli ST648(n  15)

E. coli non-ST
131/648b (n  36)

ST131 vs
ST648

ST131 vs non-ST
131/648

ST648 vs non-ST
131/648

Aminoglycosideaac(3)-IIaaac(3)-IIdaph(3=)-Ia
aph(3=)-Ic
aadA1
aadA2
aadA5
strA/B37 (35.2)
3 (2.9)2 (1.9)1 (1)3 (2.9)10 (9.5)
59 (56.2)
32 (30.5)

15 (27.8)
1 (1.9)2 (3.7)00
8 (14.8)
31 (57.4)
20 (37)

12 (80)00000
12 (80)1 (6.7)10 (27.8)
2 (5.6)0
1 (2.8)3 (8.3)2 (5.6)16 (44.4)
11 (30.6)

0.001
0.999
0.999
NA
NA
0.186
0.14
0.027

0.999
0.561
0.515
0.4
0.061
0.305
0.283
0.652

0.001
0.999NA
0.9990.54
0.9990.03
0.083

Fluoroquinolone
qnrB4
qnrS1
qepA

1 (1)
3 (2.9)2 (1.9)

0
0
0

0
0
0

1 (2.8)3 (8.3)2 (5.6)

NA
NA
NA

0.4
0.061
0.157

0.9990.546
0.999Aminoglycoside andfluoroquinolone
aac(6=)Ib-cr

69 (65.7)

39 (72.2)

14 (93.3)

16 (44.4)

0.163

0.015

0.001

Beta-lactams
blaCTX-M-15
blaCTX-M-27
blaOXA-1
blaTEM-1B
blaSHV-12
blaCMY-42
blaDHA-1

99 (94.3)
2 (1.9)69 (65.7)
41 (39)
1 (1)
1 (1)
1 (1)

51 (94.4)
2 (3.7)38 (70.4)
20 (37)00
0

15 (100)0
14 (93.3)
12 (80)1 (6.7)0033 (91.7)
0
17 (47.2)
9 (25)0
1 (2.8)1 (2.8)

0.999
0.999
0.093
0.004
0.217
NA
NA

0.68
0.515
0.046
0.258
NA
0.4
0.4

0.546
NA
0.004
<0.0010.294
0.999
0.999Macrolide
mphA
ermB

76 (72.4)5 (4.8)46 (85.2)
2 (3.7)

12 (80)1 (6.7)18 (50)2 (5.6)

0.694
0.527

0.001
0.9990.064
0.999Chloramphenicol
catA1
catB3

13 (12.4)
66 (62.9)

037 (68.5)

9 (60)
14 (93.3)

4 (11.1)
15 (41.7)<0.001
0.093

0.023
0.016

0.001
0.001

Sulfonamide
sul1
sul2

70 (66.7)
32 (30.5)

39 (72.2)
20 (37)

12 (80)1 (6.7)19 (52.8)
11 (30.6)

0.743
0.027

0.074
0.652

0.115
0.083

Trimethoprim
dfrA12
dfrA17
dfrA1
dfrA5

10 (9.5)
58 (55.2)
2 (1.9)1 (1)8 (14.8)
31 (57.4)
000
12 (80)002 (5.6)15 (41.7)
2 (5.6)
1 (2.8)

0.186
0.14
NA
NA

0.305
0.197
0.157
0.4

0.9990.016
0.999
0.999Tetracycline
tetA
tetB
tetD

50 (47.6)
31 (29.5)
1 (1)40 (74.1)
1 (1.9)0

2 (13.3)
14 (93.3)
0

8 (22.2)
16 (44.4)
1 (2.8)<0.001<0.001NA<0.001<0.001
0.4

0.703
0.001
0.999ab

Downloaded from http://aac.asm.org/ on August 11, 2015 by guest

Drug and resistance gene

Bold P values represent statistically significant results. NA, data not available.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.

We also searched for mobile acquired elements carrying resistance genes in the isolates (Tables 6 and 7). We identified fivemobile acquired elements carrying multiple resistance genes:
pEC958 (E. coli O25b:H4-ST131 strain EC958, plasmid pEC958),
pKF3-140 (Klebsiella pneumoniae strain KF3, plasmid pKF3-140),
IS26 (E. coli DNA, insertion sequence IS26, insertion sequence
June 2015 Volume 59 Number 6

ISEcp1, blaCTX-M-27), p6234 (K. pneumoniae strain 6234, plasmid p6234), and pEK499 (E. coli strain A, plasmid pEK499). The
pEC958 element was the most common, and was identified in 70
isolates, followed by p6234, which was identified in 64 isolates,and pKF3-140, which was identified in 20 isolates. Eleven isolates
carried three mobile acquired elements (pEC958, pKF3-140, andAntimicrobial Agents and Chemotherapy

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TABLE 4 Virulence-associated traits among extended-spectrum--lactamase-producing Escherichia coli isolates in Nepal
P valuebNo. (%) of isolates
Whole cohort(n  105)

E. coli ST131(n  54)

E. coli ST648(n  15)

E. coli non-ST
131/648c(n  36)

ST131 vs
ST648

ST131 vsnon-ST
131/648

ST648 vsnon-ST
131/648

Adhesin
papA
papG IIsfa/foc
focG
iha
hra

68 (64.8)
60 (57.1)
1 (1)
1 (1)
70 (66.7)
24 (22.9)

54 (100)
35 (64.8)
0053 (98.1)
11 (20.4)

4 (26.7)
10 (66.7)
00
4 (26.7)
1 (6.7)10 (27.8)
15 (41.7)
1 (2.8)1 (2.8)13 (36.1)
12 (33.3)<0.001
0.999NANA<0.0010.44<0.001
0.051
0.4
0.4
<0.0010.219

0.9990.132
0.999
0.999
0.746
0.076

Toxin
hlyD
cnf1
sat
vat

28 (26.7)
12 (11.4)
71 (67.6)
3 (2.9)9 (16.7)
9 (16.7)
54 (100)0

8 (53.3)
05 (33.3)
0

11 (30.6)
3 (8.3)
12 (33.3)
3 (8.3)

0.007
0.189
<0.001
NA

0.131
0.349
<0.001
0.061

0.203
0.546
0.9990.546

Siderophore
iroN
fyuA
ireA
iutA

6 (5.7)92 (87.6)
6 (5.7)83 (79)053 (98.1)
2 (3.7)54 (100)0
14 (93.3)
0
14 (93.3)

6 (16.7)
25 (69.4)4 (11.1)
15 (41.7)NA
0.39
0.9990.217

0.003<0.0010.213<0.001

0.162
0.083
0.307
0.001

CapsulekpsM IIK1 kpsM
K5 kfiC

3 (2.9)2 (1.9)14 (13.3)

00
11 (20.4)

00
1 (6.7)3 (8.3)2 (5.6)2 (5.6)

NA
NA
0.44

0.061
0.157
0.067

0.546
0.999
0.999Miscellaneous
usp
traT
ompT
H7 fliCmalX

58 (55.2)
87 (82.9)
71 (67.6)
2 (1.9)104 (99)

54 (100)
47 (87)54 (100)054 (100)0
15 (100)
11 (73.3)
0
15 (100)

4 (11.1)
25 (69.4)
6 (16.7)
2 (5.6)35 (97.2)<0.001
0.333
0.002
NA
NA

<0.001
0.059<0.001
0.157
0.4

0.307
0.022
<0.001
0.9991.0Median virulence score (IQR)d

9 (6–10)9 (9–10)

7 (5–8)5 (3–7)<0.001<0.0010.074

apapG III, pic, and ibeA were tested, with no positive isolates detected.
bBold P values represent statistically significant results. NA, data not available.
c
ESBL-producing E. coli isolates with ST other than ST131 and ST648.
d
The virulence score represents the number of virulence genes detected, with pap elements counting collectively as a single trait (17).

p6234), 10 of which belonged to ST131. All 64 isolates that carried
p6234 also carried pEC958. All isolates which had these mobile
elements had 95% identity at the nucleotide level and 80%coverage level.
DISCUSSION

To our knowledge, this is the first study to reveal the microbiological and clinical epidemiology of ESBL-producing E. coli in Nepal.
We discovered that more than 90% of the ESBL-producing E. coli
isolates in Nepal were CTX-M-15 positive, and more than half
were ST131 isolates. All 54 ESBL-producing E. coli ST131 isolates
were identified as the O25b-ST131 clone, subclone H30-Rx, whichhave been reported to be more antibiotic resistant and to havevirulence profiles (16). These findings correlate with the multiple
reports on a global spread of CTX-M-15-positive O25b-ST131
clones (21).
Another key finding of this study is the discovery that ST648
isolates made up 14% of ESBL-producing E. coli in Nepal and that
their microbiological characteristics are distinct from those of
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ST131 isolates. Human isolates of CTX-M-producing E. coli
ST648 have been reported from various geographical regions,such as Europe, North and South America, Africa, and Asia; themajority of these isolates belong to phylotype D (22–25). CTX-Mproducing E. coli ST648 strains have also been isolated from wild
birds in Mongolia and Germany (24, 26–28) and from companion
and livestock animals in European countries (24, 29, 30). A recent
study on ESBL-producing E. coli isolates (n  1,152) sampled inEurope, predominantly from dogs, cats, and horses, identified 40(3.5%) ESBL-producing E. coli strains as ST648 (phylotype D,
CTX-M-15 positive; 72.5%), whereas ST131 isolates (phylotype
B2, CTX-M-15-positive; 46.9%) occurred less frequently (2.8%)(30). The authors also found that a higher proportion of ST648
strains showed resistance to most non--lactam antibiotics, andvirulence genes were less abundant in ST648 strains than in
ST131strains (30). These findings are concordant with the current
study. To our knowledge, this is the first study to systematicallyevaluate the microbiological and clinical characteristics of humanisolates of CTX-M-producing ST648 strains, all of which were

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Virulence-associated trait andgenea

Attachment

Epidemiology of ESBL-Producing E. coli in Nepal

Downloaded from http://aac.asm.org/ on August 11, 2015 by guest

Underlying conditions
Benign prostatic hyperplasia
Urolithiasis
Uterine prolapse
Pregnancy
Malignancy
Diabetes mellitus
Chronic obstructive pulmonary disease

Departments
Medicine
Surgery
Obstetrics and gynecology
Pediatrics

Demographics
Mean age, yr (SD)Male patients
InpatientsParameter

10 (9.5)

6 (15.4)
6 (5.7)6 (5.7)16 (24.2)
3 (2.9)3 (2.9)8 (7.6)40 (38.1)
21 (20)22 (21)
11 (10.5)

40.7 (23.2)
39 (37.1)
65 (61.9)

Whole cohort(n  105)

4 (7.5)6 (11.1)

4 (21.1)
2 (3.7)4 (7.4)9 (25.7)
2 (3.7)2 (3.7)2 (3.7)20 (37)12 (22.2)
13 (24.1)
6 (11.1)

41.7 (22.8)
19 (35.2)
35 (64.8)

E. coli ST131(n  54)

10 (6–14)

2 (13.3)

2 (13.3)

1 (6.7)00
2 (22.2)
01 (6.7)
2 (13.3)

7 (46.7)
1 (6.7)
2 (13.3)
2 (13.3)

45.7 (27.3)
6 (40)
10 (66.7)

E. coli ST648(n  15)

11 (9–16)

8 (22.9)2 (5.6)1 (7.1)4 (11.1)
2 (5.6)5 (22.7)
1 (2.8)0
4 (11.1)

13 (36.1)
8 (22.2)
7 (19.4)
3 (8.3)37 (22.1)
14 (38.9)
20 (55.6)

E. coli non-ST
131/648b (n  36)

0.38

0.607

0.999

0.999
0.9990.57
0.999
0.999
0.527
0.204

0.558
0.27
0.494
0.9990.541
0.767
0.999

ST131 vs
ST648

0.412

0.057

0.468

0.366
0.213
0.999
0.999
0.9990.515
0.213

0.999
0.999
0.796
0.736

0.266
0.824
0.388

ST131 vs non-ST
131/648

0.202

0.702

0.571

0.521
0.307
0.999
0.999
0.9990.294
0.999

0.539
0.251
0.709
0.624

0.251
0.9990.543

ST648 vs non-ST
131/648

P value

Severity of illness
Sepsis14 (13.6)

10 (8–13)

Valuea for:TABLE 5 Bivariate analysis of clinical characteristics of patients with isolation of extended-spectrum--lactamase producing Escherichia coli as a function of sequence type

Antimicrobial therapy
Appropriate empirical antimicrobial therapy

10 (8–14)
Values are number (%) unless otherwise indicated.
ESBL-producing E. coli isolates with ST other than ST131 and ST648.

Median duration of hospitalization, days (IQR)
a
b

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TABLE 6 Mobile acquired elements carrying resistance genes among
ESBL-producing E. coli isolates in Nepal

Isolateno.

pEC958

ST38

77
81




ST167
ST361

74
105




ST410

6
55







ST44
ST517
ST617
ST1312

27
97
46
10









New ST

34
35
39
56
100













1
2
3
5
8
14
16
19
23
24
25
26
28
29
30
37
38
43
45
47
50
52
59
60
61
63
64
67
68
69
70
72
75
78
79
80
85
86
87
90
92
96
99
101
102
104









7
15
22
36
40
42
51
57
62
65
76
82
95
98
















12
31
58
71






pKF3-140

















p6234

pEK499


































IS26



































































No. (%) carrying resistance genesa

Sequence
type

pEC958

pKF3-140

IS26

p6234

pEK499

ST131
ST648
ST405
ST38
ST167
ST361
ST410
ST44
ST517
ST617
ST1312
New ST43 (80)14 (93)4 (80)
2 (67)
1 (50)
1 (50)
2 (100)1 (100)1 (100)1 (100)1 (100)5 (63)

17 (31)

2 (4)

4 (7)

1 (20)
1 (33)

1 (20)

36 (67)14 (93)3 (60)
1 (33)

1 (13)

Total

76

20

1 (20)

2 (100)1 (100)1 (100)1 (100)5 (63)
3

64

5

a





pEK499TABLE 7 Numbers of mobile acquired elements carrying resistancegenes in each ST








p6234

positive for CTX-M-15. The fact that ST648 isolates, which show ahigher drug resistance than ST131 isolates and contain profuse
virulence genes, comprised 14% of all ESBL-producing E. coli isolates in Nepal is a concerning observation. In addition, previous
reports have suggested the spread of CTX-M-producing ST648
strains among humans and animals worldwide (24). These find-












IS26

pEC958, Escherichia coli O25b:H4-ST131 strain EC958 plasmid pEC958; National
Center for Biotechnology Information (NCBI) accession no. HG941719.1. pKF3-140,
Klebsiella pneumoniae strain KF3 plasmid pKF3-140; NCBI accession no. FJ876827.1.
IS26, E. coli DNA, insertion sequence IS26, insertion sequence ISEcp1, blaCTX-M-27;
NCBI accession no. AB976590.1. p6234, K. pneumoniae strain 6234 plasmid p6234;
NCBI accession no. CP010390.1. pEK499, E. coli strain A plasmid pEK499; NCBI
accession no. EU935739.1.



pKF3-140a

pEC958

pEC958, Escherichia coli O25b:H4-ST131 strain EC958 plasmid pEC958; National
Center for Biotechnology Information (NCBI) accession no. HG941719.1. pKF3-140,
Klebsiella pneumoniae strain KF3 plasmid pKF3-140; NCBI accession no. FJ876827.1.
IS26, E. coli DNA, insertion sequence IS26, insertion sequence ISEcp1, blaCTX-M-27;
NCBI accession no. AB976590.1. p6234, K. pneumoniae strain 6234 plasmid p6234;
NCBI accession no. CP010390.1. pEK499, E. coli strain A plasmid pEK499; NCBI
accession no. EU935739.1.

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ST131

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Sequence
type

Isolateno.

ST405

Mobile acquired elements carrying resistance genesa

Mobile acquired elements carrying resistance genesa

Sequence
type

ST648

TABLE 6 (Continued)

Attachment

Epidemiology of ESBL-Producing E. coli in Nepal

ACKNOWLEDGMENTS
This study was supported by Grants for International Health Research(24S-5 and 26S-101) from the Ministry of Health, Labor, and Welfare ofJapan.
We have no conflict of interest to declare.

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Sokurenko E, Tchesnokova V, Price LB, Johnson JR. 2013. Molecularepidemiology of Escherichia coli sequence type 131 and its H30 and
H30-Rx subclones among extended-spectrum-beta-lactamase-positiveand -negative E. coli clinical isolates from the Chicago region, 2007 to
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GG. 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob Agents Chemother 53:2733–2739. http://dx.doi.org/10.1128/AAC.00297-09.
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MR, Nordmann P, Ruppe E, Sarthou JL, Frank T, Vimont S, Arlet G,
Branger C, Woodford N, Denamur E. 2009. Rapid detection of theO25b-ST131 clone of Escherichia coli encompassing the CTX-M-15producing strains. J Antimicrob Chemother 64:274 –277. http://dx.doi.org/10.1093/jac/dkp194.
19. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K,
Madden TL. 2009. BLAST: architecture and applications. BMC Bioinformatics 10:421. http://dx.doi.org/10.1186/1471-2105-10-421.
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O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640 –2644. http://dx.doi.org/10.1093/jac/dks261.
21. Oteo J, Perez-Vazquez M, Campos J. 2010. Extended-spectrum -lactamase producing Escherichia coli: changing epidemiology and clinical impact. Curr Opin Infect Dis 23:320 –326. http://dx.doi.org/10.1097/QCO.0b013e3283398dc1.
22. Sidjabat HE, Paterson DL, Adams-Haduch JM, Ewan L, Pasculle AW,
Muto CA, Tian GB, Doi Y. 2009. Molecular epidemiology of CTX-Mproducing Escherichia coli isolates at a tertiary medical center in western

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ings raise the concern that ST648 strains could present anotherpandemic clone of ESBL-producing E. coli.
Other STs found in isolates from Nepal in this study have also
been reported to be associated with ESBL production in E. coli andhave been found in wildlife, livestock, and humans (24). The
spread of ESBL-producing E. coli ST405, ST38, ST315, and ST410
has been reported in animals in Europe, as well as in humans
worldwide (24). ESBL-producing E. coli ST393 was isolated fromlivestock animals in Germany and from humans globally (24).
Global spread of ESBL-producing E. coli ST10, ST617, and ST167
has also been reported (24).
Due to the limited information available from medical charts,
we could not evaluate detailed clinical variables in the current
study. Nevertheless, it is noteworthy that more than 38% of ESBLproducing E. coli strains were isolated from the outpatient clinic,
and about a quarter of patients with ESBL-producing E. coli werepregnant women. This would suggest a wide spread of CTX-Mproducing E. coli across the communities in Nepal. Combinedwith the high resistance to orally available antibiotics, these community-isolated ESBL-producing E. coli strains present an emerging challenge for community practitioners and hospitals worldwide.
Data on the effectiveness of treatment using cephamycins andoxacephems for the ESBL-producing organisms are scarce (31,32), and further studies regarding the optimal clinical uses of thesedrugs for the treatment of ESBL-producing E. coli are warranted.
In conclusion, we revealed that the high resistance of ESBLproducing E. coli to multiple classes of antibiotics in Nepal isdriven mainly by CTX-M-producing ST131 and ST648 strains.
Their prevalence in communities is a matter of great concern, andfurther studies are necessary to identify the epidemiology of CTXM-ST648 to control the spread of ESBL-producing E. coli.

