Vol.34
No.04
DECEMBER 2006

ISSN 1348-8945

Vol.34 No.04 DECEMBERISSN 1348-8945

2006

Tropical Medicine and Health

Tropical Medicine
and Health【CONTENTS】
Reviews
Emerging diseases in Indonesia: control and challenges
Kandun IN

141

Roles of Multi-Country Networking in Prevention and Control of Emerging and Re-emerging
Infections
Sawanpanyalert P
149

Vol.34 No.04 DECEMBER 2006

Exploring Fresh Collaborative Initiatives for Combating Infectious Diseases in the Philippines
Elio-Villa LP
153
Original articles
A new species of Simulium (Nevermannia) from the Ogasawara (Bonin) Islands,
Japan (Diptera: Simuliidae)
Takaoka H and Saito K

155

Uvulectomy and other traditional healing practices:
Traditional healers’ perceptions and practices in a Congolese refugee camp in Tanzania
Kunii O, Tanaka Y, Lewis A and Wakai S

159

Population Polymorphism of Trypanosoma cruzi in Latin America indicated byProteome analysis and by in vitro amastigote proliferation
Mu J, Sone T, Yanagi T, Tada I, Kikuchi M and Hirayama K

167

Tropical Medicine
and HealthJAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 稲 平島 宮副 宮副

背は３mm（決定：宮副）Vol.34
No.04
DECEMBER 2006

ISSN 1348-8945

Vol.34 No.04 DECEMBERISSN 1348-8945

2006

Tropical Medicine and Health

Tropical Medicine
and Health【CONTENTS】
Reviews
Emerging diseases in Indonesia: control and challenges
Kandun IN

141

Roles of Multi-Country Networking in Prevention and Control of Emerging and Re-emerging
Infections
Sawanpanyalert P
149

Vol.34 No.04 DECEMBER 2006

Exploring Fresh Collaborative Initiatives for Combating Infectious Diseases in the Philippines
Elio-Villa LP
153
Original articles
A new species of Simulium (Nevermannia) from the Ogasawara (Bonin) Islands,
Japan (Diptera: Simuliidae)
Takaoka H and Saito K

155

Uvulectomy and other traditional healing practices:
Traditional healers’ perceptions and practices in a Congolese refugee camp in Tanzania
Kunii O, Tanaka Y, Lewis A and Wakai S

159

Population Polymorphism of Trypanosoma cruzi in Latin America indicated byProteome analysis and by in vitro amastigote proliferation
Mu J, Sone T, Yanagi T, Tada I, Kikuchi M and Hirayama K

167

Tropical Medicine
and HealthJAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 稲 平島 宮副 宮副

背は３mm（決定：宮副）Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 141-147
Copyright! 2006 by The Japanese Society of Tropical Medicine

１
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EMERGING DISEASES IN INDONESIA:CONTROL AND CHALLENGES
I NYOMAN KANDUN
Recieved 31, October, 2006
ABSTRACT: Infectious diseases remain an important cause of morbidity and mortality in Indonesia. The reduction, elimination, and eradication of infectious diseases have been the subject of numerous meetings and publichealth initiatives for decades. The malaria, yaws and other communicable disease eradication programs of earlier
years, although unsuccessful, contributed greatly to an understanding of the difficulties faced in trying to achievegoal of disease control. The reemergence of old infectious diseases, along with the emergence of new diseases suchas SARS, Avian Influenza and the development of antimicrobial resistance, pose significant challenges to publichealth.

BACKGROUND
The control, elimination and eradication of human diseases have been the subject of numerous public health interventions and discussions for decades. The eradication ofsmallpox was declared on May 8, 1980 at the 33rd World
Health Assembly, and was followed soon after by poliomyelitis and other eradicable diseases. Although many diseases eradication program in the past were unsuccessful, thelessons learnt contributed to an understanding about thecomplexities and difficulties faced in trying to achieve theultimate goal of disease control. An understanding of thenatural history of a disease, including multiple causation
and biological, sociopolitical and economic issues sheds
light on the public health interventions available in dealing
with communicable diseases and their containment.
Indonesia stretches from west to east, with an area of
1.9 million km２and a population of 230 million. In developing countries like Indonesia, health resources have always
been limited, and decisions as to the most preferable andcost effective intervention programs have to be targeted topriority diseases. Disease control can be defined as the reduction of diseases in a defined geographical area as a resultof deliberate control efforts. This definition should be further quantified to indicate the level of disease reduction to
be achieved. Disease elimination and eradication are the ultimate goals of any public health intervention starting from
disease control. Communicable diseases are still serious
public health problems, killing and causing suffering formillions of people in Indonesia, especially the most vulnerable groups, i.e. the poor, women and children. Communi-

cable diseases exert a negative effect on development andplace a burden on the economy of the individual and thecountry as a result of the huge costs of treatment and control.
Technical solutions combined with strategies to mobilize all levels of society from high level decisions-makers tocommunities and families will ensure the effective control
and prevention of communicable diseases. The following isa brief description of re-emerging diseases, newly emerging
diseases, and the challenges encountered in their control.
DENGUE HEMORRHAGIC FEVER
An epidemic of dengue fever / dengue hemorrhagic fever (DF/DHF) started in the Southeast Asia region after theSecond World War. The first case of DF/DHF in Indonesia
was reported in 1968 from Jakarta and Surabaya. Since thenthe frequency and magnitude of DF/DHF outbreaks have increased dramatically. As the principal mosquito vector,
Aedes aegypti and the viruses (D１, D２, D３, D４）that cause DF/DHF, expanded their geographical and age distribution nationally. Figure 1 presents the distribution of DHF by province while Figure 2 presents the seasonal variation of DHF
cases and deaths in 2004−2005 and a comparison of DHF
cases in 2005 and 2006.
The epidemiologic trends in recent decades demonstrate that the prevention and control of dengue virus transmission have failed. There is no vaccine available for dengue viruses nor effective mosquito control programs dealing
with breeding places. Emphasis has been placed on disease
surveillance and immediate response using space sprays targeting adult mosquitoes in the affected focal areas. Now

Disease Control and Environmental Health, Ministry of Health, Jakarta, Indonesia

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Figure 1. INCIDENCE OF DENGUE HEMORRHAGIC FEVER (DHF)
BY PROVINCE IN 2005
a. Trends of Dengue Hemorrhagic Fever Cases & Deaths in
2004-2005

b. NUMBER OF DENGUE HEMORRHAGIC FEVER
CASE IN 2005-2006 (UP TO 19 June 2006)

Figure 2.

strategies are being revised so as to focus on community involvement in the elimination of breeding places and to improve partnership and professionalism among program
managers at all levels.
A decentralized integrated approach that targets larval
mosquitoes is being implemented for effective Aedes aegypti control. Along with high-level political commitment,
community involvement is an important prerequisite forvector control.
TUBERCULOSIS (TB)
Tuberculosis (TB) is a bacterial disease caused by My-

cobacterium tuberculosis transmitted primarily by airborne
droplets. Infection occurs when susceptible persons inhale
infected droplets produces by coughs and sneezes of persons with active lung TB. TB causes suffering for millions
of people, particularly the poor, women, children, and HIV/AIDS patients. More than half a million new TB cases are
estimated to occur every year, with 300−400 TB deaths
daily. To cope with this problem, the DOTS strategy has
been implemented since 1998 and aims to achieve a casedetection rate (CDR) of 70% and cure rate or success rate(SR) of 85% by the end of 2006. Figure 3 and 4 show thedate on CDR and SR from 1997 to 2005.

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TB is diagnosed mainly if acid-fast- bacilli (AFB) arefound in the sputum, body fluids or tissue in combination
with clinical symptoms. In special cases, chest radiographand PPD skin test abnormalities are also taken into consideration The principal challenges in TB control include obtaining and continuing the political commitment of decision
makers, and international funding for the support of TB
control efforts. Multiple drug resistance is also a challengethat will have to be addressed in the near future.

Figure 3. Number of Cases Detected 1987-2005 in the TBC
Control Program

Figure 4. CDR and SR of AFB Positive Cases in Indonesia
1997-2005

MALARIA
Most malaria eradication programs in the past havebeen unsuccessful. Figure 5 shows the malaria endemicity
in 2005 and figure 6 the malaria situation in Indonesia from1989 to 2005.
Deforestation, mining, active rapid population migra-

Figure 6. MALARIA SITUATION IN INDONESIA 19892005

Figure 5. MALARIA ENDEMICITY DISTRIBUTION IN 2005

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tion and other development activities have contributed to
the resurgence of malaria in Indonesia. The roll back malaria strategy, which was implemented as GEBRAK Malaria
in 2000, focuses on partnership. Malaria control strategy
primarily focuses on vector and disease surveillance, early
diagnosis and prompt treatment, integrated vector management and community participation.
The principal challenges to malaria control programs
are effective coordination, long-term sustainability of vector
control efforts, population migration and environmental
changes. Anti-malaria drug resistance and insecticide resistance are also primary challenges for the future.
POLIO
Polio eradication has been targeted for the year 2008.

Figure 7. EPI TARGET DISEASES Polio epidemic in Indonesia, weeks 11-22

The polio eradication campaign is a good example of multipartner leadership in that partners come from a broad arrayof organizations such as Rotary International, WHO
UNICEF, CDC-Atlanta, USAID, AUSAID, and WorldBank.
Poliomyelitis is caused by the poliovirus (P１, P２, P３)transmitted via the orofecal route due to poor personal hygiene and environmental sanitation. Potent oral polio vaccine and injectable vaccine are available to combat the disease, and the former was included in the Routine Immunization Program in Indonesia in 1980. Universal Child Immunization coverage (!80%) was achieved in 1990. National Immunization Days were designated in 1995, 1996
and 1997 consecutively as part of the global polio eradication campaigns launched in 1988 by WHO. A wild
poliovirus-free status continued for 10 years from 1996, but
an imported polio case was reported from Cidahu, Sukabumi in March 2005. This wild poliovirus was imported
from Nigeria via Yemen and Saudi Arabia. Figure 7, 8, and9 show the spread of wild poliovirus and efforts made tocontrol the outbreak.
Since the outbreak in March 2005, there have been 305
lab confirmed WPV cases (303 in 2005 and 2 in 2006), and
46 VDPV cases. The most recent wild polio case was reported on 20 February 2006 from Aceh Tenggara district,
NAD province
Since the outbreak in March 2005, the total number oflab confirmed polio cases was 305, including 303 in 2005

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Figure 8. Wild Poliovirus Cases in Indonesia, March 2005- present

Figure 9. Wild Polio Cases by Week of Onset, Indonesia 2005-2006

and 2 cases in 2006, and 46 VDPV (vaccine derived polio
virus) cases. After various efforts including mopping up,
National Immunization Days (NID) and Sub NID, the lastcase was reported in 20 February 2006 from Southeast
Aceh. A future challenge to EPI is the sustainability of theroutine immunization, including both managerial and financial sustainability. A long-term plan is being developed toaddress future challenges.
HIV/AIDS
The Human Immunodeficiency Virus (HIV) causes acquired immunodeficiency syndrome (AIDS). The first HIV/AIDS case in Indonesia was reported in the late 80’s from
Bali, after which the disease spread rapidly to 32 out of the
33 provinces of Indonesia. The cumulative number of HIV

Figure 10. CUMULATIVE AIDS CASES UP TO THE END
OF SEPTEMBER 2006

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through partnership. The principal challenges to HIV/AIDS
control programs are intersector coordination, behavior
changes, and human resources.

