Annual Report 2014

Annual Report
2014

IOM-NCGM
Research Collaboration Oﬃce

March 2015
Kathmandu, Nepal
Tokyo, Japan

Annual Report
2014

IOM-NCGM
Research Collaboration Office

March 2015
Kathmandu, Nepal
Tokyo, Japan
ISBN 978-4-909675-25-5

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.
Eighteen 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. Two 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. In December 2014, “the 2nd Joint Conference on Infectious
Diseases with Growing Concern in Recent Years in Nepal” was held at IOM and such outcomes
were presented.
It is a great pleasure for us to summarize the outline report of our new collaboration as
Annual Report 2014 following the first publication of Annual Report 2013. We also would liketo thank all those who worked hard for the success of the project and toward the realization of
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 2014

IOM-NCGM Research Collaboration Office Annual Report 20143

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		

Tribuban University Teaching Hospital

WHO		

World Health Organization

WPRO		

Western Pacific Regional Office

IOM-NCGM Research Collaboration Office Annual Report 2014

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 and expected outcomes......... 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 2014......... 14
III. Research activities
1. General information........................................................................................................ 15
2. Progress of research activities up to FY 2014.................................................................. 16
3. Outline of each research................................................................................................. 23
Table 2: Collaborative researches in NCGM-IOM collaboration.................................. 24
Researches No.1- No.6 ................................................................................................ 25
IV. Other activities
1. Joint Conference............................................................................................................

36

2. Presentation on collaborative research results.............................................................. 36
3. Overview of the Medical Education Project and current IOM/TUTH............................. 36
4. Enhancement of Human Resource Development.......................................................... 36
5. Overview of current medical situation in Nepal............................................................. 37
Attachment-1: 2nd Joint Conference on Infectious Diseases with Growing Concern
in Recent years in Nepal (program and abstracts).......................................

39

Attachment-2: Publications on Scientific Papers.................................................................. 53
Table 3: List of papers published on international journals................................................... 54
Scientific papers.................................................................................................................. 55

IOM-NCGM Research Collaboration Office Annual Report 2014

5

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
2-1

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 2014

Tribuvan University Institute of Medicine (IOM) 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 the first phase (1980-1989), strengthening education, clinical practice, and the research infrastructure
for basic medicine and clinical medicine were mainly implemented. In the second phase (1989-1996), a
technical cooperation project was implemented, focusing mainly on improving medical education to haveinternational accreditation, enhancing the function of basic medicine, clinical medicine, and research, aswell as improving hospital management function.

Technical guidance in nephrology (1996)

International Symposium on Infectious
and Tropical diseases (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
IOM-NCGM Research Collaboration Office Annual Report 20147

Medical Education project Phase 2 (1989-1996)•

Dispatch of Japanese Experts

86•

Invitation for Nepalese Trainees 28•

Equipment Provision

Local and International Seminar/ Symposium held by the Project•

Nepal-JICA Joint Symposium on Cholelithiasis

			•Congress on Altitude Medicine and Physiology

		•

Nursing Research Conference•

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

				

6-8 January 1991
8 April 1992
2-4 November 1993

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.
2-2

After Completion of the Technical Cooperation Project (1996-2013)
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 and
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IOM-NCGM Research Collaboration Office Annual Report 2014

healthcare 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.
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 greaterawareness of the importance of infection control andresearch collaboration. At the conference, successful
technical cooperation in nosocomial infection
control in Vietnam was introduced inviting directorof Bach Mai Hospital in Hanoi. In 2013, “the 1st Joint
Conference on Infectious Diseases with Growing

Joint Symposium on nosocomicao infection
Control (September 2009)

Concern in Recent Years in Nepal” was held at IOM.
3.

Conclusion of MOU and start of new cooperation (2013- )On January 18, 2013, NCGM concluded an agreement of cooperation with IOM in Nepal for researchand related human resource development, and IOM became the fifth overseas platform for NCGM. The
Memorandum of Understanding (MOU) was signed by the President of NCGM in Tokyo firstly, and then,was signed by the Dean of 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 diseases, and development of human resources related to these 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
for the smooth and efficient implementation of collaborations including related institutions.
IOM-NCGM Research Collaboration Office Annual Report 20149

Signing of MOU at NCGM and IOM (January 2013)

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 andclinical medicines are the professors for the departmentof Microbiology and Internal Medicine, respectively. In
addition, there is a Research Department to manage
research, which serves as a counterpart.

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.
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IOM-NCGM Research Collaboration Office Annual Report 2014

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.
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.

IOM-NCGM Research Collaboration Office Annual Report 201411

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 2014, 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 1,700 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 2014
2.

Clinical Medicine
TUTH is a general hospital which has 750 beds (as of December 2014) under 20 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 aswell as essential medical care and examinations. TUTH has
been conferred duties and roles as the most important
top referral medical institution in Nepal. Since 1990,
Cardiovascular Center, Obstetrics & Gynecology Centerand Emergency Center have been constructed by Nepalese
Government.

Tribuvan University Teaching Hospital (TUTH)

The number of out-patients, in-patients, and surgical operation has been ever increasing since its
establishment, which means that the importance of TUTH in Nepalese medical care has been increasing
year by year.

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IOM-NCGM Research Collaboration Office Annual Report 2014

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 internationallyacknowledged journal. Besides the 8 basic medicine
departments, Research department, Department ofInformation Technology and National Center for HealthProfessional Education were set up after the project

Academic building on IOM

termination.
4.

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. 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 201413

Table 1		14Situation and activities of IOM/TUTH : Comparison between 1985 and 2014
1985

2014

1

Total No. of beds

401

740

2

No. of charity beds

40

63

3

No. of outpatients served (per year)

229,516

367,252

4

No. of in-patients served (per year)

11,97322,0505

Average length of hospitalization (days)

NA

6.73

6

No. of operations (per year)

5,237

12,228

7

Average bed occupancy rate (%)

NA

77.2

8

Number of Clinical Departments

13

20

9

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

8

8

10

No. of students (per school year grade)

40

75

11

Research department

-

01

12

Department of Information Technology

-

01

13

National Center for Health Professional Education-

01

IOM-NCGM Research Collaboration Office Annual Report 2014

III. Research Activities

1.

General information
To implement collaborative activities and strengthen the IOM-NCGM Research Collaboration Office,the following grants from the International Health Cooperation Research, grants from the Ministry of
Health, Labor and Welfare of Japan were utilized in 2014.
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 project
3. 25-7: Fact-finding survey of nosocomial infection control in major hospitals in Nepal and planning ofeffective improvement²² 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)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 diseases

IOM-NCGM Research Collaboration Office Annual Report 201415

²² 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, 24-5)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 Office2.

Progress of research activities up to FY 2014

Research Grant: 24-5
The purpose of this study is to analyze the latest situation of the infectious diseases which havegrowing 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 andpartly in Vietnam with the aim of contribution to effective control of these diseases.

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IOM-NCGM Research Collaboration Office Annual Report 2014

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:
1. 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.
2. 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.
3. 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.
4. 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.)
5. 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.

IOM-NCGM Research Collaboration Office Annual Report 201417

Major achievements of collaborative studies on these subjects up to now are shown below. These
have been published in conference presentations, scientific papers and reports. Furthermore, throughjoint 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.
The significance of the 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 ofeach country concerned can be considered as great progress. We aim to contribute to proper measures byfurther analyzing factors of growing issues regarding these infectious diseases (those studied by this study
group).
Major achievement up to FY 2014ØØ 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).

18•“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).

IOM-NCGM Research Collaboration Office Annual Report 2014ØØ 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
025b-ST131 clone, which is of growing global concern, was shown to have a high frequency in Nepal.
ØØ 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.
Joint conferences and information sharing carried out in this study have contributed to increased
awareness of medical personnel in each country concerned (especially in Nepal) regarding emerging healthpriorities of infectious diseases (multi-drug resistant bacteria, equity in the distribution of bed-nets formalaria prevention, and double burden of infectious diseases and non-communicable diseases).
Especially in Nepal, the use of antibiotics, poor countermeasures for nosocomial infections, healthknowledge 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.
This study 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.

Collaborative research on multi drug resistant bacteria in IOM

Data collection on dual burden of tuberculosis
and diabetes at a clinic in Kathmandu

IOM-NCGM Research Collaboration Office Annual Report 201419

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 (IOMNCGM 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 medical care. At IOM, studies have been carried out with the aim of sharing the benefits of medicalcare through human resource development, and to contribute to the improvement of medical standards
in Nepal and the 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 areasof specialization (internal medicine and public health). In FY 2014, the following studies and activities werecarried out:
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 the office andliaison with other study groups have been carried out in collaboration with Department of Microbiology,
Institute of Medicine, Tribhuvan University. Of particular note is the well-established framework forcooperation and the continuing good relationship with departments at the Institute of Medicine,
Tribhuvan University.
The summary of JICA’s “Medical Education Project”, carried out for IOM from 1980 to 1996 was
prepared. Based on that summary, a comparison of achievements at hospitals and basic medical fields atthe end of the project and now was carried out.
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 andTraining, 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.
20

IOM-NCGM Research Collaboration Office Annual Report 2014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 tobe conducted at Nursing Department was carried out.
In December, 2014, the 2nd joint conference of three study groups (24-5, 25-5, and 25-7) using the
Nepal office was held at IOM. Release of the 2014 Annual Report by March 2015 was announced.

Guidance on radiologic interpretation in TUTH

Opening address of the Dean of IOM at the 2nd Joint Conference(IOM, December 2014)

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 hasa meaning of a negative control in contrast to the countries where training has already been provided by
the above JICA’s training course.
In FY 2014, the following two surveys were carried out, followed by some advice for improvement.
Further analysis of the results are planned in order to clarify the issues and points which need to be
improved and to determine effective measures for improvement together with the Nepal side.
IOM-NCGM Research Collaboration Office Annual Report 201421

1) Examination of the Actual Condition of Measures for Nosocomial Infections in TUTH
The actual condition of nosocomial infection control were examined at the TUTH and other hospitals
in Kathmandu using a questionnaire survey prepared in advance. 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 and in 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 addressed included improving the quality of the control system and training
program, providing training to a wider range of staff members, improving waste disposal system,
updating manuals, etc.
2) 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 havea 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-washingin 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.

Training course on nosocomial infection
control for newly recruited nurses (TUTH)

22

IOM-NCGM Research Collaboration Office Annual Report 2014

3.

Outline of each research
Table 2 shows overview of the collaborative researches between NCGM and IOM. Progress statusof each research (No. 1-6) is described on pages 23-35. We have already obtained some results, and theseresults were published at medical conferences and on scientific papers as shown on pages 57- (papers
published in 2014 were put in full paper, whereas only abstracts are put regarding papers published in2012,-2013 and in press papers).
Among these researches it is particularly outstanding that new strain of New Delhi metalo-βlactactamase producer was identified among the nosocomial infection cases and named NDM-8 and 12.

IOM-NCGM Research Collaboration Office Annual Report 201423

Table 2		

Collaborative researches in NCGM-IOM Collaboration Center, Nepal
Affiliation in Nepal

Subject

Sourceof fundNo.

Chief Researchers in Japan and Nepal

1• Hiroshi Ohara (Bureau of International• Institute of Medicine,
Medical Cooperation, NCGM)
Assessment of the interface
Tribhuvan University• Jeevan B Sherchand (Dept. of
between malaria control program• Faculty of Medicine,
Public Health, Insitutie of Meidicne,
and health system strengthening
Kathandu University
Tribhuvan University)

24-5*

2

• Te r u o K i r i k a e ( R e s e a r c hInstitute,NCGM)
Molecular epidemiology of• Institute of Medicine,• Bharat M. Pokhrel (Dept. of
nosocomial pathogens in
Tribhuvan University
Microbiology, Institute of Medicine,developing countries
Tribhuvan University)

24-5*

3

Evaluation of changing• Norio Ohmagari (Disease Control ande p i d e m i o l o g y o f i n fe c t i o u sPrevention Center, NCGM)• School of Medicine,diseases in a developing country:• Jatan Sherchan (School of Medicine,
Kathmandu University
The role of healthcare associatedKathmandu University)
opportunistic infections

24-5*

4

• Institute of Medicine,• S h i n s a k u S a k u ra d a ( B u r e a u o f
Tribhuvan University,
Study on double burdenInternational Medical Cooperation,• School of Medicine, tuberculosis (TB) and nonNCGM)
Kathmandu Univeristy, communicable diseases (NCD) in• Jeevan B Sherchand (Tribhuvan• 3 clinics in Kathmandu Nepal
University, Kathmandu)
City

24-5

5

• Hiroshi Ohara (Bureau of International
Fact-finding survey of nosocomial
Medical Cooperation, NCGM)• Institute of Medicine, i n f e c t i o n c o n t r o l i n m a j o r• Bharat M. Pokhrel (Dept. of
Tribhuvan University
hospitals in Nepal and discussion
Microbiology, Institute of Medicine,on effective improvement plans
Tribhuvan University, Nepal)

24-5*
25-7*

6

• Hiroshi Ohara (Bureau ofStudies on enhancement of humanInternational, Medical Cooperation, • Institute of Medicine, resource development utilizing the
NCGM)
Tribhuvan University,
IOM-NCGM Collaboration Center• B h a r a t M . P o k h r e l ( D e p t . o f • S c h o o l o f M e i c i n e , - taking into consideration of theMicrobiology, Institute of Medicine,
Kathmandu University
dissemination of the benefits of
Tribhuvan University)
ODA project25-5** grants from the International Health Cooperation Research, a grant from the Ministry of Health, Labor and Welfare of Japan

24

IOM-NCGM Research Collaboration Office Annual Report 2014

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 20148.

Publications in FY
2014

Oral presentation
Successful factors contributed to malaria control and key challenges in Vietnam
29th Annual Meeting of the Japan Association for International Health, November
2014, Tokyo
1. Assessment of Health systems in relation to interface between malaria control
programs and health system strengthening: comparative study among Lao PDR,
Nepal and Viet Nam

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, 5) Low incentive for health workers, 6) existence of inequity of bednets
distribution.

IOM-NCGM Research Collaboration Office Annual Report 201425

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.

26

IOM-NCGM Research Collaboration Office Annual Report 2014

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 2014

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 two novel NDM-type metallo-β-lactamase variant, NDM-8 and NDM-

Antimicrob Agents Chemother. 2014, 58(10):6324-6327
Antimicrob Agents Chemother. 2014, 58(10):6302-6305
BMC Infect Dis. 2014, 14:56

12. A novel New Delhi metallo-β-lactamase variant, NDM-8 has substitutions at positions 130 (Asp to Gly) and 154(Met to Leu) compared with NDM-1. Expression of the blaNDM-8 and blaNDM-1 genes in E. coli DH5α conferred
resistance or reduced susceptibility to all cephalosporins, moxalactam, and carbapenems. NDM-8 showed
enzymatic activities against β-lactams similar to those of NDM-1. A novel New Delhi metallo-β-lactamase variant,
NDM-12, was identified in a carbapenem-resistant E. coli clinical isolate obtained from a urine sample of a patientin Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions (M154L and G222D). The profiles of
enzymatic activities of NDM-12 against β-lactams tested were similar to those of NDM-1, although NDM-12 hadlower kcat/Km ratios for all β-lactams tested except for doripenem. The lower kcat/Km ratios were likely to be causedby the lower kcat values of NDM-12 compared with those of NDM-1, as the values of NDM-12 were 11.4 to 73.6%of those of NDM-1. The blaNDM-12 gene was located in a plasmid of 160 kb. The sequence surrounding blaNDM-12 wassimilar to that of the pGUE-NDM plasmid from the E. coli strain GUE, which was isolated in India.
We reported that multidrug-resistant Klebsiella pneumoniae clinical isolates with Various Combinations of
Carbapenemases (NDM-1 and OXA-72) and 16S rRNA Methylases (ArmA, RmtC and RmtF) were disseminating
in a medical setting in Nepal. Of 25 clinical isolates Klebsiella pneumonia obtained in 2012, 17 were resistantto imipenem and meropenem with MICs ≥4 mg/L, and 20 were highly resistant to arbekacin, amikacin and

IOM-NCGM Research Collaboration Office Annual Report 201427

gentamicin with MICs ≥512 mg/L. The carbapenemase encoding genes blaNDM-1 and blaOXA-72 were observed in
17 and 9 isolates, respectively; and the 16S rRNA methylase encoding genes armA, rmtC and rmtF were observed
in 9, 5, and 9 isolates, respectively.
Of S. maltophilia clinical isolates, we detected 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′)-Iakacetylated amikacin, arbekacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin and tobramycin.
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.

28

IOM-NCGM Research Collaboration Office Annual Report 2014

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)5.

Resource of fund

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

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
2014

Epidemiology of Extended-spectrum β-Lactamase (ESBL)-producing Escherichia coliin Nepal: Predominance of CTX-M-15-type ESBL. Sherchan JB, Hayakawa K, Ohmagari N,
Kirikae T, Nagamatsu M, Tojo M, Miyoshi-Akiyama T, Ohara H, Sherchand JB, Tandukar S.
Presented as a poster presentation at Interscience Conference on Antimicrobial Agentsand Chemotherapy, Washington DC, Sep, 2014.
Differences in the clinical and microbiological characteristics of extended-spectrum
β-lactamase (ESBL)-producing Escherichia coli between Japan and Nepal. Hayakawa K,
Sherchan JB, Kirikae T, Nagamatsu M, Tojo M, Miyoshi-Akiyama T, Ohara H, Sherchand JB,
Tandukar S, Ohmagari N, Presented as a poster presentation at Interscience Conference
on Antimicrobial Agents and Chemotherapy, Washington DC, Sep, 2014.
Clinical epidemiology and molecular analysis of Extended-spectrum β-Lactamase(ESBL)-producing Escherichia coli in Nepal: Characteristics of E. coli Sequence Type ST131
and ST648
Sherchan JB, Hayakawa K, Miyoshi-Akiyama T, Ohmagari N, Kirikae T, Nagamatsu M,
Tojo M, Ohara H, Sherchand JB, Sarmila Tandukar S. Manuscript under review.

9.