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Lubke-Becker A, Guerra B, Wieler LH, Ewers C. 2010. CTX-M-15-type
extended-spectrum beta-lactamases-producing Escherichia coli from wild
birds in Germany. Environ Microbiol Rep 2:641– 645. http://dx.doi.org/10.1111/j.1758-2229.2010.00148.x.
29. Cortes P, Blanc V, Mora A, Dahbi G, Blanco JE, Blanco M, Lopez C,
Andreu A, Navarro F, Alonso MP, Bou G, Blanco J, Llagostera M.
2010. Isolation and characterization of potentially pathogenic antimicrobial-resistant Escherichia coli strains from chicken and pig farms in
Spain. Appl Environ Microbiol 76:2799 –2805. http://dx.doi.org/10.1128/AEM.02421-09.
30. Ewers C, Bethe A, Stamm I, Grobbel M, Kopp PA, Guerra B, Stubbe M,
Doi Y, Zong Z, Kola A, Schaufler K, Semmler T, Fruth A, Wieler LH,
Guenther S. 2014. CTX-M-15-D-ST648 Escherichia coli from companion
animals and horses: another pandemic clone combining multiresistanceand extraintestinal virulence? J Antimicrob Chemother 69:1224 –1230.
http://dx.doi.org/10.1093/jac/dkt516.
31. Doi A, Shimada T, Harada S, Iwata K, Kamiya T. 2013. The efficacy of
cefmetazole against pyelonephritis caused by extended-spectrum betalactamase-producing Enterobacteriaceae. Int J Infect Dis 17:e159 –163.
http://dx.doi.org/10.1016/j.ijid.2012.09.010.
32. Lee CH, Su LH, Tang YF, Liu JW. 2006. Treatment of ESBL-producing
Klebsiella pneumoniae bacteraemia with carbapenems or flomoxef: a retrospective study and laboratory analysis of the isolates. J Antimicrob Chemother 58:1074 –1077. http://dx.doi.org/10.1093/jac/dkl381.

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Pennsylvania. Antimicrob Agents Chemother 53:4733– 4739. http://dx.doi.org/10.1128/AAC.00533-09.
23. Zong Z, Yu R. 2010. Escherichia coli carrying the blaCTX-M-15 gene of
ST648. J Med Microbiol 59:1536 –1537. http://dx.doi.org/10.1099/jmm.0.022459-0.
24. Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. 2012. Extendedspectrum beta-lactamase-producing and AmpC-producing Escherichia
coli from livestock and companion animals, and their putative impact on
public health: a global perspective. Clin Microbiol Infect 18:646 – 655.
http://dx.doi.org/10.1111/j.1469-0691.2012.03850.x.
25. Mshana SE, Imirzalioglu C, Hain T, Domann E, Lyamuya EF,
Chakraborty T. 2011. Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary
hospital in Tanzania. Clin Microbiol Infect 17:1279 –1282. http://dx.doi.org/10.1111/j.1469-0691.2011.03518.x.
26. Guenther S, Aschenbrenner K, Stamm I, Bethe A, Semmler T, Stubbe
A, Stubbe M, Batsajkhan N, Glupczynski Y, Wieler LH, Ewers C. 2012.
Comparable high rates of extended-spectrum-beta-lactamase-producing
Escherichia coli in birds of prey from Germany and Mongolia. PLoS One
7:e53039. http://dx.doi.org/10.1371/journal.pone.0053039.
27. Guenther S, Ewers C, Wieler LH. 2011. Extended-spectrum betalactamase producing E. coli in wildlife, yet another form of environmental
pollution? Front Microbiol 2:246. http://dx.doi.org/10.3389/fmicb.2011.00246.
28. Guenther S, Grobbel M, Beutlich J, Bethe A, Friedrich ND, Goedecke A,Attachment

11

Original article

Assessment of Health Systems in Relation to Interface
Between Malaria Control Programs and Health System
Strengthening: Comparative Study Between Nepal andViet Nam
Ohara H1, Sherchan JB4, Pokhrel BM2, Hirayama T1, Vu Huy Nam3, Sherchand JB2
1

Bureau of International Medical Cooperation, National Center for Global Health and Medicine, Japan

2

Institute of Medicine, Tribhuvan University, Nepal

3

National Institute of Malariology, Parasitology and Entomology, Viet Nam.

4

Kathmandu University, School of Medical Sciences, Kavre. Nepal

Correspondence: Dr. Hiroshi Ohara, MD; PhD.
Email: ohara52jp@gmail.com
Abstract
Introduction: Malaria control has been a major health issue with high priority in endemic
countries and various efforts have been made with the support of foreign assistant partners.
In order to implement efficient and sustainable control, integration of the control program
into general health system or effective interactions between them is one of the important
strategies.
Methods: Studies were conducted in Nepal and Viet Nam. Information obtained fromdocument reviews, interviews, and field surveys were analyzed from the viewpoint ofinterface between malaria control program and the health system in accordance with six
building blocks of a health system, with special emphasis on good practices and challenges
in the implementation of the malaria control program.
Results: Among good practices, strong government commitment towards the control
programs to strengthen facilities and capacity of health workers at the primary level,utilization of health volunteers, setting up mobile team and intensified education for residents
were noteworthy. Key challenges mainly involved remote areas. Introduction of malaria due
to population movement and the emergence of new endemic areas have become growing
issues. While strengthening of the vertical health program appeared to have some impact on
the general health system, particularly at the primary level, dissociation between the verticalcontrol program and horizontal general health system still remains.
Conclusion: It is crucial to implement an effective and equitable malaria control program that
responds to these existing challenges and can create a sustainable health system. Addressingthese issues will lead to further strengthening of the health system there and eventually leadto the effective implementation of various health programs.
Key words: Malaria control, Health system, Nepal, Viet Nam

Introduction
Over the past decades malaria control has been implemented
intensively with the support of Global Fund against AIDS,
Tuberculosis and Malaria (GFATM)and other assistantpartners as one of the most important disease control
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programs, and good results have been obtained in someendemic countries.1-3 Malaria control programs havebeen implemented under global malaria control strategies
although key strategies have varied over times: spraying
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Ohara H et al.,12of pesticide, community-based control, school healthbased control, Roll Back Malaria etc. In recent years, theimportance of disease control measures in relation to healthsystem strengthening (HSS), particularly the integration ofa control program (vertical health system) into the general
health system (horizontal health system) with the aim ofachieving a synergic effect among health programs, hasbeen stressed as an important strategy.4-6
In the process of disease control, good practices have beenrecognized, but interaction between disease specific control
programs and HSS has been debated, namely whether
disease specific programs have actually contributed to thestrengthening of the general health system and whether
disease control programs are well integrated into thegeneral health system.7, 8
In the program implementation process, smooth
implementation of the disease specific control program hasoften been hindered by challenges or bottle necks that exist
in the health system. Through the effective intervention
to these challenges/bottlenecks,expansion of the healthprograms and strengthening of the general health systemare expected, bringing a synergistic effect on other disease
specific programs and furthermore general health system.
This study was undertaken in order to assess the interface
between disease specific programs supported by GFATM
and HSS, with special reference to interaction between
malaria control programs and general health systems.

Methods
The primary surveys were conducted for Viet Nam(2009) and Nepal (2012), by setting the National Malaria
Control Programs supported by GFATM as entry points
with special emphasis on good practices and challenges/
bottlenecks in implementing malaria control program in
relation to the components of health system. Data from thetwo countries was updated as long as possible, based on
information obtained by the supplemental survey during
2012-14 and by documents. In each country, the survey
was conducted at various levels (from central to primarylevel) by document reviews, key informant interviews, andobservation of facilities. The results of the primary surveys
were analyzed, summarized in reports and submitted to
WHO Western Pacific Regional Office.9

Document reviews
Documents related to health systems, National Malaria
Control Programs (NMCP), health statistics, GFATM
supports, etc. were collected and reviewed.10-15

Key informant interviews
Key informant interviews were conducted with thehealth staff at each level (central, regional, district, andcommunity level) of the health facility, health managers at
the district and provincial levels, and program managersof Ministry of Health (MOH in Viet Nam) or Ministry of
Health and Population (MOHP in Nepal),WHO, NGOs,
and other partner agencies. Leading contents of theinterview included an outline of the health system, general
information on malaria control programs in relation to thehealth system. Good practices and strengths to overcome
challenges/bottlenecks, and on-going interventions forexisting bottlenecks, integration between NMCP andgeneral health system, etc., were also considered

Field surveys
In addition to surveying the capital cities of each country,
malaria endemic areas, where Malaria Control Programs
have been implemented with the support of GFATM, wereselected (Thanh Hoa and Dien Bien Provinces in Viet Nam,and Dhanusha District and Hetauda City in Nepal). General
information on health and medical care, information on
health system and health program implementation, etc.
were collected at Provincial Health Offices and District
Health Offices, followed by surveys at health facilities atthe primary level (or community level).

Analysis
Information obtained from the interviews, field surveys,
and documents were summarized from the viewpointof interface between malaria control programs and thegeneral health system in accordance with the six building
blocks (components) of a health system (Leadership andgovernance, Service delivery, Workforce, Information
system, Medical products and technology, and Financing)proposed by WHO.16 Good practices and bottlenecks/
challenges in the implementation of the control programs
were identified, and possible solutions for the challenges/
bottlenecks were discussed.

Results
1. Overview of malaria conditions in Nepal
and Viet Nam
In the two countries in this survey, Viet Nam and Nepal,
malaria morbidity and mortality rates were considerablyhigh in the past and malaria was given the highest priority
in the health policy by the governments. Since the mid1990s, malaria controls were actively implemented in thesecountries based on the National Malaria Control Programs(NMCPs) and the principles of Roll Back Malaria, whichwww.jiom.com.np

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consisted of strategic priorities including vector control
and personal protection through the distribution of bednets(insecticide-treated bed nets; ITNs and long lasting
insecticidal nets; LLINs), early diagnosis and prompt
treatment (EDPT)including rapid diagnosis tests (RDTs)
and artemis in combination therapies (ACTs), malaria
surveillance and epidemic preparedness, behavioral change
communication (BCC), and improvement in program
management along with setting up targets for control. In
particular, since the early 2000s, GFATM has contributed
a large budget to malaria control programs (60-65% inViet Namand70-78% in Nepal of the total malaria control
budget). As a result, malaria in these countries has decreased
remarkably in recent years, reaching pre-elimination levels.

2. Outline of general health systems in Nepal
and Viet Nam
Health networks in the two target countries have beencreated at the central to commune level in accordance withthe administrative strata (e.g. in Nepal; central－regional－
district－commune levels,in Viet Nam; central－provincial－district－commune levels). Treatment caresystems as well as preventive care systems have also beenbasically constructed in accordance with the administrative
strata. (i.e. central, provincial (or regional) and district
hospitals and commune health stations, etc.).A referral
system, as well as health information and supply systems,
function based on this network. In addition, some healthcontrol programs have their own systems (e.g. national
institutes, provincial or regional control centers, district
control centers).
Commune Health Stations (CHSs) provide health care atthe primary level and under the CHSs, there are health posts(HPs) and sub-health posts (SHPs). These CHSs along with
HPs and SHPs have the tasks of providing primary healthcare services, first-aid and treatment, implementation ofnational health programs, assisting in normal deliveries,
family planning practices, health promotion, etc. Thereare commune (village) health workers in each commune,
under the direct management and direction of the CHSs
and commune leaders.

3. Characteristics of malaria control in
relation to the health system
A: Characteristics of general health system (horizontal
health system)
B: Characteristics of malaria control (vertical healthsystem)www.jiom.com.np

3-1 Nepal
Leadership and governance
A: The current long-term health plan (1997-2017) aims
to provide health services throughout the country,
particularly extending the primary health care system
to the rural population and improving the health statusof vulnerable populations, such as women and children,
the rural population, the poor, the underprivileged andthe marginalized. In recent years, the government has
attached high importance to the promotion of education
among residents and many primary schools have been
constructed.
B: Since the launch of the Insect Borne Disease Control
Program in 1954, the government has given malaria
control high priority, and since 1993, the Epidemiology
and Disease Control Division (EDCD) under MOHP
has taken the lead in the malaria control program.
Currently, the control program is basically implemented
using the existing health system.

Service delivery
A: Efforts have been made to design a health systemhierarchy from MOPH to the primary level, in orderto ensure that the majority of the population has
access to public health care facilities and can receive
minor treatment at affordable prices. The government
has strengthened the HPs and SHPs, which serve as
first contact to basic health services and the venue
for community-based activities with the support of
GFATM. The pull system for essential drug supply was
expanded to all 75 districts in 2010.
B: Delivery of LLINs is managed by the Population
Service International (PSI).At local level, distribution is
managed by NGOs/other partners and implementation
is carried out by a broad range of community basedorganizations. Monitoring teams for ITNs and LLINs
were organized at the district level. Mobile teams wereorganized, and are responsible for prevention, diagnosis
and treatment, in endemic areas. RDTs and microscopy
have become available as diagnostic tools for malaria
by HPs due to the support of GFATM, anda cold chain
for vaccination was used for storage of RDTs.

Workforce

A: Since the establishment of the first school of medicine
in 1980, the number has markedly increased (22schools of medicine in 2014). The increased number of
medical doctors, nurses, and other co-medical staff has
contributed to increased health and medical care of the
Nepalese people.

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Ohara H et al.,14
B: Female Community Health Volunteers (FCHVs)
were organized at the commune level for integration
activities including malaria control. Training on malaria
control for the health staff has been integrated withother disease control and training has been conductedwith the support of GFATM at the peripheral level.

Information system
A: The routine monitoring system has been improved
over the years. The Health Management Information
System (HMIS), Logistics Management Information
System (LMIS) and Fiscal Management Information
System (FMIS) have also been well developed over thelast 10 years. Health related activities are recorded andreported from the lowest health unit right on up to the
district hospitals. In addition to HMIS, other individual
programs are also providers of information.
B: Sentinel sites were set up for malaria outbreaksurveillance. Reporting and monitoring systems in the
public sector, from the peripheral level up to MOHP
level, have been strengthened.

international community has also cooperated with
policy implementation at various levels. For National
Health Programs, National Steering Committees areorganized and programs are managed more intensively
and efficiently with strong leadership and inter sectoralcollaboration. Related major national institutes are
responsible for the executive centers of the respective
health programs, as well as the provision of technicaladvice, operational research and staff training.
B: The government attached highest priority to malaria
control in all health programs. NMCP, which began
in 1991, has been reinforced by the strong leadershipof the Government and National Steering Committee,
which consists of multi-sector members,utilizing thevertical malaria control system along with general
health system. The general health system has also beenstrengthened and used as a malaria control program.
Recently, high priority has been attached to control
in frontier areas. A high literacy rate, effective use ofschool health education, education for residents inendemic areas, and preparation of guidelines have also
facilitated the smooth implementation of the program.

Medical products and technologies

Service delivery

A: In 2007, the National Drug Strategy was revised andthe National Essential Drug List was established. Inorder to manage the above processes effectively,HMIS
is used.

A: The local administration is organized into provincial,
district and communal political units which areresponsible for the implementation of the healthprograms. All medicines and medical equipment aresupplied by the government through the administrative
strata. Participation by health facilities under the
military, police and other sectors providing medical
services to the population has helped increase healthcare coverage. The referral system among medical
institutions was strengthened in collaboration withforeign assistant partners.

B: A considerable amount of ITNs, LLINs, RDTs, ACTs,
and slide glasses for microscopic testing were provided
at the peripheral level with the support of GFATM.

Financing
A: The amount of the budget for health programs fundedby the government, and the percentage of the healthbudget within the total budget, were increased (from5.1% in 1998 to 6.3% in 2013).
B: Since 2004, GFATM has greatly contributed to theprevention and treatment of malaria, particularly
through the distribution of LLINs, ACTs and RDTs
in high risk areas, along with the training of healthworkers and BCC activities for the residents. Treatment
drugs are administered to patients free of charge atpublic medical facilities nationwide and diagnostic
services for malaria are provided free of charge at all
public sector health facilities in high endemic areas.

3-2Viet Nam
Leadership and governance
A: Since the 1990s the government has worked
hard to strengthen the general health system. The

B: Service delivery in malaria control was basicallycarried out utilizing the general health system. The
Army Medicine, People’s Committee, Women’s Unionand other local organizations helped deliver bed nets
and other services in the control program.

Workforce
A: Since the 1990s, human resources in health care havebeen trained both quantitatively and qualitatively.
Improvement in training capacity to increase humanresources has been observed. Commune health workers
have participated in various programs.
B: The government has implemented a policy to train
health workers for malaria control with the cooperationof foreign donors. The role of public organizations,such as women’s unions and youth unions, in the

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implementation of malaria control programs andcollaboration with the military in hard-to-reach areas,
are also noteworthy.

Information system
A: The reporting and information system functions
efficiently, and reports from the primary level aretransmitted to upper levels. The role of the mobile teamis outstanding in information transmission and guidancein program implementation.
B: Reports on malaria from the primary level aretransmitted to upper levels and then feedback isprovided. Currently, considerable parts of these systems
are integrated into the general health system. There aremany examples of prompt and appropriate responses incases of disease outbreak. The role of the mobile teamis outstanding in the transmission of information and in
the provision of guidance for program implementation.

Medical products and technologies
A: Development of the pharmaceutical industry has
contributed considerably to the implementation of
essential drug policies targeting primary health care.
Drug quality is managed following the good practice
criteria based on standards and guidelines for drug
production, quality control, storage and distribution.
Currently, all facilities are to follow the standards ofGood Manufacturing Practice (GMP)-WHO.
B: Production of drugs used for malaria treatment,
insecticides, and bed nets within the control program
has gradually shifted to local sources and the drugs
are supplied to the peripheral level under the proper
guidance of the government. Widespread distribution ofartemis in suppositories at the primary level has greatly
contributed to a lower mortality rate.

expansion of the malaria program by strengthening
activities for high risk groups.

4. Good practices in malaria control
Table 1 summarizes the good practices in the general healthsystem which are regarded as having a good effect onmalaria control as shown in table. Table 2 summarizes thegood practices in the malaria control programs.