Figure 11. SEX DISTRIBUTION OF AIDS CASESUP TO THE END OF SEPTEMBER 2006

Figure 12. RISK FACTOR OF AIDS CASES BY MODE
OF TRANSMISSION UP TO SEPTEMBER
2006

infection and AIDS cases reported up to September 2006 is4617 and 6987, respectively. Figure 10 shows the trend of
AIDS cases from 1997 to 2006 and figure 11 shows the sex
distribution. The AIDS cases were predominantly male(82%), with females accounting for only 16% and unknown
for 2%. Risk factors by mode of transmission are shown infigure 12. Intense and rapid spread of HIV was documented
among injecting drug users (IDU) (52%), followed by heterosexuals (37.2%). The cumulative number of AIDS cases
is expected to reach 93,968 to 130,000 by the year 2010. In
Papua, pregnant women and newborn babies were infectedby HIV. The main strategy to combat HIV infection includes efforts to prevent new infection, to promote comprehensive care, and to increase coverage of HIV infection

SARS (Severe Acute Respiratory Syndrome)
During the SARS outbreak in 2003, there were only 7suspected and 2 probable cases admitted to hospitals in Indonesia. No confirmed case was reported. Strategies and actions taken during the SARS outbreak included the screening of incoming passengers from affected countries withthermo scanners, establishment and capacity building of 35
referral hospitals and 45 port health offices, public awareness and regional networking (ASEAN +3).
AVIAN INFLUENZA
Outbreaks of H5N1 infection in the poultry population
were first reported in 2003. Human cases were first reported
in July 2005. To date, 32 of 33 provinces of Indonesia haveexperienced H5N1 infection among the poultry population,
while H5N1 infection in humans has been reported fromonly 9 provinces. Figure 13 shows the distribution of avian
influenza outbreaks in animals and humans.
Strategies to control H5N1 transmission include theprevention of H5N1 transmission in the poultry population,
surveillance, bio-security, case management, public awareness/risk communication, research and development. The
main challenges to H5N1 control are coordination and resources.
NEGLECTED DISEASES
Leprosy, lymphatic filariasis, yaws, rabies, and Japanese encephalitis are considered to be neglected diseases.
Very little attention has been paid to these diseases, and insufficient resources have been allocated for their control.
Fortunately, international agencies and the international
community are interested in leprosy and lymphatic filariasis
and as a result we aim to eliminate leprosy and lymphatic
filariasis by 2020. An integrated approach is being implemented to cope with the limited resources available.

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Figure 13. AVIAN INFLUENZA OUTBREAK IN ANIMALS AND HUMANS
H5N1 in animals: 30 provinces, 202 districts/munics. H5N1 in humans: 9 provinces, 24 districts/munics.

Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 149-152
Copyright! 2006 by The Japanese Society of Tropical Medicine

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ROLES OF MULTI-COUNTRY NETWORKING IN PREVENTION AND
CONTROL OF EMERGING AND RE-EMERGING INFECTIONS
Pathom Sawanpanyalert
Recieved 13, December, 2006
ABSTRACT: The emergence of new and the re-emergence of old infectious diseases reverses our previous belief
that communicable diseases have been brought under human control. The development of antibiotic-resistant bacterial and fungal infections and the apparent lack of vaccines for many infectious diseases remind us of how vulnerable we are. Infections with SARS coronavirus, Nipah virus, and, more recently, H5N1 influenza virus in humans
are just a few reality checks and more are expected. The problems of these emerging and re-emerging infections(ERI) give us several warnings. First, there are a number of pathogens, most of which are viral, that pose potential
risks to human health and we know quite little about them. Second , many of these ERI are zoonotic. Effective control of zoonoses needs involvement and collaboration from the non-health sector. Third , since these ERI can easily
spread across geopolitical boundaries, countries with a good public health infrastructure are not risk-free andshould not be complacent. Surveillance and response efforts cannot be limited within one national boundary.
Fourth, aside from the fundamental tools in disease control that we have, e.g. basic sanitation, personal hygiene,
isolation and quarantine, and the newly-revised International Health Regulations, we have little other choices, e.g.
drugs and vaccines. This limitation suggests that we could eventually be defenseless. Fifth, recent occurrences ofcertain ERI in some countries have demonstrated that damages caused in non-health terms, e.g. economic losses,
can be significant. As a consequence, ERI may be dealt with as an economic problem while the human and healthdimensions are ignored.
There are a number of guiding principles that we may have to adopt. First, no country can or is allowed tofight ERI alone, no matter how well-developed its economic condition and public health infrastructure. However, it
should be noted that ERI is a national health security problem and that national sovereignty must be recognizedwhen addressing the ERI issues. Second , ERI problems need a lot of non-health partners, e.g. business and agriculture. Third , we need to improve our capacity to do surveillance and to respond. Surveillance without response ispointless. Fourth, we need specific tools to help tackle ERI.
Networking is a mechanism through which countries can work together to fight ERI. A number of forums exist to address the problems of ERI, e.g. meetings of WHO and other UN agencies including FAO/OIE, ASEAN＋3,
and ACMECS. In addition, several bilateral frameworks are in place to address ERI. However, it is important tonote the particular nature of ERI and follow the above-mentioned guiding principles to avoid failure.

INTRODUCTION
Emerging and re-emerging infections, by definition,
are new threats to human beings, although many of thepathogens causing the infections may have been with us fora long time without our due attention. In the course of human development, we are constantly challenged by threats,
including those from infectious diseases. Discovery of effective antibiotics and the rapid progress of virology may
instill a complacency that infectious diseases are under ourcomplete control and we do not have to be wary of communicable diseases. However, the emergence of HIV/AIDS
National Institute of Health
Department of Medical Sciences
Ministry of Public HealthThailandepidemic some twenty years ago and the recent outbreaks of
SARS coronavirus, Nipah virus, and H5N1 influenza virus
in humans reverse this belief. The threat is further aggravated by long-recognized-but-largely-ignored problems of
antimicrobial resistance not only among bacteria but also
viruses, fungi and even parasites. In other words, not only
are we unable to control existing enemies, we are facedwith more and stronger enemies. In addition, our most effective tools, e.g. antimicrobials, are becoming less effective.
There are several lessons to be leaned from our recent
encounters with emerging and re-emerging infections (ERI).
First, the list of emerging and re-emerging infections is１５０

long and growing. If we visit the first issue of the journal“Emerging Infectious Diseases or EID” published in 1980,
we can see the introductory paper by David Satcher of the
US Centers for Disease Control and Prevention on ERI, as
shown in Figure 1[1]. Although the paper is now over 20
years old, the list of EID in the paper was already long and,I am sure, if the list is revised, it will grow. Most of the diseases or syndromes on the list and the ones we have faced
recently are quite strange to us. That means we know relatively little about them. Our relative ignorance of ERI is the
major drawback in our fight against them.
Second, the origins of many recent ERI can be traced
back to animal reservoirs or carriers or cases, e.g. civet catsin SARS, pigs in Nipah viral infection, and birds in H5N1
infection. Many ERI exist in animals long before they aretransmitted to humans. Increased contact between humanbeings and animals increases the chance of infections jumping onto humans. Such includes raising domesticated or
farm animals, contacting migratory animals such as birds,
and hunting and eating exotic animals. Human medicine
does not prepare doctors or public health professionals tohandle diseases of animal origin. Therefore, there is astrong and urgent need for better and closer coordination
between the veterinary sector and human sector.
Third, the readily transmissible nature of most ERI
puts all countries at risk. SARS is a particularly contagious
disease. Nipah encephalitis spreads rapidly among pigs andcould potentially do so in humans if effective control in animals is not put in place rapidly. Migratory birds help spread
H5N1 infections to all corners of the globe. The rate of the

Figure 1: What emerging and re-emerging infections (ERI) to expect?

Figure 2: Global meat prices as a consequence of HPAI outbreaks (Source: FAO)１
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１

spread is virtually independent of geopolitical boundaries
and economic, social and public health development status,
although countries with better-developed public health systems and infrastructures may be able to control the diseases
more effectively and efficiently. This is a reminder that ERI
cannot be fought in one country alone.
Fourth, most ERI not only take lives but also result in
reduced or lost livelihoods of human beings. SARS reduced
global air travel. Nipah viral infection resulted in the cullingof a large number of pig reservoirs and cases. H5N1 diseases among poultry and in humans resulted in the massiveculling of domesticated chickens and ducks. There is evidence that the livelihood of agricultural workers has been
affected and the price of chicken meat (as the direct impact)and of other meats such as pork and beef (as a compensatory mechanism) increased significantly as a result of theculling, as shown in Figure 2. Since direct and indirect economic losses from ERIoutbreaks are enormous, many people and organizations are concerned that economic issues
may eclipse problems related to the health of the people.
Fifth, we do not have specific interventions for most
ERI. Most ERI are viral, and there are no specific antivirals
nor vaccines for the infections. Although efforts are underway to develop a vaccine for SARS, it is not yet available.
We have specific vaccines for circulating strains of human
influenza viruses, e.g. type A (H1, H3), type B, but specificvaccines need to be developed annually to match circulatingstrains. Furthermore, although we believe that vaccines produced ion the same principle as seasonal influenza vaccines
will be effective against H5N1 and other pandemic influenza strains, we are yet to come to terms with how to makean adequate amount of effective vaccines for a pandemic.

Mechanisms available to combat ERI

Given the above characteristics of ERI, it is almost impossible to discuss the prevention and control of ERI in an
isolated single-country context. Most prevention and control efforts involve other countries. International collaboration is a much-welcomed term in current public health practices. Multi-national mechanisms are currently available at
the global level, e.g. World Health Organization (WHO;http://www.who.int/csr/en/), World Organization for Animal
Health (OIE; http://www.oie.int/eng/en_index.htm), andFood and Agriculture Organization (FAO; http://www.fao.
org/ag/againfo/subjects/en/health/diseases_cards/special_avian.html). A number of regional economic forums also
address the issue of ERI, e.g. Asia-Pacific Economic Cooperation (APEC; http://www.apec.org), Association of Southeast Asian Nations (ASEAN; http://www.aseansec.org), andAyeyawady-Chaophraya-Mekong Economic Cooperation
Strategy (ACMECS;: http://www.geis.fhp.osd.mil/aboutGEIS.asp). In addition, there are a number of bilateral collaborations that specifically address ERI-related issues, e.g.
those organized by the US Centers for Disease Control andPrevention and the US Department of Defense.