Summary:
The objective of this study is to evaluate the clinical epidemiology of opportunistic healthcare associatedinfections as well as infections due to drug-resistant pathogens in a developing country. The information would be
useful to seek for the effective preventing methods for healthcare associated infections and/or infections due todrug-resistant pathogens. Information pertaining to opportunistic healthcare associated infections in developing
countries are limited, and thus, this research would provide the valuable information. In addition, infections due
to drug-resistant pathogens pose serious public threat worldwide. The results from Nepal where there has beensparse information on clinical epidemiology of drug resistant pathogens would be beneficial in terms of providing
IOM-NCGM Research Collaboration Office Annual Report 201429

the epidemiology of global spread of resistant pathogens and their impact.
We completed the analyses of the epidemiology of drug-resistant pathogens, especially on world pandemic
Extended-spectrum β-Lactamase (ESBL)-producing Escherichia coli in Nepal. We combined the information ofclinical and microbiological characteristics of this pathogen in Nepal. The results were presented as a featured
poster-walk abstract at Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, Sep,
2014. The manuscript is currently under review. The major findings are that total of 105 patients with ESBL-E.
coli isolation were identified, and majority (90%) of the ESBL-E. coli isolates were CTX-M-15 positive. The mostdominant ST was ST131 (n=54 [51.4%]), followed by 15 (14.3%) cases of ST648, on which there has been few
literature. Among the 54 ST131 isolates, all 54 (100%) were identified as O25b-ST131 clone. Higher proportion of
ST648 isolates had resistance to non-beta lactam antibiotics than ST131 or non-ST131/648, and possessed drug
resistant genes more frequently than ST131 or non-ST131/648. More than 38% of ESBL-E. coli were isolated fromoutpatient clinic, and pregnant patients consisted 24% of ESBL-E. coli cases.
In addition, we are currently analyzing the data on the comparison of epidemiology of the resistant organisms
between in Nepal and Japan. These analyses would enable to identify the key epidemiological factors associatedspecifically to opportunistic healthcare associated infections due to drug-resistant pathogens in a developing
country.

30

IOM-NCGM Research Collaboration Office Annual Report 2014

Research No.4
1.

Title(in English)

Study on double burden tuberculosis (TB) and non-communicable diseases (NCD) in
Nepal

2.

Title(in Japanese)

ネパールにおける結核と非感染性疾患の二重負荷に関する研究

3.

Main researcher

Shinsaku Sakurada (Bureau of International Medical Cooperation, NCGM)

4.

Co-Researcher(s)Takanori Hirayama (JATA/RIT), Yuko Tsuda (Health Bureau of Osaka City), Shyam K
Shrestha (FSB Clinic, Kathmandu), Jatan B Sherchan (Kathmandu University School of
Medicine, Dhulikhel), Kishor S Manandhar (CF Clinic, Kathmandu), Jeevan B Sherchand(Tribhuvan University, Kathmandu)

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, Kathmandu,
Nepal

7.

Period of the research

September, 2012-March, 20148.

P u b l i c at i o n s i n F Y
2014Oral Presentation:
Joint Conference on Infectious Diseases with growing Concern in Recent Years in Nepal,
December 2014.

9.

Summary:
NCD in developing countries has increased in recent decades and may affect control of communicable diseases
in particular TB. In Nepal incidence of TB has recently increased in elderly populations (>65 years old) but
decreased in young populations. This trend may inevitably lead Nepali people to double burden of TB and NCD.
Our objective is to survey the present status of TB and NCD in Kathmandu by epidemiological and sociologicalmethods.
We conducted a clinic-epidemiological study at three clinics in Kathmandu city. One sampling site was a clinicof respirology expert, another two sampling sites were general clinics but one of those was located in the thickly
populated area with Tibetan refugees. We used use simple questionnaires with written informed consent to detectdouble burden cases. Then, we conducted focus group discussion (FGD) with heath officials, medical doctors,
public health nurses and DOTS providers in Kathmandu. We collected 414 TB samples and 649 NCD samples
from three sites. Preliminary analysis has shown high prevalence of TB among NCD patients in one general clinic
located in the commercial area of Kathmandu city. While the estimated prevalence of TB in Nepal, 2010 was 238
per 100,000 populations, but that in the general clinic was 2,800 per 100,000 populations (the number of newly
diagnosed patients was 8 in 282 NCD patients). Among NCDs diabetes mellitus (DM) is most common except
chronic respiratory diseases. There was ten-folds higher TB prevalence among NCD patients. In FGD medical
doctors agreed that the awareness of double burden is important in the both sides of patients and health careproviders. They have a plan to distribute posters to clinics in Kathmandu to aware the double burden. The resultsof this study might contribute to measures against TB control in a new era with double burden with NCD.
Since November in 2014, we have conducted a new study over the prevalence of DM in the both new and relapse
cases of TB to evaluate the effect of DM in relapse of TB. We have performed OGTT for 258 new and 139 relapse
cases after getting informed consent. The study will be completed by the end of March in 2015.
IOM-NCGM Research Collaboration Office Annual Report 201431

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 (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 (24-5, 25-7)

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
2014Oral presentation
88 th 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

32

IOM-NCGM Research Collaboration Office Annual Report 2014

addressed included improving the quality of the control system and training program, providing training to a wider
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.
3)

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 201433

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
2014

Annual Report of the IOM-NCGM Collaboration Office in 2014 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 conducted to local staff. Management of theoffice and liaison with other study groups have been carried out.
2.

3.

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

34

IOM-NCGM Research Collaboration Office Annual Report 2014

••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.
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.

•

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.

•

Planning of “Training for Newly Employed Nurses on Measures for Nosocomial Infections” scheduled tobe conducted at Nursing Department was carried out.

IOM-NCGM Research Collaboration Office Annual Report 201435

IV. Other Activities in FY 2014

1.

Joint Conference

The “2nd Joint Conference on Infectious Diseases with Growing Concern in Recent Years in Nepal” washeld at IOM (December 5, 2014). Following inaugural speeches by representatives of IOM and NCGM, 12
presentations on collaborative research results were made followed by questions & answers and active
discussions. A total of 54 participants (Tribhuvan University counterparts, doctors and nurses from related
fields, university teaching staff in Kathmandu, a professor from Australia) including four doctors from Japan
were attended. The program and abstracts are shown on pages 39-53.
2.

Presentation on Collaborative Research Results
A research group completed the analyses of theepidemiology of drug-resistant pathogens, especially on
world pandemic Extended- spectrum β- Lactamase (ESBL)- producing Escherichia coli in Nepal. The results werecombined with the information of clinical and microbiologicalcharacteristics of this pathogen in Nepal. These results were
presented as a featured poster-walk abstract at Interscience
Conference on Antimicrobial Agents and Chemotherapy,
Washington DC, U.S.A. in September 2014.
Regarding the study on “Assessment of the interface between malaria control program andhealth system strengthening”, which are conducted in both Nepal and Vietnam, a counterpart researcher
was invited from Vietnam in November 2014 and gave a presentation at the 29 th Japan Association
for International Health Congress, Tokyo. (In 2012 and 2013, a counterpart researcher in Nepal madepresentations in Japan on this subject)
3.

Overview of the Medical Education Project and current IOM/TUTH

The overview of the Medical Education Project, which was implemented as Official Development Assistance(ODA) by JICA from 1980 to 1996, was summarized and the situations of IOM/TUTH at the end of the
project and the present time were compared.
4.

Enhancement of Human Resource Development

After consulting with Research Department, Department of Microbiology, Department of Public Healthand Department of Internal Medicine, Department of Pulmonology the following activities were initiated,focusing mainly on improving research ability (25-5, 24-5).
36

IOM-NCGM Research Collaboration Office Annual Report 2014

1) Research Department: Hearing on the overall impact of JICA project and current problems was
conducted and it found that research methods, especially, epidemiological methods and diagnostic
imaging methods were weak.
2) Department of Microbiology:
Training for analysis of drug-resistant bacteria wasconducted to the staff members of Department ofMicrobiology as a part of human resource development
activities. In addition to technical instruction at
IOM, NCGM invited two graduate students from thedepartment to the Department of Infection Control at
NCGM and gave technical instructions from October
to November in 2014 (24-5). These 2 graduates willcontinue to do research based on techniques learned,

Technical guidance at NCGM to the staff of Department ofMicrobiology, IOM

with the goal of obtaining PhD degrees.
3) Department of Internal Medicine, Department of Pulmonology:
Following provision of the necessary equipment such as
computers and scanners, NCGM in collaboration with
National Center for Disaster Medicine started to giveassistance to conduct a research entitled “Case study ofinterstitial pneumonia at Tribhuvan University Teaching
Hospital”. Japanese Doctor provided technical guidanceon medical imaging diagnosis method (chest X-ray andchest CT) to the department staff.
5.

Ward round in Pulmonology Dept.

Overview of current medical situation in Nepal
Nepal is divided into 14 administrative zones and 75 districts and zonal and district hospitals arelocated by these administrative divisions. Primary healthcare centers and health posts are located underthese hospitals, constructing a system that promotes medical care and hygiene among residents.
Major diseases are infectious diseases, perinatal disorders, and malnutrition. However, thefrequency of non-communicable diseases such as diabetes, cardiovascular diseases, hypertension, andcancer is also increases in recent years. Common infectious diseases include diarrheal diseases, respiratory
IOM-NCGM Research Collaboration Office Annual Report 201437

infections, tuberculosis, hepatitis, HIV/AIDS, etc. Vector borne infectious diseases such as malaria, dengue
fever, visceral leishmaniasis, and Japanese encephalitis are mainly distributed in the southern plains. Malaria
has decreased remarkable during the past 15 years, however dengue fever is on the increase. In the capital
Kathmandu, chronic obstructive respiratory diseases have been increasing along with the progress of air andwater pollution caused by the rapid increase in population and automobiles.
Maternal mortality ratio decreased from 530 (1996) to 281 (2006) per 100,000 births, and mortality
ratio of infants under five years decreased by 48% from 118 to 61 per 1,000 births between 2001 and 2005.

38

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1
2nd Joint Conference on Infectious Diseases with Growing Concern
in Recent Years in Nepal
Date: 		December 5, 2014

Venue: 		

Institute of Medicine (IOM), Tribhuvan University, Kathmandu, Nepal		Registration: 8:00- 8:30

		

(Master of Ceremony: Ms. Shovita Shrestha, Dept. of Microbiology)

8:30 		

Introduction of participants

8:40 		

Inauguration by lighting the lamp: Prof. Rakesh P. Shrivastav, Dean, IOM

8:45 		

Remarks: Prof. Rakesh P. Shrivastav, Dean, IOM

8:50 		

Remarks: Prof. Jeevan K. Shrestha, Campus Chief

8:55 		

Remarks: Prof. Bimal K. Sinha, Asst Dean, IOM

9:00 		

Remarks: Prof. Bharat M. Pokhrel, Asst Dean, IOM

9:05 		

Remarks: Prof. Sarala Shrestha, Asst Dean, IOM

9:10 		

Remarks: Prof. Sharad R. Onta, Asst Dean, IOM

9:15 		

Remarks: Prof. Dipak P. Mahara, Executive Director, TUTH

9:20 		

Remarks: Dr. Teruo Kirikae, Director, Dept. of Infection Control, NCGM

Session A:

Overview of Collaboration and Health Issues

		(Chairperson: Prof. Bharat M. Pokhrel)

9:25-9:45

A-1: Overview of collaboration between IOM and NCGM		(Dr. Hiroshi Ohara, NCGM)

9:45-10:05

A-2: Multidrug resistance- an emerging health issue in Nepal		(Prof. Basista Rijal IOM)

Session B :Multi-Drug Resistant Bacteria		(Chairpersons: Prof. Basista Rijal, Dr. Kayoko Hayakawa)

10:20-10:35

B-1: Emergence of multidrug-resistant Gram-negative nosocomial pathogens in Nepal

		(Dr. Teruo Kirikae, NCGM)

10:35-10:55

B-2: Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in a 				

university hospital in Nepal		(Shovita Shrestha, IOM)

10:55-11:15

B-3: Molecular epidemiology of multidrug-resistant Escherichia coli isolates in hospitals in 		

		

Nepal

		

(Basudha Shrestha, IOM)

11:15-11:35

B-4:  Epidemiology of Extended-spectrum β-Lactamase (ESBL)-producing

		Escherichia coli in Nepal: Predominance of CTX-M-15-type ESBL		(Dr. Jatan B. Sherchan, Kathmandu Univ.)
IOM-NCGM Research Collaboration Office Annual Report 201439

Attachment-1

Session C:

Healthcare Associated Infection Control

		(Chairperson: Dr. Teruo Kirikae)

11:35-11:50

C-1: Fact-finding survey of nosocomial infection control in leading hospitals in Kathmandu 2014

		(Prof. Bharat M. Pokhrel, IOM)

11: 50-12:05

C-2: Current situation and challenges of trainings on infection control for newly recruited 				nurses in TUTH

		

(Ns. Dharma L. Shrestha, TUTH)

12:05-13:00

Lunch

Session D:

Diarrheal Diseases, Malaria, Double Burden, Other Important Issues

		(Chairpersons: Prof. Jeevan B. Sherchand, Dr. Hiroshi Ohara)

13:00-13:15

D-1: Rotavirus gastroenteritis in children in Nepal		(Prof. Jeevan B. Sherchand, IOM)

13:15-13:30

D-2: Assessment of the malaria control program in relation to general health system

		(Dr. Hiroshi Ohara, NCGM)

13:30-13:50

D-3: The series of studies from “DOUBLE BURDEN OF TUBERCULOSIS AND NON-COMMUNICABLE DISEASES” to “PREVELANCE OF DIABETES IN TUBERCULOSIS CASES,” in Kathmandu valley,
Nepal
		

(Dr. Takanori Hirayama, Japan Anti-Tuberculosis Association)

13:50-14:05

D-4: Promotion of Research Collaboration between Nepal and Japan - Clinical Study Setting in 				Pulmonology		(Dr. Mitsuhiro Kamimura, National Disaster Medical Center)

14:05-14:20

Discussion

		(Chairpersons: Prof. Bharat M. Pokhrel, Dr. Teruo Kirikae, Dr. Hiroshi Ohara )14:20		

Closing remarks: Prof. Rakesh P. Shrivastav, Dean, IOM

Joint Conference at IOM (December 2014)40

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1(A-1)

Overview of Collaboration between IOM and NCGM
Hiroshi Ohara
Bureau of International Medical Cooperation
National Center for Global Health and Medicine, Tokyo, Japan

Tribhuvan University /Institute of Medicine (IOM) and National Center for Global Health and Medicine(NCGM) have a long history of cooperation starting technical cooperation project supported by JICA. In view ofthe successful results of the project along with the intimate and reliable relationship, IOM and NCGM agreed toenter into the Memorandum of Understanding (MOU) to promote collaboration between the two institutions
focusing on researches and related activities. Currently, the following researches have been conducted basedon the research grants from the Ministry of Health, Labor and Welfare, Japan.
1. 24-5: Studies on causative 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 Center ‐ taking into consideration of the dissemination of the benefits of ODA project
3. 25-7: Fact-finding survey of nosocomial infection control in major hospitals in Nepal and planning ofeffective improvement
As the results of preliminary study, we recognized some emerging health priorities in Nepal andthe following subjects were selected to conduct collaborative studies (mainly infectious diseases): Diarrhea
caused by emerging pathogens, Multi-drug resistant bacteria, Healthcare associated infections, Dual burden of
infectious diseases and non-communicable diseases, Malaria control in relation to health system etc.
It is a great pleasure that “the 2nd Joint Conference on Infectious Diseases with Growing Concern in Recent
Years in Nepal” is held in Kathmandu on December 5, 2014. In this conference we will present the results of ourcollaborative studies. We are strongly convinced that our collaboration will contribute to promotion of healthalong with medical science in both countries.
Key words: IOM, NCGM, Collaboration, MOU, Emerging health priorities

IOM-NCGM Research Collaboration Office Annual Report 201441

Attachment-1(A-­‐2)  Multi-­‐drug  Resistance-­‐  an  Emerging  Health  Issue  in  Nepal  B  P.  Rijal,  S.K.  Mishra,  H.  Kattel,  R.Dahal,  S.  Sharma,  N.Shah,  K  Parajuli,  S.Khatiwada,  S.Khadka,    N.  parajuli,  S.  
Dhital,                  S.  Dahal,  B  .shrestha,  S.S.  Dhakal,  J.B.Sherchand  and  BM  Pokherel    Department  of  Microbiology,  Tribhuvan  University  Teaching  Hospital  and  Maharajganj  Medical  college,    Institute  of  Medicine  (IOM),  Tribhuvan  University,  Kathmandu    Correspondence  to:  Basista  Rijal,  email:  basistarijal@gmail.com  Introduction:	
  Antimicrobial	
  resistance,	
  especially	
  Multi-­‐drug	
  resistance	  (MDR)	
  is	
  a	
  global	
  and	  National	
  Health	  problem.	
  MDR	  increases	
  morbidity,	
  treatment	
  failure,	
  cost	  of	
  treatment	
  and	  mortality.	
  The	  present	
  study	  has	
  analyzed	
  the	
  trend	  of	
  antimicrobial	
  resistance	
  of	
  the	
  clinical	
  isolates	
  with	  special	
  consideration	
  to	
  MDR	
  and	
  also	  to	
  appropriateness	
  of	
  such	  study.	
  Method:	
  The	
  data	  were	  collected	
  and	
  analyzed	
  from	  many	  published	
  and	
  current	
  ongoing	
  studies	
  at	
  the	  IOM	
  and	  in	
  Kathmandu,	
  mostly	
  within	
  the	  10	
  years.	
  Results:	
  The	
  study	  revealed	
  that	  	  increase	  in	
  resistance	
  of	
  	
      Escherichia  coli	
  over	  a	
  period	  	
  with	  	  	
  different	
  classes	
  of	
  drug;	  	
  Cephalexin	
  (82-­‐100%),	
  Ceftazidime	  (2-­‐100%),	
  Cefotaxime	
  (15-­‐100%),	
  Ciprofloxacin	
  (55-­‐82%),	  	
  Gentamycin	
  35-­‐58%)	
  and	  ,	  Amikacin	
  (	  0-­‐84%	
  ).The	  study	  also	
  showed	
  that	  	
  High	  level	  of	
  MDR	
  E.coil	  (	  49-­‐100%)	
  and	  	
  ESBL	  (	
  16-­‐83%).	
  	