5. Bottlenecks/challenges
Table 1 Good practices in the general health system(Strengthening of General Health System), which
contributed to malaria control
Leadership and governance1

Effort of the government to strengthen the general
health system

2

Efficient management of the National Health Programswith strong leadership (V)3High priority given to the promotion of education
Service delivery

4

Strengthening of health posts and sub-health posts

5

Effective service delivery in accordance with theadministrative strata (V)6Support of military and police to service delivery (V)7Contribution of the mobile teams

8

Expansion of the pull system for essential drug supply
Workforce9

Increased training opportunities for health workers.

10 Marked increase of the number of doctors and nurses (N)11 Improved skills by commune
conducting various programs

healthworkers

Information system
12 Improvement in the routine monitoring system

Financing
A: A broad orientation of health financing was set in the
1990s through the development of health insurance, thepartial user fee policy, and the Government’s resolution
on “social mobilization” in areas of education, healthand culture. The government also focused on subsidies
to users of health services, such as health care for thepoor and children under 6 years of age. The healthbudget has continued to increase in line with theeconomic development of the country.
B: Initially, the governments ought to increase the malaria
program budget, and the People’s Committee of Viet
Nam and international community financially supported
the program. GFATM has greatly contributed to the

13 Contribution by the mobile teams (V)Medical products and technologies
14 Management of drug quality based on the standards
and guidelines (V)15 Development of the pharmaceutical industry and its
contribution to the essential drug policy (V)16 Revision of the National Drug Strategy (N)Health financing
17 Substantial support from the GFATM
18 Increase funding of the health budget by the government
19 Development of the health insurance, partial user fee
policy, subsidies to users of health services (V)(V): Outstanding in Viet Nam, (N): Outstanding in Nepal

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Ohara H et al.,Table 2 Good practices in malaria control
Leadership and governance
1

High priority of malaria control by the government

2Strong leadership of the government and the National Steering Committee (V)3

Utilization of the general health system in the malaria control program
Service delivery

4

Participation by the Army Medicine, People’s Committee, Women’s Union and other local organizations(V)5

Monitoring teams for ITNs and LLINs at the district level (N)6Contribution by the mobile teams
Workforce

7

Increased training opportunities for health workers

8Contribution bywomen’s unions, youth unions and military in hard-to-reach areas(V)9Contribution by Female Community Health Workers (N)Information system

10 Monitoring visits and periodical submission of reports
11 Setting up the sentinel sites for malaria outbreak surveillance(N)12 Appropriate transmission of the information and feedback to primary level (V)Medical products and technologies
13 Provision of considerable amount of ITNs, LLINs, RDTs, ACTs
14 Domestic production of treatment drugs, insecticides and bed nets(V)Health financing
15 Considerable financial supportfor malaria control by GFATM
16 Treatment drugs provided free of charge at public facilities(V): Outstanding in Viet Nam, (N): Outstanding in Nepal
ITNs: insecticide-treated bed nets, LLINs: long lasting insecticidal nets,
RDTs: rapid diagnosis tests, ACTs: artemisinin combination therapies

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Table 3 Existing challenges and bottlenecks in malaria control
Viet
Nam

A. Leadership and governance

Nepal

1

Weak program management capacity

2

Introduction of malaria associated with population movement

++

++

3

Weak health system in remote (frontier & border) areas

+

++

4

Weak coordinationbetween medical institutions, public-private sectors and laboratories

+
Viet
Nam

++

++

B. Service delivery
567Inequality in the distribution of bed nets (to vulnerable people)

Nepal
++

Many hard-to-reach areas
Weak coordination between local government and GFATM in the distribution of bed
nets+

C. Workforce

Viet
Nam

8

Shortage of health workers and manpower in remote areas

9

Low skill level of health workers in remote areas

10

Frequent changes in health workers and manpower in remote areas

11

Limited number of entomologists

++
+
+
Viet
Nam

D. Information system
12

Weak private health sector

13

Poorly developed reporting system from the private health sector

14

Inadequate disease surveillance system

+
++
Viet
Nam
+

E. Medical products and technologies
15

Inadequate quality assurance system for malaria testing16

Weak function of the National Reference Laboratory

17

Difficulty in treatment due to increasing anti-malaria drug resistance ofP. Falciparum

++
++
Nepal
++
+
++
++
Nepal
+
++
+
Nepal
++
++

18

Sustainable supply of health products (ACT, RDT, LLIN)

++
Viet
Nam
+

19

Low incentive for health workers

++

++

20

Heavy dependence on GFATM (sustainability is a challenge)

+
Viet
Nam
++

++
Nepal

+

++

F. Health financing

Other
21

22

New endemic areas have been reported (environmental and social factors are suspect)
Increased number of cases of imported malaria

++
Nepal
++

++

++: major challenges/bottlenecks, +: intermediate, No mark: minor

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Ohara H et al.,18

Discussion
The general health system in Nepal used to be fragile
in the past, but has gradually been strengthened andhas been utilized in greater part in the malaria control
program.17During the period of political instability (19962006), health systems were affected, but malaria control
was minimally affected compared to other disease control
programs due to its high governmental priority and thecontinuous support of the international community.
The Vietnamese government has worked hard to strengthen
the existing health systems since the 1990s (both malaria
specific and general health systems). The international
community has also cooperated with policy implementationof Viet Nam at various health system levels.18Malaria
control measures were effectively implemented under thestrong leadership of the National Steering Committee,
which has further strengthened and utilized the existing
health system and mobilized public organizations.
Generally, collaboration among disease specific programs
at upper levels is limited, and a health staff, as well as an
infrastructure, is dedicated to each program. However,health care at lower levels has greater integration(both in
Nepal and Viet Nam). By coordinating with communityand social organizations, health workers carry out various
tasks, such as primary health care, implementation ofnational health programs, preventive medicine, IEC
activities, etc. GFATM and other assistant partners provide
support by promoting training and supplying essential
medicine and equipment. Malaria control has gradually
become integrated with the primary health care system.
Best practices were identified from survey results. Amongthese, intensified education for residents focusing on disease
prevention, strengthening of facilities at the primary levelsuch as health posts and training health workers, utilizationof health volunteers at the primary level, giving frontierareas high priority, and setting up mobile teams, werenoteworthy and were held in in common by both countries.
In addition, effective implementation under the strong
leadership of the National Steering Committee could beseen in Viet Nam, utilization of the existing heath system
was outstanding.
A synergetic effect of disease specific programs(vertical
health programs) such as malaria control on the general
health system could be seen to some degree, particularly
at the primary level. The management system of vertical
health programs appeared to have a good impact on
the general health system at various levels. However,dissociation between vertical malaria control programand horizontal general health system also seems to exist.

In addition, similar to other reports,coordination between
malaria control programs and other disease specificprograms is limited in many cases.5,6 It is true that carryingout a vertical health system is important in implementing
a malaria control program, but intensification of themalaria control program does not automatically lead tostrengthening of the general health system.19More effort
is needed to realize maximum synergy between disease
specific programs and the general health system, as well as
among different health programs.
As seen in the results of a similar study in Laos, if thegeneral health system appears weak, a strong vertical healthsystem supported by GFATM can function separatelyfrom the general health system.20,21 These findings werealso observed in Nepal and Viet Nam, particularly at theearly stage of support by GFATM, where disease specificprograms utilized procurement, information, monitoring
systems, etc., outside of the MOH (MOHP), with varying
levels of support and input provided by the disease specificdivisions.
One of the current leading challenges/bottle necks is thelimited coverage and quality of malaria control measuresamong populations living in remote areas. Most healthworkers are concentrated in large cities and towns, while
the health personnel and/or medical supplies of many healthfacilities at the primary level and at some district hospitals
are still insufficient. A poorly developed reporting systemfrom the private health sector, inadequate quality assurance
system for malaria testing, and weak coordination between
the local government and GFATM in the distribution of bed
nets were also pointed out (particularly in Nepal). In Nepal
it was suggested that bed nets are not always distributed
to the vulnerable populations, as similarly reported insome African countries.22, 23In addition, the current heavy
dependence on GFATM undermines the assurance ofsustainable malaria control.
Introduction of malaria due to population movement,
increased drug resistant malaria, and the emergenceof new endemic areas have become growing issues inmalaria control in recent years in many endemic countries.
Although not always health system related, theseissues do affect health systems and active health systemstrengthening seems to be crucial for their control. Suchgrowing challenges are often related to political issues,poverty, and a changing environment due to indiscriminate
development, global warming, etc.24,25 In order to addressthese growing challenges, strong government leadership, a
sector-wide approach, and inter sectoral collaboration are
required.

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Assessment of Health Systems

It is crucial to implement effective malaria control
programs which address these challenges and bottlenecks,
seeking their elimination. Particularly, emphasis on
strengthening the health systems in remote areas, trainingof the health staff at the peripheral level, diagnosis basedon accurate quality assurance, promotion of public-private
relationship and addressing the issue of imported malaria,
are desired. To create sustainable health systems, serious
consideration of issues regarding the availability ofdomestic resources, including workers, supplies and local
participation, as well as budgetary resources, are needed.
Moreover, good practices which have been identified inthis survey are expected to provide useful lessons in theeffective implementation of malaria control in endemic
countries. Addressing these issues will directly lead tofurther strengthening of the health systems and eventuallyto the effective implementation of various health programs.

Acknowledgements
This assessment, and the primary surveys in Nepal and Viet
Nam were conducted by the National Center for Global
Health and Medicine, as the WHO Collaborating Center for
Health System Research, with the Grants for International
Health Research (22-3, 24-5) from the Ministry of Health,Labour and Welfare, Japan. We wish to express our sincere
gratitude to all the interviewees who took part in theseassessments and surveys.
Conflict of interest: None declared

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International Journal of Antimicrobial Agents 46 (2015) 526–531

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agentsjournal homepage: http://www.elsevier.com/locate/ijantimicag

Molecular epidemiology of multidrug-resistant Acinetobacterbaumannii isolates in a university hospital in Nepal reveals the
emergence of a novel epidemic clonal lineage
Shovita Shrestha a,1 , Tatsuya Tada b,1 , Tohru Miyoshi-Akiyama c , Hiroshi Ohara d ,Kayo Shimada b , Kazuhito Satou e , Kuniko Teruya e , Kazuma Nakano e , Akino Shiroma e ,Jeevan Bdr. Sherchand a , Basista Psd. Rijal a , Takashi Hirano e , Teruo Kirikae b,∗ ,Bharat Mani Pokhrel aaDepartment of Microbiology, Institute of Medicine, Tribhuvan University, Kathmandu, Nepal
Department of Infectious Diseases, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japanc
Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japand
Department of International Medical-Cooperation, National Center for Global Health and Medicine, Tokyo 162-8655, Japane
Okinawa Institute of Advanced Science, Okinawa 904-2234, Japanba r t i c l e

i n f oArticle history:
Received 30 April 2015
Accepted 15 July 2015
Keywords:
Multidrug-resistant Acinetobacterbaumannii
16S rRNA methylase
Carbapenemase
Molecular epidemiology
Clonal complex

a b s t r a c t
The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical
problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains
isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patientswere screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the
MDR A. baumannii isolates were sequenced by MiSeqTM (Illumina), and the complete genome of one
isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single
nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistancegenes were identiﬁed. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, withthe majority being resistant to aminoglycosides, carbapenems and ﬂuoroquinolones but not to colistin
and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as blaNDM-1 , blaOXA-23
or blaOXA-58 . MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel
clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured
genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA).
© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction
The emergence of multidrug-resistant (MDR), extensively
drug-resistant (XDR) and pandrug-resistant (PDR) Acinetobacterbaumannii has become a serious medical problem worldwide [1].
MDR strains are deﬁned as isolates not susceptible to at least one
agent in three or more antimicrobial categories, XDR strains aredeﬁned as isolates not susceptible to at least one agent in all but
two or fewer antimicrobial categories, and PDR strains are deﬁned
as isolates not susceptible to all classes of agent, including aminoglycosides, antipseudomonal carbapenems, antipseudomonal∗ Corresponding author. Tel.: +81 3 3202 7181 (ext. 2838); fax: +81 3 3202 7364.
1

E-mail address: tkirikae@ri.ncgm.go.jp (T. Kirikae).
These two authors contributed equally to this study.

ﬂuoroquinolones,
antipseudomonal
penicillins/-lactamase
inhibitors, extended-spectrum cephalosporins, folate pathway
inhibitors, penicillins/-lactamase inhibitors, polymyxins andtetracyclines [2]. MDR isolates are frequently involved in nosocomial outbreaks, mainly in intensive care units [3]. Nosocomial
pneumonia caused by MDR isolates may affect critically ill patients,
making them more difﬁcult to treat [1].
Most MDR A. baumannii isolates harbour either genes or mutations in speciﬁc genes that are associated with resistance toantimicrobial agents [4]. For example, aminoglycoside-resistantstrains may harbour genes encoding aminoglycoside-modifying
enzymes (AMEs) and 16S rRNA methylases [5], whereas -lactamresistant isolates have been found to harbour genes encoding
carbapenemases for all -lactams except for monobactams [4]. Fluoroquinolone resistance has been associated with mutations in thequinolone resistance-determining region, which includes the gyrA

http://dx.doi.org/10.1016/j.ijantimicag.2015.07.012
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and parC genes that encode DNA gyrase and topoisomerase IV,
respectively [6].
Most MDR A. baumannii isolates resistant to aminoglycosides
produce AMEs, including aminoglycoside N-acetyltransferases andaminoglycoside O-nucleotidyltransferases [7]. Increasing numbersof MDR A. baumannii isolates have been reported to produce 16S
rRNA methylases [8]. To date, 10 types of plasmid-encoded 16S
rRNA methylase genes have been identiﬁed in aminoglycosideresistant clinical isolates (armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF,
rmtG, rmtH and npmA), with isolates harbouring armA reported
worldwide [8].
Carbapenemase-producing A. baumannii isolates have also
shown increased global spread [1], with these carbapenemases
identiﬁed as belonging to Ambler classes B and D [9]. Most ofthese isolates produce acquired carbapenem-hydrolysing class D-lactamases (CHDLs), with others producing class B metallo-lactamases (MBLs) [4]. Carbapenem resistance in A. baumannii ismainly due to the production of CHDLs, including OXA-23, OXA-58,
OXA-40 and OXA-143 [1,10]. Some carbapenem-resistant A. baumannii isolates produce an intrinsic OXA-51-like carbapenemase,whereas others produce class B MBLs, including IMP, NDM, SIM andVIM types [1].
This study analysed the molecular epidemiology of MDR A. baumannii isolates obtained from a university hospital in Nepal. The
complete genome of one of these isolates, which belonged to anovel clonal complex 149 (CC149) lineage (ST622), was sequenced.
2. Materials and methods
2.1. Bacterial strains
A total of 246 Acinetobacter spp. isolates were obtained from 246
different patients treated at a university hospital in Kathmandu,
Nepal, from September 2013 to June 2014. Clinical information on
all patients and isolates is shown in Supplementary Table S1. One
isolate (A. baumannii IOMTU433) was obtained in September 2013
from a sputum sample of a patient in a respiratory ward of the hospital. This patient had a history of acute exacerbation of chronic
obstructive pulmonary disease with hypertension, type II diabetes
mellitus and hypothyroidism. Acinetobacter spp. isolates were identiﬁed by biochemical methods and by the sequences of their 16S
rRNA, gyrB and blaOXA-51-like genes.
Supplementary Table S1 related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015.
07.012.
2.2. Antimicrobial susceptibility
Antimicrobial susceptibility testing was performed by the disk
diffusion method according to the guidelines of the Clinical andLaboratory Standards Institute (CLSI) [11]. The disk method wasused to screen MDR Acinetobacter spp. strains for susceptibility to amikacin, ampicillin/sulbactam, ciproﬂoxacin, cefepime,
ceftazidime, ceftriaxone, doxycycline, gentamicin, imipenem,
levoﬂoxacin, meropenem, polymyxin, piperacillin/tazobactam
and trimethoprim/sulfamethoxazole as described previously [2].
Carbapenem-resistant isolates were further screened for MBL
production by a combination disk method using imipenem
and ethylene diamine tetra-acetic acid (EDTA) [12]. Minimum
inhibitory concentrations (MICs) were determined using the
microdilution method as described in CLSI guidelines [11].
2.3. Whole-genome sequencing
Whole genomes of all of the MDR A. baumannii isolates wereextracted using a DNeasy Blood & Tissue Kit (QIAGEN, Tokyo,

78

527

Japan) and were sequenced by MiSeqTM (Illumina, San Diego, CA).
More than 10-fold coverage was archived for each isolate. The
whole-genome sequences of all 122 isolates have been depositedat GenBank as accession nos. DRX035998–DRX036119.
The whole genome of A. baumannii IOMTU433 was extractedusing the cetyltrimethylammonium bromide DNA extraction procedure. Its entire genome was sequenced using the PacBio RS II
platform (Paciﬁc Biosciences, Menlo Park, CA) with a 20-kb insert
library and P5-C3 chemistry. Prior to sequencing, libraries of >7 kb
were selected using BluePippin targeted size selection system (Sage
Science, Beverly, MA). De novo assembly was performed using thehierarchical genome assembly process (HGAP) workﬂow, including consensus polishing with Quiver. The resulting contigs weremerged using Minimus2. The ﬁnal, circularised assembly for thestrain was polished again to ﬁnish the genome. Annotation of the
genome sequences of the strain was accomplished by primarycoding DNA sequence (CDS) extraction and initial functional assignment using the RAST automated annotation servers. The results
were compared to verify the annotations and were corrected manually by in silico molecular cloning (In Silico Biology Inc., Kanagawa,Japan).
2.4. Phylogenetic analysis
Single nucleotide polymorphisms (SNPs) of the genome
sequences of all MDR isolates tested were identiﬁed by comparison with the sequence of A. baumannii strain MDR-TJ (GenBank
accession no. CP003500) [13], with all reads of each isolate aligned
against the MDR-TJ sequence using CLC Genomics Workbench
v.8.0.1 (CLC bio, Tokyo, Japan). SNP concatenated sequences werealigned using MAFFT (http://mafft.cbrc.jp/alignment/server/). Phylogenetic trees were constructed from the SNP concatemers. Two
methods were used to evaluate robustness: a maximum-likelihood
approach, PhyML 3.0; and the Bayesian MCMC framework, BEAST1.
For PhyML, HKY and gamma were chosen for a nucleotide substitution model, and tree robustness was evaluated by an approximate
likelihood-ratio test (aLRT). For BEAST, MCMC chains were run for
10 million generations, with sampling every 1000 generations. Convergences and effective sample sizes (ESSs) of the estimates werechecked using Tracer v1.4 (http://beast.bio.ed.ac.uk/Tracer). Threeof the phylogenetic trees constructed in this study had ESS values>200, suggesting sufﬁcient mixing of the Markov chain.
2.5. Multilocus sequence typing (MLST) and drug resistance genes
MLST sequence types were deduced as described in the protocols of the Institut Pasteur MLST databases (http://pubmlst.
org/abaumannii/). Clonal complexes were determined by eBURST
v.3 (http://eburst.mlst.net). The sequences of drug resistancegenes, including -lactamase-encoding genes (http://www.lahey.
org/studies), aminoglycoside resistance genes and quinolone resistance genes, were determined using CLC Genomics Workbench
v.8.0.1.
3. Results
3.1. Screening for multidrug-resistant Acinetobacter baumannii
Of the 246 Acinetobacter spp. isolates tested, 129 unique
MDR/XDR isolates were detected, including 122 A. baumannii
isolates, 6 Acinetobacter calcoaceticus isolates and 1 Acinetobacter bereziniae isolate. No PDR isolate was detected. Of the 122
A. baumannii isolates, 109 and 13 were XDR and MDR, respectively (Table 1), including 60 (49.2%) from respiratory tract
specimens, 30 (24.6%) from pus and wounds, 13 (10.7%) from