Guiding principles for dealing with ERI
The operation of international collaborations on ERI
requires a few common principles. The following principles
are proposed as overarching principles that could guide international collaboration, especially in fighting ERI.
First, no country can or is allowed to fight ERI alone,
no matter how well-developed its economic condition or
public health infrastructure. The rapid transmissibility
across geopolitical boundaries demands that we all work together. No country is immune and no one can be complacent. It is therefore a wise strategy for wealthy countries, e.
g. countries in North America and Western Europe, to comeFigure 3: International (global and regional) forums for emerging and re-emerging infections

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to Asia to fight H5N1 at the forefront. However, care should
be taken to avoid infringement on national sovereignty. A
delicate balance has to be maintained.
Second, ERI problems call for a lot of non-health partners, e.g. business and agriculture, and effective control is
not possible without involvement of these partners. It is encouraging to see, at the global revel, a close working relationship between human health and animal health sectors infighting H5N1 diseases. However, this working relationshipmay not be seen at the country and sub-country levels. Lackof such close collaboration at all levels could easily fail us.
Therefore, mechanism(s) must develop to forge and foster
such collaboration in the fight against ERI.
Third, we need to improve our capacity to conduct surveillance and to respond. The importance of surveillancecannot be stressed enough. However, we should not forgetthat the definition of surveillance is “information for action”.
This means that surveillance without response is pointlessand may be useless. At the same time, response without surveillance is blind and can be really harmful.
To respond to ERI more effectively, we need specifictools to tackle ERI. Although basic health sanitation, e.g.
hand wash, cough etiquette, and other improvements ingeneral hygiene, is effective in reducing human-to-humantransmission of ERI, we need more effective interventions.
Development of vaccines and new drugs are tedious andtime-and resource-consuming, but we need to invest in them.
If “we”, the private vaccine and drug manufacturers, are notready, then “we”, the governments, need to kick in. The rolethat governments can play ranges from providing sufficient
supplies of drugs and vaccines (from available channels) to
support for research, development and production of drugs
and vaccines. Unfortunately, most governments do not possess the capacity nor carry the leverage to enhance research,development and production of the capacity necessary.
Most of the capacity to produce drugs and vaccines belongs
to multi-national private companies. In order for a country
at risk of ERI epidemics to be self-sufficient in the vaccinesand drugs needed to combat ERI, the government may needto promote technology transfer from private vaccine-anddrug-manufacturing companies. This technology transfer
may require the cooperation of international organizations,such as the World Health Organization.

REFERENCES
１．Satcher D. Emerging Infections: Getting Ahead of theCurve. Emerg Infect Dis [serial online] 1995 Jan-Mar[cited2006 Dec 10] :1.Available from: URL: ftp://ftp.cdc.
gov/pub/EID/vol 1 no 1/adobe/satcher.vol 1no1. pdf

Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 153-154
Copyright" 2006 by The Japanese Society of Tropical Medicine

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EXPLORING FRESH COLLABORATIVE INITIATIVESFOR COMBATING INFECTIOUS DISEASES IN THE PHILIPPINES
LUNINGNING P. ELIO-VILLA
Recieved 27, October, 2006

Eight of the 10 leading causes of morbidity in the Philippines are infectious in nature, including pneumonia, diarrhea, bronchitis, influenza, tuberculosis, malaria, chicken
pox and measles. Pneumonia and tuberculosis continue tocause a significant number of deaths across the country andpersist to be among the 10 leading causes of mortality. The
diminishing burden of communicable diseases as major
causes of death may be attributed to improved health technology and health care delivery systems. Appropriate strategies and technologies include immunization, improved sanitation and personal hygiene, better nutrition, early treatment
and steady supply of antibiotics made available at the community or at first level health facilities. However, the persistence of communicable diseases as major causes of morbidity points to the fact that much still needs to be done to reduce the occurrence of illness through preventive and promotive health measures.
A 20-year (1981-2000) trend analysis reveals a generaldecline in number of cases, and number of deaths from
communicable diseases of public health importance, which
include, among others, tuberculosis, malaria, schistosomiasis, tetanus, diphtheria, pertussis, measles, rabies, diarrheal
diseases and pneumonia.

munity and appropriate management with oral rehydration
resulted in the reduction of deaths from diarrhea. Reductionof mortality from pneumonia, especially among children,
may be attributed to early recognition of the public andearly diagnosis and treatment with appropriate antibiotics.
HIV/AIDS is “slow and low” in the Philippines, whichmeans that transmission is slow and prevalence is low.
However, it threatens to grow into a major epidemic in the
country.
Although malaria is no longer a leading cause of death,it has remained among the leading causes of morbidity inthe country, especially in rural areas. The fatal consequences and cyclical occurrence of outbreaks of dengue
every three to five years is a public health concern. Efforts
to eliminate filariasis are hindered by limited resources forannual mass treatment in endemic areas. Although theprevalence is declining, it is a source of concern that forsome endemic infectious diseases such as schistosomiasis,areas previously identified as non-endemic areas have beenfound to have new cases.

Although tuberculosis remains to be a major publichealth concern in recent years, effective case finding, disease management with DOTS strategy, and partnership withthe private sector have led to significant improvements inthe prevention and control of the disease.

Emerging infectious diseases, i.e. newly identified orpreviously unknown infections including re-emerging IDs,
cause serious public health problems if not contained as
close as possible to their source. The inherent unpredictability of a variety of previously known infections and unknown diseases can limit the responsiveness of even themost organized health system.

Successful nationwide immunization campaigns undertaken over the years have resulted in the eradication ofpoliomyelitis and a continuous decline in the incidence ofmeasles, tetanus, diphtheria and pertussis. I mproved sanitation, better nutrition and increased awareness of the com-

In 2003, the sudden and unexpected emergence of
SARS presented an opportunity to initiate measures tostrengthen local and international surveillance for emerging
and re-emerging infections as well as to strengthen quarantine and isolation measures, build capacity in laboratory di-

Medical Specialist!
Program Manager for Emerging Infectious Diseases
National Center for Disease Prevention and Control
Department of HealthPhilippines

１
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agnosis and clinical management, strengthen structure, systems and procedures for triaging, infection control, surveillance and epidemiologic investigation, hospital referral, advocacy and risk communication.
Avian influenza (AI) or bird flu due to the highly
pathogenic influenza virus, H5N1, is a grave threat to humanity. The unpredictability of the influenza virus and theserious possible consequences of a pandemic warrant constant vigilance and good planning in order to reduce the impact of a pandemic.
The unexpected and unusual increase in meningococcal disease, with meningococcemia as a predominant form,in the Cordillera Autonomous Region led to the death of atleast 50% of cases in the early stage of this occurrence,causing public anxiety and seriously affecting the economy,
particularly in Baguio City. There is a need to develop theregional capability for laboratory diagnosis and to conduct
research studies on population carriage and serogroups predominant in the Philippines.
Hepatitis C is an emerging infection in the Philippines
with high prevalence among IV drug users studied. There isa need for more epidemiological data among contacts ofpositive cases to determine the extent of transmission and tomonitor and avert the progression of the disease.
Leptospirosis increases during the rainy season as theaftermath of flooding but cases are also recognized in regions with vast farmlands. There is a need to establish alaboratory-based surveillance for leptospirosis, to determine
the burden of disease in the Philippines, and to identify thefactors for susceptibility and severity and the circulating serovars causing various degrees of severity of disease.
Tuberculosis has been identified as a leading cause of
CNS infections. Other etiologies: varicella, herpes zoster,
Japanese encephalitis. There is a need for a continuinglaboratory-based surveillance of meningitis and encephalitis
as identification of a specific etiology will serve as a basis
for specific treatment and vaccination programs.
The current data on Japanese encephalitis may be justthe tip of an iceberg. The national and regional incidence
and burden of disease of Japanese encephalitis and the regional distribution among its animal hosts need to be determined as a basis for national policy and programs.
Despite the continuing decline in mortality from pneumonia, more efforts can be done to strengthen measures for

its prevention and control. There is a need to strengthenlaboratory capabilities to conduct etiologic identification
and antimicrobial resistance surveillance for appropriate
management of cases. Infection control in the health caresetting also needs to be strengthened.
The following areas for possible technical cooperation
may be considered: strengthening systems for early recognition of disease and response of local units, establishment of
real-time surveillance and information systems, building diagnostic and management capacities for infectious diseases
in designated national and sub-national facilities, epidemiological studies on emerging infectious diseases such as
meningococcal disease, melioidosis, Legionella, Hepatitis C,
etiologies of CNS, diarrhea and pneumonia, burden of disease as basis for public health programs for influenza, JE,
leptospirosis, developing well-trained and well-equipped response teams from the national to the local level, preparedness assessment through simulation and other similar exercises, operational studies: evaluation of preparedness of
LGUs, of hospitals, public awareness on various EIDs.

Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 155-158
Copyright! 2006 by The Japanese Society of Tropical Medicine

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A NEW SPECIES OF SIMULIUM (NEVERMANNIA) FROM THE
OGASAWARA (BONIN) ISLANDS, JAPAN (DIPTERA: SIMULIIDAE)
HIROYUKI TAKAOKA1 and KATSUMI SAITO2
Accepted 5, December, 2006
ABSTRACT: Simulium (Nevermannia) satakei sp. nov. is described on the basis of the pupa and mature larvae
collected from the Ogasawara (Bonin) Islands in Japan. This new species, tentatively (due to lack of the adultstage) assigned to the vernum species-group of the subgenus Nevermannia, is characterized in the pupa by four gill
filaments lacking transverse ridges, and in the larva by a small, M-shaped postgenal cleft, antenna without hyaline
bands, and simple rectal papilla. The morphological differences among this new species and the two known species,
S. (N.) uemotoi from Japan and S. (N.) karzhantacum from Uzbekistan and Turkmenistan, are noted. This is thesecond species of the family Simuliidae from the Islands.
Key words: Nevermannia, Simuliidae, Simulium, black fly, Ogasawara Islands, new species

Until now, Simulium (Nevermannia) bonninense(Shiraki), a member of the vernum species-group of thesubgenus Nevermannia Enderlein, was the only species ofthe family Simuliidae so far recorded from the Ogasawara(Bonin) Islands located in the Pacific Ocean, ca. 1,000 km
south-southeast of Tokyo (Shiraki, 1935; Stone, 1964; Saito
et al., 1974; Takaoka et al., 1999). Recently, a pupa and a
few larvae of an unknown species were collected together
with some pupae and larvae of S. (N.) bonninense from asmall stream in Hahajima, one of the Ogasawara Islands.
This is described here as a new species and is tentatively (due to lack of the adult stage) assigned to the vernum
species-group within the subgenus Nevermannia.
The terms for morphological features used here follow
those of Takaoka (2003). Holotype and paratype specimensof the new species are deposited at the Department of Infectious Disease Control, Faculty of Medicine, Oita University,
Oita, Japan.
Simulium (Nevermannia) satakei sp. nov.
DESCRIPTION. Pupa. Body length 2.0 mm. Head . Integument (Fig. 1A) yellowish, moderately covered withsmall tubercles; antennal sheath (Fig. 1B) sparsely covered
with small tubercles; frons with 2 medium-long slender
trichomes (Fig. 1C) on each side; face with 1 long stout
trichome (Fig. 1D) on each side, which is 1.3―1.7 times aslong as those of frons. Thorax. Integument yellowish, moderately covered with small tubercles, and on each side with1