  	
  	
  	
  Similarly,	
  the	  resistance	
  pattern	
  of	
  Klebsiella  Pneumoniae  was  	
  	
  
also	
  on	
  alarming;	
  Ceftazidime	  (21-­‐100%),	
  	  	
  gentamycin	
  (	  29-­‐8	  %),	  	
  Amicacin	
  (	  19-­‐81%),	
  Meropenum	  	
  (0-­‐
47%).	
  The	
  high	  proportion	
  of	
  of	
  K.pneumoniae	
  	
  were	  MDR	  (67-­‐100%),	
  ESBL	
  (66%)	
  and	
  MBL	  (52%).	
  Majority	
  of	
  the  Acinetobacter  bumennii,	
  a	
  emerging	
  pathogen	
  were	  resisistant	
  	
  to	
  Ceftazidime	  (82-­‐
100%),	
  Gentamycin	
  (62-­‐100),	
  Amikacin	
  (54-­‐82%)	
  ,	
  Piperacillin	
  (77-­‐100%)	
  and	
  Meropenum	
  (	  35-­‐79%),.	
  The	
  high	  level	  of	
  MDR	  (9.6-­‐10%),	
  ESBL	
  (12%)	
  and	
  MBL	  (66%)	
  was	
  observed.	
  Moreover,	
  the	  percentage	
  of	
  MRSA	  was	  (13-­‐66%)	
  also	
  shocking.	
  However,	
  Vancomycin	
  resistant	
  Staphylococcus  aureus	
  was	  not	
  observed.	
  	
  Likewise,	
  the	
  rising	
  pattern	
  of	
  drug	  resistance	
  was	
  observed	
  to	
  Streptococcus  pneumoniae.	
  The	
  S.pneumoniae	
  were	  resistant	  to	
  Penicillin	
  (2-­‐10	  %),	
  Ampicillin	
  (2-­‐11%),	
  Erythromycin	
  (0-­‐12	
  %)	
  .Furthermore,	
  High	  antimicrobial	
  resistance	  	
  and	  MDR	  was	
  also	  observed	
  in	
  other	  Gram	
  negative	  and	  Gram	  positive	  bacteria	
  including	
  enteropathogens,	
  Pseudomonas	
  and	
  other	  pathogens.	
  Conclusion:	
  The	
  rising	
  pattern	
  of	
  antimicrobial	
  resistance	
  and	  MDR	  is	
  a	
  serious	
  threat.	
  	  	
  Strong	
  measures	  to	
  control	
  and	  prevention	
  of	
  drug	  resistance	
  is	
  urgently	
  needed.	
  To	
  cope	  the	
  problem,	
  the	  resistance	  surveillance	
  should	
  be	
  done	  in	  a	
  regular	
  basis	  by	
  following	
  a	
  standard	
  method.	
  The	  result	
  of	
  the	  surveillance	
  and	
  its	
  impact	
  should	
  be	
  disseminated	
  to	
  policy	
  makers,	
  prescribers	
  and	  to	
  communities.	
  A	
  strong	  infection	
  control	
  and	  prevention	
  practice	
  should	
  be	
  implemented	
  in	
  the	
  hospitals.	
  	
  	
  	
  
Key	
  words:	
  Trends,	
  antimicrobial	
  resistance,	
  Nepal	  42IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1(B-1)

Emergence of multidrug-resistant Gram-negative nosocomial pathogens in Nepal
Teruo Kirikae1, Hiroshi Ohara1 , Basista Rija2, Jeevan B Sherchand2 and Bharat M Pokhrel2
1: National Center for Global Health and Medicine, Japan
2: Department of Microbiology, Institute of Medicine, Tribhuvan University, Nepal
Since the first antibiotic, penicillin introduced in 1940s, anti-infective drugs to prevent mortality andmorbidity arising from infections have been one of the most effective health interventions in the historyof modern medicine. Drug-resistant pathogens were gradually emerging at first, but suddenly they weredisseminating rapidly in medical settings. The current situation is becoming serious with an increasing
incidence of detection of highly drug-resistant pathogens to all known drug treatments. An example is thatof common bacterial infections in hospital settings. Infections with carbapenem-resistant pathogens are on
the rise, and have recently become resistant to ‘last-resort antibiotics’. These bacteria are an increasing causeof mortality in many countries. According to a WHO report the “…world is heading towards a post-antibiotic
era in which many common infections will no longer have a cure and once again kill unabated”. The selection
pressure exerted by the antibiotic gets intensified with the overuse, misuse or underuse of the antimicrobial
agents. Another mechanism of drug resistance is acquisition of drug resistance mediated through plasmids
or mobile genetic elements. Such resistance can be horizontally transferred from one organism to anotherorganism, including from animal infectious agents to human pathogens. This is very common with entericbacteria such as Escherichia coli, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter and Enterococcus.
Professors Pokhrel and Sherchan and their colleagues at Institute of Medicne, and Teaching Hopspital,
Tribhuvan University, noticed the existence of multidrug-resistant nosocomial pathogens in hospitals in Nepal.
They did surveillance of drug-resistant pathogens at first time in Nepal. Since 2012, Drs. Kirikae and Ohara at
National Center for Global Health and Medicine, join their research in Nepal.
We have conducted an international collaboration study on molecular epidemiology of multi-drugresistant nosocomial pathogens. During the study period, Dr.s Kirikae and Ohara visited IOM more than ten
times and Professors Pokhrel and Sherchan visited Japan twice and once, respectively. An instructor, Rajan
Kumar Dahal, an M.Sc. student, Manoj Kumar Shah stayed in NCGM, 2013. Two PhD students, Basudha
Shrestha and Shovita Shrestha stayed in NCGM twice 2014. We successfully achieved our goals set for our firststudy project. We will show a part of the results of the study in this conference.

IOM-NCGM Research Collaboration Office Annual Report 201443

Attachment-1(B-2)

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in auniversity hospital in Nepal
Shovita Shrestha1, Tatsuya Tada2, Tohru Miyoshi-Akiyama3, Kayo Shimada2, Kazuhito Satou4, Kuniko Teruya4,
Kazuma Nakano4, Akino Shiroma4, Jeevan Bdr. Sherchand1, Basista Psd. Rijal1, Takashi Hirano4, Teruo Kirikae2
and Bharat Mani Pokhrel1
1:Department of Microbiology, Institute of Medicine, Tribhuvan University, Kathmandu, Nepal
2:Department of Infectious Diseases
3:Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo
162-8655, Japan;
4:Okinawa Institute of Advanced Science, Okinawa 904-2234, Japan
Objectives: To clarify the genetic and epidemiological properties of MDR Acinetobacter baumannii clinicalisolates obtained from patients in Nepal and determine a complete genome sequence of a MDR A. baumannii
isolates.
Methods: Acinetobacter spp. isolates obtained from single patients were screened for MDR A. baumannii
with antimicrobial disk susceptibility tests. The whole genomes of the MDR isolates were sequenced by MiSeq(Illumina). The complete whole genome of an isolate, IOMTU433, were sequenced by PacBio RS II. Phylogenic
trees were constructed from the SNP concatemers. MLSTs were deduced and drug resistant genes weredetected.
Results: Fifty percent of Acinetobacter spp. isolates (122/246) were MDR A. baumanni, of which the majority
were resistant to aminoglycosides, carbapenems, fluroquinolones but not colistin and tigecycline. These
isolates harbored a 16S rRNA methylase gene, armA, and either blaNDM-1, blaOXA-23 or blaOXA-58. 49.1% of the
MDR A. baumannii are isolated from sputum. Most of the A. baumannii are resistant to most of the antibiotic
tested. The multidrug-resistant A. baumannii harboured different β-lactamase genes and carbapenemase
genes. All the 122 MDR A. baumannii isolates belonged to 15 different types of MLST. eBURST analysis ofthem together with Institut Pasteur's MLST database found that 5 of the 15 STs were grouped into 3 clonal
complexes (CCs), including CC1 (ST1and ST623), CC2 (ST2) and CC149 (ST149 and ST622). The phylogenetic tree
revealed 3 clades, CC1, CC2 and CC149, respectively. To our knowledge, the CC149 is a novel clonal complex of A.
baumannii, to which MDR isolates are spreading in medical settings in Nepal.
Conclusions: This is the first report of a novel clonal complex, CC149, of A. baumannii, to which MDR isolates
are spreading in medical settings in Nepal.

44

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1(B-3)

Molecular epidemiology of carbapenem-resistant Escherichia coli in Nepal
Basudha Shrestha1, Tatsuya Tada2, Tohru Miyoshi-Akiyama3, Kayo Shimada2, Jeevan Bdr. Sherchand1, Basista
Psd. Rijal1, Teruo Kirikae2* and Bharat Mani Pokhrel1
1: Department of Microbiology, Institute of Medicine, Tribhuvan University, Nepal
2: National Center for Global Health and Medicine, JapanThe emergence of carbapenem-resistant Enterobacteriaceae (CRE) isolates producing class B metallo-βlactamases (MBLs) has become a major concern in medical settings worldwide. MBLs are produced by many
species of Gram-negative bacteria. MBLs confer resistance or reduced susceptibility to almost all β-lactams
including carbapenems. New Delhi metallo-β-lactamase (NDM) -1 producing Escherichia coli and Klebsiellapneumoniae isolates was initially obtained from an Indian patient in 2008 in Sweden. Since then, NDM-1producing Enterobacteriaceae has been reported in 29 countries until December 2012. Till date, 12 NDM
variants producers have been found in several countries; including NDM-8 and NDM-12 producing E. coli from
Nepal.
The purpose of this study is to determine phenotypic and genotypic properties of carbapenem-resistant
E. coli isolates obtained from hospitalized patients in Nepal. E. coli isolates were screened with antimicrobial
disk susceptibility tests and MICs of carbapenems were determined with the microdilution method. Whole
genome sequences of carbapenem-resistant E. coli isolates were determined by next generation sequencers.
Of 250 E. coli isolates tested, 39 (16%) were resistant to carbapenems. Of the 39 CRE isolates, 32 produced
various NDM variants, including NDM-1 (10 isolates), NDM-3 (2), NDM-4 (1), NDM-5 (11), NDM-7 (7) and NDM12 (1). Of the 32 NDM producing carbapenem-resistant E. coli isolates, 17 also produced 16S rRNA methylases,
including, ArmA (8), RmtB(7) and RmtC (2), which confer high resistance against all aminoglycosides.
These results strongly suggest that carbapenem-resistant E. coli isolates producing both NDM-type metallo-βlactamases and 16S rRNA methylases disseminates in medical settings in Nepal. In this study, we discovered anovel NDM variant, NDM-12 in a clinical isolate in Nepal.
Key words: Carbapenem-resistant Enterobacteriaceae (CRE), New Delhi Metallo-β-lactamase (NDM), 16S rRNA
methylase

IOM-NCGM Research Collaboration Office Annual Report 201445

Attachment-1(B-4)

Epidemiology of Extended-spectrum β-Lactamase (ESBL)-producing Escherichia coli in
Nepal: Predominance of CTX-M-15-type ESBL
J. B. Sherchan1, K. Hayakawa2, N. Ohmagari2, T. Kirikae2, M. Nagamatsu2, M. Tojo2, T. Miyoshi-Akiyama2, H.
Ohara2, J. B. Sherchand3, S. Tandukar3
1:Kathmandu University School of Medical Sciences, Dhulikhel. Nepal
2:National Center for Global Health and Medicine, Tokyo, Japan
3:Tribhuvan University Teaching Hospital, Kathmandu, Nepal
Background: Extended-spectrum β-lactamase (ESBL)-producing bacteria are a major threat to antimicrobialresistant organisms worldwide. In particular, the spread of CTX-M-type ESBL-producing Escherichia coli (ESBLEC)
is a significant public health concern. Epidemiology reports on ESBLEC in Asia are limited. Therefore, we
examined the molecular and clinical epidemiology of ESBLEC in large university hospitals in Nepal.
Methods: Unique cases with isolation of ESBLEC from 2/2013 –7/2013 were included. ESBLEC isolates werescreened for pabB and CTX-M β-lactamase genes by using O25b-ST131 clone allele-specific PCR. E. coli
phylogenetic groups were determined by PCR.
Results: Totally, 107 ESBLEC strains were isolated during the study period. All ESBLEC isolates were collectedfrom urine. The mean age of patients with ESBLEC was 40.8 ± 23.2 years, and 66 (61.7%) were female. In
female patients, ESBLEC were more frequently identified at outpatient clinics than male patients (female
patients with ESBLEC isolation at outpatient clinics: n=33 [50%] vs male patients: n=7 [17.1%]) (p<0.001). Malepatients with ESBLEC often had underlying urological conditions such as benign prostate hypertrophy (n = 7[17.1%]) and urolithiasis (n = 3 [7%]). Fifteen (22.7%) female patients with ESBLEC were pregnant. Of the 107
isolates, 96 (89.7%) were positive for CTX-M-15 and 55 (51.4%) were identified as E. coli O25b-ST131 clones.
Clinical and microbiological characteristics are presented in Table 1.
Conclusion: Most of the ESBLEC isolates in Nepal were CTX-M-15 positive, and more than half were of E. coliO25b-ST131. The clinical and microbiological characteristics did not differ between the ST131 and non-ST131
groups. The resistance of ESBLEC to multiple classes of antibiotics as well as its prevalence in communities,
especially among pregnant females, is of great concern.
46

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1(C-1)

Fact-finding survey of nosocomial infection control in leading hospitals
in Kathmandu 2014
Bharat M. Pokhrel1, Hiroshi Ohara2, Teruo Kirikae2, Shyam K. Mishra1,Hari P. Kattel3, Sangita Sharma1, Dharma L.
Shrestha3, Yumiko Haneishi2, Keshav P. Parajuli1, Basista P. Rijal1, Jeevan B. Sherchand1
1:Department of Microbiology,Institute of Medicine, Tribhuvan University, Nepal
2: Bureau of International Medical Cooperation, National Center for Global Health and Medicine, Japan
3:Tribhuvan University Teaching Hospital, Institute of Medicine, Nepal
Nosocomial infection is worldwide problem both in developed and developing countries. In view to assess theactual scenario of health care associated infection, 5 referral hospitals of capital of Nepal were surveyed at one
time in the year 2014. Survey methods included standard questionnaire, site visits and interviews.
Briefly, questionnaire format consisted method of infection control, training situation, personal protection
equipment (PPE) etc.
These hospitals had nurses ranging from 70-530 and doctors 20-250 as per bed capacity. The bed capacity ofthese hospital ranged 100-460. Two hospitals had infectious diseases department. Four hospitals had protocal
for hospital associated infection control. Two hospitals had WHO/ CDC guide line for infection control. Threehospitals had infection control committee. One hospital holds meeting bimonthly or as per demand. Two
hospitals did not have N95 mask. Whereas others responded poor availability of N95 mask. Regarding personal
protective equipments; gloves, gowns and masks were sufficiently available where as goggles were poorly
available. Respondents of all hospital were expecting infection control training, although in some hospitals they
had in-house training.
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.
Key words: Fact-findings survey, nosocomial infection control, Leading hospitals, Kathmandu,2014

IOM-NCGM Research Collaboration Office Annual Report 201447

Attachment-1(C-2)

Current situation and challenges of trainings on infection control for
newly recruited nurses in TUTH
Dharma Laxmi Shrestha
Nursing Department, TUTH
Tribhuvan University Teaching Hospital (TUTH) is a tertiary care hospital in Kathmandu. It provides
specialty care in an academic setting to patients who are referred from around Nepal. TUTH provides
outpatient and inpatient care and patients tend to have complex problems requiring consultation from medical
and surgical subspecialties as well as intermediate and intensive levels of care (for example, ICU).
Infection control is significant challenge at TUTH for nursing staff as nursing staff provide a critical rolein implementation of infection control practice. In reality, there are many challenges to effective infection
control practice which results in inadequate implementation. Proper hand hygiene is not effectively practiced.
Patients with transmissible infectious diseases are not able to be properly isolated. Both of these simple
infection control practices contribute to nosocomial infections. Infection control training of nursing staff isperformed. However, frequent turn over of nursing staff means that training has to be frequently repeated
which is not always feasible or implemented. Even when nursing staff are educated regarding proper infection
control practice, insufficient infrastructure for infection control is a problem. Only high care areas (ICU and
MICU) have alcohol hand wash is possible the other wards, soap and water is available. There may only be
one sink in an entire ward to wash hands, making it inconvenient and difficult for nursing staff to perform handhygiene during clinical practice. Drying of hands is often used by a towel which may serve as a reservoir for
microorganisms and contaminate health care worker’s hands right after hand washing.
At present, there is no regular monitoring of infection control practice in the hospital. Without propermonitoring, it is difficult to understand how well we perform infection control activities .Recently hospital has
going to establish a health care waste management system as recommended by WHO protocol.