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Table 1
Summary of the drug susceptibility proﬁles for 122 Acinetobacter baumannii isolates.
Drug resistancea

No. of isolates

No. of drug
categoriesb

Resistancepatternc

XDR (109 isolates)

54
55

8
7

1
5

MDR (13 isolates)

11
2

6
5

5
2

XDR, extensively drug-resistant; MDR, multidrug-resistant.
a
MDR strains are deﬁned as isolates not susceptible to ≥1 agent in ≥3 drug categories, and XDR strains are deﬁned as isolates not susceptible to ≥1 agent in all but≤2 categories [2].
bNumbers of drug categories in which the isolate is not susceptible to at least one
agent.
c
Number of drug susceptibility patterns of the isolates.
Table 2
Minimum inhibitory concentrations (MICs) and percent antimicrobial resistance(%R) of Acinetobacter baumannii clinical isolates (n = 122).
Antimicrobial
agent

AmikacinArbekacin
Ceftazidime
Ciproﬂoxacin
Colistin
Meropenem
Tigecyclineb

Resistancebreakpoint(g/mL)a

≥64
–
≥32
≥4
≥4
≥16
≥4

%R

98
–
100
99
0
98
11

MIC (g/mL)

Range

MIC50

MIC90

4 to >512
1 to >51264 to >512
1 to >5120.13–2
1–512<0.031–8

>512
>512
>512
64
0.5
64
1

>512
>512
>512
512
1
128
4

MIC50/90 , MIC that inhibits 50% and 90% of the isolates, respectively.
a
Breakpoints for antimicrobial resistance were determined according to theguidelines of the Clinical and Laboratory Standards Institute [11]. The breakpoint
for tigecycline resistance was provided for Enterobacteriaceae but not for Acinetobacter spp. by the European Committee on Antimicrobial Susceptibility Testing(EUCAST) and the US Food and Drug Administration (FDA), although it was proposedas ≥4 g/mL [14].
bThe MICs to tigecycline were 8 g/mL for 4 isolates, 4 g/mL for 9 isolates,
2 g/mL for 19 isolates, 1 g/mL for 61 isolates, 0.5 g/mL for 21 isolates, 0.25 g/mL
for 7 isolates and <0.031 g/mL for 1 isolate.

urinary tract specimens, 9 (7.4%) from blood, 7 (5.7%) from cerebrospinal ﬂuid and 3 (2.5%) from other sources (Supplementary
Table S1).
3.2. Antimicrobial susceptibility
Most of the 122 XDR/MDR A. baumannii isolates were resistant to amikacin, ceftazidime, ciproﬂoxacin and meropenem(Table 2), with 109 (89.3%) and 97 (79.5%) having MICs ≥ 512 mg/L
to amikacin and arbekacin, respectively (data not shown). All
isolates were resistant to ceftazidime and 119 (97.5%) wereresistant to meropenem. All of the isolates were sensitive tocolistin with MICs ≤ 2 mg/L. Moreover, 90 isolates (73.8%) had
MICs ≤ 1 mg/L to tigecycline, whereas 4 isolates (3.3%) had MICs of
8 mg/L (data not shown). One XDR isolate (IOMTU433) was resistant to amikacin (MIC ≥ 512 mg/L), ceftazidime (MIC ≥ 512 mg/L),
ciproﬂoxacin (MIC = 32 mg/L), meropenem (MIC = 128 mg/L) andarbekacin (MIC ≥ 512 mg/L) but was susceptible to colistin(MIC = 0.25 mg/L) and tigecycline (MIC = 0.25 mg/L).
3.3. Multilocus sequence typing and phylogenetic analysis
The 122 MDR A. baumannii isolates belonged to 15 different
MLST sequence types (Fig. 1). eBURST analysis of these isolates,
together with the Institut Pasteur MLST database, showed that 5 of
the 15 sequence types could be grouped into three clonal complexes, CC1 (ST1 and ST623), CC2 (ST2) and CC149 (ST149 andFig. 1. Molecular phylogeny of the multidrug-resistant (MDR) Acinetobacter baumannii strains. A maximum-likelihood phylogenetic tree was constructed from the
122 MDR A. baumannii isolates, which were clustered into three clonal complexes(CC1, CC2 and CC149).

ST622) (Fig. 1). CC1 contained 21 isolates (4 ST1 and 17 ST623),
CC2 contained 36 isolates (all ST2) and CC149 contained 44 isolates(38 ST149 and 6 ST622). A maximum-likelihood phylogenetic tree
constructed from the 122 MDR isolates (Fig. 1) was congruent withthe tree constructed by the Bayesian method (Supplementary Fig.
S1). These phylogenetic trees revealed three clades, corresponding
to CC1, CC2 and CC149. All 122 isolates contained intrinsic blaADC
as well as encoding blaOXA-51-like variants speciﬁc to STs. Of the 21
isolates belonging to CC1, the four ST1 isolates had blaOXA-69 andthe 17 ST623 isolates had blaOXA-371 . All 36 CC2 isolates belonging
to ST2 had blaOXA-66 , and all 44 CC149 isolates belonging to ST149
and ST622 had blaOXA-104 (Fig. 1; Table 3). Most CC1 isolates harboured blaOXA-23 and blaOXA-420 (a novel blaOXA-58 variant), mostCC2 isolates harboured blaOXA-23 , aac(6� )-Ib and aadA1, and mostCC149 isolates harboured blaNDM-1 , blaOXA-23 , blaPER-7 and the 16S
rRNA methylase gene armA (Table 3).
Supplementary Fig. S1 related to this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015.07.
012.
3.4. Complete genome sequencing of IOMTU433
One isolate (IOMTU433) was selected for sequencing of thecomplete genome because it belonged to a novel sequence type
(ST622) and a novel clonal complex (CC149) and harboured several drug resistance genes. The complete genome sequences of
IOMTU433 and of the plasmid pIOMTU433 have been depositedin GenBank with the accession nos. AP014649 and AP014650,
respectively. The complete genome sequence of IOMTU433 had
1030-fold coverage for a chromosome and 974-fold coverage fora plasmid. IOMTU433 consisted of a single circular chromosomeof 4 000 970 bp, with an average GC content of 39.15%, whereas
pIOMTU433 consisted of a single circular plasmid of 189 354 bp
with an average GC content of 39.53% (Fig. 2). The chromosome
was found to contain 3724 protein-encoding genes, including 74
tRNA genes and 1 tmRNA gene for all amino acids. The plasmid wasfound to contain 203 protein-encoding genes. MLST of IOMTU433
assigned it to ST622 belonging to CC149. IOMTU433 harboured four-lactamase-encoding genes, including blaNDM-1 encoding a MBL,
blaOXA-23 encoding a class D carbapenemase, blaOXA-104 encoding

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Table 3
Multilocus sequence typing (MLST) and drug resistance genes in Acinetobacter baumannii isolates.
Clonal complex

MLST

No. of isolatesa

Carbapenemase- and
ESBL-encoding genes

Aminoglycosideacetyl/adenylyltransferase-encoding genes

CC1

ST1
ST623

4
17

blaNDM-1 , blaOXA-23 , blaOXA-69
blaOXA-23 , blaOXA-371 ,blaOXA-420 (15/17), blaPER-7(2/17), blaPSE-1 (3/17)

aacC1 (1/4), aadA1
armA (4/17), aadA2 (3/17),
aadB (9/17)

CC2

ST2

36

blaOXA-23 (35/36), blaOXA-66 ,blaPER-7 (4/36), blaTEM-1(6/36)

armA (2/36), aac(6 )-Ib (26/36),
aac(6 )-Im (1/36), aacC1(17/36), aadA1 (30/36), aadB
(1/36)

CC149

ST149

38

ST622

6

blaNDM-1 (19/38), blaOXA-23 ,blaOXA-104 , blaPER-7 (35/38)
blaNDM-1 (4/6), blaOXA-23 ,blaOXA-104 , blaPER-7 (4/6)

armA (29/38), aacC1 (1/38),
aadB (1/38)
armA (3/6)

ST10
ST15

7
2

armA (5/7)
armA

ST25

4

ST32

2

ST52
ST85
ST103
ST624
ST625

2
1
1
1
1

blaOXA-23 , blaOXA-68 , blaPER-7
blaOXA-23 , blaOXA-51 (1/2),
blaOXA-132 (1/2), blaPER-7(1/2), blaTEM-166 (1/2)
blaOXA-23 (3/4), blaOXA-64 ,blaPER-7
blaNDM-1 , blaOXA-32 ,blaOXA-420 (1/2), blaPSE-1
blaPER-7 , blaOXA-98 , blaPSE-1
blaNDM-1 , blaOXA-94
blaOXA-70 , blaPER-8 , blaPSE-1
blaOXA-23 , blaOXA-203
blaOXA-23 , blaOXA-58 ,blaOXA-90

Other

armA (3/4), aacC1 (2/4), aacC2(1/4)
armA (1/2), aacA2 (1/2)
armA, aadB
aadB
armA
aadB
aacC2

ESBL, extended-spectrum -lactamase.
a
Total number of isolates belonging to the same sequence type.

a variant of an OXA-51-like intrinsic carbapenemase, and blaPER-7
encoding an extended-spectrum -lactamase (ESBL), as well as
one aminoglycoside resistance gene (armA) encoding a 16S rRNA
methylase. Three of these genes (blaNDM-1 , blaOXA-23 and blaOXA-104 )were located in various regions of the chromosome (Fig. 2A), whilst
two (armA and blaPER-7 ) were located on the plasmid pIOMTU433(Fig. 2B).

IOMTU433 was found to have two gyrA mutations resulting in
the amino acid substitutions Ser83Leu and Asp87Glu as well as
a mutation in parC (Ser80Leu) resulting in ﬂuoroquinolone resistance. The blaNDM-1 was found to be located between two copies
of ISAba125, from nucleotide 132 336 to nucleotide 142 373 (GenBank accession no. PRJDB3154) (Fig. 3A). This sequence showed
100% homology with that of the Citrobacter freundii plasmid

Fig. 2. Circular representation of (A) the genome of Acinetobacter baumannii IOMTU433 and (B) the plasmid pIOMTU433. The outermost circle indicates the coordinates of thecomplete genome. The second and third circles show annotated coding DNA sequences (CDS) encoded by the forward (dark grey) and reverse (light grey) strands, respectively.
The fourth circle (black) shows antibiotic resistance genes, including blaNDM-1 , blaOXA-23 , blaOXA-104 , armA and blaPER-7 . The G+C plots are indicated in the innermost circle,
ranging from below (black) to above (grey) average.

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Fig. 3. Genetic environments of (A) blaNDM-1 in Acinetobacter baumannii IOMTU433 and (B) armA in pIOMTU433 and pNDM-CIT. The sequence of pNDM-CIT was obtained
from GenBank (accession no. JX182975).

pNDM-CIT (GenBank accession no. JX182975.1) [15] and of theKlebsiella oxytoca E718 plasmid pKOX R1 (GenBank accession no.
CP003683) [16] and 99% homology with A. baumannii 161/07 (GenBank accession no. HQ857107) [17]. The NDM-1 in A. baumannii
161/07 was integrated into the mfs gene adjacent to a homoserine
lactone synthase gene (cepI) on the chromosome [17]. In contrast,
the NDM-1 in A. baumannii IOMTU433 was integrated into a different site, in ISAba125, which was located between two housekeeping
genes. Speciﬁcally, NDM-1 was downstream of the vgrG gene,which encodes part of a type IV secretion system, and upstream ofparE, which encodes a topoisomerase IV gene. The sequence of thegenetic environment surrounding armA, from nucleotide 188 552
to nucleotide 6544, showed 100% homology with the sequence ofC. freundii pNDM-CIT from nucleotide 33 435 to nucleotide 25 178.

4. Discussion
To our knowledge, this is the ﬁrst molecular epidemiologicalanalysis of MDR A. baumannii clinical isolates in Nepal. CC149 isapparently a novel clonal complex of A. baumannii, to which onethird of MDR isolates in Nepal belong. An A. baumannii ST149 isolate
was ﬁrst identiﬁed in Japan (http://pubmlst.org/abaumannii/). Of
the 122 isolates analysed in this study, 44 (36.1%) belonged to
CC149, indicating that MDR A. baumannii isolates belonging toCC149 are spreading in other medical settings in Nepal. Further
studies are needed to determine whether MDR isolates belonging to
ST149 are emerging and spreading in other South Asian countries.
In addition to CC149 isolates, most of the ST623 isolates belonging to CC1 harboured the novel blaOXA-51 variant blaOXA-371 and the
blaOXA-58 variant blaOXA-420 , indicating that MDR A. baumannii may
evolve in a unique manner in South Asian countries.
In contrast to CC149, CC1 and CC2 are clonal complexes of MDR
isolates more common in Europe (European clones I and II) as
well as globally (International clonal complexes 1 and 2) [18]. The
isolates belonging to ST10, ST15, ST25, ST32 and ST52 belonged
to international clone III, members of which have disseminatedthroughout Europe, including in the Czech Republic, Denmark,Germany, Greece, Italy, The Netherlands, Norway, Portugal, Spainand Sweden [19].

High levels of drug resistance in these MDR A. baumannii isolates may be due to their harbouring various drug resistance genes.
NDM-1, blaOXA-23 and blaPER-7 may be associated with high levels of ceftazidime resistance, and blaNDM-1 , blaOXA-23 and blaOXA-104
may be associated with high levels of carbapenem resistance. Overexpression of the blaOXA-51-like gene has been reported to cause
carbapenem resistance [18], whereas armA can result in high levels of amikacin and arbekacin resistance. Producers of ArmA havebeen shown to be resistant to most clinically used aminoglycosides,
including amikacin and arbekacin [8].
This study strongly suggests that A. baumannii isolates producing NDM-1 and 16S rRNA methylase ArmA have been spreading
throughout medical settings in Nepal. Bacteria producing NDM1 and 16S rRNA methylases are resistant to clinically important
carbapenems [9] and aminoglycosides [8], respectively. Gramnegative pathogens producing NDM enzymes and 16S rRNA
methylases, including Escherichia coli, Klebsiella pneumoniae andProvidencia rettgeri, have spread throughout medical settings in
Nepal [20–23]. Although the subject of this study was MDR and XDR
A. baumannii isolates, it is also necessary to survey aminoglycosideand carbapenem-resistant A. baumannii isolates in Nepal.
Funding: This study was supported by grants from the Kanae
Foundation for Asia-Oceania Collaborative Research and International Health Cooperation Research [24-S-5 and 26-A-103,
respectively] and a grant of the Research Program on Emerging andRe-emerging Infectious Diseases from the Japan Agency for Medical
Research and Development (AMED).
Competing interests: None declared.
Ethical approval: This study was reviewed and approved by theInstitutional Review Board of the Institute of Medicine at Tribhuvan University (Kathmandu, Nepal) [ref. 6-11-E] and the Biosafety
Committee at the National Center for Global Health and Medicine(Tokyo, Japan) [approval nos. 26-D-088 and 26-D-089].

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[5] Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin Infect Dis 2007;45:88–94.
[6] Lopes BS, Amyes SG. Insertion sequence disruption of adeR and ciproﬂoxacin
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[8] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases
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[9] Queenan AM, Bush K. Carbapenemases: the versatile -lactamases. Clin Microbiol Rev 2007;20:440–58, table of contents.
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[14] Piewngam P, Kiratisin P. Comparative assessment of antimicrobial susceptibility testing for tigecycline and colistin against Acinetobacter baumannii

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[16] Liao TL, Lin AC, Chen E, Huang TW, Liu YM, Chang YH, et al. Complete genome
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[19] Zarrilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multidrugresistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents2013;41:11–9.
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Basudha Shrestha,a Tatsuya Tada,b Tohru Miyoshi-Akiyama,c Kayo Shimada,b Hiroshi Ohara,d Teruo Kirikae,b Bharat M. Pokhrela
Department of Microbiology, Institute of Medicine, Tribhuvan University, Maharajgunj, Kathmandu, Nepala; Department of Infectious Diseasesb and Pathogenic Microbe
Laboratory,c Research Institute, and Bureau of International Medical Cooperation,d National Center for Global Health and Medicine, Toyama, Shinjuku, Tokyo, Japan

A novel New Delhi metallo--lactamase, NDM-13, was identified in a carbapenem-resistant Escherichia coli clinical isolate obtained from the urine of a patient in Nepal. The enzymatic activity of NDM-13 against -lactams was similar to that of NDM-1.
However, NDM-13 displayed significantly higher kcat/Km ratios for cefotaxime. The genetic environment of blaNDM-13 was determined to be tnpA-IS30-blaNDM-13-bleMBL-trpF-dsbC-cutA-groES-groL, with blaNDM-13 located within the chromosome.