3 long stout simple trichomes (Fig. 1E) mediodorsally, 2long simple trichomes (1 somewhat shorter and more slender than the other) (Fig. 1F) anterolaterally, 1 medium-longsomewhat stout simple trichome (Fig. 1G) posterolaterally,and 3 short slender simple trichomes (Fig. 1H) [though 1
additional medium-long slender trichome (Fig. 1I) was present on the right side] ventrolaterally. Gill (Fig. 1J) with 4
slender thread-like filaments arranged in dorsal and ventral
pairs arising from short common basal stalk; stalk of ventral
pair slightly shorter than common basal stalk but slightly
longer than the stalk of dorsal pair; dorsalmost filament andventralmost one basally diverged vertically at a right angle
when viewed laterally; all filaments subequal in thickness to
one another; lengths of all filaments not measurable due toloss of apical portion except dorsal filament of dorsal pair(2.7 mm long) and ventral filament of ventral pair (2.1 mmlong) of right gill; all filaments light yellow, gradually tapered toward apex, furnished with annular furrows but lacking ridges (Fig. 1K), and densely covered with minute tubercles on outer surface. Abdomen. Dorsally, all segments
weakly sclerotized and pale yellow; segments 1 and 2
sparsely or moderately covered with small tubercles (Fig. 1
L); segment 1 with 1 medium-long simple slender seta (Fig.
1M) on each side; segment 2 with 1 medium-long simple
slender seta and 5 short dark spinous setae (Fig. 1N) on
each side; segments 3 and 4, each with 4 dark stout hooks
and 1 short spinous seta on each side; segments 5―9 eachwith spine-combs and comb-like groups of minute spines
lying transversely along anterior margin (Fig. 1O) on each

Department of Infectious Disease Control, Faculty of Medicine, Oita University, Hasama, Yufu City, Oita, 879-5593 Japan
Laboratory of Medical Zoology, College of Environmental Health, Azabu University, 1-17-71, Fuchinobe, Sagamihara City, Kanagawa, 2298501 Japan

2

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Fig.1. Pupa of Simulium (Nevermannia) satakei sp. nov.
A, frons and part of face (left half); B, antennal sheath; C, frontal trichome; D, facial trichome; E―I, thoracic trichomes (E, mediodorsal; F, anterolateral; G, posterolateral; H and I, ventrolateral); J, gill filaments (right side; lateral view); K, basal part of gill filamentshowing lack of transverse ridge; L, dorsal surface of abdominal segments 1 and 2 (right half); M, medium-long simple slender seta on
dorsal surface of 1st abdominal segment; N, medium-long simple slender seta and short spinous seta on dorsal surface of abdominal
segment 2; O, spine-combs and comb-like groups of minute spines on abdominal segment 9 (right half); P, terminal hooks (end view);
Q, cocoon (dorsal view). Scales. 0.5 mm for Q; 0.1 mm for J; 0.04 mm for A, B and L; 0.02 mm for C―I, K, M, N, O and P.

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Fig.2. Mature larva of Simulium (Nevermannia) satakei sp. nov.
A, mandible; B, hypostoma; C and D, ventral surfaces of head capsules showing postgenal clefts of different sizes and shapes. Scales. 0.05 mm for C and D; 0.02 mm for B;
0.01 mm for A.

side; segment 9 with pair of cone-shaped terminal hooks(Fig. 1P). Ventrally, segments 3―8 nearly transparent andsegment 9 weakly sclerotized and pale yellow; segment 3
with 3 short simple setae on each side; segment 4 with 1
simple dark hooklet (slightly shorter and smaller than thoseon segments 5―7) and 3 short simple setae on each side;
segment 5 with 2 bifid dark hooks and a few short simple
setae on each side; segments 6 and 7 each with 1 bifid dark
inner hook and 1 simple or bifid dark outer hook, and a few
short simple setae on each side; segments 4―8 with comblike groups of minute spines. Segment 9 with short simple
seta on each lateral side. Cocoon (Fig. 1Q). Simple, wallpocket-shaped, moderately woven, with anterior margin
somewhat thickly woven, and extending ventrolaterally;
floor woven on posterior 2/5; individual threads visible; 2.5mm long by 2.0 mm wide.
Mature larva. Body length 3.5―4.0 mm. Body color
creamy yellow. Cephalic apotome yellow; head spots all
positive and medium brown. Lateral surface of head capsuleyellowish except eye-spot region yellowish-white, with eyebrow light brown; 2 large and１small spots just before posterior margin, as well as 2 small spots below eye-spot re-gion all positive and medium brown. Ventral surface of headcapsule yellow; 1 elongate spot on each side of postgenal
cleft positive and medium brown. Cervical sclerites composed of 2 rod-like small pieces, not fused to occiput,
widely separated from each other. Antenna composed of 3
segments and apical sensillum, much longer than stem oflabral fan; proportional lengths of 1st, 2nd, and 3rd segments 1.00: 0.62―0.78: 0.83―0.93. Labral fan with about 34
rays. Mandible (Fig. 2A) with 1st comb-tooth longest; 2nd
and 3rd comb-teeth subequal in length to each other; mandibular serrations composed of 2 teeth (1 large and 1 small);large tooth at a right angle to mandible on apical side; 2―4
supernumerary serrations present. Hypostoma (Fig. 2B)with a row of 9 apical teeth, the median tooth and corner
teeth being most prominent, and median tooth of 3 intermediate teeth on each side smallest; lateral margins with welldeveloped teeth; 3 or 4 hypostomal bristles in a row, subparallel to, or slightly diverging from, lateral margin on eachside. Postgenal cleft (Fig. 2C,D) small, M-shaped, 0.41―
0.64 times as long as postgenal bridge. Thoracic and abdominal cuticle almost bare except dorsal surface of a few
posterior segments sparsely to moderately covered with col-

１５８

orless minute setae and areas on both sides of anal sclerite
moderately covered with colorless short setae. Rectal scales
absent. Rectal papilla simple, without secondary lobules.
Anal sclerite X-shaped, anterior arms 0.8 times as long as
posterior ones; accessory sclerite absent. Ventral papillae
present ventrolaterally. Posterior circlet of hooks with about
64 rows of up to 15 hooks per row.
Female and Male. Unknown.
TYPE SPECIMENS. Holotype pupa with its associated cocoon, collected from a small shaded stream (width about 10cm) slowly flowing in a forest, located on the right side of
Chibusa Dam, Hahajima, Ogasawara Islands, Tokyo, Japan,
18.VI.2005, by K. Satake. Paratypes: 2 mature larvae and 1
immature larva, same locality and data as those of the holotype.
ETYMOLOGY. The species name satakei honors Dr. K.
Satake, who collected this new species.
REMARKS. This new species is tentatively assigned to thevernum species-group of the subgenus Nevermannia by
having the four gill filaments per side in the pupal stage, theantennae without any transverse hyaline bands, the mandible with supernumerary serrations, the main tooth of themandibular serrations at a right angle on the apical side to
the mandible, the hypostoma with serrated lateral margins,
and the ventral papillae well developed in the larval stage.
The larva of this new species is very similar to that ofS . (N .) uemotoi of the vernum species-group from Japan(Sato et al., 2004): it shares several characteristics including
the small, M-shaped postgenal cleft and the simple rectal
papilla. There are some differences, however, in the relative
length of the three segments of the larval antennae (1.00:
0.62―0.78: 0.83―0.93 versus 1.0: 1.2―1.4: 0.8―1.0) between
the two species. On the other hand, the pupa of this new
species is easily distinguished from that of S . (N .) uemotoi
by the following characteristics (those of S . (N .) uemotoi
are shown in parentheses): frontal trichomes in two pairs (inthree pairs), antennal sheath sparsely covered with small tubercles (bare), transverse ridges on the gill filaments absent(present), dorsal surface of the abdominal segments 1 and 2
sparsely covered with minute tubercles (densely and neatly
covered with minute tubercles), and spine-combs on the abdominal segment 9 distinct (absent or indistinct if any).
This new species is also similar to S . (N .) karzhantacum (Rubtsov, 1956) from Uzbekistan and Turkmenistan, which has a similarly shaped larval postgenal
cleft, but differs in the pupal stage from the latter species bylacking the transverse ridges on the surface of the gill filaments.

Simulium (Nevermannia) satakei sp. nov. represents
the second species recorded from the Ogasawara Islands. It
should be noted that this new species is not closely related
to S . (N .) bonninense, which to date had been the only species prevalent in the islands, because there are distinct differences in the relative length of the first and second segments of the larval antennae and in the shape of the larval
postgenal cleft as well as in the presence or absence ofspine-combs on the dorsal surface of the fifth and ninth segments of the pupal abdomen between the two species.

ACKNOWLEDGEMENTS
We are grateful to Dr. K. Satake, National Institute forEnvironmental Studies, Tsukuba, Japan, for kindly providing his black fly specimens for our examination.

REFERENCES
Rubtsov, I.A. 1956. Blackflies (fam. Simuliidae) [Moshki (sem.
Simuliidae)]. Fauna of the USSR. 859pp., New Series No.64,
Insects, Diptera 6 (6). Akademii Nauk SSSR, Leningrad [=St.
Petersburg], Russia. In Russian. [English translation: 1990.
Blackflies (Simuliidae). 1,042 pp., 2nd Ed. Fauna of the USSR.
Diptera, 6 (6). E.J. Brill, Leiden].
Saito, K., Hori, E. and Ogata, K. 1974. Simuliidae of Ogasawara
Islands. Jpn. J. Sanit. Zool., 24: 338 (Japanese abstract only).
Sato, H., Takaoka, H. and Fukuda, M. 2004. A new species ofSimulium (Nevermannia) (Diptera: Simuliidae) from Japan.
Med. Entomol. Zool., 55: 201―210.
Shiraki, T. 1935. Simuliidae of the Japanese Empire. Mem. Fac.
Sci. & Agr. Taihoku Imp. Univ., 16: 1―90.
Stone, A. 1964. Diptera: Simuliidae. Insects of Micronesia, 12:
629―635.
Takaoka, H. 2003. The Black Flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. xxii+581pp., Kyushu University
Press, Fukuoka.
Takaoka, H., Saito, K. and Suzuki, H. 1999. Simulium (Nevermannia) bonninense from the Ogasawara (Bonin) Islands, Japan(Diptera: Simuliidae): taxonomic assignment to the vernumgroup and descriptions of male, pupa and mature larva. Jpn. J.
Trop. Med. Hyg., 27: 189―194.

Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 159-166
Copyright! 2006 by The Japanese Society of Tropical Medicine

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UVULECTOMY AND OTHER TRADITIONAL HEALING PRACTICES:
TRADITIONAL HEALERS’ PERCEPTIONS AND PRACTICESIN A CONGOLESE REFUGEE CAMP IN TANZANIAOSAMU KUNII1, YASUO TANAKA2, ALYSON LEWIS3, SUSUMU WAKAI2
Accepted 17, December, 2006
ABSTRACT: Little is studied about traditional healers’ perceptions toward and practice of uvulectomy, which is
known as a traditional surgical practice mainly in Africa and which sometimes results in severe complications. This
study aimed to clarify the perceptions toward and practice of uvulectomy and the other traditional healing practicesof traditional healers in a Congolese refugee camp in Tanzania. Interviews were conducted with 149 traditional
healers, comprised of 59 registered, 68 non-registered and 22 faith healers.
A total of 1.7% of the registered healers and 8.8% of the non-registered healers had ever conducted uvulectomy on children (a median of 2 months to a median of 3 years of age) and had received cash or domestic fowls
equivalent to US$1-3 per operation. Although over 80% of the respondents believed traditional treatments to be
more effective than modern medicine, less than 20% considered uvulectomy beneficial and in fact about 40% considered it to be harmful. The respondents raised cough, vomiting, appetite loss and other symptoms as an indication
for uvulectomy, and death, bleeding, throat pain and other symptoms as harmful effects associated with uvulectomy. In this camp, the healers also performed other surgical procedures, such as male and female circumcision,
tattoos and scarification.
In conclusion, only a limited number of the traditional healers believed that uvulectomy is beneficial and performed it on infants and young children, and these were mainly non-registered healers who had relatively little collaboration with modern health professionals. In refugee settings where modern health professionals might not befamiliar with traditional healing, it is considered crucial to assess the risks of ongoing traditional practices and to
strive to achieve more strategic communication between modern and traditional health providers.
Keywords: traditional healing, healers, uvulectomy, perception, Congolese refugees, Tanzania

INTRODUCTION
The persisting conflicts in the Great Lakes region of
Africa have caused the flow of a large number of refugees
into the United Republic of Tanzania. Tanzania has maintained an open-door policy since its independence, and, as aresult, it hosted approximately 520,000 refugees including
more than 370,000 from Burundi and about 140,000 from
Congo-Kinshasa, 3,500 from Somalia, and 2,700 from
Rwanda by the end of 2002 [1].
In response to the extremely poor health status of theserefugees, modern health care services have been provided
intensively in refugee camps, when possible, by skilled ortrained refugees and persons from the host community andaid organizations. However, there are still many refugeecommunities where traditional healing practices arestrongly preferred.
While modern health intervention has contributed
1greatly to the improvement of the heath status in manycamps, efforts to reduce infant mortality have stagnated insome camps [2]. Although no survey was conducted there,
modern health providers in these camps suspected a traditional surgical practice called ‘uvulectomy’ to be one of thecauses. Uvulectomy is a procedure in which the uvula issevered. It is a traditional healing practice used mainly in
Africa, sometimes leading to serious complications [3, 4, 5].
However, no study has shed light on the perceptions of traditional healers as to its beneficial and adverse effects or its
actual practice, especially in refugee settings. In the refugee
camps, moreover, little was known about perceptions toward or practices of traditional healing methods other thanuvulectomy.
This study aimed to determine the traditional healers’
perceptions and their implementation of traditional healing
practices, with special reference to uvulectomy.

Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
Department of International Community Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
3
International Federation of Red Cross and Red Crescent Societies, Geneva, Switzerland
2

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PARTICIPANTS AND METHODS
Study area
We selected Lugufu Camp, one of the biggest refugee
camps in Kigoma Province, western Tanzania. It accommodated about 50,000 refugees from Congo-Kinshasa at thetime of our study, and it continued to grow by an average of
1,000 refugees per month due to persistent armed conflicts,
political instability, and deteriorating humanitarian conditions in that country. The camp was in the post-emergency
phase with a crude mortality rate of 0.65 deaths/10,000 persons/day and an under-five mortality rate of 1.88/10,000
persons/day in 2000, thus indicating that the relief programs
had successfully kept the health situation under control. [6]Participants
Since there was no reliable document or registration
method to identify traditional healers in the study site, wegathered information through preliminary interviews withmodern health professionals and community health workers
in the camp prior to the study. The results indicated that
there were three types of healers, namely, registered traditional healers, non-registered traditional healers and faith
healers. In the local language, the three types of healer are
called ‘MFUMU’ or ‘MTEE’, ‘MLAKO’, and ‘BAYUMBE’ or ’MAHA WA ASA’O’, respectively The ‘MFUMU’or ‘MTEE’ was registered with the Ministry of Health inCongo-Kinshasa and was certified to examine and treat patients, supposedly with herbs and other medicinal subjects.
The ‘MLAKO’ provided traditional healing practices without certification. The ‘BAYUMBE’ or ’MAHA WA ASA’O’
was a faith healer, also referred to as a ‘prayer leader’,‘elder prayer’, ‘father/mother of prayer’ or ‘a director of
prayer’, who usually organized religious gatherings andprovided healing mainly through prayers.
Study Preparation
Permission to carry out the study was obtained fromthe UNHCR and Tanzania Red Cross Society, with the support from the International Federation of Red Cross and
Red Crescent Societies, organizations that took responsibility for health and other related activities in the camp. We
made a list of traditional healers that had been identified in
the camp by Congolese community health workers, and invited all of them to participate in our study. Informed consent was obtained, and finally all the healers in the list, 149
individuals in total, agreed to participate.
Eighteen Congolese refugees engaged in community
health services speaking and writing English and Swahili
were recruited and trained as interviewers for the study. We
also recruited and trained Tanzanian health personnel com-

petent in speaking and writing both English and Swahili as
study supervisors. The authors were responsible for the
training of both interviewers and supervisors and checkedand confirmed the quality of supervisions. The study was
conducted from May to July 2001.
Questionnaire
Semi-structured interviews were designed to explore
traditional healers’ perceptions toward and use of uvulectomy and other traditional healing practices, and their attitude toward modern health care services. Background information gathered from each respondent included religion,
years of education, and the person from whom the healing
practices were learned. Special efforts were made to obtain,in the respondent’s own words, of the healing practices andtheir indications, and the perceived benefits and harm ofuvulectomy. The respondents who used herbal medicine
were further asked about the type of herbal medicine, their
preparation, usage and indications.
To quantify the perceived benefits and harm of traditional practices, the interview also included the following
questions: “In comparison to modern medicine, how effective do you think your traditional treatments are?” with fourchoices “All of my treatments are more effective than modern medicine”, “Some of my treatments are more effective
than modern medicine”, “None of my treatments are more
effective than modern medicine” and “I don’t know”; “To
what extent do you trust modern doctors?” with fourchoices “very much”, “somewhat”, “not at all”, and “I don’t
know.”; “To what extent do you think uvulectomy is beneficial or effective?” with four choices “very much”, “somewhat”, “not at all”, and “I don’t know”; “To what extent doyou think uvulectomy is harmful or results in a negative effect?” with four choices “very much”, “somewhat”, “not atall”, and “I don’t know”. The interview also involved somequestions on the practice of uvulectomy such as the age ofthe patients and the amount of money or gifts received fromthe patients undergoing the uvulectomy procedure.
Analysis
The chi-square test was used to compare categoricalvariables using the SPSS statistical software package version 10.0 for Windows. The descriptive data and their interpretations were anonymously examined for specific meanings and clustered into meaningful groups. To quantify indications for healing practices and harms of uvulectomy, one
common term was labeled in each group. We also used
qualitative data by selecting typical expressions in eachgroup to complement or interpret the results of analyses ofquantitative data.

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all of the non-registered healers used herbs for treatment,but only 13.6% of the faith healers did so. Various parts ofmedicinal plants and trees including fruit peel and sap wereused, but the most popular choices were the roots, leaves
and flowers of plants and tree bark. The healers mainly prepared concoctions for oral administration from these plants,
some of which were used as a laxative to purge the body of‘impure spirits’ thought to cause illnesses. Some herbs wereground into paste or powder and then applied to sites affected by fractures, cancer and other illnesses. Concoctions
were also used as enemas to cure various illnesses, andpaste from herbs was used as a suppository to treat hemorrhoids. Other treatments included steam inhalation of theherbs and application of herbal powder/paste to tattoos or
scarified areas. Plant roots combined with tree bark wereused for the treatment of post-abortion problems, sometimes by insertion into the vagina.
Most of the healers selected a specific treatment foreach illness, sometimes using a combination of treatments
and trying an alternative treatment when no results wereseen. In addition to herbs, the respondents used bones andother parts of animals, honey, salt, oil and certain types ofsoil for treatment.
As shown in Table 3, the indications perceived to betreated effectively by traditional healing practices andherbal medicine ranged from acute illnesses such as cel-RESULTS
Profile of the respondents
Among the 149 traditional healers, 59 (39.6%) wereregistered healers, 68 (45.6%) were non-registered healers,and 22 (14.8%) were faith healers. As shown in Table 1,
there was no significant difference in the age distribution or
years of education among the three types of healers. Males
were dominant among the registered healers, while morethan half of the faith healers were women. The religion of
the three groups of healers was significantly different: Muslims made up most of the registered healers, while Christians comprised most of the non-registered and faith healers.
Some of the registered and non-registered healers followed
other traditional or indigenous religions or beliefs such as
Baha’i, Kimbansiste, and Kitawara orthodox religions.
The persons from whom the respondent had learned
healing practices also significantly differed among the threegroups as follows: about 80% of the registered healers hadlearned from family members, about 40% of the nonregistered from traditional healers, and about 60% of thefaith healers from other individuals such as ‘inspiration’,‘God’ and ‘clergy persons’.
Traditional healing practices and their indications
As shown in Table 2, all of the registered and almost

Registered healers

Total (N=149)
Age (years)
20-29
30-39
40-49
50-59
Sex
male
femaleReligion
ChristianMuslimothers
Education (years)
0
1-6
7-14
Who taught healingfamilytraditional healer
by oneself
others