48

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Attachment-1(D-1)

Rotavirus gastroenteritis in children: Hospital associatedmolecular epidemiology in Nepal
Jeevan B. Sherchand1 and Hiroshi Ohara2
1: Tribhuvan University Teaching Hospital, Institute of Medicine, Department of Microbiology and Public HealthResearch Laboratory, Kathmandu, Nepal
2: National Center for Global Health and Medicine, Tokyo, Japan
ABSTRACT
Rotavirus is the major pathogens of community acquired acute gastroenteritis in children, but their role inhospital acquired gastroenteritis is not fully understood. The aim of the study was to assess the incidence of
hospital associated gastroenteritis and molecular evidence among hospitalized children less than 5 years ofage.
Stool samples were collected from 1721 hospital admitted children under 5 years of age attending Children’s
Hospital with acute watery diarrhea during 2010 and 2011 and findings will be presented. But the presentation
will be highlighted from suspected children with hospital acquired acute gastroenteritis. Acute gastroenteritis
was classified as hospital acquired infection if diarrhea developed in 48 hours or more after admission. The
incidence of hospital acquired infection due to rotavirus was 31.2% (48/ 154) by ELISA. The distribution ofrotavirus genotypes G and P, serotype G12 represented 48% of rotavirus strains characterized by reverse
transcription-polymerase chain reaction genotyping during the study, and was associated with P-types P[6],P[8] and P[4]. Further, a total of nine G/P type combination were identified, with G12 P [6] 30% being themost commonly detected rotavirus strain type. Most of the children who had hospital acquired rotavirus
gastroenteritis found symptoms of diarrhea, vomiting, fever, poor sucking and dehydration. Additional findings
showed that 2% cases of rotavirus co-infection with bacterial pathogens of Esch coli and Shigella species.
The study revealed that G 12 and G12P [6] were found major genotypes causing hospital acquired rotavirus
gastroenteritis in Nepal. Introduction of rotavirus vaccine along with strengthening hygienic measures could
substantially reduce the incidence of hospital acquired acute gastroenteritis in children of Nepal.
Key word: Rotavirus, Gastroenteritis, Hospital associated, Nepal

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Attachment-1(D-2)

Assessment of the malaria control program in relation to general health system
Hiroshi Ohara1, Jeevan B. Sherchand2, Jatan B. Sherchan3, Takanori Hirayama1
1: Bureau of International Medical Cooperation, National Center for Global Health and Medicine
2: Public Health Laboratory, Institute of Medicine, Tribhuvan University
3: Department of Clinical Microbiology, Kathmandu University
Malaria control has been a major health issue with high priority in endemic countries. In this study,
successful factors in malaria control and key challenges were analyzed, particularly in relation to health system.
Studies were conducted in Nepal and Vietnam. Information obtained from the document reviews,
interviews, and field surveys were analyzed from the viewpoint of interface between malaria control program(vertical health system) and the general health system with special emphasis on good practices and key
challenges.
Leading good practices include: 1)Strong government commitment for malaria control (Particularly
in Vietnam), 2)National strategy for rural development and intensified education for residents, 3)Effective
vertical system from national to village level for malaria surveillance and service delivery, 5)Strengthening thecapacity of health workers along with mobilization of mass organizations, and 6)Support from internationalorganizations.
Key challenges include: 1)Large number of population at risk in remote and mountainous areas, 2)
Uncontrolled seasonal migrants and cross border movement, 3)Lack of human resources for malaria network,and 4)Increasing drug resistance of malaria parasite, 5)Inequality in distribution of bed nets (Particularly in
Nepal).
Strengthening of the vertical health program appeared to have some impact on the general healthsystem, particularly at the primary level. More efforts are requested for strengthening of the health system
in remote areas, training of health staff at peripheral level, accurate quality assurance, promotion of publicprivate relationship and equity in bed net distribution. These tackling will directly lead to further strengtheningof the general health system, and eventually effective implementation of various health programs andcontribution to UHC.
Key words: Malaria, Nepal, Vietnam, Challenges, Equity
50

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-1(D-3)

The series of studies from “DOUBLE BURDEN OF TUBERCULOSIS AND NONCOMMUNICABLE DISEASES” to “PREVELANCE OF DIABETES IN TUBERCULOSIS CASES,”
in Kathmandu valley, Nepal
Takanori Hirayama1, Jatan B Sherchand2, Shayam Shrestha3, Kishor Manadhar4, Yoko Tsuda5, Keshav Parajuli6,
Jeevan B Sherchand6, Sharan Gopali7, Shinsaku Sakurada8
1: Japan Anti-Tuberculosis Association
2: Kathmandu Medical University
3: Friends of Shanta Bhawan Clinic
4: Chhatrapati Free Clinic
5: Osaka city Public Health Office 6: Institution of Medicine, Tribhuvan University
7: Japan-Nepal Health & TB Research Association
8: National Center for Global Health and Medicine
Non-communicable diseases (NCD) patients have increased and tuberculosis (TB) patients haveincreased in elderly populations in recent decades in Nepal. This trend may inevitably lead Nepali people todouble burden of TB and NCD. To understand the situation, interviews about NCD situations based on thequestionnaire format had been conducted for TB patients and NCD patients who have history of TB treatment
at clinics. A single disease of NCD could not be probed epidemiologically as a risk of TB in this study. However,the double burden of TB and NCD was observed at high ratio. And the TB patients with NCD were older than
TB patients without NCD. TB screening for NCD patients at clinics might be worth considering as an opportunity
for early diagnosis.
Focus Group Discussion was conducted in January 2014. Participants were 4 Doctors, 1 Government
officer, 1WHO TB Expert, and 6 Public Health Nurses. The main theme was the present situation of TB and
NCDs double burden in Nepal and its associated problems. Their conclusions :1) The problem of double burden
is yet unrecognized at the national level. 2) DM might be a screening target for TB patients and diabetics might
be a target group for TB screening.
To study the association between DM and TB, the study about prevalence of diabetes in new andrelapse tuberculosis cases had been started from November 2014.

IOM-NCGM Research Collaboration Office Annual Report 201451

Attachment-1(D-4)

Promotion of Research Collaboration between Nepal and Japan --- Clinical Study
Setting in Pulmonology
Mitsuhiro Kamimura1, Hiroshi Ohara2
1: Department of Pulmonology, National Hospital Organization Disaster Medical Center, 2: Bureau ofInternational Medical Cooperation, NCGM
Key words: Pulmonology, JICA Project, Kathmandu, Nepal
The institute of medicine (IOM) of Tribhuvan University Teaching Hospital (TUTH) was supported by JICA projectfrom 1980 to 1996, during that period IOM accepted many experts from National Center for Global Healthand Medicine (NCGM). Even after the termination of JICA Project, joint research studies have been carried out
by several researchers of IOM and NCGM, mainly focused on infectious diseases, gastrointestinal diseases,
etc. In January 2013, MOU was concluded to expand the activities of joint researches and human resource
development between IOM and NCGM. Based on this MOU, the project of “The research for strengthening of
human resource development at IOM in Nepal TUTH” was started. The Pulnomology Department of National
Hospital Organization Disaster Medical Center (DMC), which has strong relationship with NCGM, also has
its history of mutual communication with TUTH focusing on pulmonary rehabilitation, study on bacterial
contamination of endoscopies, etc. This time DMC started the joint study with Pulmonology Department of
TUTH under the observation of NCGM to establish and to expand the development of better co-operation
for the conduction of joint clinical studies between TUTH and DMC, NCGM and extract the factors whichshould be improved for better communication. The joint study is focused on the current status of interstitial
pneumonitis (IP) in Nepal. As the infectious diseases have been overcome gradually in developing countries,
non-communicable diseases, such as hypertension, diabetes mellitus or COPD are now emerging as new healthproblems. Though the prevalence is much less, IP must be a big health issue in developing countries and exact
reality of the disease is not yet understood. This might be the first report in Nepal and also the factors to besolved for better performance of joint study between TUTH and Japan will be clarified through this study. Thus,
we believe that this study might contribute the purpose of the project which includes activation of researchcommunications and human resource development between Nepal and Japan.

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IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-2
Publications of scientific papers
Table 3 shows the list of list of papers published on international journals as Achievement in NCGMIOM Collaboration Office
Papers published on international medical journals are shown on pages 55- (papers published in 2014 were
put in full paper, whereas only abstracts are put regarding papers published in 2012,-2013 and in press
papers)Nurses in TUTH

NCGM researchers with the Dean andAssistant Deans of IOM, Tribhuvan University

Entrance of the IOM-NCGM Research Collaboration Officein Academic Building of IOM,IOM-NCGM Research Collaboration Office Annual Report 201453

Attachment-2

Table 3				

List of papers published on international journals (papers in press are included)
2012-2014: Achievement in NCGM-IOM Collaboration Office◎ Full papers are put in this annual report○ Only abstract is put in this annual report
Papers54

1

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.
Moleculare evidence based hospital acquired rotavirus gastroenteritie in Nepal. Prime J Mirobobiol Res
2012; 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.

○

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.

○

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◎

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.

◎

9

Tada T, Shrestha B, Miyoshi-Akiyama T, Shimada K, Ohara H, Kirikae T, Pokhrel BM. NDM-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.

◎

10

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.

◎

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.

◎

12

Sherchan JB, Hayakawa K, MD, 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 E. coli Sequence Type ST131 and ST648.
Antimicrob Agents and Chemother (in press)○

13

Ohara H, Matsumoto M, Hirayama T, Akashi H, Sherchand JV, Nam VH, Sherchan JB. Assessment of healthsystems in relation to interface between malaria control programs and health system strengthening:
Comparative study between Nepal and Viet Nam. Tropical Med Health (in press)○

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-2

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, fungi
Page 14and 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 of

IOM-NCGM Research Collaboration Office Annual Report 201455

Attachment-2

Prime Journal of Microbiology Research (PJMR)
ISSN: 2251-127X. Vol. 1(2), pp. xxx-xxx
October 27th, 2011www. primejournal. org/PRE

Full Length Research

Hospital acquired Rotavirus Gastroenteritis andmolecular evidence in Nepal
Sherchand JB1, Ohara H2, Sherchan JB3, Tandukar S1, Sakurada S2, Gurung B4, Ansari S1,
Rijal BP1, and Pokhrel BM1
1

Tribhuvan University Teaching Hospital, Institute of Medicine, Department of Microbiology and Public Health ResearchLaboratory, Kathmandu, Nepal
2
National Center for Global Health and Medicine, Tokyo, Japan3
Department of Medical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel.
4
Kanti Children’s Hospital, Kathmandu, Nepal;thAccepted 4 October, 2011

Rotavirus is the major pathogens of community acquired acute gastroenteritis in children, but their rolein hospital acquired gastroenteritis is not fully understood. The aim of the study was to assess theincidence of hospital acquired gastroenteritis and molecular evidence among hospitalized children lessthan 5 years of age. A total of 154 children with hospital acquired acute gastroenteritis in children’s
hospital of Kathmandu were enrolled between January and December 2010. Acute gastroenteritis wasclassified as hospital acquired infection if diarrhea developed in 48 hours or more after admission. The
incidence of hospital acquired infection due to rotavirus was 31.2% (48/ 154) by ELISA. The distribution ofrotavirus genotypes G and P, serotype G12 represented 48% of rotavirus strains characterized by reverse
transcription-polymerase chain reaction genotyping during the study, and was associated with P-types
P[6], P[8] and P[4]. Further, a total of nine G/P type combination were identified, with G12 P [6] 30% being
the most commonly detected rotavirus strain type. Most of the children who had hospital acquired
rotavirus gastroenteritis found symptoms of diarrhea, vomiting, fever, poor sucking and dehydration.
Additional findings showed that 2% cases of rotavirus co-infection with bacterial pathogens of Esch coliand Shigella species. The study revealed that G 12 and G12P [6] were found major genotypes causing
hospital acquired rotavirus gastroenteritis in Nepal. Introduction of rotavirus vaccine along withstrengthening hygienic measures could substantially reduce the incidence of hospital acquired acutegastroenteritis in children of Nepal.
Key word: Rotavirus, gastroenteritis, hospital acquired, Nepal
INTRODUCTION
Rotavirus is the major pathogen of community acquired
acute gastroenteritis in children, but their role in hospitalacquired gastroenteritis and molecular evidence is not
fully understood. Although rotavirus is the most frequentcause of gastroenteritis in children under 5 years of age,but, the virus can cause severe diarrhea and dehydration,
especially in children aged 6 to 24 months. In developing
countries, acute gastroenteritis due to rotavirus infection
causes the death of approximately 440,000 children
every year (Parashar et al. 2009; Festini et al. 2010 and*Corresponding author. E-mail: jeevansherchand@hotmail.com

56

IOM-NCGM Research Collaboration Office Annual Report 2014

Parashar et al. 2006).The rotavirus genus of theReoviridae family is very diverse, as it consists ofdifferent groups (A-G) and of different types based on thecharacteristics of the surface proteins VP7 (G =glycoprotein) and VP4 (P = protease-sensitive protein).
To date, at least 23 G types and 31 P types of group A
rotavirus, the group which most commonly infects
humans, have been differentiated (Festini et al. 2010;
Kang et al. 2006 and Ursu et al. 2009).
The virus is mainly transmitted by feco-oral route or bydirect contact, but it can occasionally be transmitted
through droplets. Since the virus is stable in theenvironment, transmission can occur through theingestion of contaminated water and food, and throughAttachment-2

IOM-NCGM Research Collaboration Office Annual Report 201457

Attachment-2

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

58

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

IOM-NCGM Research Collaboration Office Annual Report 2014

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

May 2013 Volume 57 Number 5Attachment-2

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

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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
260

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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 2014

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62

SputumSputumPus

SputumIOMTU4IOMTU91IOMTU94IOMTU99

NLRTI

SSI

NLRTI

NLRTI

SSI

128

512

TZP

>1,024>1,024

CAZ

256

64

FEP

>1,024

1,024

512

4

>1,024>1,024128

256>1,024 1,024 >1,024 1,024

1,024

1,024

PIP

64

2

64

16

32

32

1

32

16

16

64

1

64

32

64

IPM DPM MEM

1,024>1,024

1,024

1,024

1,024

AMK

GEN
128

CIP
>128

CST

1,024

>1,024

256

256

128

>128 1,024

128

512

512

FOF

>1,024 >1,024 >1,024 >256 >128 1,024

1,024>1,024 >1,024 >1,024>1,024 >1,024 >1,024 >256 >128

>1,024 >1,024 >1,024

ABK

MIC (mg/L)
ATM

4

4

4

4

4

TIG

blaNDM-1, blaVEB-1, blaOXA-10, blaTEM-1, blaADC-67,
armA, aadA1

blaOXA-10, blaVEB-1, blaTEM-1, blaADC-67,
armA, aadA1

blaNDM-1, blaOXA-10, blaVEB-1, blaTEM-1, blaADC-67,
armA, aadA1

blaOXA-72, blaOXA-10, blaVEB-1, blaTEM-1, blaADC-67,
armA, aadA1

blaNDM-1, blaOXA-10, blaVEB-1, blaTEM-1, blaADC-67,
armA, aadA1, aadA2

Antibiotics resistant genes

SSI, surgical site infection; NLRTI, nosocomial lower respiratory tract infection PIP, piperacillin; TZP, piperacillin/tazobactam; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; DPM, doripenem; MEM, meropenem; ATM,aztreonam; ABK, arbekacin; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; CST, colistin; FOF, fosfomycin; TIG, tigecycline.

Pus

Tissue Infection
sourcesIOMTU1

Strains

Table 1 Summary of the characteristics of the 5 P. rettegeri strains, including antimicrobial resistance profiles and resistant genes

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Methods
Bacterial strains

Five P. rettgeri clinical isolates were obtained from May
to July 2012 from 5 patients at Tribhuvan University
Teaching Hospital in Kathmandu, Nepal. Three isolates
were from sputum and 2 from pus at surgical sites.
Samples were obtained as part of standard patient care.
Phenotypical identification [17] was confirmed by API
32GN (BioMérieux, Mercy l'Etoile, France) and 16S rRNA
sequencing (1,497 bp) [18,19].
Antimicrobial susceptibilities

MICs were determined using the microdilution method,
according to the guidelines of the Clinical Laboratory
Standards Institute (CLSI) [20]. Breakpoints to antibiotics
were determined. The modified Hodge test, the meropenem-sodium mercaptoacetic acid double-disk synergy test
(Eiken Chemical, Tokyo, Japan) and E-test (imipenem/
EDTA) (AB Biodisk, Solna, Sweden) were performed.
Entire genome sequencing

The entire genomes of these isolates were extracted andsequenced by MiSeq (Illumina, San Diego, CA). CLC
genomics workbench version 5.5 (CLC bio, Tokyo,
Japan) was used for de novo assembly of reads and to
search for 923 drug-resistance genes, including genes
encoding β-lactamases, 16S rRNA methylases andaminoglycoside-acethyl/adenylyltransferases; point mutations in the gyrA, parC and pmrCAB operons; and point
mutations in the fos genes, including fosA, fosA2, fosA3,
fosC and fosC2.
Pulsed-field gel electrophoresis (PFGE) and southernhybridization

PFGE analysis was performed as described [3]. An
813 bp probe for blaNDM-1 was synthesized by PCR
amplification using the primers 5′-atggaattgcccaatattatgcac-3′ (forward) and 5′-tcagcgcagcttgtcggccatgcggg-3′(reverse), and a 780 bp probe for blaOXA-72 was synthesized using the primers 5′-agtttctctcagtgcatgttcatctat-3′(forward) and 5′-agaaccagacattccttctttcatttc-3′ (reverse).
Southern hybridization to detect blaNDM-1 and blaOXA-72
was performed using these probes, which were detectedusing DIG High Prime DNA labeling and detection
starter kit II (Roche Diagnostics, Mannheim, Germany).

Tribhuvan University (ref. 6-11-E) and the Biosafety Committee, National Center for Global Health and Medicine(approval number: 23-M-49).

Results
Antimicrobial susceptibilities

Four of the 5 isolates were resistant to carbapenems(doripenem, imipenem and meropenem) and piperacillin/tazobactam, whereas the fifth was susceptible topiperacillin/tazobactam, doripenem and meropenem andshowed intermediate resistance to imipenem (Table 1).
All 5 isolates were highly resistant to cephalosporins(ceftazidime and cefepime), aztreonam, aminoglycosides(arbekacin, amikacin and gentamicin), ciprofloxacin, colistin and fosfomycin, and all 5 showed intermediate
resistance to tigecycline. The four isolates resistant tocarbapenems were negative with the modified Hodge
test, but three of the four isolates were positive with themeropenem-sodium mercaptoacetic acid double-disk
synergy test and E-test/EDTA.
Drug-resistant genes

All 5 isolates tested had several genes associated with
β-lactam and aminoglycoside-resistance (Table 1). These
isolates had blaVEB-1, blaOXA-10, blaTEM-1, blaADC-67(ampC), armA and aadA1; 3 had blaNDM-1; and 1 had
blaOXA-72. None of these isolates had any other βlactamase encoding genes, including the class A genes
blaSHVs and blaCTX-Ms; the class B genes blaAIM, blaDIM,
blaFIM, blaGIM, blaIMPs, blaINDs, blaKHM, blaSIM, blaSMB,
blaSPM, blaTMBs, and blaVIMs; or the class D gene
blaOXAs except for blaOXA-10 and blaOXA-72. None had
other genes encoding 16S rRNA methylases or aminoglycoside acetyl/adenylyltransferases. All 5 isolates had
point mutations in the quinolone-resistance-determining
regions of gyrA and parC, with amino acid substitutionsof S83I and D87E in GyrA and S80I in ParC, but none
had any mutations in the pmrCAB operon and fos genes.
All sequences of the drug-resistant genes tested wereidentical to those registered in GenBank.