The emergence of metallo--lactamases (MBLs) and increased
carbapenem resistance among Gram-negative pathogens has
become a serious problem worldwide (1). MBLs, which are produced by many Gram-negative bacterial species (1) and by Grampositive Bacillus spp. (2, 3), confer resistance or reduce bacterial
susceptibility to carbapenems, cephalosporins, and penicillins, except for monobactams (1). NDM-1 was initially isolated in Sweden from Klebsiella pneumoniae and Escherichia coli in 2008 (4).
Subsequently, 16 NDM variants (www.lahey.org/studies) havebeen reported in several countries (5–17).
E. coli IOMTU558 was isolated from a urine sample obtained
from an inpatient at a university hospital in Nepal in 2013. The
bacterial species was identified by biochemical analysis and confirmed by 16S rRNA sequencing (18). MICs were determined using the microdilution method (19). The MBL genes (blaDIM,
blaGIM, blaIMP, blaNDM, blaSIM, blaSPM, and blaVIM) were amplified
by PCR (20, 21), and the sequence of the entire genome was determined by MiSeq (Illumina, San Diego, CA). Sequences of all
drug resistance genes registered at the Lahey Clinic website (www.lahey.org/studies), including MBL genes, were determined using
CLC genomics workbench version 5.5. Multilocus sequence typing (MLST) was performed according to protocols appropriate forE. coli sequences in the MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). E. coli transformants expressing blaNDM genes
were constructed as described previously (22), with blaNDM-1,
which originated from Pseudomonas aeruginosa IOMTU9 (17),used as the reference gene. Recombinant NDMs were purified as
described previously (22), with -lactamase activities during thepurification process monitored using nitrocefin (Oxoid, Ltd., Basingstoke, United Kingdom). Initial rates of -lactamase activities
were determined as described previously (22), with some modifications. In the modified protocol, the substrate solutions wereprewarmed to 37°C in a water bath to minimize fluctuations in
the temperature of the reaction mixture. Km values, kcat values,
and kcat/Km ratios were determined by analyzing -lactam hydrolysis using Lineweaver-Burk plots. Previously reported
wavelengths and extinction coefficients were used for analysisof -lactam substrates (23–25). Km and kcat values were determined using triplicate analyses. A DNA plug, digested for 3 hwith I-CeuI, was separated by pulsed-field gel electrophoresisand subjected to Southern hybridization as described previously (26). Probes for 16S rRNA and blaNDM were prepared as
described previously (27, 28).
September 2015 Volume 59 Number 9

The MICs of -lactams for E. coli IOMTU558 are listed inTable 1. The MICs of the other antibiotics were as follows: amikacin, 16 g/ml; arbekacin, 32 g/ml; ciprofloxacin, 1,024 g/ml;
colistin, 1 g/ml; fosfomycin, 8 g/ml; gentamicin, 256 g/ml;
kanamycin, 256 g/ml; levofloxacin, 32 g/ml; minocycline, 4g/ml; tigecycline, 2 g/ml; and tobramycin, 128 g/ml. PCR
showed that the isolate was positive for blaNDM and negative forthe other genes. Sequencing of the PCR product showed that
it was a novel blaNDM variant. This new variant was designated
blaNDM-13, and the sequence was deposited in GenBank.
IOMTU558 belonged to sequence type 101 (ST101). Based on thepredicted amino acid sequence, NDM-13 had two amino acidsubstitutions (D95N and M154L) compared to NDM-1 and one
amino acid substitution each compared to NDM-3 (M154L) and
NDM-4 (D95N). The isolate harbored blaCTX-M-15, blaTEM-166,
and ampC. The promoter region of ampC (nucleotides [nt] 42
to 1) included a mutation at nt 18 (G to A), but there were noother mutations or nucleotide insertions compared with E. coli
strain K-12. The isolate harbored ompC and ompF and their positive regulator, ompR. The ompR gene had no mutation comparedwith that of the K-12 strain. IOMTU588 harbored efflux pumpgenes (the acrAB-tolC operon), the marR repressor gene, and thegene (yedS) encoding the putative outer membrane protein. The
marR gene had a mutation resulting in an amino acid substitution(Y137H).
E. coli DH5 expressing blaNDM-1 or blaNDM-13 showed a significant reduction in susceptibility to all tested -lactams, except
for aztreonam, compared with the DH5 strain expressing a vector control (Table 1). E. coli DH5 expressing blaNDM-13 showed a
Received 9 February 2015 Returned for modification 22 March 2015
Accepted 6 July 2015
Accepted manuscript posted online 13 July 2015
Citation Shrestha B, Tada T, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T,
Pokhrel BM. 2015. Identification of a novel NDM variant, NDM-13, from amultidrug-resistant Escherichia coli clinical isolate in Nepal. Antimicrob Agents
Chemother 59:5847–5850. doi:10.1128/AAC.00332-15.
Address correspondence to Teruo Kirikae, tkirikae@ri.ncgm.go.jp.
B.S. and T.T. contributed equally to this article.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00332-15

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on January 27, 2016 by Kohnodai Hospital, National Center for Global Health and Medicine

Identification of a Novel NDM Variant, NDM-13, from a MultidrugResistant Escherichia coli Clinical Isolate in Nepal

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TABLE 1 MICs of various -lactams for E. coli strain IOMTU558 and E. coli DH5 transformed with plasmids encoding NDM-1 or NDM-13
IOMTU558DH5(pHSG398/NDM-1)

DH5(pHSG398/NDM-13)

DH5(pHSG398)

Ampicillin
Ampicillin-sulbactam
Aztreonam
Cefepime
CefoselisCefotaxime
Cefoxitin
Cefpirome
Ceftazidime
Ceftriaxone
Cephradine
Doripenem
Imipenem
Meropenem
Moxalactam
Penicillin G

1,024
1,024
1,024
1,024
1,024
1,024
1,024
1,024
1,024
1,024
1,024128
32
128
1,024
1,024256
256
0.063
1
16
16
64
4
256
16
512
0.25
1
0.25
32
256

512
256
0.063
1
16
64
64
4
512
32
256
0.25
2
0.25
32
256

4
4
0.063
0.063
0.063
0.063
0.063
0.063
0.063
0.06316
0.0630.125
0.0630.25
32

4-fold-higher MIC of cefotaxime than E. coli DH5 expressing
blaNDM-1 (Table 1).
The recombinant NDM-1 and NDM-13 hydrolyzed all tested
-lactams, except for aztreonam (Table 2). The enzymatic activity
profiles of NDM-13 against the tested -lactams were similar to
those of NDM-1; however, NDM-13 showed a higher kcat/Km ratio
for cefotaxime, which may have been due to NDM-13 having alower Km value than NDM-1 (Table 2). The kinetic parameters for
NDM-1 (Table 2) differed from those of our previous report (22).
The modified protocol utilized in this study may have affected theresults of the kinetic assays.
The genomic environment of blaNDM-13 was tnpA-IS30blaNDM-13-bleMBL-trpF-dsbC-cutA-groES-groL, and the sequence
was deposited in GenBank. This genomic structure, except for
blaNDM-13, was identical to that of pPMK1 expressed by K. pneumoniae PMK1 (isolated in Nepal) (29), an Enterobacter hormaechei CCHB10892 plasmid (from Brazil) (30), pKPX-1 from K.
pneumoniae KPX (from Taiwan) (31), and pNDM-MAR isolated
from K. pneumoniae (from Morocco) (32, 33). However, theblaNDM-13 gene was located on the chromosome (Fig. 1).

The two substitutions in NDM-13 compared to NDM-1 (i.e.,
D95N and M154L) increased the affinity of the enzyme for cefotaxime and affected the catalytic activity of the enzyme against thisdrug (Table 2). The D95N amino acid substitution was reported toreduce the kcat values of NDM-3 compared with NDM-1 for all-lactamases tested (28). An NDM-4 mutant containing M154L
was found to have increased hydrolytic activity toward carbapenems and several cephalosporins compared with that of NDM-1(12). NDM-13 with the D95N and M154L substitutions did notshow increased hydrolytic activity against the tested carbapenems,
cephalosporins, and penicillins, except for cefotaxime. Among the
known NDM variants (NDM-1 to NDM-13), amino acid substitutions were observed at 13 amino acid positions: 28, 32, 36, 69,
74, 88, 95, 130, 152, 154, 200, 222, and 233. Positions 28, 32, and 36
are located in the signal peptide region. Positions 95, 130, and 154
have been reported to affect -lactam hydrolyzing activity (12, 17,28), although they are not located at the active site of NDM-1 or inamino acid residues that bind to zinc ions (34, 35). The effects of
the remaining 10 substitutions on hydrolyzing activity have notyet been reported. Determination of the hydrolyzing activities of

TABLE 2 Kinetic parameters of NDM-1 and NDM-13 enzymes
Result fora:
NDM-1
1 1

-LactamKm (M)

kcat (s )kcat/Km (M

Ampicillin
Aztreonam
Cefepime
Cefotaxime
Cefoxitin
Ceftazidime
Cephradine
Doripenem
Imipenem
Meropenem
Penicillin G

76  6NHb
129  849  6
13  354  414  339  256  721  419  630  0.6NH
9.9  0.4
41  2
1.0  0.03
8.5  0.2
23  113  0.3
14  121  119  1

0.40
NH
0.077
0.85
0.076
0.16
1.8
0.34
0.26
1.1
1.1

a
b

s )

Km (M)

kcat (s1)kcat/Km (M1 s1)

140  26NH
83  1122  212  230  328  449  471  637  637  552  3NH
8.2  0.6
38  1
1.1  0.1
7.5  0.3
27  115  117  126  149  2

0.38
NH
0.099
1.7
0.093
0.25
0.98
0.31
0.24
0.73
1.3

The proteins were initially modified by a His tag, which was removed after purification. The Km and kcat values are means  standard deviations from 3 independent experiments.
NH, no hydrolysis was detected under conditions with substrate concentrations up to 1 mM and enzyme concentrations up to 700 nM.

5848

84

NDM-13
1

aac.asm.org

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IOM-NCGM Research Collaboration Office Annual Report 2015

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MIC (g/ml) for:
Antibiotic(s)Attachment

Novel NDM-13 Metallo--Lactamase-Producing E. coli

ACKNOWLEDGMENTS
This study was reviewed and approved by the Institutional Review Boardof the Institute of Medicine at Tribhuvan University (reference no.
6-11-E) and the Biosafety Committee at the National Center for Global
Health and Medicine (approval no. 26-D-088 and 26-D-089).
The study was supported by grants from the Kanae Foundation for
Asia-Oceania Collaborative Research and International Health Cooperation Research (24-S-5 and 26-A-103, respectively) and a grant from the
Research Program on Emerging and Re-Emerging Infectious Diseases
from Japan Agency for Medical Research and Development, AMED.
FIG 1 Localization of the blaNDM-13 gene on the chromosome of E. coli strain
IOMTU558, separated by pulsed-field gel electrophoresis (PFGE). Lanes: A,
contour-clamped homogeneous electric field (CHEF) DNA size marker (Saccharomyces cerevisiae; Bio-Rad, Hercules, CA); B, I-CeuI-digested total DNA
of E. coli IOMTU558; C, lambda ladder pulsed-field gradient (PFG) marker(New England BioLabs, Ipswich, MA); D, hybridization of I-Ceu-I-digested
total DNA of IOMTU558 with a 16S rRNA-specific probe; and E, hybridization of I-Ceu-I-digested total DNA of IOMTU558 using a blaNDM-13-specificprobe.

all NDM variants is critical to understanding the mechanisms underlying the molecular evolution of the NDMs.
The profile of -lactam resistance in IOMTU558 (Table 1)
could be explained by the presence of blaNDM-13, as well as
blaCTX-M-15, blaTEM-166, and ampC. Among these genes,
blaCTX-M-15 confers high resistance against most -lactams(except carbapenems), including aztreonam, cefepime, cefotaxime, and ceftriaxone (36). ampC could not be overexpressed in
the isolate. The -lactam-susceptible E. coli wild-type strain has
been found to express ampC at low levels due to degenerated promoter boxes upstream of ampC (37). Two mutations in the ampC
promoter sequence, at positions 42 (C to T) and 18 (G to A),
generate two alternative promoter boxes, resulting in high-level
expression of ampC (37). IOMTU558 had a mutation at position
18 (G to A) but not at position 42. It is unclear whether the
mutation at position 18 alone could affect the ampC expression
level in IOMTU558. The other mechanisms of -lactam resistanceare decreased cell permeability due to loss or alteration of theompC and ompF porins and activation of efflux systems (38). Lossor alteration of porins was not detected in IOMTU558. The marR
repressor gene of marAB and the yedS gene encoding a putativeouter membrane protein were reported to be involved in carbapenem resistance via downregulation of efflux pump genes (theacrAB-tolC operon). The marR mutation and yedS could contribute to carbapenem resistance in IOMTU558 (39).
NDM-producing E. coli belonging to ST101 seems to be an
epidemic strain in several regions of the world, although ST101 E.
coli strains are not recognized as a cause of pandemics, as is E. coliclone ST131 producing CTX-M types of -lactamases (40).
NDM-1-producing E. coli isolates belonging to ST101 have beenreported in Bulgaria (41), Canada (42), England (43), India (44),Pakistan (43), South Korea (45), and the United States (46).
This is the first report describing an NDM-13-producing E. coli
isolate. NDM-type MBLs have evolved under the pressure of antibiotic usage. Therefore, NDM-producing pathogens must be
monitored.
September 2015 Volume 59 Number 9

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Nucleotide sequence accession number. The sequence of
blaNDM-13 has been deposited in GenBank under accession no.
LC012596.

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Shrestha et al.

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Attachment

MECHANISMS

MICROBIAL DRUG RESISTANCE
Volume 00, Number 00, 2015ª Mary Ann Liebert, Inc.
DOI: 10.1089/mdr.2015.0126

A Novel 6¢-N-Aminoglycoside Acetyltransferase,
AAC(6¢)-Ial, from a Clinical Isolate of Serratia marcescensInte ersor nd onR ed alep fo Uro r D se
du is Oct tri nl
io bu yntionTatsuya Tada,1 Tohru Miyoshi-Akiyama,2 Kayo Shimada,1 Rajan K. Dahal,3 Shyam K. Mishra,3Hiroshi Ohara,4 Teruo Kirikae,1 and Bharat M. Pokhrel 3

Serratia marcescens IOMTU115 has a novel 6¢-N-aminoglycoside acetyltransferase-encoding gene, aac(6¢)-Ial.
The encoded protein AAC(6¢)-Ial has 146 amino acids, with 91.8% identity to the amino acid sequence ofAAC(6¢)-Ic in S. marcescens SM16 and 97.3% identity to the amino acid sequence of AAC(6¢)-Iap in
S. marcescens WW4. The minimum inhibitory concentrations of aminoglycosides for Escherichia coli expressing AAC(6¢)-Ial were similar to those for E. coli expressing AAC(6¢)-Ic or AAC(6¢)-Iap. Thin-layer
chromatography showed that AAC(6¢)-Ial, AAC(6¢)-Ic, or AAC(6¢)-Iap acetylated all the aminoglycosides
tested, except for apramycin, gentamicin, and lividomycin. Kinetics assays revealed that AAC(6¢)-Ial is afunctional acetyltransferase against aminoglycosides. The aac(6¢)-Ial gene was located on chromosomal DNA.

Introduction

T

Materials and Methods

Bacterial strains, plasmids, and antimicrobial
susceptibility

he main mechanisms underlying resistance to aminoglycosides are the production of aminoglycosidemodifying enzymes12 and 16S rRNA methylases.22 The
aminoglycoside 6¢-N-acetyltransferases [AAC(6¢)s] modify anumber of clinically important aminoglycosides. The AAC(6¢)-I
type confers resistance to amikacin, dibekacin, isepamicin,
kanamycin, netilmicin, sisomicin, and tobramycin, whereas theAAC(6¢)-II type acetylates dibekacin, gentamicin, kanamycin,
netilmicin, sisomicin, and tobramycin. To date, 45 genes designated as aac(6¢)-Ia to -Iaj, which encode AAC(6¢)-I enzymes,
have been cloned and characterized.10,12,17,20
Serratia marcescens is well recognized as a nosocomial
pathogen and has been responsible for outbreaks in neonatal
and pediatric intensive care units21 and outbreaks of bloodstream infection associated with intravenous drug solutions.15
Moreover, respiratory colonization with S. marcescens
has been a risk factor for outbreaks of bloodstream infection.18 Drug-resistant S. marcescens isolates have been
emerging in medical settings; these isolates produce aminoglycoside modification enzymes [AAC(6¢)-Ib, AAC(6¢)Ib-cr, AAC(6¢)-Ic, AAC(6¢)-Iid, and ANT(3")-Ii],3,4,13,19 16S
rRNA methylases (ArmA and RmtB),1,2 and metallo-blactamases,9,11,23 although S. marcescens is naturally resistantto tetracycline, amoxicillin, amoxicillin-clavulanate, cephalothin, and colistin.14

N

ot

Fo

rP

S. marcescens IOMTU115 was isolated from an endotracheal aspirate of a patient in a medical ward of a hospital in
Nepal in 2012. S. marcescens WW4 was originally isolated
from environmental water samples in a paper factory in
Taiwan.6 Escherichia coli DH5a (Takara Bio, Shiga, Japan)
and E. coli BL21-CodonPlus (DE3)-RIP (Agilent Technologies, Santa Clara, CA) were used as hosts for recombinant
plasmids and proteins, respectively. Plasmids pSTV28 (Takara) and pQE2 (Qiagen, Tokyo, Japan) were used for cloning of aac(6¢)s and purification of recombinant AAC(6¢)s,respectively, as described previously.16 Minimum inhibitory
concentrations (MICs) for S. marcescens IOMTU115 andE. coli transformants with aac(6¢) genes were determinedusing the microdilution method.8
Genome sequencing and cloning

Genomic DNA of S. marcescens IOMTU115 was extracted using the DNeasy Blood and Tissue kit (Qiagen) andsequenced with MiSeq (Illumina, San Diego, CA). Morethan 20-fold coverage was achieved for IOMTU115. A new
6¢-N-aminoglycoside acetyltransferase variant was designated as aac(6¢)-Ial. aac(6¢)-Ial and its genetic environment

1

Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
Department of Microbiology, Institute of Medicine, Tribhuvan University, Kathmandu, Nepal.
4
Department of International Medical-Cooperation, National Center for Global Health and Medicine, Tokyo, Japan.
2
3

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FIG. 1. Alignment of AAC(6¢)-Ic, AAC(6¢)-Ial, and AAC(6¢)-Iap amino acid sequences. Identical residues are marked
with black boxes.

Analysis of aminoglycoside acetyltransferase activity

The open reading frames (ORFs) of aac(6¢)-Ic, aac(6¢)-Ial,
and aac(6¢)-Iap were cloned into the pQE2 expression vector
with the same primer sets as described above. Purification of
the recombinant AAC(6¢)-Ic, AAC(6¢)-Ial, and AAC(6¢)-Iap
proteins and thin-layer chromatography analysis were performed as described previously.17
Results

ml; fosfomycin, 32 mg/ml; gentamicin, 2 mg/ml; imipenem,
8 mg/ml; kanamycin, 16 mg/ml; meropenem, <0.125 mg/ml;
penicillin, >1,024 mg/ml; tigecycline, 2 mg/ml; and tobramycin, 4 mg/ml.

r
In Pe
te rs
or nd onR ed alep fo Uro r D se
du is Oct tri nl
io bu yntion(315,403 bp) was deposited in GenBank with the Accession
Nos. AB871481 and AB894481, respectively. A putativeaac(6¢) gene of S. marcescens WW4 was designated asaac(6¢)-Iap and deposited in the GenBank with the Accession No. AB979699.
The aac(6¢)-Ic gene was synthesized by Funakoshi, Tokyo,
Japan. aac(6¢)-Ic, aac(6¢)-Ial, and aac(6¢)-Iap genes werecloned into the multiple cloning site of pSTV28, as described
previously.17 E. coli DH5a was transformed with pSTV28-aac(6¢)-Ic, -aac(6¢)-Ial, and -aac(6¢)-Iap to determine the
MICs for aminoglycosides.
Amino acid sequence alignment was calculated using the
CLUSTAL W2 program. Genetic environments surrounding
drug-resistant genes were annotated and compared using
the BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi?
CMD = Web&PAGE_TYPE = BlastHome).