Non-registeredhealersN%

Faith healers

p-value

N

%

N

%

59

100

68

100

22

100

14
16
15
14

23.7
27.1
25.5
23.7

16
26
14
12

23.5
38.2
20.6
17.7

5
9
7
1

22.7
40.9
31.8
4.6

0.459

43
16

72.9
27.1

40
28

58.8
41.2

9
13

40.9
59.1

0.025

15
24
20

25.4
40.7
33.9

25
22
21

36.8
32.3
30.9

18
2
2

81.8
9.1
9.1

<0.001

10
18
31

17
30.5
52.5

10
26
32

14.7
38.2
47.1

3
11
8

13.6
50
36.4

0.6

47
10
2
0

79.7
16.9
3.4
0

34
27
3
4

50
39.7
4.4
5.9

4
0
5
13

18.2
0
22.7
59.1

<0.001

Table 1. Profile of the three groups of traditional healers１６２

lulites, diarrhea and wounds, to chronic illnesses such as
cancer and diabetes. The registered and non-registered healers treated similar indications, but the faith healers also
treated other indications related to mental problems and sterility.
The exorcism of evil spirits was practiced by 30.5% of
the registered and 25.0% of the non-registered healers, but
not by the faith healers. As shown in Table 3, the registeredand non-registered healers exorcised evil spirits as a treatRegistered healers
N (=59)%Use herbs
yesno
Exorcise evil spirits
yesnoEver performed uvulectomyyesnoEffectiveness of traditional healing
compared to modern medicine
All are more effective
Some are more effective
None is more effective
Don’t know
How beneficial is uvulectomy
Very much
Somewhat
Not at all
Don’t know
Do you know the benefits of uvulectomyyesnoHow harmful is uvulectomy
Very much
Somewhat
Not at all
Don’t know
Do you know the harmful effects ofuvulectomyyesnoTrust modern doctors
Very much
Somewhat
Not at all
Don’t know
Cooperate with modern doctors
Very much
Somewhat
Not at all
Don’t know

ment for what they called ‘madness’ or ‘impure spirits’, ‘invisible’ illnesses, sterility, and so forth, in addition to examining such illnesses. The healers who answered ‘impure
spirits’ explained that they caused not only mental but also
physical problems.
Prayer was the main treatment procedure, and for somethe only treatment procedure, among the faith healers, who
explained that their healing prayers were different from theexorcising of evil spirits.
Nonregistered healers
N (=68)%Faith healers
N (=22)%p-value

59
0

100
0

67
1

98.5
1.5

3
19

13.6
86.4

<0.001

18
41

30.5
69.5

17
51

25
75

0
22

0
100

0.015

1
58

1.7
98.3

6
62

8.8
91.2

0
22

0
100

0.088

3
47
6
3

5.1
79.6
10.2
5.1

8
41
4
15

11.8
60.3
5.9
22

1
15
0
6

4.5
68.2
0
27.3

0.028

1
4
14
40

1.7
6.8
23.7
67.8

2
12
13
41

3
17.6
19.1
60.3

0
4
4
14

0
18.2
18.2
63.6

0.009

14
45

23.7
76.3

18
50

26.5
73.5

2
20

9.1
90.9

0.235

16
2
0
41

27.1
3.4
0
69.5

12
15
2
39

17.6
22.1
2.9
57.4

3
7
0
12

13.6
31.8
0
54.6

0.013

13
46

22
78

25
43

36.8
63.2

10
12

45.5
54.5

0.074

35
24
0
0

59.3
40.7
0
0

41
23
1
3

60.3
33.8
1.5
4.4

17
5
0
0

77.3
22.7
0
0

0.302

36
21
1
1

61
35.6
1.7
1.7

32
27
5
4

47.1
39.7
7.4
5.9

14
5
2
1

63.6
22.7
9.1
4.6

0.344

Table 2. Traditional healers’ practices and perceptions of healing and uvulectomy and attitudes toward modern doctors

１
６
３

The practice of uvulectomy
Uvulectomy is called ‘ELEMI’ in Kibembe, the refugees’ local language. Only 7 (4.8%) of the healers had everconducted uvulectomy: 1 (1.7%) of the registered healers, 6(8.8%) of the non-registered and none of the faith healers; 1(1.7%) of the 58 Christians, 2 (4.2%) of 48 Muslims and 4(9.3%) of the 43 other religious beliefs. The above sevenhealers had performed uvulectomy repeatedly, for a total of
47 cases: one registered healer had treated 2 cases and thesix non-registered healers had conducted an average of 7
cases each (range: 2-12cases).
The recipients of uvulectomy were mostly infants andchildren, starting from a median of 2 months of age (range:
1week-4months) up to a median of 3 years (range: 2-5
years). Five of the healers received 1,375 Tanzanian shillings (TZS) in cash (equivalent to US$1.5 as of May 2001)
on average (range: TZS 1,000-2,000; US$1.1-2.2) per
uvulectomy, while the other two healers were given a hen(equivalent to TZS 1,400; US$1.6) and a duck (equivalent
to TZS 2,000; US$2.2) per uvulectomy.

Top five indications
treated effectively
by traditional healing

Top five indications
for herbs

Top five indications
for exorcising

Indications foruvulectomy

Harmful effects
of uvulectomy

Perceived effects of traditional healing and attitudes towardmodern medicine
Over 80% of the traditional healers believed that ‘all orsome of the traditional treatments were more effective thanmodern medicine’. However, 11.8% of non-registered healers believed that ‘all the traditional treatments were more
effective than modern medicine’, while 10.2% of the registered healers believed that ‘none of the traditional treatments were more effective than modern medicine’.
Over 98% of all respondents trusted modern doctors‘very much’ or ‘somewhat’. In practice, 96.6% of the registered healers cooperated with modern doctors either ‘very
much’ or ‘somewhat’. However, more than 10% of the nonregistered and the faith healers did not cooperate with modern doctors at all or did not know to what extent they cooperated.
Perceived effects of uvulectomyMore than half of the respondents did not knowwhether uvulectomy was effective or harmful. In all three

Registered healers(n)Non-registered healers(n)Faith healers(n)fracture
diabetes
epilepsycancerhemorrhoid

19
17
10
9
8

fracture
diabetesblisters
cellulites
diarrhea

10
8
7
6
6

madnesssterility
epilepsy
impure spirits
mental trouble

7
7
5
2
2

diabetes
fracturecancerepilepsycellulites

22
19
16
12
9

cellulites
sterility
diarrhea
fracture
wound

12
11
11
10
9

sterility
abortion
impure spirits
mental trouble
paralysis

3
1
1
1
1

impure spirits
madness
examination
sterility
invisible illness

8
4
3
2
2

madness
examination
impure spirits
cellulites
epilepsy

6
6
5
1
1

cough
vomiting
appetite loss
baby’s crying
throat pain
death

2
1
1
1
1
1

cough
vomiting
appetite loss
throat pain
baby’s crying
fever

5
4
2
2
1
1

cough
appetite loss
throat pain
fever

3
1
1
1

death
bleeding
throat pain/swelling
nerve injury

8
7
2
1

bleeding
death
throat pain/swellingcoughappetite loss

15
11
10
3
1

bleeding
death
throat pain/swelling
appetite loss

7
4
3
1

Table 3. Indications for healing practices and the harmful effects of uvulectomy as perceived by traditional healers１６４groups of healers, those who thought uvulectomy ‘very
harmful’ were greater in number than those who thought it‘very effective’.
Those who considered uvulectomy ‘very effective’ or‘somewhat effective’ were more likely to be non-registeredhealers than the others, while those who considered uvulectomy ‘very harmful’ or ‘somewhat harmful’ were more
likely to be faith healers.
None of those who considered uvulectomy ‘very effective’ cooperated ‘very much’ with modern doctors, while
25.6% of those who considered uvulectomy ‘very harmful’cooperated ‘very much’ with modern doctors.
As shown in Table 3, the three groups of healers gave
similar answers regarding the indications for uvulectomy,such as cough, appetite loss and sore throat. They described
the indications as follows: “Uvulectomy is effective forcough, vomiting, and throat dryness that stops the passageof air or oxygen.” “If uvulectomy is done, patients can theneat food, because a uvula prevents food from passing
through throat.” “Uvulectomy can help babies when they
have a cough, sore throat or high fever and when they can’teat.” “If uvulectomy is not done in time, a patient will die
because of blocked respiration and swelling of the throat.”
Several responses were observed regarding the harmful
effects of uvulectomy among the three groups of healers.
The healers pointed out bleeding, death, sore throat, appetite loss, nerve injury, and responded as follows: “I haveseen somebody die from cutting the uvula because the patient’s nerve was cut and throat swelling occurred.” “Blood
discharge can cause death.” “Throat inflammation andblood discharge can cause death.” “A patient may get thinner, vomit frequently and develop a sore throat.”
Thirteen respondents responded that uvulectomy was
harmful only if inappropriately done. “Uvulectomy can be
dangerous if the person who does it is a charlatan. The patient may die.” “Uvulectomy is safe if it is done by an experienced nurse in the dispensary.” “When done poorly, thehealer may cut the tonsils and the patient may bleed todeath.” “When uvulectomy is done poorly, it causes a sorethroat and the patient is not able to eat.” “If one doesn’tknow how to cut properly, the procedure will hurt the throat
and the patient cannot eat.”Six of the seven healers who had performed uvulectomy in the past responded that the procedure was ‘somewhat effective’ and also ‘somewhat harmful’, and responded
as follows: “If one doesn’t know how to cut the uvula, thepatient may develop a sore throat. But I have done it in theCongo and had no problems.” “A person may develop asore throat after uvulectomy. It is dangerous if one doesn’tknow how to cut it properly. It is particularly dangerous forbabies. I performed uvulectomy in the Congo, but here I do

not have the proper instruments.” “If it is done by someone
who doesn’t know how to do it, it can cause serious problems.” “A sore throat and bleeding occur after uvulectomy,
but there is a type of root which I can use as a medicine tostop such problems.” “One must have sufficient experiencebut I think there is no problem for adults.” “Uvulectomy affects the throat. If the healer doesn’t know how to cut, it cancause respiratory problems.” The one remaining healer who
had performed uvulectomy said, “Uvulectomy is not goodat all. Actually it is very harmful. It causes bleeding anddeath.”
Other surgical practices
Some surgical procedures other than uvulectomy were
performed by the healers in the camp, such as male and female circumcision, tattoo, scarification and hemorrhoidectomy.
The female external genitalia, especially the clitoris,
and the male hemorrhoid were both called ‘EHANYA’ inthe local language. They were described as follows:“Women with EHAYA cannot get pregnant. So traditional
birth attendants cut the EHANYA of women who want to
get pregnant.” “EHANYA is cut away with a razor blade or
knife. After the operation, traditional medicine is applied tothe site.” “It is usually effective, but some people say it is
not good because many recipients get sexual diseases.”“About one in 10 women receive this procedure and the results are good. But sometimes it causes severe bleeding andinfection from sexual diseases and HIV/AIDS. “Male circumcision is called “BOTENDE”. “The prepuce of the penis is removed with a machete because in ourtribe you are considered to be a child if you still have it.”
“Most men receive BOTENDE and it is necessary to win respect as an adult or grown-up.” “It is difficult to do. If you
perform it improperly, you will kill many people.”
The herbal application used to treat scarified areas ofskin is called “EMBE”. “If someone is suffering from local
pain, the skin in the painful region is cut with a razor bladeor knife and then the resin of a tree is applied to the bleeding site.” “It cures diseases. But it can also cause excessive
bleeding and scaring.” “If one razor blade is used on many
patients, it can also cause the transmission of HIV.”DISCUSSIONS
Uvulectomy was widely performed in African nations
such as Tanzania [7-9], Ethiopia [10-12], Sudan [13], Nigeria [14, 15, 16], Morocco [17], Cameroon [18], Niger [19],and in Middle Eastern countries including the South Sinai[20, 21] and Saudi Arabia [22]. Although it was reported as
a common practice [5, 12, 23], its prevalence varied among