Nucleotide sequence accession numbers

The nucleotide sequences surrounding blaNDM-1 andblaOXA-72 have been deposited in GenBank with the accession number AB828598 and AB857844, respectively.
Ethical approval

The study protocol was reviewed and approved by theInstitutional Review Board of the Institute of Medicine,

Figure 1 PFGE profiles obtained following SfiI digestion of
P. rettgeri chromosomes.

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PFGE and southern hybridyzation

Of the 5 P. rettgeri isolates, 4 had identical PFGE patterns
and the fifth showed 95.7% similarity (Figure 1). Three ofthese isolates had a plasmid harboring blaNDM-1 and one
had a plasmid harboring blaOXA-72, with plasmid sizes
ranging from 9.42 to 23.1 kbp (data not shown).
Genomic structures surrounding blaNDM-1 and blaOXA-72

The genetic environments surrounding blaNDM-1 (Accession no. AB828598) was blaNDM-1-bleMBL-trpF-dsbC-cutA1.
All 3 isolates harboring blaNDM-1 (IOMTU1, 91 and 99)
had the same genetic environments. The blaOXA-72
gene was flanked by conserved inverted repeats at the
XerC/XerD binding sites [21], indicating mobilization
by site-specific recombination mechanisms. The rep1
gene was located downstream of blaOXA-72 (Accession
no. AB857844).

Discussion
The relatively high MICs to piperacillin/tazobactam andcarbapenems of the five P. rettgeri isolates were likely
due to the presence of blaNDM-1 or blaOXA-72. The
enzymatic activities of metallo-β-lactamases, including
NDM-1, were not inhibited by tazobactam [22], a βlactamase inhibitor, in agreement with the MIC profilesof these isolates to piperacillin/tazobactam. The highMICs of all 5 isolates to ceftazidime, cefepime and aztreonam were likely due to the presence of blaVEB-1 [23],and the presence of armA in these isolates was likely
associated with their extremely high resistance to allaminoglycosides tested [11]. Point mutations in the quinolone-resistance-determining regions of gyrA and parC
have been associated with high resistance to quinolones[24]. Point mutations in pmrCAB operon have been associated with the resistance of Acinetobacter spp. [25] andPseudomonas aeruginosa [26] to polymxyin and colistin;
and the presence of fos genes, including fosA, fosA2, fosA3,
fosC and fosC2, has been associated with resistance tofosfomycin in Gram-negative bacteria [27-29].
Plasmids containing blaNDM-1 or blaOXA-72 may be
disseminated among Gram-negative pathogens in Nepal.
The genetic environments surrounding blaNDM-1 in our
P. rettgeri strains (blaNDM-1-bleMBL-trpF-dsbC-cutA1)
were also observed in other plasmids, including A. baumannii plasmid pAbNDM-1 from China (Accession no.
JN377410), Citrobacter freundii plasmid pYE315203
from China (Accession no. JX254913), E. coli plasmid
pNDM102337 from Canada (Accession no. JF714412),
K. pneumoniae plasmid pKP-NCGM18-1 from Nepal(Accession no. AB824738) [30], K. pneumoniae plasmids
pKPX-1, pKPN5047 and pNDM-HN380 from China(Accession nos. AP012055, KC311431 and JX104760,
respectively), and P. rettgeri plasmid pFR90 (Accession no.
JQ362415) from China. In addition, the genetic structures

64

Page 4 of 5

of OXA-72 producing Acinetobacter spp [31-34] and K.
pneumoniae (Accession no. JX268653 and AB825955
deposited in 2012 and 2013, respectively) had thesame genetic structure (blaOXA-72-rep1) as our strainof P. rettgeri.

Conclusions
To our knowledge, this is the first report describing P.
rettgeri strains harboring blaNDM-1 or blaOXA-72 andarmA isolated from patients in Nepal. These 5 strains
were highly resistant to both β-lactams and aminoglycosides and expanded in a clonal manner in the hospital.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
TT: Performed PCR and sequencing, analyzed data and drafted themanuscript. TMA: Performed entire genome sequencing. RKD and MKS:
Performed drug susceptibility tests. HO: Supervised this study. KS: Performed
pulsed-field gel electrophoresis and its pattern analysis. TK and BMP:
Designed protocols and supervised this study. All authors read andapproved the final manuscript.
Acknowledgements
The authors thank emeritus professor Masayasu Nakano, Jichi Medical
University, for comments on the manuscript. This study was supported by
grants [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 Number 24790432].
Author details
1
Department of Infectious Diseases, Research Institute, National Center for
Global Health and Medicine, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655,
Japan. 2Department of International Medical-Cooporation, National Center
for Global Health and Medicine, Shinjuku, Tokyo, Japan. 3Department ofMicrobiology, Institute of Medicine, Tribhuvan University, Maharajgunj,
Kathmandu, Nepal.
Received: 1 May 2013 Accepted: 27 January 2014
Published: 3 February 2014
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Gram-negative bacteria: an update. Drug Resist Updat 2012, 15:133–148.
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Wade WG: Design and evaluation of useful bacterium-specific PCRprimers that amplify genes coding for bacterial 16S rRNA. Appl Environ
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Simmon KE, Croft AC, Petti CA: Application of SmartGene IDNS software
to partial 16S rRNA gene sequences for a diverse group of bacteria in aclinical laboratory. J Clin Microbiol 2006, 44:4400–4406.
National Committee for Clinical Laboratory Standards: Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 9th
ed. In Approved standard M07-A9. 8th edition. Wayne, Pa: Clinical andLaboratory Standards Institute; 2012.
D'Andrea MM, Giani T, D'Arezzo S, Capone A, Petrosillo N, Visca P, Luzzaro F,
Rossolini GM: Characterization of pABVA01, a plasmid encoding the
OXA-24 carbapenemase from Italian isolates of Acinetobacter baumannii.
Antimicrob Agents Chemother 2009, 53:3528–3533.
Bush K, Jacoby GA: Updated functional classification of beta-lactamases.
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Poirel L, Naas T, Guibert M, Chaibi EB, Labia R, Nordmann P: Molecular andbiochemical characterization of VEB-1, a novel class A extendedspectrum beta-lactamase encoded by an Escherichia coli integron gene.
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Bonomo RA: Resistance to colistin in Acinetobacter baumannii associatedwith mutations in the PmrAB two-component system. Antimicrob Agents
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system of Pseudomonas aeruginosa that modulates resistance to cationicantimicrobial peptides and addition of aminoarabinose to lipid A.
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28. Xu H, Miao V, Kwong W, Xia R, Davies J: Identification of a novel
fosfomycin resistance gene (fosA2) in Enterobacter cloacae from the
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29. Wachino J, Yamane K, Suzuki S, Kimura K, Arakawa Y: Prevalence offosfomycin resistance among CTX-M-producing Escherichia coli clinicalisolates in Japan and identification of novel plasmid-mediatedfosfomycin-modifying enzymes. Antimicrob Agents Chemother 2010,
54:3061–3064.
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T, Pokhrel BM: Dissemination of 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 Agents 2013, 42:372–374.
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Vila J, Quinn JP, Villegas MV: First identification of OXA-72 carbapenemasefrom Acinetobacter pittii in Colombia. Antimicrob Agents Chemother 2012,
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doi:10.1186/1471-2334-14-56
Cite this article as: Tada et al.: NDM-1 Metallo-β-Lactamase and ArmA
16S rRNA methylase producing Providencia rettgeri clinical isolates in
Nepal. BMC Infectious Diseases 2014 14:56.

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IOM-NCGM Research Collaboration Office Annual Report 201465

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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

66

Dissemination 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 2014

>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-2

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373

IOM-NCGM Research Collaboration Office Annual Report 201467

Attachment-2

374

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

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

68

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 2014

Attachment-2

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

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.
6302 aac.asm.org

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

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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

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

Antimicrobial Agents and Chemotherapy

p. 6302– 6305

October 2014 Volume 58 Number 10

IOM-NCGM Research Collaboration Office Annual Report 2014

69

Attachment-2

NDM-12 Metallo--Lactamase Variant-Producing E. coli

TABLE 1 MICs of various -lactams for E. coli IOMTU388.1 and E. coli DH5 transformed with plasmids encoding NDM-12 or NDM-1
MIC (g/ml) for strain:
IOMTU388.1

DH5(pHSG398/NDM-1)

DH5(pHSG398/NDM-12)DH5(pHSG398)

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

1,024
1,02464
512
1,024
1,024
1,024512
1,024
1,024
1,02432
1664
1,024
1,024256
128
0.063
0.5
16
8
64
2
256
16
512
0.063
0.5
0.25
16
256

512
128
0.063
1
8
16
16
2
256
16
256
0.063
0.25
0.125
4
256

4
2
0.063
0.0631
0.063
0.063
0.063
0.063
0.06316
0.063
0.063
0.0630.125
32

-lactam substrates have been reported previously (34–36). The
Km and kcat values (means  standard deviations) were obtained
from three individual experiments. The enzymatic activities of
NDM-1 were measured in parallel with those of NDM-12.
The plasmid harboring blaNDM-12 was extracted (37) and sequenced using MiSeq (Illumina, San Diego, CA). The size of theplasmid harboring blaNDM-12 was determined using pulsed-field
gel electrophoresis (PFGE) and Southern hybridization. A probe
for blaNDM-12 from IOMTU388.1 was amplified by PCR using theprimer sets for EcoRI-NDM-F and PstI-NDM-R. Signal detection
was carried out using the digoxigenin (DIG) High Prime DNA
labeling and detection starter kit II (Roche Applied Science, Indianapolis, IN).
Mating-out assays between the parental strain IOMTU388.1
and the chloramphenicol-resistant E. coli strain BL21 were performed in LB broth using a 1:4 donor/recipient ratio for 3 h at
37°C. Transconjugants were selected on Muller-Hinton agar
plates containing ceftazidime (100 g/ml) and chloramphenicol(30 g/ml). Selected transconjugants harboring blaNDM-12 wereconfirmed by PCR with the primer set EcoRI-NDM-F and PstINDM-R.
E. coli DH5 harboring blaNDM-1 or blaNDM-12 showed reduced
susceptibility to moxalactam and all penicillins, cephalosporins,and carbapenems tested compared with DH5 harboring a vector
control (Table 1). The MICs of the -lactams cefoxitin and moxalactam for DH5 harboring blaNDM-12 were 4-fold less than thosefor DH5 harboring blaNDM-1 (Table 1).
As shown in Table 2, recombinant NDM-1 and NDM-12hydrolyzed all -lactams tested except for aztreonam. The profilesof enzymatic activities of NDM-12 against -lactams tested weresimilar to those of NDM-1, although NDM-12 had lower kcat/Km
ratios for all -lactams tested except for doripenem. The lower
kcat/Km ratios were likely to be caused by the lower kcat values of
NDM-12 compared with those of NDM-1, as the values of
NDM-12 were 11.4 to 73.6% of those of NDM-1 (Table 2). The
profiles of enzymatic activities of NDM-1 except for cefoxitin weresimilar to those of NDM-1 that we reported previously (29). The
kcat/Km ratio for cefoxitin in Table 2 was 10-fold higher than that

TABLE 2 Kinetic parameters of the NDM-1 and NDM-12 enzymesa
NDM-1b

NDM-12b

-LactamKm (M)

kcat (s1)bkcat/Km(M1 s1)

Km (M)

kcat (s1)bkcat/Km(M1 s1)

Ampicillin
Aztreonam
Cefepime
Cefotaxime
Cefoxitin
Ceftazidime
Cefradine
Doripenem
Imipenem
Meropenem
Moxalactam
Penicillin G

231  33
NHc
162  7102  16
13  1202  727  3201  27
249  4381  104.5  2.3
67  6249  22
NH
31  1137  7
6.7  0.156  172  1114  944  2139  107.6  0.3104  1

1.1
NH
0.19
1.1
0.50
0.28
2.7
0.57
0.34
1.7
2.0
1.6

126  4
NH
103  645  426  253  457  488  2125  2291  867  564  8136  2NH
11.1  0.238  10.66  0.01
5.7  0.1
16  153  122  253  26.0  0.242  2

1.1
NH
0.11
0.84
0.02
0.11
0.28
0.60
0.18
0.58
0.09
0.66

a

The proteins were initially modified by a His tag, which was removed after purification.
The Km and kcat values shown represent the means from 3 independent experiments  standard deviations.
cNH, no hydrolysis was detected under conditions with substrate concentrations up to 1 mM and enzyme concentrations up to 700 nM.
bOctober 2014 Volume 58 Number 1070

IOM-NCGM Research Collaboration Office Annual Report 2014aac.asm.org 6303

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Antibiotic(s)Attachment-2

Tada et al.

FIG 1 Localization of blaNDM-12 on a plasmid from E. coli strain IOMTU388.1

separated by PFGE. Left lane, MidRange PFG marker (New England BioLabs,
Tokyo, Japan); middle lane, plasmids from E. coli strain IOMTU388.1; rightlane, hybridization of the plasmid with a probe specific for blaNDM-12.

ACKNOWLEDGMENTS
This study was supported by grants from International Health Cooperation Research (24-S-5 and 26-A-103) and a grant (H24-Shinko-ippan010) from the Ministry of Health, Labor and Welfare of Japan.

REFERENCES

in our previous study (see Table 2 in reference 29). The difference between the ratios may be explained by the use of different
buffer solutions in the kinetics assays (Tris buffer and phosphate buffer, respectively). It was reported that phosphate ionsaffected the enzymatic activities of metallo--lactamase IMP-1(38). Phosphate ions may affect the enzymatic activities of
NDM-1 against cefoxitin.
The MBL gene blaNDM-12 in E. coli IOMTU388.1 was detected in a plasmid, pIOMTU388-NDM (accession no.
AB926431), with a size of 160 kb (Fig. 1). The sequence surrounding blaNDM-12 was blaNDM-12-bleMBL-trpF-dsbC-tnpA-sulI-qacEI.
This plasmid showed more than 99.9% identity at the nucleotide
sequence level to the sequence located from bp 70978 to 77904 in the
pGUE-NDM plasmid (accession no. JQ364967) from E. coli strain
GUE, which was isolated in India (39), and also showed 99.9%
identity at the nucleotide sequence level to the sequence locatedfrom bp 372 to 7298 in the pEC77-NDM plasmid (accession
no.AB898038) from E. coli strain NCGM77, which was isolated inJapan (40). The plasmid harboring blaNDM-12 belonged to the IncF
incompatibility group and was conjugated from IOMTU388.1 toE. coli BL21 at a conjugative frequency of 1.63  103.
The 2 substitutions M154L and G222D in NDM-12 (comparedwith NDM-1) affected the activity of the enzyme (Table 2). Nordmann et al. (24) reported that a mutant containing M154L(NDM-4) possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to NDM-1. Unexpectedly, NDM-12, which contains the M154L substitution, did
not show an increase in hydrolytic activities. The substitution at
position 222 found in NDM-12 has been not reported in othervariants, to our knowledge. Although we did not directly compare
the enzymatic activity of NDM-12 with those of NDM-4, the substitution of G222D in NDM-12 may be associated with a decreasein hydrolytic activities toward these antibiotics (Table 2). Position
222 is located in loop L10 of NDM-1, which forms the active site of
NDM-1 with L3 at the bottom of a shallow groove (41–44).
Among all known 11 NDM-1 variants, amino acid substitutions
were found at 13 amino acid positions, including positions 28, 32,
36, 69, 74, 88, 95, 130, 152, 154, 200, and 233. Positions 28, 32, and36 were in the signal peptide region. Positions 95, 130, and 154
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have been reported to affect -lactam-hydrolyzing activities, although whether the activities are affected by the other 6 substitutions has not been reported. Residue 95 is located in 1 on theprotein surface, and the amino acid substitution at position 95
affected the kcat values of NDM-3 (40). The substitution at position 130 (Met to Leu) showed increased hydrolytic activity toward
carbapenems and several cephalosporins compared to NDM-1(24, 29).
This is the first report describing NDM-12-producing E. coli in
Nepal. NDMs seem to evolve rapidly; therefore, careful monitoring of NDM-producing pathogens is required.
Nucleotide sequence accession number. The plasmid sequence including blaNDM-12 has been deposited in GenBank underaccession no. AB926431.

Antimicrobial Agents and Chemotherapy

IOM-NCGM Research Collaboration Office Annual Report 201471

Attachment-2

NDM-12 Metallo--Lactamase Variant-Producing E. coli

28. Gottig S, Hamprecht AG, Christ S, Kempf VA, Wichelhaus TA. 2013.
Detection of NDM-7 in Germany, a new variant of the New Delhi metallobeta-lactamase with increased carbapenemase activity. J. Antimicrob.
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8:e61322. http://dx.doi.org/10.1371/journal.pone.0061322.