Drug resistance profile of S. marcescens IOMTU115

AAC(6¢)-Ial consists of 146 amino acids. Multiple sequence alignments among AAC(6¢) enzymes revealed
that AAC(6¢)-Ial had 97% identity to AAC(6¢)-Iap fromS. marcescens,6 92% identity to AAC(6¢)-Ic from S. marcescens,13 38% identity to AAC(6¢)-Iag from Pseudomonas
aeruginosa,5 and 37% identity to AAC(6¢)-Iy from Salmonella enterica.7 Both AAC(6¢)-Ial and AAC(6¢)-Iap are
newly described in this study, although AAC(6¢)-Iap was
deposited as a putative protein in a complete genome sequence of S. marcescens WW4 (Accession No. NC020211).
Biological properties of AAC(6¢)-Iap had not been analyzed.
As shown in Fig. 1, amino acid substitutions existed at
positions 4, 18, 23, 24, 25, 26, 28, 56, 64, 84, 86, 95, and
122 among the three AAC(6¢) variants.
Aminoglycoside susceptibility of E. coli transformants

As shown in Table 1, a vector control of E. coli DH5a/pSTV28 was susceptible to all the aminoglycosides tested,
whereas E. coli DH5a/pSTV28-aac(6¢)-Ic, aac(6¢)-Ial, andaac(6¢)-Iap were resistant to all aminoglycosides, except for
apramycin, gentamicin, and lividomycin, with 2- to 64-foldhigher MICs than those of the vector control. The MICs ofapramycin, gentamicin, and lividomycin in E. coli expressing
aac(6¢)-Ic, aac(6¢)-Ial, and aac(6¢)-Iap were the same as
those in the vector control. The MIC profiles were essentiallysimilar among the E. coli expressing aac(6¢)-Ic, aac(6¢)-Ial,
and aac(6¢)-Iap (Table 1). E. coli expressing aac(6¢)-Ial andot

Fo

The MICs of S. marcescens IOMTU115 were as follows:
ampicillin, 32 mg/ml; ampicillin–sulbactam, 16 mg/ml; arbekacin, 8 mg/ml; amikacin, 8 mg/ml; aztreonam, <0.125 mg/
ml; ceftazidime, 0.25 mg/ml; cephradine, 1,024 mg/ml; cefepime, 0.25 mg/ml; cefotaxime, 0.25 mg/ml; cefoxitin,
32 mg/ml; ciprofloxacin, <0.125 mg/ml; colistin, >1,024 mg/

Amino acid sequence of AAC(6¢)-Ial enzyme

Table 1. MICs of Aminoglycosides for Escherichia coli Transformants

N

MIC (lg/ml)
ABK

AMK

APR

DIB

GEN

ISP

KAN

LIV

NEO

NET

SIS

TOB

E. coli DH5a/pSTV28
E. coli DH5a/pSTV28-aac(6¢)-Ic
E. coli DH5a/pSTV28-aac(6¢)-Ial
E. coli DH5a/pSTV28-aac(6¢)-Iap

0.5
4
4
4

0.5
1
1
2

2
2
2
2

0.25
8
16
16

0.125
0.125
0.125
0.125

0.125
0.5
0.5
0.25

0.5
4
8
4

2
2
2
2

0.5
2
2
2

0.5
8
8
8

0.25
4
4
2

0.5
8
8
8

Straina

MICs of aminoglycosides for E. coli DH5a transformants with aac(6¢)-Ic, aac(6¢)-Ic, and aac(6¢)-Ic were determined using the
microdilution method.8aMICs for E. coli strains were determined with Mueller–Hinton broth preparations and individual aminoglycosides.
ABK, arbekacin; AMK, amikacin; APR, apramycin; DIB, dibekacin; GEN, gentamicin; ISP, isopamicin; KAN, kanamycin; LIV,
lividomycin; MIC, minimum inhibitory concentration; NEO, neomycin; NET, netilmicin; SIS, sisomicin; SPM, spectinomycin; STM,
streptomycin; TOB, tobramycin.

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AAC(6¢)-IAL IN A S. MARCESCENS CLINICAL ISOLATE

3

rP

In
te ersor nd onR ed alep fo Uro r D se
du is Oct tri nl
io bu yntionFIG. 2. TLC analysis of acetylated aminoglycosides by AAC(6¢)Ic (A), AAC(6¢)-Ial (B), andAAC(6¢)-Iap (C). AAC(6¢) andvarious aminoglycosides were incubated in the presence (+) or absence (-) of acetyl-coenzyme A.
The arrow indicates the directionof development. ABK, arbekacin;
AMK, amikacin; APR, apramycin;
DIB, dibekacin; GEN, gentamicin;
ISP, isopamicin; KAN, kanamycin;
LIV, lividomycin; NEO, neomycin;
NET, netilmicin; SIS, sisomicin;
TOB, tobramycin.

Foaac(6¢)-Iap were resistant to dibekacin, with MICs 64-foldhigher than those of the other aminoglycosides.

specificity will be associated with an amino group at position 1 of ring I. Dibekacin has an amino group, whereas
amikacin has a hydroxyl group at position 1 of ring I.

TLC analysis and kinetics against aminoglycosides

Not

The results of TLC analysis of aminoglycosides acetylated by AAC(6¢) enzymes were shown in Fig. 2. Lividomycin was used as a negative control because it has ahydroxyl group instead of an amino group at the 6¢ position
and, therefore, cannot be acetylated by AAC(6¢). In total, 9out of the 12 aminoglycosides tested were acetylated by
AAC(6¢)-Ic, AAC(6¢)-Ial, and AAC(6¢)-Iap, with the exception of apramycin, gentamicin, and lividomycin (Fig. 2).
Kinetics parameters of AACs(6¢) against the nine aminoglycosides were determined, and the results are shown in
Table 2. The profiles of the kinetics parameters were essentially similar among the three enzymes. The kcat/Km
values against dibekacin were relatively high compared withthose against other aminoglycosides, whereas the values
against amikacin were relatively low (Table 2). The substrate specificity of AAC(6¢)-Ial is similar to that ofAAC(6¢)-Ic and AAC(6¢)-Iap. The kinetics parameters of thethree enzymes against dibekacin were relatively high among
the three enzymes, whereas those against amikacin wererelatively low (Table 1). These differences in substrate

Genetic environments surrounding aac(6¢)-Ial
and aac(6¢)-Iap

As shown by the genome rearrangement map in Fig. 3, a
genetic region of S. marcescens IOMTU115 from nucleotide(nt) 1 to 315,403 (Accession No. AB894481) had 97%
identity to a region of S. marcescens WW4 from nt
4,490,655 to 4,842,041 (Accession No. CP003959) (Fig. 3).
Bacteriophage-derived genes with 33,097 bp were inserted
from nt 4,583,432 to 4,616,528 in WW4. The downstream
region of the genetic environment surrounding aac(6¢)-Ial
was similar to the one surrounding aac(6¢)-Iap (Fig. 3). The
downstream region of the genetic environment surrounding
aac(6¢)-Ial contained several housekeeping genes, including
dnaG, rpoD, and mug (Fig. 3), indicating that aac(6¢)-Ial
was located in the chromosome.
Discussion

Amikacin and isepamicin share a characteristic structureof an l-hydroxyaminobuteroyl amide moiety at N-3 in the

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Table 2. Kinetics Parameters of Aac(6¢)-Ic, Aac(6¢)-Ial, and Aac(6¢)-Iap Enzymes
AAC(6¢)-Ic
Aminoglycosidekcat(s-1)a152 – 59
10 – 455 – 18 0.24 – 0.05
405 – 136 44 – 11
149 – 27
10 – 2
185 – 53
2.8 – 0.5
465 – 58
6.3 – 0.7
20 – 5
0.6 – 0.1
16 – 7
0.6 – 0.1
20 – 4
0.8 – 0.1

kcat /Km(lM-1s-1)
0.064
0.005
0.11
0.069
0.015
0.013
0.029
0.041
0.039

Km
(lM)a

kcat
(s-1)a399 – 36
17 – 286 – 48
0.2 – 0.1
582 – 182 85 – 24
404 – 125 25 – 6
322 – 73
10 – 2
470 – 27
7.8 – 0.4
23 – 60.73 – 0.06
12 – 30.49 – 0.03
28 – 30.67 – 0.05

AAC(6¢)-Iap
kcat /Km(lM-1s-1)
0.048
0.002
0.15
0.061
0.032
0.025
0.032
0.041
0.024

Km
(lM)a

kcat
(s-1)a210 – 134
9–5
24 – 40.27 – 0.03
487 – 84
48 – 7551 – 205 26 – 7
280 – 104
6–2
696 – 155 12 – 2
7–1
0.15 – 0.01
0.6 – 0.1
15 – 3
12 – 3
0.4 – 0.1

kcat /Km(lM-1s-1)
0.042
0.011
0.10
0.048
0.020
0.017
0.022
0.043
0.037

N Fo
ot r
In Pe
te rs
or nd onR ed alep fo Uro r D se
du is Oct tri nl
io bu y
n
tio
n

ABK
AMK
DIB
ISP
KAN
NEO
NET
SIS
TOB

Km
(lM)a

AAC(6¢)-Ial

The proteins were initially modified by a His-tag, which was removed after purification.
a
Km and kcat values represent the means of three independent experiments – standard deviations.

2-deoxystreptamine ring II, whereas the remaining aminoglycosides do not. This moiety will affect the substratespecificities between the three enzymes. As shown in Table 2,
kinetics parameters against dibekacin were high comparedwith those against other aminoglycosides, whereas thoseagainst amikacin were low. The differences of the kinetics
parameters between dibekacin and amikacin will be associated with an amino group at position 1 of ring I. Dibekacin has
an amino group, whereas amikacin has a hydroxyl group at
position 1 of ring I. A total of 13 amino acid substitutions,found among the 3 AAC(6¢)-Ic, AAC(6¢)-Iai, and AAC(6¢)Iap, may not be associated with the aminoglycoside modifications because of no difference between these enzymatic
properties (Table 2).

These intrinsic aac(6¢) variants in S. marcescens may be
evolved while mutating in their unique ways in the conserved
genetic environment, because these genes were not observedin other organisms. Our study suggests that aac(6¢) variants in
S. marcescens have potential to become more resistant toaminoglycosides by causing further genetic selection. Shawet al. reported that expression of the AAC(6¢)-I aminoglycoside resistance profiles can be due to (1) mutation of a geneencoding a negative regulator, (2) insertion upstream orwithin the 5¢ coding region of the aac(6¢)-I gene in S. marcescens, or (3) point mutations that create a new promoter.13
The aac(6¢) genes may contribute to aminoglycoside resistance in S. marcescens. The aac(6¢)-Ic, aac(6¢)-Ial, andaac(6¢)-Iap were detected in clinical and environmental

FIG. 3. A genome rearrangement map between Serratia marcescens IMOTU115 (Accession No. AB894481) and S.
marcescens WW4 (Accession No. CP003959), and the comparison of the genomic environments surrounding aac(6¢)-Ial in
IOMTU115 and aac(6¢)-Iap in WW4. The genetic map from nt 1 to 315,403 in S. marcescens IMOTU115 had 97% identity
compared to that from nt 4,490,655 to 4,842,041 in S. marcescens WW4. The upstream region of genomic environment
surrounding aac(6¢)-Ial was similar to that surrounding aac(6¢)-Iap. The genomic environment surrounding aac(6¢)-Ial
contained several housekeeping genes, including dnaG, rpoD, and mug.

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AAC(6¢)-IAL IN A S. MARCESCENS CLINICAL ISOLATE

isolates of S. marcescens.6,13 Therefore, further monitoringand investigation of aminoglycoside-resistant S. marcescens
are necessary to understand the diversity and evolution of
S. marcescens intrinsic aac(6¢) genes.
Acknowledgments

8. National Committee for Clinical Laboratory Standards.
2015. Performance standards for antimicrobial susceptibility testing; 25th informational supplement, M100S25. 8th ed. Clinical and Laboratory Standards Institute,
Wayne, PA.
9. Queenan, A.M., and K. Bush. 2007. Carbapenemases: theversatile beta-lactamases. Clin. Microbiol. Rev. 20:440–
458, table of contents.
10. Rather, P.N., H. Munayyer, P.A. Mann, R.S. Hare, G.H.
Miller, and K.J. Shaw. 1992. Genetic analysis of bacterial
acetyltransferases: identification of amino acids determining
the specificities of the aminoglycoside 6¢-N-acetyltransferase
Ib and IIa proteins. J. Bacteriol. 174:3196–3203.
11. Rieber, H., A. Frontzek, and Y. Pfeifer. 2012. Emergence of
metallo-beta-lactamase GIM-1 in a clinical isolate of Serratia
marcescens. Antimicrob. Agents Chemother. 56:4945–4947.
12. Shaw, K.J., P.N. Rather, R.S. Hare, and G.H. Miller.
1993. Molecular genetics of aminoglycoside resistancegenes and familial relationships of the aminoglycosidemodifying enzymes. Microbiol. Rev. 57:138–163.
13. Shaw, K.J., P.N. Rather, F.J. Sabatelli, P. Mann, H.
Munayyer, R. Mierzwa, G.L. Petrikkos, R.S. Hare, G.H.
Miller, and P. Bennett. 1992. Characterization of thechromosomal aac(6¢)-Ic gene from Serratia marcescens.
Antimicrob. Agents Chemother. 36:1447–1455.
14. Stock, I., S. Burak, K.J. Sherwood, T. Gruger, and B.
Wiedemann. 2003. Natural antimicrobial susceptibilitiesof strains of ‘unusual’ Serratia species: S. ficaria, S. fonticola, S. odorifera, S. plymuthica and S. rubidaea. J. Antimicrob. Chemother. 51:865–885.
15. Sunenshine, R., M. Schultz, M.G. Lawrence, S. Shin, B.
Jensen, S. Zubairi, A.M. Labriola, A. Shams, J. NobleWang, M.J. Arduino, F. Gordin, and A. Srinivasan.
2009. An outbreak of postoperative gram-negative bacterial
endophthalmitis associated with contaminated trypan blue
ophthalmic solution. Clin. Infect. Dis. 48:1580–1583.
16. Tada, T., T. Miyoshi-Akiyama, R.K. Dahal, S.K. Mishra, K. Shimada, H. Ohara, T. Kirikae, and B.M.
Pokhrel. 2014. Identification of a novel 6¢-N-aminoglycosideacetyltransferase, AAC(6¢)-Iak, from a multidrug-resistantclinical isolate of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 58:6324–6327.
17. Tada, T., T. Miyoshi-Akiyama, K. Shimada, M. Shimojima, and T. Kirikae. 2013. Novel 6¢-N-aminoglycosideacetyltransferase AAC(6¢)-Iaj from a clinical isolate of
Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
57:96–100.
18. Takahashi, H., M.H. Kramer, Y. Yasui, H. Fujii, K.
Nakase, K. Ikeda, T. Imai, A. Okazawa, T. Tanaka, T.
Ohyama, and N. Okabe. 2004. Nosocomial Serratia
marcescens outbreak in Osaka, Japan, from 1999 to 2000.
Infect. Control Hosp. Epidemiol. 25:156–161.
19. Tran van Nhieu, G., and E. Collatz. 1987. Primarystructure of an aminoglycoside 6¢-N-acetyltransferase
AAC(6¢)-4, fused in vivo with the signal peptide of theTn3-encoded beta-lactamase. J. Bacteriol. 169:5708–5714.
20. Vakulenko, S.B., and S. Mobashery. 2003. Versatility ofaminoglycosides and prospects for their future. Clin. Microbiol. Rev. 16:430–450.
21. Voelz, A., A. Muller, J. Gillen, C. Le, T. Dresbach, S.
Engelhart, M. Exner, C.J. Bates, and A. Simon. 2010.
Outbreaks of Serratia marcescens in neonatal and pediatricintensive care units: clinical aspects, risk factors andmanagement. Int. J. Hyg. Environ. Health 213:79–87.

Fo
ot r
In Pe
te rs
or nd onR ed alep fo Uro r D se
du is Oct tri nl
io bu yntionThis study was approved by the Institutional Review
Board of the Institute of Medicine at Tribhuvan University(6-11-E) and the Biosafety Committee of the Research Institute of the National Center for Global Health and Medicine (Approval Nos. 26-D-088 and 26-D-089). This study
was supported by a grant from Japan Agency for Medical
Research and Development (AMED), a grant (26-A-103)
from International Health Cooperation Research, and a grantfrom Kanae Foundation for Asia-Oceania Collaborative
Research.

5

Author Disclosure Statement

No competing financial interests exist.

ReferencesN

1. Doi, Y., K. Yokoyama, K. Yamane, J. Wachino, N.
Shibata, T. Yagi, K. Shibayama, H. Kato, and Y. Arakawa. 2004. Plasmid-mediated 16S rRNA methylase in
Serratia marcescens conferring high-level resistance toaminoglycosides. Antimicrob. Agents Chemother. 48:491–
496.
2. Kang, H.Y., K.Y. Kim, J. Kim, J.C. Lee, Y.C. Lee, D.T.
Cho, and S.Y. Seol. 2008. Distribution of conjugativeplasmid-mediated 16S rRNA methylase genes among
amikacin-resistant Enterobacteriaceae isolates collected in
1995 to 1998 and 2001 to 2006 at a university hospital inSouth Korea and identification of conjugative plasmids
mediating dissemination of 16S rRNA methylase. J. Clin.
Microbiol. 46:700–706.
3. Kim, C., D. Hesek, J. Zajicek, S.B. Vakulenko, and S.
Mobashery. 2006. Characterization of the bifunctionalaminoglycoside-modifying enzyme ANT(3†)-Ii/AAC(6¢)IId from Serratia marcescens. Biochemistry 45:8368–8377.
4. Kim, S.Y., Y.J. Park, J.K. Yu, Y.S. Kim, and K. Han.
2009. Prevalence and characteristics of aac(6¢)-Ib-cr in
AmpC-producing Enterobacter cloacae, Citrobacter
freundii, and Serratia marcescens: a multicenter study from
Korea. Diagn. Microbiol. Infect. Dis. 63:314–318.
5. Kobayashi, K., I. Hayashi, S. Kouda, F. Kato, T.
Fujiwara, S. Kayama, H. Hirakawa, H. Itaha, H. Ohge,
N. Gotoh, T. Usui, A. Matsubara, and M. Sugai. 2013.
Identification and characterization of a novel aac(6¢)Iag associated with the blaIMP-1-integron in a multidrugresistant Pseudomonas aeruginosa. PLoS One 8:
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6. Kuo, P.A., C.H. Kuo, Y.K. Lai, P.L. Graumann, and J.
Tu. 2013. Phosphate limitation induces the intergenericinhibition of Pseudomonas aeruginosa by Serratia marcescens isolated from paper machines. FEMS Microbiol.
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7. Magnet, S., P. Courvalin, and T. Lambert. 1999. Activation of the cryptic aac(6¢)-Iy aminoglycoside resistancegene of Salmonella by a chromosomal deletion generating atranscriptional fusion. J. Bacteriol. 181:6650–6655.