１６５

the tribes, regions and countries [11, 19, 20].
This study demonstrated that various types of traditional healing practices were being conducted for different
illnesses in the refugee camp and that most healers wereconfident with their effectiveness. Among the healers in thiscamp, however, uvulectomy was not a common practice andonly a limited number of registered and non-registered healers had performed it. Uvulectomy was performed on infants
and young children by the age of five in this camp, a finding
consistent with previous reports [16, 18, 24, 25]. However,in some reports the operation was performed on newborns
soon after birth [3, 17].
In our preliminary interview, modern health professionals in the study site suggested that most of the traditional healers believe in the beneficial effects of uvulectomy.
However, our results showed that many healers did notknow the effect of uvulectomy and that those who thought
it harmful outnumbered those who thought it beneficial.
Indications for and adverse effects of uvulectomyraised by the healers in our study were similar to those described in other studies [17, 26, 27, 28]. Moreover, previous
studies reported that uvulectomy was performed for its prophylactic and/or curative effect on abdominal pain, insomnia [3] and chronic diarrhea [22]. Other studies reported
bronchopneumonia, tetanus, meningitis, sepsis, dehydration,
edema of the glottis and cellulites of the neck as complications of uvulectomy diagnosed by modern professionals [3,10, 13, 29-33].
However, our study indicated that a limited number ofhealers had repeatedly conducted uvulectomy and that mostof them believed it to be effective, although some of themknew its complications. In this camp, special huts were built
within modern health compounds for traditional healers topractice their procedures and to collaborate with modern
health professionals. However, our preliminary interviews
with modern health professionals revealed that not many of
the traditional healers had used the huts or engaged in any
real collaboration. Our findings showed that the nonregistered healers had cooperated less with modern healthpersonnel and performed uvulectomy more frequently thanthe other healers.
In addition to uvulectomy, healers performed traditional surgical practices such as female genital circumcisionand scarification, which had potential complications [34,35]. However, as was the case in this study site, these risky
surgical practices were not regularly surveyed or assessed in
refugee camps.
Our study has some limitations. It could not be generalized to other refugee camps because perceptions and practices of traditional healing might differ according to countryof origin, ethnicity and culture among the displaced popula-tions. Semi-structured interviews might not be enough tocapture all the details of healers’ perceptions, beliefs andpractices, although this study attempted to gather qualitative
information to complement quantitative data.
Access to modern health services has improved, buttraditional healing may continue to be a popular or alternative among those who are accustomed to receiving suchpractices. Especially in emergency or post-emergency situations, modern health professionals might not be well acquainted with the cultures and traditions of refugees or displaced persons who have moved from different places.
When severe complications resulting from traditional practices are identified or expected, interventions could include
not only directly discouraging such practices but also reducing high-risk procedures, for example by urging people to
avoid shared instruments and promoting early contact withor referral to modern health providers.
It might seem easy to just recommend collaboration,
but in reality it is difficult to promote real collaboration between modern and traditional health providers. The firststep could be precise situation analysis, preferably with theparticipation of the healers themselves and community people. The second step could be active dialogue and close
communication of facts and analysis based on mutual respect.

CONCLUSION
In a Congolese refugee camp in Tanzania, a small
number of traditional healers considered uvulectomy tohave beneficial effects on cough, vomiting and other conditions, and in practice had performed uvulectomy on infants
and young children. However, most of the healers who had
conducted uvulectomy were non-registered healers who engaged in relatively little collaboration with modern doctors.
An increased child mortality risk due to the adverse effects
of uvulectomy was suspected. It is recommended, therefore,that the health authorities of the camp identify the healers
who performed this procedure and strive to achieve morestrategic communication and collaboration between modern
and traditional health practitioners.

ACKNOWLEDGEMENTS
This study was funded by the Foundation for Development of the Community. We wish to thank Dr. T. Sugishita,
Dr. T. Ojima and Mr. A. Kaseda for their kind assistance,
Mr. H.S. Kateranya, Mr. Kibasa, Ms. S.M. Luta, and the
Health Information Team members in Lugufu Camp of Tanzania Red Cross Society for kindly assisting us in the study,
and all the traditional healers for participating in the study.

１
６
６

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Tropical Medicine and Health Vol. 34 No. 4, 2006, pp. 167-174
Copyright! 2006 by The Japanese Society of Tropical Medicine

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Population Polymorphism of Trypanosoma cruzi in Latin America indicated byProteome analysis and by in vitro amastigote proliferation
JIAN-BING MU１＊＊, TOSHIO SONE１, TETSUO YANAGI２, ISAO TADA３,MIHOKO KIKUCHI４, and KENJI HIRAYAMA１,４＊
Accepted 26, December, 2006
Abstract: Nineteen stocks of Trypanosoma cruzi originating from several endemic countries for Chagas’ disease inCentral and South America were subjected to two-dimensional protein electrophoresis analysis. The presence orabsence of a total of 492 polypeptide spots among 19 gel profiles was determined. The stocks were classified into
three major distinctive groups derived from (I) Central America and the northern part of South America; (IIa) Central America and the northern part of South America; and (IIb) central and southern parts of South America, whichshowed perfect concordance with the previously reported classification based on isozyme and DNA sequence
analyses. Late log phase of each epimastigote was inoculated to human cell lines WI-38 and Hs 224.T originatingfrom the lung and muscle, respectively, and the number of trypomastigotes released was counted. The number oftrypomastigotes from T . cruzi in group I released from the two cell lines was significantly higher than that in group
III (p<0.05). The findings suggested that the phenetic distance appearing within the T . cruzi may, to some extent,
be associated with the intracellular growth of T . cruzi, one of the characteristic features of growth found in the species.
keywords: Trypanosoma cruzi; Two-dimensional gel electrophoresis; in vitro amastigote proliferation, genetic
polymorphism, Latin America

INTRODUCTION
Chagas’ disease, caused by infection of the flagellate
protozoan Trypanosoma cruzi, is an important health problem in Central and South America, since 16 to 18 million
people in the endemic area suffer from this disease and an
additional 90 million are exposed to the risk of acquiring
the infection [1]. The illness may remain an asymptomatic
infection throughout life or develop into overwhelming
acute myocarditis in infants or cardiomyopathy in patientsin all endemic area and/or digestive forms predominantly inthe southern part of South America [2]. Although the factors influencing this variable clinical course have not been
elucidated, it has been suggested that the association ofclinical symptoms and the severity of the disease in certain
geographic regions may be related to genetic factors in thehuman population and the variability within T . cruzi species [2-9].
The extent of genetic variability within the species T .
１cruzi was studied in terms of its isozyme patterns (zymodemes) [2, 4-10], kinetoplast DNA restriction fragments (schizodems) [11], karyotypes [12, 13], and random amplification of polymorphic DNA (RAPD) [8, 14, 15], leading to aconclusion that T . cruzi exhibits by nature a high genetic
variability. Recently, population-genetic approaches haveshown that T . cruzi presents a typical clonal population
structure and consists of at least two major lineages that arevery ancient events [15-17], while analyses of the population of T . cruzi isolated from humans and insect vectors inmajor endemic areas of Central and South America by zymodeme [16, 18, 19] and polymorphism of several genes[20, 21] suggest the existence of three lineages.
In the present study, the proteins extracted from 19
stocks of T . cruzi isolated in Guatemala, Ecuador, Peru,
Brazil, Paraguay, and Chile were analyzed by twodimensional polyacrylamide gel electrophoresis (2D-PAGE).
The number of shared and unshared polypeptide spots on
the profiles among the stocks indicated how many lineages

Department of Medical Zoology, Saitama Medical School, Iruma, Saitama 350-0495, Japan
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan３
Department of Parasitology, Faculty of Medicine, Kyushu University, Fukuoka, Fukuoka 812-8582, Japan
４
Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University Sakamoto, Nagasaki 852-8523, Japan*
Corresponding author: Kenji Hirayama, Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Sakamoto,Nagasaki 852-8523, Japan
hiraken@nagasaki-u.ac.jp**
Present address Jian-bing Mu, National Institute of Allergy and Infectious Diseases Bethesda, MD 20892-8132, USA
２

１
６
８

Table 1. Origin of the isolates and strains of Trypanosoma cruzi
used in this study
Locality

Zymodeme
Reference
Classification

Name of isolatesHost172, TM14, TM43,
TM47, TM51, TM52,
CL strain
119
H6, H18
H1, H20
Peru 1, Peru 2
Tulahuen
Y strain
GS, LO, RF

T . dimidiata Guatemala I

19, 21

T . infestans Brazil
I
Opossum Ecuador I
Human
Guatemala I
Human
Guatemala IIa
Human
Peru
IIa
T . infestans Chile
IIb
Human
BrazilIIb
HumanParaguay IIb

21
2119, 21
19, 21
19, 2021
2118, 19of T .cruzi exist in the endemic areas of Chagas’ disease.

MATERIALS AND METHODSParasites
Nineteen stocks of Trypanosoma cruzi from different
ecological and geographic origins were used (Table 1). Epimastigotes of individual T . cruzi were maintained at 26°
Cin liver infusion tryptose (LIT) medium supplemented with
10% heat-inactivated fetal calf serum (FCS), according to
the well-established method [22].
Sample preparation
Epimastigotes were collected by centrifugation (400 x
g for 15 min) followed by washing twice with phosphatebuffered saline (PBS). After measurement of the wet weightof the pellet,100 µl of lysis buffer (0.15 M NaCl, 4% Triton
X-100, 10 mM Tris-HCl, pH 8.0) containing 2 mM phenylmethylsulfonylfluoride and 20 µg/ml leupeptin was added
to 100 mg pellet (approximately 1.5 x 10９parasites), mixed
well by vortex, and kept on ice for 10 min. The mixture wasthen centrifuged at 8,000 x g for 10 min and the supernatant
was collected. An aliquot of the solution was drawn off andwas used for the determination of protein concentration using micro BCA protein assay reagent kit (Pierce, Rockford,
IL, USA). Finally, 120 mg urea, 10 µl 2-mercaptoethanol,and 22 µl of 40% Ampholine (pH 3.5-10, Pharmacia,
Uppsala, Sweden) was added to the supernatant. About 2
mg of protein, which corresponded to 50-80 µl of the final
mixture, was used as a sample for 2D-PAGE.
2D -PAGE
2D-PAGE was performed according to the method ofO’Farrell [23] with slight modifications. Briefly, the prepared sample was electrofocused in a tube gel containing
9.2 M urea, 2% Triton X-100, and 4% Ampholine (pH3.510) for a total of 6875 Vh under the condition that 0.01 M

Figure 1. 2D-PAGE profiles of the proteins extracted from 6 isolates of T . cruzi. 2D-PAGE was performed as described under Materials and Methods. Nineteen profiles were divided broadly into 3 groups. A representative profile of each group is shown. Group 1; TM172,
group IIa; H20, group IIb; Y strain.