Attachment-2

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

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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. Pokhrelca

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.
b6324

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p. 6324 – 6327

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Attachment-2

Novel AAC(6=)-Iak-Producing S. maltophilia in Nepal

shi Co. Ltd. (Tokyo, Japan). The aac(6=)-Iak and aac(6=)-Iz genes
were cloned into the corresponding sites of pSTV28 using primers
SalI-aac(6=)-Iak-F (5=-ATGCGTCGACATGACCGGCAGCGCG
GCCACGATCCGCCCG-3=) and PstI-aac(6=)-Iak-R (5=-ATCTG
CAGTCACGCCGATGGCTCCAGTGGCATGCGGAAA-3=) andSalI-aac(6=)-Iz-F (5=-ATGGTCGACATGATCGCCAGCGCGCC
CACGATCCGCC-3=) and PstI-aac(6=)-Iz-R (5=-ATCTGCAGTCACGCCGATGGCTCCAGCGGCATGCGGAA-3=), respectively.
E. coli DH5 was transformed with pSTV28-aac(6=)-Iak or
pSTV28-aac(6=)-Iz to assess aminoglycoside resistance.
The open reading frame of AAC(6=)-Iak was cloned into thepQE2 expression vector using the primers SalI-aac(6=)-Iak-F and
PstI-aac(6=)-Iak-R for protein expression. Purification of the recombinant AAC(6=)-Iak protein and thin-layer chromatography(TLC) analysis were performed as previously described (7). The
kinetic activities of AAC(6=)-Iak were determined as previously
described (8).
AAC(6=)-Iak consists of 153 amino acids. As shown by thedendrograms of AAC(6=) based on amino acid sequences in Fig. 1,
AAC(6=)-Iak is close to AAC(6=)-Iz and AAC(6=)-Iam (accession
no. AB971834) from S. maltophilia (10). Multiple sequence alignments among AAC(6=) enzymes revealed that AAC(6=)-Iak had
86.3% identity to AAC(6=)-Iz from S. maltophilia (11), 84.3%

identity to AAC(6=)-Iam from S. maltophilia (10), 47.7% identity
to AAC(6=)-Iag from Pseudomonas aeruginosa (8), 43.1% identity to AAC(6=)-If from Enterobacter cloacae (12), and 42.8% identity to AAC(6=)-Iy from Salmonella enterica (13).
We compared the enzymatic properties of AAC(6=)-Iak withthose of AAC(6=)-Iz because they had similar amino acid sequences and were detected in S. maltophilia. As shown in Table 1,
E. coli expressing AAC(6=)-Iak or AAC(6=)-Iz showed decreased
susceptibility to all aminoglycosides tested except for apramycin,
gentamicin, and lividomycin. The MICs of netilmicin and tobramycin for E. coli expressing AAC(6=)-Iak were significantly lower
than those for E. coli expressing AAC(6=)-Iz. The MICs of theother aminoglycosides tested were not significantly different between E. coli isolates expressing AAC(6=)-Iak and AAC(6=)-Iz (Table 1).
S. maltophilia IOMTU250 was highly resistant to all aminoglycosides tested, whereas E. coli expressing aac(6=)-Iak was susceptible to all aminoglycosides tested except for dibekacin (Table 1).
The discrepancy of aminoglycoside susceptibilities between S.
maltophilia and E. coli could be explained by the presence of efflux
pump genes specific for S. maltophilia. Whole-genome sequencing with the MiSeq system in this study showed that IOMTU250
had the efflux pump genes specific for S. maltophilia, including

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FIG 1 Dendrogram of 6=-N-aminoglycoside acetyltransferases [AAC(6=)s] for comparison with AAC(6=)-Iak. The dendrogram was calculated using the
CLUSTAL W2 program. Branch lengths correspond to numbers of amino acid exchanges for the proteins. EMBL/GenBank/DDBJ accession numbers of theproteins are as follows: AAC(6=)-Ia, M18967-1; AAC(6=)-Ib, M23634; AAC(6=)-Ic, M94066; AAC(6=)-Id, X12618; AAC(6=)-Ie, M13771; AAC(6=)-If, X55353;
AAC(6=)-Ig, L09246; AAC(6=)-Ih, L29044; AAC(6=)-Ii, L12710-1; AAC(6=)-Ij, L29045; AAC(6=)-Ik, L29510; AAC(6=)-Im, CAA91010; AAC(6=)-Iq, AF047556-1;
AAC(6=)-Ir, AF031326; AAC(6=)-Is, AF031327; AAC(6=)-It, AF031328; AAC(6=)-Iu, AF031329; AAC(6=)-Iv, AF031330; AAC(6=)-Iw, AF031331; AAC(6=)-Ix,
AF031332; AAC(6=)-Iy, AF144880; AAC(6=)-Iz, AF140221; AAC(6=)-Iad, AB119105; AAC(6=)-Iae, AB104852; AAC(6=)-Iaf, AB462903; AAC(6=)-Iag, AB472901;
AAC(6=)-Iai, EU886977; AAC(6=)-Iaj, AB709942; AAC(6=)-Iak, AB894482; AAC(6=)-Ial, AB871481; AAC(6=)-Iam, AB971834; AAC(6=)-IIa, M29695; AAC(6=)IIb, L06163; AAC(6=)-IIc, AF162771; AAC(6=)-29a, AF263519; AAC(6=)-29b, AF263519; AAC(6=)-30, AJ584652; AAC(6=)-31, AJ640197; AAC(6=)-32,
EF614235; AAC(6=)-33, GQ337064; AAC(6=)-I30, AY289608; AacA7, U13880; AacA8, AY444814; AacA29, AY139599; AacA34, AY553333; AacA35, AJ628983;
AacA37, DQ302723; AacA40, EU912537; and AacA43, HQ247816.

Attachment-2

Tada et al.

TABLE 2 Kinetic parameters of AAC(6=)-Iak and AAC(6=)-Iz enzymesa
AAC(6=)-Iak

ABK
AMK
DIB
ISP
KAN
NEO
NET
SIS
TOB

36  1332  1724  344  930  7
10  270  64116  4abc

AAC(6=)-Iz
kcat (s1)ckcat/Km(M1 s1)

Km
(M)c

kcat (s1)ckcat/Km(M1 s1)0.57  0.10
0.11  0.03
0.25  0.04
0.05  0.01
0.04  0.01
0.32  0.01
0.30  0.02
0.16  0.01
0.08  0.01

0.016
0.004
0.010
0.001
0.001
0.034
0.004
0.038
0.006

17  624  531  549  844  913  225  87112  20.57  0.06
0.26  0.02
0.46  0.05
0.11  0.01
0.05  0.01
0.29  0.01
0.34  0.05
0.20  0.03
0.25  0.03

0.036
0.011
0.015
0.002
0.001
0.023
0.013
0.028
0.021

The proteins were initially modified by a His tag, which was removed after purification.
ABK, arbekacin; AMK, amikacin; DIB, dibekacin; ISP, isepamicin; KAN, kanamycin; NEO, neomycin; NET, netilmicin; SIS, sisomicin; TOB, tobramycin.
Km and kcat values represent the means of results from 3 independent experiments  standard deviations.

smeABC, smeDEF, smeZ, smeJK, and the pcm-tolCsm operon.
These genes are known to be associated with aminoglycoside resistance (14–16), although it is difficult to clarify whether or notthese efflux pump genes contribute to aminoglycoside resistance
together with aac(6=)-Iak in S. maltophilia IOMTU250.
As shown in Table 2, recombinanttk;1 AAC(6=)-Iak andAAC(6=)-Iz acetylated arbekacin, amikacin, dibekacin, isepamicin, kanamycin, neomycin, netilmicin, sisomicin, and tobramycin. The profile of enzymatic activities of AAC(6=)-Iakwere similar to those of AAC(6=)-Iz, although AAC(6=)-Iak had
higher kcat/Km ratios for neomycin and sisomicin (Table 2).
To examine the acetyltransferase activity of AAC(6=)-Iakagainst aminoglycosides, we performed TLC using the purified
recombinant AAC(6=)-Iak. Lividomycin was used as a negative
control because it has a hydroxyl group instead of an amino groupat the 6= position and therefore cannot be acetylated by AAC(6=).
As shown in Fig. 2, all aminoglycosides tested except for apramycin, gentamicin, and lividomycin were acetylated by AAC(6=)-Iak,and amikacin, isepamicin, kanamycin, and tobramycin were partially acetylated by AAC(6=)-Iak under the experimental conditions used. The TLC data for apramycin, gentamicin, and lividomycin were consistent with the MICs of the aminoglycosides for E.
coli with pSTV28-aac(6=)-Iak and E. coli with the control vector(Table 1).
The substrate specificity of AAC(6=)-Iak was similar to that ofAAC(6=)-Iz, although some kinetic parameters of AAC(6=)-Iakwere different from those of AAC(6=)-Iz; i.e., the Km for netilmicin
and the kcat for tobramycin of AAC(6=)-Iak were different fromthose of AAC(6=)-Iz (Table 2). The chemical structure of netilmi-

cin is similar to that of sisomicin except for a residue at position 1
in 2-deoxystreptamine ring II (position R2; ethylamino and aminogroups, respectively). The ethylamino group at position R2 in
netilmicin, therefore, must be critical for the substrate affinity ofAAC(6=)-Iz but not that of AAC(6=)-Iak. The chemical structureof tobramycin is similar to that of dibekacin, but tobramycin has ahydroxyl group at position 4= in ring I (position R1), whereas
dibekacin does not, indicating that the hydroxyl group of tobramycin at position R1 negatively affects the turnover rate (kcat) of
AAC(6=)-Iak but not that of AAC(6=)-Iz.
The structure of the genetic environment surrounding aac(6=)Iak was similar to that of a region surrounding aac(6=)-Iam in S.
maltophilia K279a, obtained from a patient in the United Kingdom (10) (Fig. 3). The genetic environment surrounding aac(6=)Iak from nucleotides (nt) 23623 to 46040 had 92% identity to a
genetic region in S. maltophilia K279a (accession no. AM743169)
from nt 3660929 to 3683268 (10). The genetic environment surrounding aac(6=)-Iak in S. maltophilia IOMTU250 contained atleast 5 housekeeping genes, including purA, acnA, aroK, aroB, andthrA, indicating that aac(6=)-Iak was located in the chromosomal
genome.
All the aac(6=)-Iz, aac(6=)-Iak, and aac(6=)-Iam genes were detected in clinical isolates of S. maltophilia (10, 11, 17). These genes
contributed to decreased susceptibility to 2-deoxystreptamine
aminoglycoside antibiotics, such as neomycin, netilmicin, sisomicin, and tobramycin but not gentamicin (Table 1) (17). The deletion of aac(6=)-Iz in a clinical isolate of S. maltophilia resulted in
the increased susceptibility of the isolate (17).
This study was approved by the Institutional Review Board of

Downloaded from http://aac.asm.org/ on November 6, 2014 by NATIONAL CENTER FOR GLOBAL

Aminoglycosideb

Km
(M)c

FIG 2 Analysis of acetylated aminoglycosides by thin-layer chromatography. AAC(6=)-Iak and various aminoglycosides were incubated in the presence () or
absence () of acetyl coenzyme A. ABK, arbekacin; AMK, amikacin; APR, apramycin; DIB, dibekacin; GEN, gentamicin; ISP, isopamicin; KAN, kanamycin; LIV,
lividomycin; NEO, neomycin; NET, netilmicin; SIS, sisomicin; TOB, tobramycin.

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Antimicrobial Agents and Chemotherapy

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Attachment-2

Novel AAC(6=)-Iak-Producing S. maltophilia in Nepal

the Institute of Medicine at Tribhuvan University (approval
6-11-E) and the Biosafety Committee of the Research Institute of
the National Center for Global Health and Medicine (approval
25-M-038).
Nucleotide sequence accession number. The sequence foraac(6=)-Iak and its genetic environments (76,559 bp) was deposited in GenBank under accession number AB894482.
ACKNOWLEDGMENTS
This study was supported by grants from International Health Cooperation Research (26-A-103 and 24-S-5) and a grant from the Ministry of
Health, Labor and Welfare of Japan (H24-Shinko-Ippan-010).

REFERENCES
1. Brooke JS. 2012. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin. Microbiol. Rev. 25:2– 41. http://dx.doi.org/10.1128/CMR.00019-11.
2. Denton M, Kerr KG. 1998. Microbiological and clinical aspects of infection associated with Stenotrophomonas maltophilia. Clin. Microbiol. Rev.
11:57– 80.
3. Shaw KJ, Rather PN, Hare RS, Miller GH. 1993. Molecular genetics ofaminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138 –163.
4. Wachino J, Arakawa Y. 2012. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gramnegative bacteria: an update. Drug Resist. Updat. 15:133–148. http://dx.doi.org/10.1016/j.drup.2012.05.001.
5. Ramirez MS, Tolmasky ME. 2010. Aminoglycoside modifying enzymes.
Drug Resist. Updat. 13:151–171. http://dx.doi.org/10.1016/j.drup.2010.08.003.
6. Partridge SR, Thomas LC, Ginn AN, Wiklendt AM, Kyme P, Iredell JR.
2011. A novel gene cassette, aacA43, in a plasmid-borne class 1 integron.
Antimicrob. Agents Chemother. 55:2979 –2982. http://dx.doi.org/10.1128/AAC.01582-10.
7. Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T.
2013. Novel 6=-N-aminoglycoside acetyltransferase AAC(6=)-Iaj from aclinical isolate of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 57:96 –100. http://dx.doi.org/10.1128/AAC.01105-12.
8. Kobayashi K, Hayashi I, Kouda S, Kato F, Fujiwara T, Kayama S,
Hirakawa H, Itaha H, Ohge H, Gotoh N, Usui T, Matsubara A, Sugai

M. 2013. Identification and characterization of a novel aac(6=)-Iag associated with the blaIMP-1-integron in a multidrug-resistant Pseudomonas
aeruginosa. PLoS One 8:e70557. http://dx.doi.org/10.1371/journal.pone.0070557.
9. Clinical and Laboratory Standards Institute. 2012. Performance standards for antimicrobial susceptibility testing; 23rd informational supplement, M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA.
10. Crossman LC, Gould VC, Dow JM, Vernikos GS, Okazaki A, Sebaihia
M, Saunders D, Arrowsmith C, Carver T, Peters N, Adlem E, Kerhornou A, Lord A, Murphy L, Seeger K, Squares R, Rutter S, Quail MA,
Rajandream MA, Harris D, Churcher C, Bentley SD, Parkhill J, Thomson NR, Avison MB. 2008. The complete genome, comparative andfunctional analysis of Stenotrophomonas maltophilia reveals an organism
heavily shielded by drug resistance determinants. Genome Biol. 9:R742008-9-4-r74. http://dx.doi.org/10.1186/gb-2008-9-4-r74.
11. Lambert T, Ploy MC, Denis F, Courvalin P. 1999. Characterization of
the chromosomal aac(6=)-Iz gene of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 43:2366 –2371.
12. Teran FJ, Suarez JE, Mendoza MC. 1991. Cloning, sequencing, and use
as a molecular probe of a gene encoding an aminoglycoside 6=-Nacetyltransferase of broad substrate profile. Antimicrob. Agents Chemother. 35:714 –719. http://dx.doi.org/10.1128/AAC.35.4.714.
13. Magnet S, Courvalin P, Lambert T. 1999. Activation of the cryptic
aac(6=)-Iy aminoglycoside resistance gene of Salmonella by a chromosomal deletion generating a transcriptional fusion. J. Bacteriol. 181:6650 –6655.
14. Chang LL, Chen HF, Chang CY, Lee TM, Wu WJ. 2004. Contributionof integrons, and SmeABC and SmeDEF efflux pumps to multidrug resistance in clinical isolates of Stenotrophomonas maltophilia. J. Antimicrob.
Chemother. 53:518 –521. http://dx.doi.org/10.1093/jac/dkh094.
15. Gould VC, Okazaki A, Avison MB. 2013. Coordinate hyperproductionof SmeZ and SmeJK efflux pumps extends drug resistance in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 57:655– 657. http://dx.doi.org/10.1128/AAC.01020-12.
16. Huang YW, Hu RM, Yang TC. 2013. Role of the pcm-tolCsm operon in
the multidrug resistance of Stenotrophomonas maltophilia. J. Antimicrob.
Chemother. 68:1987–1993. http://dx.doi.org/10.1093/jac/dkt148.
17. Li XZ, Zhang L, McKay GA, Poole K. 2003. Role of the acetyltransferase
AAC(6=)-Iz modifying enzyme in aminoglycoside resistance in
Stenotrophomonas maltophilia. J. Antimicrob. Chemother. 51:803– 811.
http://dx.doi.org/10.1093/jac/dkg148.

October 2014 Volume 58 Number 1076

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Downloaded from http://aac.asm.org/ on November 6, 2014 by NATIONAL CENTER FOR GLOBALFIG 3 Genetic environments surrounding aac(6=)-Iak in S. maltophilia IOMTU250 and aac(6=)-Iam in S. maltophilia K279a.

Attachment-2

38
Original article

Nosocomial Bacterial Infection and Antimicrobial
Resistant Pattern in a Tertiary Care Hospital in Nepal
Sah MK1, Mishra SK2, Ohora H3, Kirikae T3, Sherchan JB1, Rijal BP2 and Pokhrel BM2
Department of Microbiology, Kathmandu University and 1Kantipur Dental College, Teaching Hospital & ResearchCenter, Kathmandu, Nepal.

1

Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, Nepal.

2

National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan.

3

Correspondence: Manoj Kumar Sah
E-mail: yourmanoj_sah@yahoo.com.