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Address correspondence to:
Teruo Kirikae, MD, PhD
Department of Infectious Diseases
Research InstituteNational Center for Global Health and Medicine
1-21-1 Toyama
Shinjuku
Tokyo 162-8655
JapanE-mail: tkirikae@ri.ncgm.go.jp

N

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22. Wachino, J., and Y. Arakawa. 2012. Exogenously acquired
16S rRNA methyltransferases found in aminoglycosideresistant pathogenic Gram-negative bacteria: An update.
Drug Resist. Updates 15:133–148.
23. Wachino, J., H. Yoshida, K. Yamane, S. Suzuki, M.
Matsui, T. Yamagishi, A. Tsutsui, T. Konda, K. Shibayama, and Y. Arakawa. 2011. SMB-1, a novel subclass B3
metallo-beta-lactamase, associated with ISCR1 and a class
1 integron, from a carbapenem-resistant Serratia marcescens clinical isolate. Antimicrob. Agents Chemother. 55:
5143–5149.

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bs_bs_banner

Hepatology Research 2015; 45: 1163–1169

doi: 10.1111/hepr.12482

Original Article

Use of nucleoside analogs in patients with chronic hepatitis B
in Nepal: A prospective cohort study in a single hospital
Naohiko Masaki,1* Pradeep Krishna Shrestha,3 So Nishimura,2* Kiyoaki Ito,1*
Masaya Sugiyama1 and Masashi Mizokami1
1

The Research Center for Hepatitis and Immunology, 2Kohnodai Hospital, National Center for Global Health andMedicine, Chiba, Japan, and 3Department of Medicine, Tribhuvan University, Kathmandu, Nepal

Aim: There still remain many concerns about the present sta-

Results: The younger, especially female patients of reproduc-tus of antiviral therapy for chronic hepatitis B in developing
countries in Asia, where the monitoring systems of virologicalmarkers have not been well established, despite the high prevalence of hepatitis B virus (HBV) infection. To investigate it in Nepal, this prospective cohort study was conducted at the
Teaching Hospital of Tribhuvan University in Kathmandu.

tive age were more frequently prescribed with these NA, and anincreased preference for the use of TDF was observed over time.
However, there was insufﬁcient follow up of the NA-treated patients in this cohort, and not a few patients developed emergenceof NA-resistant HBV: known resistance to LMV in 3 patients andincidental resistance to non-administrated NA in four patients.

Methods: From 2007 to 2012, 65 patients were consecutively

Conclusion: The results of the present study indicate that edu-

enrolled, 44 of whom received nucleoside analogs (NA), suchas lamivudine (LMV), adefovir or tenofovir (TDF), on the decisionof the local hepatologist. Virological determinations were performed in Japan, by using the serially collected serum samples
at the Teaching Hospital. Statistical analysis was performed,
using Mann–Whitney U-test or Fisher’s exact test.

cation for physicians as well as for infected patients regarding
the proper use of NA, together with establishment of appropriate monitoring systems for virological markers, is warranted toprevent an increase in NA-resistant HBV infections in Nepal.

INTRODUCTION

deaths related to liver cirrhosis or hepatocellular carcinoma caused by chronic HBV infection occur annually.1
HBV infection is endemic, particularly in Asian and Africancountries, and the prevalence is reported to be as high as
20% of the general population in some countries.2 In therural areas of Nepal, one of the poorest countries in theworld, the prevalence of HBV and hepatitis C virus(HCV) among blood donors was reported to be 0.86%
and 0.52%, respectively, in 2001–2002.3 Our epidemiological study conducted between 1997 and 2002 also demonstrated that HBV and HCV carrier rates among 540
blood donors in the urban area of Nepal were 2.4% and1.1%, respectively (e.g. N. Masaki, unpubl. data, 2003).
The recent introduction of nucleoside analogs (NA)such as lamivudine (LMV), adefovir (ADF), entecavir(ETV) and tenofovir (TDF) into clinical settings has significantly attenuated the progression of liver ﬁbrosis andhepatocarcinogenesis,4 leading to improvements in the

A

CCORDING TO ESTIMATES by the World Health Organization, almost 240 million people are infected byhepatitis B virus (HBV) worldwide, and up to 780 000

Correspondence: Dr Naohiko Masaki, The Research Center for Hepatitis
and Immunology, National Center for Global Health and Medicine,
Kohnodai 1-7-1 Ichikawa, Chiba 272-8516, Japan. Email:nmasaki0808@gmail.com*
Author contribution: N. M. designed the research protocol, obtained
funding and wrote the ﬁrst draft of the manuscript. P. K. S. was thecounterpart of this research in Nepal and contributed to the collection ofserum samples as well as clinical information. S. N. and K. I. contributed tothe acquisition of data and the analysis. M. S. carried out the molecularanalyses of hepatitis B virus. M. M. supervised the entire research project. All
authors read and approved the ﬁnal manuscript.
*
Present addresses: S. N. and K. I. are now afﬁliated with Saiseikai Fukuoka
General Hospital, Fukuoka, Japan, and Department of Gastroenterology,
Aichi Medical University School of Medicine, Aichi, Japan, respectively.
Received 14 August 2014; revision 23 December 2014; accepted 5 January
2055.

Key words: antiviral therapy, chronic hepatitis B, hepatitis B
virus, nucleoside analogs, resistance© 2015 The Authors.
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Hepatology Research published by Wiley Publishing Asia Pty Ltd on behalf of The Japan Society of Hepatology
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made.

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prognosis of HBV-related liver diseases. However, because
the pharmacological nature of these NA is chieﬂy conﬁned
to competitive inhibition of HBV DNA polymerase, semipermanent use of NA is principally necessary to preventthe relapse of hepatitis accompanying HBV proliferation.
However, the long-term use of NA may result in emergenceof NA-resistant HBV,5 therefore, it is necessary to regularly
monitor the serum viral load during treatment.
Although the HBV and HCV carrier rates in Nepal appear to be relatively lower than in other developing countries in Asia, it is important to understand the current useof therapeutic strategies. However, little information about
the present status of antiviral therapy for chronic HBV isavailable in this country, especially in terms of NA use. Furthermore, there are several serious socioeconomic problems to be addressed in Nepal. In relation to chronic
HBV, domestic laboratories in Nepal are not able to perform virological determinations of HBV genotypes or
HBV viral load, and sending serum samples to overseas
commercial labs is costly. For example, the measurementof HBV viral load in India costs approximately 8000 Nepalese rupees (i.e. $US 127). In contrast, NA are relatively
inexpensive in Nepal, because of an inﬂux of generic drugs
produced by foreign pharmaceutical companies. One tablet of LMV, ADF, ETV or TDF costs 18 ($US 0.28), 30($US 0.48), 80 ($US 1.26) and 43 Nepalese rupees($US 0.68), respectively. As a result, NA may be disorderly
administrated in this country. This study aimed to document NA use for HBV and discuss the socioeconomic
problems that need to be addressed in most of the developing countries in Asia.

METHODSC

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N. Masaki et al.

ONSECUTIVE NEPALESE PATIENTS with chronic
HBV infection in the outpatient clinic of the
Tribhuvan University Teaching Hospital in Kathmandu
were enrolled between August 2007 and August 2012,
and the observation period extended from the ﬁrst day ofenrollment to February 2013. The Teaching Hospital,
which was established in 1983 with the support of theJapanese Government as an integral part of the Instituteof Medicine of Tribhuvan University, provides academic
training programs (basic, graduate and postgraduate) for
the Institute and, as a national hospital, renders medicalcare and services to the Nepalese people.
Nucleoside analog administration was based on the decision of the local hepatologist (P. K. S.), and the clinicalcourse was prospectively analyzed. NA treatment was categorized into three groups: LMV-, ADF- and TDF-based. The
authors assert that all procedures contributing to this work

comply with the ethical standards of the relevant national
and institutional committees of the National Center for
Global Health and Medicine, Japan, on human experimentation and with the Declaration of Helsinki of 1975,
as revised in 2008. At that time, when we had started thisproject, the ethics committee of Tribhuvan University
Teaching Hospital was not organized yet; hence, oral or
written informed consent was obtained by the local
hepatologist (P. K. S.), and this study was conducted aspart of routine clinical practice.

Determination of HBV markers
Only a qualitative determination of hepatitis B surface
antigen (HBsAg), hepatitis B surface antibody (HBsAb),
hepatitis B e-antigen (HBeAg) and hepatitis B e antibody(HBeAb) was available in Nepal, therefore, quantitative determination of the virological parameters was performed inJapan using serum samples collected at the Teaching Hospital. In addition to the samples collected at enrollment,
the local hepatologist was advised to collect serial serum
samples at least every 6 months in NA-treated patients.
Hepatitis B surface antigen, HBeAg and HBeAb were determined by chemiluminescent microparticle immunoassay (ARCHITECT; Abbot Japan, Tokyo, Japan). The HBVviral load was determined by transcription-mediated ampliﬁcation (Chugai Diagnostics Science, Tokyo, Japan) or
AccuGene m-HBV (Abbott RealTime HBV; Abbot Japan;
lower limits of detection, 1.7 log copies/mL), according
to the manufacturers’ instructions. HBV genotype was determined by restriction fragment length polymorphism,
as described previously.6 NA-resistant HBV was determined by INNO-LiPA HBV DR version 2, according to
the manufacturer’s instructions (INNOGENETICS, Ghent,
Belgium). This line-probe assay can simultaneously detect
HBV mutations resistant to LMV, ADF, ETV and TDF.

Statistical analysis
Statistical analysis was performed using the Mann–
Whitney U-test for comparison of the means of continuous variables or Fisher’s exact test for categorical variables.
A two-tailed P-value of less than 0.05 was considered statistically signiﬁcant.

RESULTS
Demographic features and laboratory data at
enrollment

T

ABLE 1 PROVIDES the characteristics of the 65 patients (72% male) who were enrolled during thestudy period. The average ± standard deviation age was
33 ± 13 years, and 32% and 28% were aged in their© 2015 The Authors.
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Table 1 Demographic features and laboratory data at theenrollment

Table 2 Proﬁles of the NA-treated 44 patients enrolled in thisprospective studyn (%)†Sex (men/women)
Age (years)
Serum ALT (IU/mL)
HBeAg/HBeAb
+/
+/+
/
/+
HBV viral load (log copies/mL)≤3.9
4.0–6.9≥7.0
HBV genotype
Aa (A)C
C/D recombinantD
Not detected

1165

No. of patients†

47 (72%)/18 (28%)
33 ± 13‡ (range, 16–71)
34 ± 32‡ (range, 2–176)

Sex (male/female)Age (years)

28/16
31 ± 12‡(range: 16–71)

Treatment history of NA at enrollment
Naive cases
22for LMV/for ADF/for TDF
7/3/12On NA treatment
19
of LMV/of ADF/of LMV + ADF/of TDF
8/3/2/6
Previous history of NAs
3
of LMV/of LMV + ADF
2/1
Treatment proﬁles of NA during observation period§
LMV-based
12
on LMV alone/LMV alone→stop
9/3
ADF-based
6
on ADF alone/on LMV + ADF/LMV + ADF→stop
2/3/1
TDF-based
23
LMV→TDF/ LMV→ADF→TDF/on TDF alone
4/2/1729 (46.0%)
1 (1.6%)
1 (1.6%)32 (49.2%)
24 (36.9%)
16 (24.6%)
25 (38.5%)
12 (18.5%)
14 (21.5%)
1 (1.5%)33 (50.8%)
5 (7.7%)†

Unless otherwise indicated,
Mean ± standard deviation.
After excluding three patients with only previous history of NA, thetreatment proﬁles were evaluated in 41 patients, as of February 2013.
ADF, adefovir; LMV, lamivudine; NA, nucleoside analogs; TDF,
tenofovir.

Unless otherwise indicated.
‡
Mean ± standard deviation.
ALT, alanine aminotransferase; HBeAb, hepatitis B e antibody; HBeAg,
hepatitis B e-antigen; HBV, hepatitis B virus.

‡

20s and 30s, respectively. The extent of liver inﬂammation was relatively mild. The proportions of HBeAg positive and HBeAb positive patients were similar, and38.5% of the patients had a viral load of 7.0 log
copies/mL or more. The grades of viral load were quiteconsistent with the HBeAg/HBeAb status, namely, 21(72%) out of 29 patients positive for HBeAg and negative for HBeAb had a higher viral load (≥7.0 log
copies/mL), while 20 (63%) out of 32 patients negativefor HBeAg and positive for HBeAb had a lower viralload (≤3.9 log copies/mL, P < 0.001). In this cohort,
HBV genotype D was the most prevalent, affecting as
many as 50% of the patients.

patients (50%) were naive cases for NA, and LMV, ADF
and TDF were administrated in seven, three and 12 patients, respectively. Nineteen patients had already been of
NA treatment at enrollment: LMV, ADF, LMV + ADF and
TDF were administrated in eight, three, two and six patients, respectively. Three patients had only previous history of NA, and received no further treatment. As for thetreatment proﬁles of the 41 patients treated with NA during the observation period until February 2013, LMV-,
ADF- and TDF-based therapies were carried out in 12, six
and 23 patients, respectively. Four patients stopped NA:
one receiving LMV alone and one receiving LMV + ADF because of HBsAg loss during the observation period, while
the remaining two stopped LMV due to the decision of
the patients. The preference for TDF as the ﬁrst or secondline of treatment was clearly demonstrated in this prospective study (56.1%).

†

Proﬁles of the NAs-treated patients
Of the 65 enrolled patients, 44 (68%) received NA during
the observation period or before enrollment. NA were prescribed in 16 of the 18 female patients (88.9%) and 28 of
the 47 male patients (59.6%, P < 0.05). The average age of
the NA-treated patients was signiﬁcantly younger than thatof the NA-untreated patients (31 ± 12 years vs 39 ± 15
years, P < 0.05).
In Table 2, the proﬁles of the NA-treated patients are detailed. Among 44 patients enrolled in this study, 22§

Follow up of the NA-treated patients andemergence of NA-resistant HBVThe number of patients who returned to the hospital forfollow-up visits decreased over time. As detailed in
Figure 1, 44 NA-treated patients were classiﬁed according
to both the time after enrollment (~every 6 months) and© 2015 The Authors.
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Figure 1 Subsequent hospital visits decreased in this prospective
cohort of patients treated with nucleoside analogs. The local
hepatologist was advised to collect serial serum samples approximately every 6 months in patients with chronic hepatitis B treatedby nucleoside analogs. The numbers of hospital visits during theobservation period were evaluated as of February 2013. For thepatients who were enrolled more than 3 years from the end of observation period, the numbers of follow-up visits to the Teaching
Hospital were signiﬁcantly less than expected. ♀ denotes the four
female patients who were lost to follow up before February 2013.

the numbers of follow-up visits to the Teaching Hospital(range, 1–6). Especially for the patients who were enrolled
more than 3 years prior from the end of the observation
period (i.e. February 2013), the numbers of follow-up
visits were signiﬁcantly less than expected. Under these circumstances, serial determinations of serum HBV DNA
levels and the emerging rate of NA-resistant HBV were difﬁcult to achieve in clinical settings.
Of those patients who were followed, seven developed
NA-resistant HBV during the observation period (Fig. 2);
as the ﬁrst line treatment, ﬁve of these patients were treatedwith LMV and two patients were treated with TDF. In threepatients (patients CHB-16, CHB-22, CHB-23) with LMVresistant HBV, the clinical course varied. A change to TDF
dramatically decreased the HBV viral load in patient
CHB-16; however, serum levels of HBV DNA rose up again
thereafter, probably due to poor adherence to TDF, considering absence of NA-resistant HBV; the M204I mutation
spontaneously disappeared in patient CHB-22 despite
the continuation of LMV; and in patient CHB-23, theM204I mutation disappeared immediately after replacement with TDF, without viral breakthrough. Interestingly,
in the remaining four patients (i.e. CHB-25, CHB-28,

CHB-38 and CHB-49), incidental and unrelated resistanceto NA that had not been administrated (i.e. ETV or ADF)
was detected; however, except in one patient in whom
LMV was withdrawn (i.e. CHB-28), the HBV DNA levels
were fully suppressed, despite the emergence of incidental
resistance. In addition, in TDF-treated patients (i.e. CHB38 and CHB-49), such incidental resistance (i.e. ADF resistance and ETV resistance, respectively) was detected justtransiently and spontaneously disappeared.
To evaluate the effects of TDF, serial changes of serum
HBV DNA were examined in 16 patients naive to TDF
treatment, for whom multiple serum samples were available. As shown in Figure 3, 14 out of 16 patients (88%)
achieved a fair response to TDF, except for two patients
whose HBV DNA levels were unchanged, likely as a resultof poor adherence. In 13 patients (81%), HBV DNA levels
decreased to less than 3.0 log copies/mL within 12 monthsof the start of TDF. As detailed above, even though two patients (i.e. CHB-38 and CHB-49 in Fig. 2) developed incidental resistance to either ADF or ETV that was not
administered, the virological response to TDF was satisfactorily maintained (Fig. 3).