Abstract
Introduction: Nosocomial infection is a global problem with multi facet outcomes. At
present, the emergence of resistance to antimicrobial agents is a global public health problem
which is well pronounced in developing countries.
Methods: The aim of this study was to determine the prevalence of bacteria causing nosocomial
infections and their antibiotics resistant pattern among the patients admitted at Tribhuvan
University Teaching Hospital (TUTH), Kathmandu, Nepal. The study was conducted during
a period of March 2011 to February 2012. Nine hundred clinical specimens which included
urine, sputum, endotracheal aspirates, pus & blood were subjected for bacterial culture andtheir antibiotics sensitivity test at the Department of Microbiology with the use of standard
method as described by American Society for Microbiology (ASM).
Results: Prevalence of bacteria causing nosocomial infection was 34.4% (n=310). Out of 310
specimens, urine 122 (39.30%), sputum 78(25.2%), pus 78(25.2%), endotracheal secreation
24 (7.7%) and blood 8(2.6%). Three hundred thirty three bacteria were isolated fromthree hundred ten specimens. The most common isolates were Escherichia coli followedby Acinetobacter species, Klebsiella pneumonia and Staphylococcus aureus. In-vitro
antibiotic susceptibility tests revealed that the Gram-negatives bacilli were only sensitive
to fluroquinolones, ceftrixone, cefepime carbapenem, polymyxin B and colistin sulphate
while the Gram-positive cocci were sensitive to fluroquinolones, Ceftroxone, cefepime andvancomycin.
Conclusion: The findings suggested the need for constant monitoring of susceptibility of
specific pathogens in different populations to commonly used anti-microbial agents to copeup this alarming situation in the hospital for the management of such patients and prevent thedissemination of such strains.
Key words: Nosocomial infections, Bacteria and Antibiotics

Introduction
Nosocomial infections, also called healthcare acquired
infections or health care-associated infections, is definedby the Center for Disease Control (CDC) as a localized orsystemic condition that results from adverse reaction to thewww.jiom.com.np

presence of an infectious agent(s) or its toxin(s) and thatwas not present or incubating at the time of admission to thehospital. For most bacterial nosocomial infections usually
become evident after 48 hours (i.e., the typical incubation

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Nosocomial Bacterial Infection

period) or more after admission. However, because the
incubation period varies with the type of pathogen and tosome extent with the patient’s underlying condition, each
infection must be assessed individually for evidence thatlinks it to the hospitalization 1.
Nosocomial infection is a problem throughout the worldboth in developed and developing countries. The changing
pattern of the bacterial isolates causing nosocomial
infection has been observed in different time period. The
impact of nosocomial infection on public health is a subjectof increasing concern, due to the increasing numbers of
hospitalized patients in crowded facilities, many of whom
have impaired immunity, the emergence of new microorganisms, and the increase in antibiotic resistance. In
many countries, strict guidelines and policies for control,
prevention, and management of nosocomial infections are
implemented but even then hospital infections do occur in
one form or another. In Nepal, there is a lack of educationin this field but other social, ethical and economic factors
also need to be considered in the control of nosocomial
infections.
Over 1.4 million people worldwide suffer from infectious
complications acquired in hospital. The highest frequencies
of nosocomial infections were reported from hospitals in
the Eastern Mediterranean and South-East Asia Regions(11.8 and 10.0%, respectively), with a prevalence of 7.7
and 9.0%, respectively in the European and Western PacificRegions. Twenty five to 50% of nosocomial infections are
due to the combined effect of the patients own flora andinvasive devices. Most infections acquired in hospital
today are caused by microorganisms which are common
in the general population, in whom they cause no or milder
disease than among hospital patients (Staphylococcus
aureus, coagulase-negative Staphylococci, Enterococci,
Enterobacteriaceae) 2.
Nosocomial infections are also important public healthproblems in developing countries, as well as in developed
countries. The socioeconomic impact, i.e. prolongationof hospitalization, mortality, and cost, of these infections
adversely affects patients and nations economic wellbeing. They are important for both patient and publichealth problem in developing countries, as well as in somedeveloped countries (3, 4). Nosocomial infections may resultin an excess length of stay in hospital for up to 10 days andan increase in the costs of hospitalization (5, 6). Nosocomial
infections pose a critical threat to patients, especially inthe high-risk departments, such as the Intensive Care Unit
(ICU) (7, 8). Risk factors for the development of nosocomial
infections in the Surgical Intensive Care Unit (SICU)
setting include poor nutritional status, exposure to multiple
antibiotics, indwelling central venous catheters; mechanical39
ventilation and length of ICU stay 9. Over the past several
decades, the frequency of antimicrobial resistance and its
association with serious infectious diseases have increased
at alarming rates. The increasing resistance rate amongnosocomial pathogens is a commonly encounter problem(10, 11).
It is estimated that in developed countries 5–10% patients
get one of these infections during hospitalizations, whereas
in developing countries rates are higher up to (25%) 12. Aninternational study covering 47 hospitals in 14 countries(Europe, Eastern Mediterranean, Southeastern Asia andWestern Pacific Region) over the period from 1983 to 1985
showed that an average prevalence rate was 8.7%, ranging
from 3 to (21%) 13.
Today, antibiotic remain the front line therapy forconquering bacterial infections. However, treatment withthese drugs is to be acknowledges as a two edged sword.
As antimicrobial agents have been misused and overused,bacteria have fought back with a selection process by which
certain strains are now no longer susceptible to one or moreagents. As a result, bacteria that once seemed to be losing
the battle for survival have re-emerged to create therapeutic
dilemmas with resulting increased risks of treatment
failure and disease complications. As the incidence ofantimicrobial resistance rises, so do costs associated with
its consequences. The worldwide emergence of multidrug
resistance (MDR) among Gram-negative and Grampositive bacteria has resulted in a great threat to conquer
against the microbes.
During invasive procedures pathogens that are present
on medical personnel hands or in the instruments or thatare acquired by the patient in the skin, respiratory tract,
genitourinary tract, gets entry into the already weakened
patients. These medical procedures bypass naturalprotective barrier against the entry of pathogens and provide
an easy route for infection. Patients already colonized withhospital strains on admission are instantly put at a greaterrisk when they undergo such invasive procedure leading to
nosocomial infections.

MethodA Prospective study was conducted from March
2011-February 2012 at intensive care unit, medical wards,
orthopedic ward, neurological ward, surgical ward,
surgical ICU and Department of Microbiology, TUTH.
A total of 900 specimens which included urine, sputum,
pus, endotracheal secretions and blood were collectedfrom patients admitted at TUTH. All the specimens
were collected, culture, identification tests were doneby according to the standard protocol by the ASM andwww.jiom.com.np

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Sah MK et al.,40
analyzed accordingly 14. The antibiotic sensitivity tests ofthe pathogens isolated from the clinical specimen against
different antibiotics were done using Mueller Hinton agar
by the standard disk diffusion technique of Kirby- Bauer
method as recommended by CLSI 15. This study wasapproved by Institutional Review Board of Institute ofMedicine. Data were analyzed by using SPSS version 17.0.
A detailed clinical examination and review of systems most
likely reveal the involved organs or systems. Investigation
should be focused on these abnormal areas such as;
bloodstream, UTI, pneumonia and surgical-site infection.
Laboratory test for nosocomial infection can be performedby taking specimens from the sites of the infection 16.
Laboratory analyses aim to identify the responsible
infectious agent, evaluation of its susceptibility to antiinfectious treatments, typing of bacterial strains etc. The
identification of common nosocomial infection sites andsimplified criteria for each infection. (Table: 1)

ResultNine hundred patients admitted between March 2011 to
February 2012 at Tribhuvan University Teaching Hospitalwere studied for prevalence of nosocomial infections. On
admission, they were carefully examined clinically aswell as microbiologically to exclude community-acquired
infections and to determine any underlying risk factors.
Out of nine hundred specimens 34.4% (n= 310) were foundto be associated with nosocomial infection. (Table 2 andFigure 1)
Table 2 Prevalence of nosocomial infection
Total no. of specimens
900
Specimens those associated with nosocomial
infection
310

Table 1 Provides common nosocomial infection sites
and Simplified criteria for each infection2.
Type ofnosocomial
infections

Simplified criteria

Surgical site
infection

Any purulent discharge, abscess, or
spreading cellulitis at the surgicalsite during the month after theoperation

Urinary infection

Positive urine culture (1 or 2
species) with at least 105 bacteria/
ml, with or without clinicalsymptoms

Respiratory
infection

Figure 1. Prevalence of nosocomial infection
Distribution of specimens associated with nosocomial
infections

Respiratory symptoms with atleast two of the following signs
appearing during hospitalization:

Out of 310 specimens, urine 122 (39.30%), sputum
78(25.2%), pus 78(25.2%), endotracheal secreation 24(7.7%) and blood 8(2.6%). ( TABLE 3)— Cough— Purulent sputum

Table 3 Distribution of specimens associated withnosocomial infections— New infiltrate on chest
radiograph consistent with infection

Specimens

Number

Percent

Vascular catheter
infection

Inflammation, lymphangitis or
purulent discharge at the insertionsite of the catheter

Urine

122

39.3

Sputum

78

25.2

Pus

78

25.2

Septicemia

Fever or rigours and at least one
positive blood culture

ET secretion

24

7.7

Blood

8

2.6

Total

310

100

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Nosocomial Bacterial Infection

41

Three hundred thirty three bacteria were isolated fromthree hundred ten specimens. Among the 122 urinary
bacterial isolates, Escherichia coli was found to be themost predominant (41.8%) followed by Enterococcus
faecalis (14.8%), Acinetobacter spp. (15.6%), Klebsiellapneumoniae (9%), Pseudomonas aeruginosa (9%),
Staphylococcus aureus (9%) and Citrobacter freundii(0.8). In case of sputum specimens (n=79), K. pneumoniae
was found to be most predominant bacteria (25.3%)
which was followed by Acinetobacter spp. (3%), E. coli
(16.5%), P. aeruginosa (10.1%), S. aureus (19%). Whereas(n=95) bacteria were isolated from pus specimens, E. coli

(31.6%) was most common pathogen which is followedby Acinetobacter spp. (20.0%), K. pneumoniae (13.7%),
P.aeruginosa (11.6%), C. freundii (4.2%), M. morgannii(2.1%) S. aureus (15.8%) and E. faecalis (1.1%).Amongthe 29 endotracheal bacterial isolates, Acinetobacter
spp was found to be more predominant (44.8%) which
was followed by P. aeruginosa (24.1%), K. pneumoniae(20.7%) and E.coli (10.3%). Moreover, eight bacteria wereisolates from blood in which Acinetobacter spp. was foundto be more predominant (50%) which was followed by C.
freundii (12.5%), E. coli (12.5%) and S. aureus (25%).
(Table 4 )Table 4 Distribution of Bacteria associated with Nosocomial Infection
Sites of Nosocomial Infection

Bacterial Isolates

Number

Percent

UTI (n=122)

Escherichia coli

51

41.8

Acinetobacter spp.

19

15.6

Klebsiella pneumoniae

119

Pseudomonas aeruginosa

11

9

Citrobacter freundii

1

0.8Enterococcus spp.

18

14.8

Staphylococcus aureus

11

9

K. pneumoniae

20

25.3

Acinetobacter spp.

20

25.3

E.coli13

16.5

P.aeruginosa

8

10.1

C.freundii

3

3.8

S.aureus

15

19

E.coli30

31.6

Acinetobacter spp.

19

20.0

K.pneumoniae

13

13.7

P.aeruginosa

11

11.6

C.freundii

4

4.2

M.morgannii

2

2.1

S.aureus

15

15.8

E.faecalis

1

1.1

Acinetobacter spp

13

44.8

P.aeruginosa

7

24.1

K.pneumoniae

6

20.7

E.coli3

10.3

Acinetobacter spp.

4

50

C.freundii

1

12.5

E.coli1

12.5

S.aureus

2

12.5

LRTI (n=79)

SSI (n=95)

VAP (n=29)

BSI

(n=8)

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Sah MK et al.,42
Table 5 Distribution of bacteria associalted with nosocomial infection
E. coliA. spp.

K. pneumoniae

P. aeruginosaE. spp.

S. aureus(n=51)(n=19)(n=11)(n=11)(n=18)(n=11)

Amoxycillin

98

100

100

-

100

100

Ciprofloxacin

96

100

100

100

77.8

81.8

Co-trimoxazole

86.3

40

100

-

-

81.8

Nitrofurantoin

39.2

94.7

81.8

-

33.3

18.2

Norfloxacin

86.3

100

100

100

83.3

81.8

Cephalexin

94.2

100

100

-

-

90.9

Ceftriaxone

86.3

47.4

100

54.5

-

-

Cefotaxime

86.3

52.6

100

54.5

-

-

Ceftazidime

86.3

47.4

100

54.5

-

-

Cefepime

68.6

36.8

90.9

36.4

-

-

Gentamycin

64.7

100

90.9

90.9

-

63.6

Amikacin

35.3

89.5

63.6

81.8

-

45.5

Ampicillin-Sulbactam

92.2

68.4

100

-

-

-

Cefoparazone -Sulbactam

17.6

36.8

54.5

45.5

-

-

Piperacillin

82.4

57.9

81.8

45.5

-

-

Piperacillin-Tazobactam

29.5

26.3

54.5

45.5

-

-

Imipenem

5.9

31.6

27.3

36.4

-

-

Meropenem

5.9

26.3

27.3

36.4

-

-

Polymyxin B

0

0

0

0

-

-

Colistin sulphate

0

0

0

0

-

-

Erythromycin

-

-

-

-

94.5

90.9

Vancomycin

-

-

-

-

0

0

Cloxacillin

-

-

-

-

-

54.5

Clindamycin

-

-

-

-

-

63.6

Antibiotics

Incidence of antibiotics resistant with E. coli to ciprofloxacin (96%), cephalosporin(86.3%), gentamycin (64.7%),
nitrofurantoin (39.2%), Acinetobacter spp. to cephalosporin (100%), ciprofloxacin (100%), Klebsiella pneumoniae
to ceftriaxone (100%), amikacin 89.5%), P. aeruginosa to ceftazidime (54.5%), piperacillin (45.5%) and piperacillintazobactam (45.5%), Enterococcus spp. to ciprofloxacin (77.8%), and S. aureus to amoxycillin (100%), ciprofloxacin(81.8%), cloxacillin (54.5%) and cefalexin (90.9%) in urinary isolates.(Table 5)

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Nosocomial Bacterial Infection

43

Table 6 Antimicrobial Resistant Pattern of Urinary Isolates Presented in Percentage
K. pneumoniae
Antibiotics(n=20)

A. spp.

E. coliP. aeruginosa

S. aureus(n=20)(n=13)(n=8)(n=15)

Amoxycillin

100

100

100

-

100

Ciprofloxacin

85

95

92.3

62.5

86.7

Ofloxacin

85

95

84.6

62.5

86.7

Co-trimoxazole

85

100

92.3

-

73.3

Cephalexin

100

100

92.3

-

93.3

Ceftriaxone

95

100

92.3

62.5

93.3

Cefotaxime

95

100

92.3

62.5

93.3

Ceftazidime

95

100

92.3

75

-

Cefepime

85

95

77

50

93.3

Gentamycin

90

95

84.6

75

80

Amikacin

65

95

53.8

62.5

73.3

Ampicillin-Sulbactam

-

100

92.3

-

-

Cefoparazone -Sulbactam

45

80

38.5

12.5

-

Piperacillin

90

95

92.3

87.5

-

Piperacillin -Tazobactam

55

95

69.2

25

-

Imipenem

15

95

23

12.5

-

Meropenem

15

95

23

12.5

-

Polymyxin B

0

0

0

0

-

Colistin sulphate

0

0

0

0

-

Erythromycin

-

-

-

-

100

Vancomycin

-

-

-

-

0

Cloxacillin

-

-

-

-

66.7

Clindamycin

-

-

-

-

66.7

Bacteria isolated from sputum specimens showed antibiotics resistant with K. pneumoniae to ceftriaxone (95%), amikacin(65%), Acinetobacter spp. to cephalosporin (100%), ofloxacin (95%), E. coli to cephalosporin (92.3%), carbapenem(23%), P. aeruginosa to ceftazidime(75%), piperacillin (87.5%) and piperacillin-tazobactam (25%) and S. aureus toamoxycillin (100%), ofloxacin (86.7%), cloxacillin (66.7%) and cefalexin (93.3%).(Table 6)

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Sah MK et al.,44
Table 7 Antimicrobial Resistant Pattern of Sputum Isolates Presented in Percentage
Antibiotics

E. coliA. spp.

K. pneumoniae

P. aeruginosa

S. aureus(n=30)
100(n=19)
100(n=13)(n=11)

Amoxycillin

100

Ciprofloxacin

96.7

100

100

100

(n=15)
100

100

Ofloxacin

86.7

100

100

Co-trimoxazole

96.7
76.7

80

100

Cephalexin

96.7

94.7

100

-

Ceftriaxone

83.3

89.5

100

81.8

67.7

Cefotaxime

83.3

89.5

100

81.8

67.7

Ceftazidime

83.3

89.5

100

81.8

-

Cefepime

76.7

80

92.3

63.6

Gentamycin

73.3

94.7

100

90.1

67.7
73.3

Amikacin

40

84.2

92.3

90.1

46.7

Ampicillin-Sulbactam

90

68.4

100

23.3

40

92.3

45.5

-

Cefoparazone -Sulbactam
Piperacillin

93.3

84.2

100

63.6

Piperacillin-Tazobactam

36.7

57.9

77

63.6

Imipenem

6.7

63.2

38.5

54.5

Meropenem

6.7

63.2

38.5

54.5

Polymyxin B

0

0

0

0

Colistin sulphate

0

0

0

0

-

-

-

-

-

-

-

-

Erythromycin
Vancomycin
Cloxacillin
Clindamycin

-

-

86.7
80
93.3

93.3
0

-

67.7

-

73.3

The rate of antibiotics resistant with E. coli to ceftriaxone (83.3%), carbapenem (6.7%), Acinetobacter spp. to cephalosporin(89.5%), ofloxacin (100%), K.pneumoniae to ceftriaxone (95%), amikacin (92.3%), P. aeruginosa to ceftazidime (81.8%),
piperacillin (63.6%) and piperacillin-tazobactam (63.6%) and S. aureus to amoxycillin (100%), ofloxacin (86.7%),
cloxacillin (67.7%) and cefalexin (93.3%) in pus isolates.(Table 7)

www.jiom.com.np

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Attachment-2

Nosocomial Bacterial Infection

45

Table 8 Antimicrobial Resistant Pattern of Pus Isolates Presented in Percentage
Antibiotics

Acinetobacter spp. (n=13)

K. pneumoniae (n=6)

Amoxycillin

100

100

Ciprofloxacin

100

100

100

Co-trimoxazole

100

16.7

-

Ofloxacin

100

100

Cephalexin

100

100

Ceftriaxone

100

100

100

Cefotaxime

100

100

100

Ceftazidime

100

100

100

Cefepime

100

16.7

71.4

Gentamycin

100

100

85.7

Amikacin

100

16.7

85.7

Ampicillin-Sulbactam

100

16.7

Cefoparazone -Sulbactam

92.2

66.7

28.6

Piperacillin

100

100

42.9

Piperacillin-Tazobactam

100

100

14.3

Imipenem

92.2

0

28.6

Meropenem

92.2

0

28.6

Polymyxin B

0

0

0

Colistin sulphate

0

0

0

Bacteria isolated from endotracheal screation revealed
antibiotics resistant with Acinetobacter spp. showed 100%
resistant to most commonly prescribed antibiotics except
carbapenem, cefoparazone-salbactam (92.2%) respectively,
K. pneumoniae to amikacin (16.7%), cefoparazonesalbactam (67.7%), P. aeruginosa to ceftazidime (100%),
piperacillin (42.9%) and piperacillin-tazobactam (14.3%)(Table 8)
The antibiogram of blood isolated, for Acinetobacter spp.
the antibiotic effect was very poor. They showed 100%
sensitive to Polymyxin B and colistin sulphate followed by
cefoperazone-sulbactam (75%), Piperacillin-tazobactam(75%), imipenem (75%). E.coli were found to be resistant

71.4

to amoxycillin, aminoglycosides and second generation of
cephalosporin, and sensitive to polymyxin B and colistin
sulphate, imipenem and meropenem and others. For
S.aureus, amoxycillin had no effect. Vancomycin were
found to be most effective antibiotic (100%) which was
followed by amkacin and cloxacillin (each 50%).

Discussion
The study was aimed to find out the current prevalence andtrend of the bacteria causing nosocomial infections andthe efficacy of drugs being used against them. The overall
prevalence of bacteria causing nosocomial infection is(34.4%), which is higher than the similar studies in theother hospitals from different countries, which werewww.jiom.com.np

84

P. aeruginosa (n=7)

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Journal of Institute of Medicine, December, 2014, 36:3

Attachment-2

Sah MK et al.,46
(13%-17.8%) (17-22). This increase in the prevalence of
nosocomial infections in this hospital may be attributed
to less attention being paid to well-established processes
for decontamination and cleaning of soiled instruments
and other items, followed by sterilization and high-level
disinfection processes and improving safety in operatingrooms and other high-risk areas where the most serious andfrequent injuries and exposures to infectious agents occur.

common bacteria causing VAP26. Gram negative bacteria,
P. aeruginosa and Acinetobacter baumanii are commonly
associated with late onset VAP 27.

In this study, the most common nosocomial infection was
found to be UTI (39.30%) followed by LRTI (25.20%),
SSI (25.20%), VAP (7.7%) and BSI (2.6%). Our results areconcurrent with the multicentic study in Greece showedthat UTI was (22.4–38.2%), LRTI (21.1–32.6%), SSI(14.6–22.7%) and BSI (9–13.2%) 23.

Currently many microorganisms have become resistant todifferent antimicrobial agents and in some cases to nearly
all agents. Resistance to antimicrobial agents is a problem
in health care facilities, but in hospitals, transmission ofbacteria is amplified because of the highly susceptible
population (WHO, 2002). The antibiotic resistant of ourstudy confirmed the alarming percentage of resistance
exhibited by pathogens to the common antibiotics in use.

The bacteria isolated in our study from patients who were
suffered from nosocomial urinary tract infections included
E. coli (41.8%) followed by Acinetobacter spp. (15.6%),
Enterococcus spp. (14.8%) and S. aureus (9%).These
results were supported by Neto et al. (2003) study which
was done among 188 patients with positive urine culture
in Brasileira and found that the most common pathogens
causing nosocomial urinary tract infections were E-coli(26%), Klebsiella spp. (15%), P. aeruginosa (15%) andEnterococcus spp. (11%)24.
In this study K. pneumoniae was found to be most
predominant bacteria (25.3%) causing nosocomial LRTI
followed by Acinetobacter spp. (25.3%), E. coli (16.5%)
and P. aeruginosa (10.1%). In a study by Singh et al,most frequent isolates causing LRTIs were Klebsiellaspp. (24.48%), followed by Proteus (18.33%) and E. coli
(12.24%) which concurrent with our study 25. This showsthat the prevalence of K. pneumoniae has increased in 2012
as compared to 2010 at TUTH. A study done by Mishra
et al showed the growth of 18.95% of K. pneumoniae inlower respiratory tract infection.
In the surgical site infection (SSI), E. coli (31.6%) were
found to be most predominant followed by Acinetobacter
spp. (20%), K. pneumonia (13.7%), P. aeruginosa (11.6%),
C. freundii (4.2%), M. morgannii (2.1%) and S. aureus(15.8%), Enterococcus spp.(1.1%). Regarding the growth
pattern, single bacterial growth was found in 10.5% of
the cases while 79.5% were multiple bacterial growths (2or more than 2). This could be because of the profoundinfluence of endogenous contamination from the bowel andhollow muscular organs of patients.
In case of ventilator associated pneumonia (VAP),
Acinetobacter spp. was found to be more predominant(44.8%) followed by P. aeruginosa (24.1%), K. pneumonia(20.7), E. coli (10.3%). A study conducted in Nepal
by Ranjit S, Bhattarai B, Acinetobacter spp.was mostwww.jiom.com.np

In case of nosocomial blood stream infection (BSI),
Acinetobacter spp. was found to be more predominant(50%) followed by C. freundii (12.5%), E. coli (12.5%)
and S. aureus (25%).

However, the present study showed a high prevalenceof resistance to the commonly prescribed antimicrobial
agents. This may be because of the intense use of
antimicrobial agent in the hospital, easy availability andindiscriminate use of these drugs outside the hospitals, andmany antibiotics are available over the counter for selfmedication. These problems, coupled with the increasechance of cross infection among inpatients, are known toaccount for circulating resistance strains.
The emergence of Gram-negative bacterial species withacquired resistance to various broad spectrum β-lactams
and other classes of antimicrobials is becoming a worldwide
clinical problem. This may be due to exposure of hospitalized
patients to different broad and extended spectrum drugs
beside multiresistant isolates are disseminated widely inthe hospital setting due to different iatrogenic mechanism
and these patients may not be immunocompetent.
This study provides insights into the problem of resistancein bacterial pathogens in TUTH. Our results demonstrated
that, in general, isolates have high rates of resistance toantibiotics commonly used in developing countries. We also
found a high rate of resistance to amoxicillin, first, second
and third generation cephalosporins, fluroqunolones,
aminoglycosides and co-trimoxazole. Therefore, cheap
antibiotics such as amoxicilline, ciprofloxacin, gentamycin,
cephalexin and co-trimoxazole are now of limited benefit
in the treatment of infections in TUTH.
The high level of ciprofloxacin resistance among E. coli,
and more generally Enterobacteriaceae, rules out the use of
ciprofloxin as empirical treatment when invasive infections
due to these pathogens are suspected. The rate of resistanceto third-generation cephalosporins is also worrisome.
The high prevalence of Acinetobacter spp. in UTI 15.5%

Journal of Institute of Medicine, December, 2014, 36:3

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Attachment-2

Nosocomial Bacterial Infection(n=19), LRTI 25.3% (n=20), SSI 20% (n=19) and in P.
aeruginosa UTI 9% (n=11), LRTI 10.1% (n=8), SSI 11.6%(n=11) may have been exacerbated by failure of infection
control in the hospitals. The overall rate of antibiotic
resistance in Acinetobacter spp. was higher than that in
P. aeruginosa, this observation contrasts with previous
results founds in South Africa28. Resistance to carbapenem(imipenem) in Acinetobacter spp. was (31.6-95) %, but(12.5-54.5) % in P. aeruginosa. This high rate of resistanceto carbapenem in Acinetobacter spp. in our study is striking
given that this antibiotic is frequently prescribed in TUTH,
Nepal. This result may be due to the clonal spread of amulti-resistant strain of A. baumanii.
The indication of antibiotic therapy for nosocomial UTIs
in acute care settings is a controversial issue. Nonetheless,the treatment of symptomatic UTIs is virtually universal.
Yet routine therapy increases not only drug costs but also
adverse drug reactions and the emergence of antibiotic–
resistant microorganisms. The increasing antimicrobial
resistance among the bacteria causing nosocomial urinary
tract infections makes therapy of this type of infectionsdifficult and leads to more use of extensive broad-spectrum
drugs.
Carbapenem resistance in Acinetobacter was widespread.
Carbapenems therefore can no longer be relied on as
empiric therapy for these organisms, leading to an increasein use of alternatives such as polymyxin B and colistin.
We found that 100% were susceptible to polymyxin B andcolistin sulphate.
Carbapenems have potent activity against multidrug
resistant Acinetobacter isolates. Acinetobacter spp.
may develop resistance to carbapenem through various
mechanisms including class B and D carbapenemase
production, decreased permeability, altered penicillin
binding proteins and rarely over expression of efflux
pumps (29, 30). The resistance of Acinetobacter spp. towards
the carbapenems is much higher in this study as compared
to different studies in Indian hospitals at All India Instituteof Medical Sciences (AIIMS) (34.7% for meropenem and27.2% for imipenem)31.

Conclusion
It is quite alarming that prevalence of bacteria causing
nosocomial infection was 34.4% in TUTH. This study
showed that Gram-negative bacilli were the predominantisolates. Polymyxin B, colistin sulphate, imipenem,
meropenem and nitrofurantoin were relatively effective
drugs for Gram-negative bacilli where as vancomycin
was relatively effective drugs for Gram-positive cocci.
However, all the bacteria isolated from nosocomial

47
infection were 100% resistance to Ampicillin. Empiricaltreatment to nosocomial infections provoke drug resistance,
therefore treatment should be based on the result of culture
and sensitivity. This study concludes that if one could not
wait the culture results in nosocomial infection amoxicillin,cloxacillin, ciproflocacin, gentamycine are quite ineffective
to treat these infections.

Acknowledgement
We would like to express my sincere gratitude to thepatients, staffs and Institutional Review Board of Instituteof Medicine, TU for their help and cooperation.
Conflict of interest: None declared.

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10. Tullu MS., Deshmukh CT., Baveja SM. Bacterial
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22. Jroundi I, Khoudri I, Azzouzi A, Zeggwagh AA,
Benbrahim NF, Hassouni F et al. Prevalence of
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11. Jones R. Resistance Patterns among Nosocomial
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23. Gikas A, Roumbelaki M, Pediaditis J, Nikolaidis
P, Levidiotou S, Kartali S, et al. Hellenic Infection
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2004; 25(4):319–24.

12. Inan D, Saba R, Yalcin AN, Yilmaz M, Ongut G,
Ramazanoglu A et al. Device-associated nosocomial
infection rates in Turkish medical-surgical intensive
care units. Infect Control Hosp Epidemiol 2006;
27(4):343–8.
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14. Henry D. Isenberg. Clinical Microbiology Procedures
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15. Clinical and Laboratory Standards Institute.
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17. Gedebou M, Kronvall G, Habte-Gabr E, Ringertz S.
The bacteriology of nosocomial infections at Tikur
Anbessa Teaching Hospital, Addis Ababa. Acta Pathol
Microbiol Immunol Scand [B] 1987; 95:331-36.
18. Dridi E, Chetoui A, Zaoui A. Investigation of theprevalence of nosocomial infection in a Tunisianregional hospital. Sante Publique 2006; 18:187-94.
19. Habte-Gabr E, Gedebou M, Kronvall G. Hospitalacquired infections among surgical patients in Tikur
Anbessa Hospital, Addis Ababa, Ethiopia. Am J InfectControl 1988; 16:7-13.
20. Gedebou M, Habte-Gabr E, Kronvall G, Yoseph S.
Hospital-acquired infections among obstetric andgynaecological patients at Tikur Anbessa Hospital,
Addis Ababa. J Hosp Infect 1988; 11:50-9.
21. Raka L, Zoutman D, Mulliqi G, Krasniqi S, Dedushaj
I, Raka N et al. Prevalence of Nosocomial Infectionsin High-Risk Units in the University Clinical Centerof Kosova. Infect Control Hosp Epidemiol. 2006;
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24. Neto JAD, Da Silva LDM and Martins ACP et al.
Prevalence and bacterial susceptibility of hospitalacquired urinary tract infection. Acta Cirurgica
Brasileira 2003; 18(Suppl 5).
25. Singh AK, Sen MR, Anupurba S, Bhattacharya P.
Antibiotic sensitivity pattern of bacteria isolated from
nosocomial infections in ICU. J Commun Dis 2002;
34:257-63.
26. Ranjit S, Bhattarai B. Incidence and Risk Factors
for Ventilator-Associated Pneumonia in Kathmandu
University Hospital. Kathmandu Univ Med J 2011;33(1)28-31.
27. Joseph NM, Sistla S, Dutta T et al. ventilator-associatedpneumonia in a tertiary care hospital in India: role ofmulti-drug resistant pathogens. J Infect Dev Ctries
2010; 4(4):218-225.
28. Das RN, Chandrashekhar TS, Toshi HS, Gurung
M, Shrestha N, Shivananda PG. Frequency andsusceptibility profile of pathogens causing urinary
tract infections at a tertiary care hospital in western
Nepal. Singapor Med J 2006; 47(4):281-5.
29. Heritier C, Poirel L, Lambert T, Nordmann P.
Contribution of acquired carbapenem hydrolyzing
oxacillinase to carbapenem resistance in Acinetobacterbaumannii. J Antimicrob Agents Chemother. 2005;
49:3198-202.
30. Quale J, Bratu S, Landman D, Heddurshetti
R. Molecular epidemiology and mechanism of
carbapenem resistance in Acinetobacter baumannii
endemic in New York City. J Clin Infect Dis 2003;
37:214-20.
31. Gupta E, Mohanty S, Sood S, Dhawan B, Das BK,
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Attachment-2

Clinical epidemiology and molecular analysis of Extended-spectrum
β-Lactamase (ESBL)-producing Escherichia coli in Nepal:
Characteristics of E. coli Sequence Type ST131 and ST648
Jatan. B. Sherchan, MD,1 Kayoko Hayakawa, MD, PhD,2# Tohru Miyoshi-Akiyama, PhD,3 Norio.
Ohmagari,	
  MD, MSc,2 Teruo Kirikae, MD, PhD,3 Maki Nagamatsu, MT2,3 Masayoshi Tojo, MD, PhD,2,3
Hiroshi Ohara,	
  MD, PhD 4 Jeevan B Sherchand,	
  PhD5 Sarmila Tandukar, MSc5
Department of Clinical Microbiology, Kathmandu University School of Medical Sciences, Dhulikhel,
Nepal1; Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo,
Japan2; Department of Infectious Diseases, Research Institute, National Center for Global Health andMedicine, Tokyo, Japan3; Department of International Medical Cooperation, National Center for Global
Health and Medicine, Tokyo, Japan;4 Public Health Research Laboratory, Institute of Medicine,
Tribhuvan University Teaching Hospital, Kathmandu, Nepal5
Key words: ESBL; Extended-spectrum β-lactamase; CTX-M; E. coli; Antimicrobial resistance, wholegenome sequence
Abstract
Background: Recently, CTX-M type extended-spectrum beta-lactamase-producing E.coli (ESBL-E. coli)
have emerged worldwide. Especially, E. coli O antigen type 25 (O25) and sequence type (ST) 131, often
associated with the CTX-M-15 ESBL, has been increasingly reported globally. However, epidemiology
reports on ESBL-E.coli in Asia are limited.
Methods: Patients who had clinical isolates of ESBL- E. coli in Tribhuvan university teaching hospital in
Kathmandu, Nepal were included. To analyze MLST, phylotypes, virulence genotypes, O25b-ST131
clone, and distribution of acquired drug resistance genes, whole genome sequence of the isolates wereconducted.
Results: During the study period, total of 105 patients with ESBL-E. coli isolation were identified, andmajority (90%) of the ESBL-E. coli isolates were CTX-M-15 positive. The most dominant ST was ST131(n=54 [51.4%]), followed by 15 (14.3%) cases of ST648. Among the 54 ST131 isolates, all 54 (100%)
were identified as O25b-ST131 clone as well as the H30-Rx subclone. Analyses comparing 3 groups(ST131, ST648, non ST131/648) were conducted. Higher proportion of ST648 isolates had resistance tonon-beta lactam antibiotics than ST131 or non-ST131/648, and possessed drug resistant genes more
frequently than ST131 or non-ST131/648. ST131 had virulence genes most frequently, followed by ST
648. Clinical characteristics were similar among groups.
More than 38% of ESBL-E. coli were isolated from outpatient clinic, and pregnant patients consisted 24%
of ESBL-E.coli cases.
Conclusions:

We identified that the highly resistant status of ESBL-E coli to multiple classes ofantibiotics in Nepal are mainly driven by CTX-M-producing ST131 and ST648. Their immense
prevalence in communities is of great concern.

Antimicrobial Agents and Chemotherapy
88

IOM-NCGM Research Collaboration Office Annual Report 2014

Attachment-2

Assessment of health systems in relation to interface between malaria
control programs and health system strengthening: Comparative study
between Nepal and Viet Nam
Hiroshi Ohara, Yasuyo Matsumoto, Takanori Hirayama, Hidechika Akashi, Jeevan B Sherchand, Vu HuyNam, Jatan B Sherchan

Bureau of International Medical Cooperation, National Center for Global Health and Medicine, Japan
Institute of Medicine, Tribhuvan UniversityFaculty of Medicine, Kathmandu University
National Institute of Malariology, Parasitology and Entomology
Abstract
Malaria control has been a major health issue with high priority in endemic countries and various
efforts have been made supported by foreign assistant partners. In order to implement efficient andsustainable control, integration of the control program into general health system or effective interactions
between them is one of the important strategies.
Studies were conducted in Vietnam and Nepal. Information obtained from the document reviews,
interviews, and field surveys were analyzed from the viewpoint of interface between malaria control
program and the health system in accordance with the 6 building blocks of a health system with special
emphasis on good practices and challenges in implementing malaria control program.	
 Among good practices, strong government commitment on the control programs strengthening offacilities and capacity of health workers at primary level, utilization of health volunteers, setting up
mobile teams, intensified education for residents were noteworthy.

Key challenges were mainly existingin remote areas. Introduction of malaria due to population movement and the emergence of new endemicareas have become growing issues. Strengthening of the vertical health program appeared to have some
impact on the general health system, particularly at the primary level. However, it can be said that
dissociation between vertical control program and horizontal general health system also exists.	
 It is crucial to implement effective and equitable malaria control program addressing existing
challenges and to create sustainable health systems. These tackling will lead to further strengthening of
the health system there and eventually effective implementation of various health programs.

IOM-NCGM Research Collaboration Office Annual Report 201489

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.

90

IOM-NCGM Research Collaboration Office Annual Report 2014

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 2014
IOM-NCGM Research Collaboration Oﬃce
©2015 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

Annual Report 2014

Annual Report
2014

IOM-NCGM
Research Collaboration Oﬃce

March 2015
Kathmandu, Nepal
Tokyo, Japan