DISCUSSIONIN THIS PROSPECTIVE study, we found that NA wereprescribed preferably in younger female patients withchronic hepatitis B and that the monitoring systems for
HBV markers were extremely insufﬁcient in this country.
As a consequence, not a few patients developed emergenceof NA-resistant HBV: known resistance to LMV in three patients and incidental resistance to non-administrated NAin four patients.
In developing countries, where health insurance systems
are not available, patients can obtain medications without
a doctor’s prescription after the ﬁrst visit to the pharmacy.
Moreover, socioeconomic problems such as persistentpoverty and insufﬁcient infrastructure may prevent regular
medical checkups,7 even with chronic liver disease, as was
conﬁrmed in the present study.
One concern in younger patients and, in particular, female patients of reproductive age, is the use of NA without
appropriate checkups. All of the approved NA have prominent warnings regarding the potential risk of fetal anomaly with their use just before and during pregnancy.
Telbivudine and TDF are considered Pregnancy Category
B drugs (i.e. no known teratogenicity or embryotoxicity
in animal studies, but inadequate human studies) by the
US Federal Drug Administration, while LMV, ADF andETV are Category C (i.e. embryotoxic or teratogenic in animal studies, but inadequate human studies).8© 2015 The Authors.
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Figure 2 Emergence of NA-resistant hepatitis B virus. Seven patients developed NA-resistant hepatitis B virus in this prospective cohort.
Patients 1–5 received LMV-based treatment, and patients 6 and 7 received TDF-based treatment. In two patients (i.e. CHB-25 and CHB28) treated with LMV and two patients (i.e. CHB-38 and CHB-49) treated with TDF, incidental resistance to unused NA such as ETV or
ADF was also detected. ADF, adefovir; ETV, entecavir; Gt, genotype; lc, log copies; LMV, lamivudine; NA, nucleoside analog; TDF,
tenofovir.

Accordingly, their use in men and women of child-bearing
age should be limited.
Also of concern is that long-term NA use may result in
NA-resistant HBV. The ﬁrst approved NA for chronic HBV
was LMV, and the cumulative incidence of LMV resistance,
including M204V/I or L180M, has been reported up to
70% at treatment year 5,4 leading to hepatitis ﬂare-up as
well as viral breakthrough. ADF add-on therapy is effective
for the prevention of these adverse events; however, additional use of medication may increase patients’ mental
and ﬁnancial burden. In contrast, ETV and TDF are potent
HBV inhibitors with a high barrier to resistance.9–13 Of thelimited number of patients with follow-up data in the current study, seven developed NA-resistant HBV; ﬁve weretreated with LMV and two with TDF. The classic YMDD
mutation was detected in three patients treated with
LMV, and, in the remaining four patients, incidental resistance to other non-administrated NA (ETV and ADF) was
found. This information is relevant to future considerationof drug choice in these patients. Because, in CHB-38 andCHB-49 treated with TDF, we did not check the NA-

resistant HBV at pretreatment, the possibility of preexisting ADF-resistant or ETV-resistant HBV could not be
excluded. According to the recent systematic review andmeta-analysis,14 the incidence of naturally occurring resistance rates in naive chronic hepatitis B was reported to be
5.39% globally (including China, Japan, Turkey, Korea,
Iran, India, Pakistan, Thailand, Australia, USA, Brazil,
Spain, Italy, France and South Africa; 95% conﬁdence interval, 4.54–6.24%); the highest incidence was 7.83%(6.48–9.18%) in China, while 1.39% (0.67–2.10%) inother countries. In subgroup analysis, it was shown that genotype C HBV infection, male and HBeAg negative patients had a slightly higher natural mutation rate,
although the precise mechanisms were not elucidated. In
contrast, in CHB-25 and CHB-28, ETV-resistant HBV ﬁrst
emerged during the observation period. In CHB-25, the virological response to LMV seemed to be relatively slow,
and in CHB-28, LMV was stopped owing to the patient’s
will at 1 year and 1 month before. Although these twocases could not yet be classiﬁed as multidrug resistanceevolution in patients failing NA,15 further follow ups© 2015 The Authors.
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In conclusion, this prospective study documented thepresent status of antiviral therapy in patients with chronic
HBV in Nepal, one of the representative developing countries in Asia. Owing to a lack of monitoring systems, there
is a risk of an increase in NA-resistant HBV infections.
Given the prevalence of HBV in these countries, this could
represent a global public health problem. In addition, education for physicians as well as for infected patients regarding the appropriate use of NA is urgently required.

ACKNOWLEDGMENTS

T

Figure 3 Serial changes in serum HBV DNA levels in patients naive to tenofovir treatment. In 16 patients naive to TDF treatment,
multiple serum samples were available for determination of serum HBV DNA levels. Fourteen of 16 patients (88%) achieved afair response to TDF, except for two patients (§) who likely had
poor adherence, considering no emergence of NA-resistant HBV.
In 13 patients (81%), HBV DNA levels decreased to less than
3.0 log copies/mL within 12 months after the start of TDF. Twopatients (i.e. CHB-38 [†] and CHB-49 [‡] in Fig. 2) developed incidental resistance to either ADF or ETV, that was not administrated, the virological response to TDF was satisfactorily
maintained (dashed line). ADF, adefovir; ETV, entecavir; HBV,
hepatitis B virus; LMV, lamivudine; NA, nucleoside analog; TDF,
tenofovir.

should be mandatory to prevent progression of chronic
liver diseases.
A recent study reported that no resistance to TDF was detected after 6 years of treatment in patients with HBeAg
positive and HBeAg negative chronic HBV, and regularuse of TDF could satisfactorily suppress HBV proliferation,16 which was also demonstrated in the present cohort.
These lines of evidences may explain the recent preference
for TDF in Nepal, even though the monitoring systems forvirological markers have not been well established.
There are several limitations in this study. First, owing to
the small sample size, we must be prudent in drawing conclusions. However, we believe that the use of antiviral
medications documented in this study are representativeof general practice at this hospital, the afﬁliated hospitalof the only national university of Nepal. Second, it is difﬁcult to determine the speciﬁc reasons other than poor drug
adherence in patients with poor response to NA, because
we could did not have enough data regarding the quality
or storage conditions of generic drugs from neighboring
countries.

HIS STUDY WAS supported by a Grant-in-Aid of the
National Center for Global Health and Medicine(no. 22A9) to N. M. The funding source had no involvement in the study design; collection, analysis or interpretation of the data; writing of this manuscript; or the decision
to submit this manuscript for publication.

REFERENCES
1 World Health Organization. Hepatitis B Fact sheet N°204 Updated July 2014. Available at: http://www.who.int/
mediacentre/factsheets/fs204/en/. Accessed August 14, 2014.
2 Chen CJ, Wang LY, Yu MW. Epidemiology of hepatitis B virus
infection in the Asia-Paciﬁc region. J Gastroenterol Hepatol
2000; 15(Suppl): E3–E6.
3 Karki S, Ghimire P, Tiwari BR, Maharjan A, Rajkarnikar M.
Trends in hepatitis B and hepatitis C seroprevalence among
Nepalese blood donors. Jpn J Infect Dis 2008; 61: 324–6.
4 Chang TT, Liaw YF, Wu SS et al. Long-term entecavir therapy
results in the reversal of ﬁbrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B.
Hepatology 2010; 52: 886–93.
5 European Association for the Study of the Liver. EASL clinicalpractice guidelines: Management of chronic hepatitis B virus
infection. J Hepatol 2012; 57: 167–85.
6 Mizokami M, Nakano T, Orito E et al. Hepatitis B virus genotype assignment using restriction fragment length
polymorphism patterns. FEBS Lett 1999; 450: 66–71.
7 Liaw YF. Antiviral therapy of chronic hepatitis B: opportunities and challenges in Asia. J Hepatol 2009; 51: 403–10.
8 Fontana RJ. Side effects of long-term oral antiviral therapy forhepatitis B. Hepatology 2009; 49(5 Suppl): S185–S195.
9 Chang TT, Gish RG, de Man R et al. A comparison of entecavirand lamivudine for HBeAg-positive chronic hepatitis B. N
Engl J Med 2006; 354: 1001–10.
10 Marcellin P, Heathcote EJ, Buti M et al. Tenofovir disoproxil
fumarate versus adefovir dipivoxil for chronic hepatitis B. N
Engl J Med 2008; 359: 2442–55.
11 Chang TT, Lai CL, Kew Yoon S et al. Entecavir treatment for up
to 5 years in patients with hepatitis B e antigen-positivechronic hepatitis B. Hepatology 2010; 1: 422–30.

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12 Heathcote EJ, Marcellin P, Buti M et al. Three-year efﬁcacy andsafety of tenofovir disoproxil fumarate treatment for chronic
hepatitis B. Gastroenterology 2011; 140: 132–43.
13 Lai CL, Shouval D, Lok AS et al. Entecavir versus lamivudine
for patients with HBeAg-negative chronic hepatitis B. N Engl
J Med 2006; 354: 1011–20.
14 Zhang Q, Liao Y, Cai B et al. Incidence of natural resistancemutations in naïve chronic hepatitis B patients: a systematic

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review and meta-analysis. J Gastroenterol Hepatol 2014. DOI:
10.1111/jgh.12831
15 Locarnini S Primary resistance, multidrug resistance, andcross-resistance pathways in HBV as a consequence of treatment failure. Hepatol Int 2008; 2: 147–51.
16 Kitrinos KM, Corsa A, Liu Y et al. No detectable resistance totenofovir disoproxil fumarate after 6 years of therapy in patients with chronic hepatitis B. Hepatology 2014; 59: 434–42.

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Editorial

Earthquake disaster-associated health effects and theneed for improved preventive measures.
Sherchand JB
Research Department, Tribhuvan University Institute of Medicine, Maharajgunj, Kathmandu Nepal
Correspondence: Dr. Jeevan B. Sherchand
Email: jeevanbsherchand@gmail.com
During the past 20 years, earthquakes alone have caused
more than a million deaths worldwide1. Nine countries(Armenia, Chile, China, Guatemala, Iran, Italy, Japan, Peru,
and Turkey) account for more than 80% of all fatalities
in this century, and almost half of the total numbers ofearthquake casualties in the world during this period haveoccurred in China alone2.
The recent Nepal earthquake of April 25, 2015 ofmagnitude 7.8 on the Richter scale had its epicenter in
the area near Barpak, a mountain village between thecapital, Kathmandu, and the tourist town of Pokhara. The
earthquake was followed by many powerful after shocks on
the same day and a very powerful one (6.7 on the Richterscale) hit Nepal on the very next day, Sunday April 26.
The earthquakes, which caused extensive damage tobuildings and thousands of deaths and injuries were evenfelt in Pakistan, India and Bangladesh.
In Nepal many historic and recently-built buildings
collapsed or were very badly damaged, temples have beenruined, roads destroyed. There were nearly 8,800 deaths
and more than 23,000 injured in Nepal and tens of deaths
in India & Tibet. The quake was followed by more than
1000 after shocks and another huge earthquake (7.3 on the
Richter scale) on May 12.
Despite remarkable scientific progress in seismology andearthquake resistant engineering during the past few years,
achieving high standards of safety against earthquakes
is a goal that has yet to be achieved in many parts of
the world. But in Nepal, due to lack of resources and an
unstable political situation, no proper safety measures
were established a head of time, resulting in great lossof innocent Nepalese (and foreigners) people’s lives andearthquake related injuries and illness.
People’s health impact: In most earthquakes disasters,
people are killed by mechanical energy as a direct resultof being crushed by falling building materials. Deaths
resulting from major earthquakes can be instantaneous,
rapid, or delayed3. Instantaneous death can be due to severe

crushing injuries to the head or chest, external or internal
hemorrhage or drowning from earthquake-induced tidal
waves (tsunamis). Rapid death occurs within minutes orhours and can be due to asphyxia from dust inhalation orchest compression, hypovolemic shock, or environmental
exposure (e.g., hypothermia). Delayed death occurs
within days and can be due to dehydration, hypothermia,
hyperthermia, crush syndrome, wound infections, or
postoperative sepsis4,5. As with most natural disasters, themajority of people requiring medical assistance following
earthquakes have minor lacerations and contusions caused
by falling elements, like pieces of masonry, roof tiles andtimber beams6. The next most frequent reasons for seeking
medical attention are simple fractures not requiring
operative intervention7.
Such light injuries usually require only outpatient
treatment and tend to be much more common thansevere injuries requiring hospitalization. Major injuries
requiring hospitalization include skull fractures withintracranial hemorrhage (e.g., subdural hematoma);
cervical spine injuries with neurologic impairment; anddamage to intrathoracic, intraabdominal, and intrapelvic
organs, including pneumothorax, liver lacerations, andruptured spleen. Most seriously injured people will sustain
combination injuries, such as pneumothorax in addition toan extremity fracture. Am-putations and chronic sequelae
of orthopedic and neurologic injuries, especially spinal
cord injuries, can be expected8. Huge amounts of dust are
generated when a building is damaged or collapses, anddust clogging the air passages and filling the lungs is a major
cause of death for many building-collapse victims2,9,10.
Fulminant pulmonary edema from dust inhalation may also
be a delayed cause of death11. Dust has hampered rescue
and clean-up operations by causing eye and respiratorytract irritation. Burn sand smoke inhalation from fires used
to be major hazards after an earthquake.
During the urbanization process, cities are laden withchemical and petroleum products that could contribute
substantially to the generation of toxic substances following
an earthquake11,12. Industrial storage facilities for hazardous

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2materials might explode or leak and damage to a nuclear
power plant could lead to widespread contamination byradioactive materials. In a major earthquake, pipelines
carrying natural gas, water, and sewage can be expected tobe disrupted.
In earthquakes, people over 60 years of age are at increased
risk for death and injury and can have a death rate fivetimes higher than that of the rest of the population. Children
between 5 and 9years of age, women, and the chronicallyill also seem to be at an elevated risk for injury and death12.
Lack of mobility to flee collapsing structures, inability towithstand trauma, and exacerbation of underlying disease
are factors that may contribute to the vulnerability of thesegroups. Mortality distribution by age will also be affected toa certain degree by the social attitudes and habits of different
communities. In general, the morbidity and mortality rates
are significantly greater among people who are indoors thanamong those who are outdoors when the tremor begins 9.
Although the probability of finding live victims diminishes
very rapidly with time, entrapped people have survived formany days. People have been rescued alive 5, 10 and even
14 days after an earthquake13, and these “miracle rescues”
are often the result of exceptional circumstances. For
example, someone with very light injuries could be trappedin a void deep in the rubble with air and possibly water
available.
What efforts need to be taken to improve preventive
measures?: Prevention and control efforts need to be
multidisciplinary and should include public education
programs, as well as better building design and improved
quality of construction in those areas most likely to suffer
an earthquake14. Avoiding unnecessary residential andcommercial construction on or near active faults and inareas subject to landslide slope failures, soil liquefaction,and rock falls is technically a secondary prevention measurefor earthquakes, but it is a primary prevention measure forearthquake related injuries15. Prompt rescue should improve
the outcome of victims, and early medical treatment should
lessen the sequelae of the primary injuries (e.g., woundcomplications, chronic neurological disabilities). Provisionof adequate food, water, and shelter should especially help
people in vulnerable age groups and those with pre-existing
diseases. Effective environmental control measures should
prevent secondary environmental health problems, suchas gas leaks, fallen or loose wires, damaged appliancesand pipelines, sewage backup, and water contamination.
Public health officials need to establish in advance how theaffected areas will be surveyed. Just as speed is required
for effective search and extrication, it is also essential for
effective emergency medical services, since the greatest
demand occurs within the first 24 hours9.The medical andwww.jiom.com.np

public health impacts of a severe earthquake may well becompounded by significant damage to medical facilities,
hospitals, clinics, and supply stores within the affected area 16.
In the worst-case scenario, a hospital building may itself
be damaged by the earthquake, and the hospital staff may
have to continue emergency treatment without using thebuildings17. Hospital emergency plans in earthquake areas
should provide for the contingency of evacuating patientsfrom the wards; safely removing critical equipment fromoperating theaters, radiology departments, and other partsof the hospital; and re-establishing routine patient-careservices18.
In addition, a comprehensive training course conducted inadvance is essential for provision of sophisticated medicalcare to victims in the first hours after a catastrophic
earthquake. In such a scenario physicians, nurses, and other
health care providers who will encounter situations forwhich they have not been prepared. Therefore, a thoroughtraining course should be required for all medical facility
staff. As outlined in the medical disaster-response model,
the subjects covered in the training course would be masscasualty triage, airway management, use of intravenous
fluids, anesthesia and analgesia in the field, crush-injury
treatment, and command and control.
Problems and challenges: The first need in earthquake
assistance is medical care and medicines for the injured
in a situation where all government hospitals in the area
have been destroyed. Teams of independent doctors fromother parts of the country, along with army medical staff,
are providing some basic access in such areas in Nepal.
However, some recently created medical camps in different
areas, mainly in rural Nepal, seem to have very few or no
bandages, gauze or painkillers. There is also a need forbasic provisions, including food, water and tents.
Earthquakes will continue to affect human populations into
the distant future. With technologic advances, increasingly
complex infrastructures, and new building designs, the built
environment will evolve over time. Shifts in population
locations and characteristics will accompany theseenvironmental changes. Every earthquake is different, as
is every population affected. Thus, despite the significant
challenges involved, there is a critical need for evidencebased prevention and preparedness efforts to ensure thebest possible chances of limiting earthquake related death,injury and destruction in the future.

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Frechette CN. Rescuing earthquake victims inArmenia. Plast Reconstr Surg.1989; 84:838–40.

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center by earthquake: initial effects on patients andstaff. Calif Med (now Western J Med). 1972; 116:63–7.
17. Arnold C, Durkin M. Hospitals and the San Fernando
earthquake of 1971: the operational experience. San
Mateo, CA: Building Systems Development, Inc.;
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preparedness: when the disaster strikes. Plant,
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10. Hingston RA, Hingston L. Respiratory injuries inearthquakes in Latin America in the 1970s: a personal
experience in Peru, 1970; Nicaragua, 1972–73; andGuatemala, 1976. Disaster Med. 1983:1: 425–6.
11. Showalter PS, Myers MF. Natural disasters in the
United States as release agents of oil, chemicals, orradiological materials between 1980–1989: analysis
and recommendations. Risk Anal. 1994; 14:169–82.
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13. Noji EK. Medical consequences of earthquakes:

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Acknowledgements

Publication of this annual report was supported by grants from the International Health
Cooperation Research (25-5), a grant from the Ministry of Health, Labor and Welfare of Japan.

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Contact details for further information
Hiroshi OHARA, M.D., Ph.D.
Bureau of International Health Cooperation, Japan
National Center for Global Health and Medicine, Japan
Email: oharah@it.ncgm.go.jp
Annual Report 2015
IOM-NCGM Research Collaboration Office© National Center for Global Health and Medicine, JapanAll rights reserved.
National Center for Global Health and Medicine, Japan
Bureau of International Health Cooperation, Japan
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655
Tel: +81-3-3202-7181 / Fax: +81-3-3205-6780
info@it.ncgm.go.jp

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Annual Report
2015

IOM-NCGM
Research Collaboration Oﬃce

March 2016
Kathmandu, Nepal
Tokyo, Japan