H３PO４ was used for upper reservoir solution and 0.02 M
NaOH for lower reservoir solution. After electrofocusing,gels were removed from each glass tube and were soaked insample buffer for SDS-PAGE for 1 h. One of the gels wascut into small pieces, soaked in water, and measured pH of
the solution. Next, SDS-PAGE containing 11% acrylamide
was performed. The gels were soaked in ethanol/acetic acid/water (4:1:5) overnight to remove Ampholine and then was
stained with coomassie brilliant blue R-250 for 1 h. After
destaining, the gel was dried using Gel Drying film
(Promega, Madison, WI, USA). The molecular mass of the１
６
９

Figure 2. A schematic representation of 492 polypeptide spots. The selected 492 spots were used to judge the presence or absence of the spots among 19 stocks of T . cruzi. Part of the spot numbers is shown. Among them, 160 spots (closed
circle) were shared among the stocks and 332 spots (open circle) were present in limited stocks.

spots on the gel was estimated using the following standards: phosphorylase b, 94 kDa; bovine serum albumin, 67
kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; and α-lactalbumin, 14.4 kDa.
Growth of epimatigotes in medium and release of trypomastigotes from human cell lines
Epimastigotes of T . cruzi were adjusted to 1 x 10５/ml
in LIT medium containing 10% FCS and cultured in 24well culture plates (Corning, Corning, NY, USA) for 8 days.
The number of epimastigotes in the medium was counted
every day at 24 h intervals during culture. The doublingtime of each stock was calculated from the number of epimastogotes on day 2-6 showing exponential growth.
Human lung fibroblast-like cell line WI-38 and humanmuscle rhabdomyocarcinoma cell line Hs 224.T were obtained from American Type Culture Collection (Rockville,
MD, USA). The cell lines were maintained in Minimum Essential Medium Eagle (MEME) medium (Sigma ChemicalCo., St. Louis, MO, USA) supplemented with 10% FCS(complete medium) at 37°
C in a humidified atmosphere
containing 5% CO2.
A suspension of the cells was obtained from the confluent monolayer culture of the cells after treatment of trypsin/EDTA. The cells (1 x 10５) were cultured with 1 ml complete medium in 24-well culture plate for 24 h. Late log
phase of epimastigotes cultured for 8 days was transferred
to a 15 ml tube, washed twice with PBS, and finally suspended in MEME medium. One ml of suspension of parasites (1 x 10６) was inoculated to the monolayer culture of

WI-38 or Hs 224.T cells (parasites: cells=10:1) [24]. After
24 h incubation of the co-culture, the T . cruzi in the medium and attached to the bottom of the plate were removed
completely by several changes of the MEME medium andfinally resuspened in 2 ml of MEME medium containing
5% FCS added to each well. The cultures were further continued for 8 days. An aliquot of the medium from the culture was taken off every day at 24 h intervals and the trypomastigotes that appeared in the medium were counted. All
cultures were set up in triplicate.
Data analyses
The similarity coefficient (S) defined by Dice [25] wascalculated from the number of shared and unshared spots
between two stocks among all combinations of T . cruzi.
The distance (D) was estimated by the following formula[14,26]:
D=1-S=1-2 a/ (2 a+b+c), wherea=the number of spots shared between profile 1 and 2;
b=the number of spots present in profile 1 but absent in 2;
c=the number of spots absent in profile 1 but present in 2.
The unpaired Student’s t-test was used to determine
the significance of differences in the cumulative number oftrypomastigotes among the groups. P values less than 0.05
were considered statistically significant.

RESULTS
The polypeptide mapping by 2D -PAGE
2D-PAGE analyses of the proteins extracted with 4%

１
７０

Figure 3. Diagram of the presence (black square) and absence (white square) of 492 spots in individual stocks of T . cruzi.
Polypeptide spots are shown in order of number. Estimated values of pI and molecular mass (M ) are indicated besides each spot number. The isolates are aligned from the left to the right as follows, TM17, TM43, TM47, TM51,
TM52, 119, 172, CL strain, H6, H18, H1, H20, Peru 1, Peru 2, Y strain, Tulahuen, GE, RF, LO１
７１

Figure 4. Matrix display of each distance between the two strain
combinations from 19 isolates examined.

Triton X-100 from 19 stocks of epimastigotes (Table 1)
were examined to evaluate the variability within T . cruzi
species. Polypeptide spots in the range from 604 to 798
could be detected on individual profiles. Some of the profiles resembled each other, while others were dissimilar. All
profiles were then roughly divided into three groups (completely corresponding to I, IIa, and IIb reported by using
isozyme profiles [19]) when the gels showing a similar pattern were collected together in one group. Representative
profiles of group I (TM172), IIa (H20), and IIb (Y strain)
are shown in Figure 1.
Detection of shared and unshared spots of all the stocks
Since it was difficult to determine whether the spots oneach profile of two stocks from intergroups were shared or
unshared, two of three extracts; TM-43 (group I), H1 (IIa),
and Y strain (IIb) or a total of three combinations weremixed and analyzed by 2D-PAGE. By comparison among
the three gels, one being a mixture of two stocks and theothers were the two individual stocks, it was possible to determine the presence or absence of the spots between stocks(data not shown).
A total of 492 polypeptide spots out of 604 to 798
spots from 19 profiles were selected when the spot could be
detected in more than two strains and the presence or absence could be judged among the 19 profiles. A schematic
representation of the numbered spots is presented in Figure
2. Among them, 160 spots were shared among the 19 stocks,
while 332 were found in limited stocks. The physicochemical properties (relative molecular mass and pI ) and the presence or absence of individual spots on the 19 profiles are
summarized (Figure 3). The sum of the spots detected on
the profiles in each stock ranged from 320 (RF) to 358 (H18).
Based on the presence or absence of the spots, thenumber of shared and unshared spots between two stocks

Figure 5. Release of trypomastigotes from human cell line WI38. Human cell line WI-38 (1 x 10５/well) cultured in
24-well culture plate was co-cultured with the late log
phase of epimastigotes (1 x 10６) of T . cruzi for 24 h.
After removal of T . cruzi with several changes of culture medium, the cells were cultured for the indicatednumber of days. The trypomastigotes released from
WI-38 cells were counted every day at 24 h intervals.

among all combinations of the 19 stocks was counted. The
number of unshared spots was calculated between thestocks and summarized in Figure 4. The results indicate thatthe 19 stocks of T . cruzi can be divided into 3 groups:
group I; TM14, TM43, TM47, TM51, TM52, 119, 172, CL
strain, H6, and H18: group IIa; H1, H20, Peru 1, and Peru
2: group IIb; Y strain, Tulahuen, GS, RF, and LO. Group I
consisted of 10 T . cruzi; 8 were isolated in Guatemala, 1 in
Brazil and 1 in Ecuador: group IIa 4 T . cruzi; 2 in Guatemala and 2 in Peru: group IIb 5 T . cruzi; 3 in Paraguay, 1 in
Brazil, and 1 in Chile.
Growth of epimatigotes in medium and release of trypomastigotes from human cell lines
The doubling time of epimastigotes of the 10 stocks ingroup I, 4 in group IIa, and 5 in Group IIb was calculated tobe 24.0±0.5 h, 23.7±0.5 h, and 24.0±0.5 h, respectively.
The values were not significantly different among the threegroups.
The time course of the release of trypomastigotes from
WI-38 cells after infection of epimastigotes in the late log

１７２

Table 2. Number of released trypomastigotes (day 7) from different cell lines infected by T . cruzi
T .cruzi isolatesGroup IGroup IIaGroup IIb*

WI-38 cells (x 10４/ml)
39.0+23.0＊
47.7+47.4
9.3+6.9＊

Hs 224 T cells (x 10４/ml)
71.2+24.4＊
47.2+47.2
26.9+27.0＊

Group I vs IIb p<0.02

phase is shown in Figure 5. Trypomastigotes appeared in
the medium after four to seven days’ culture and thereafter
the number increased as a function of the culture day. Similar results were obtained when Hs 224.T cells were used.
The number of trypomastigotes released from WI-38 andHs 224.T cells on day 7 after infection of epimastigotes isshown in Table 2. The release of trypomastigotes in group I
was significantly higher than that in group IIb (p<0.05).
However, within group IIa, two showed a high number of
released trypomastigotes while the other two were very low,making it difficult to compare between Group IIa and theother groups.

DISCUSSION
2D-PAGE is widely utilized to detect genetic variations in the population of a species, genus, or family. Thismethod was applied to identify the surface proteins andmonitor the change of expression of these surface proteinsin the differentiation of epimastigotes to amastigotes andtrypomastigotes in T . cruzi [27-30]. To our knowledge, thisis the first report on an attempt to determine the extent ofvariabilities of proteins within the T . cruzi species.
Among the 492 spots analyzed in this study, 160 spots(32.5%) were shared among the 19 stocks of T . cruzi and332 (67.5%) were present in several limited stocks (Fig.3).
The number of shared and unshared spots between two profiles in all combinations of T . cruzi was used to characterize the groups (Fig.3). At present, it is difficult to determine
whether the extent of variability observed in T . cruzi species is high or low, since there have only been reports on theestimation of variabilities by 2D-PAGE in populations inplasmodium falciparum (49%, the value being altered tospot unsharing) [31, 32] and Cryptosporidium pavum species (3%) [33] in protozoa, Drosophila (23%) [34] in insects and the cat family (15%) [35] bear (37%) [36], hominoid primates (40%) [37], and humans (3-6%) [38].
There have been a number of reports concerning thegenetic variabilities of T . cruzi species analyzed mainly by
zymodemes [4-10, 19] and DNA polymorphisms [11-19,20], suggesting the existence of several lineages of T . cruzi
within the T . cruzi species [reviews: 39-41]. Our observation is essentially consistent with both previous reports andour previous observations [16-21, 41].
As indicated in Figure 3 and 4, there are several identifiable intra-group variations, although the level of the variability is much smaller than that observed between groups.
Thus, the prototype of the genome of each group has been
well conserved during geographical expansion. It seems
there were very few genetic exchange events between
groups which might have occurred in the overlapping geographical areas, although the genetic exchange could be detected by the population study [42] as well as in vitro study[43].
The doubling time of epimastigotes of the tested isolates showed no difference, while the number of trypomastigotes released from two human cell lines, WI-38 and Hs
224.T, varied. The number of released trypomastigotes of H
18, H1, H20, GS, LO, and RF was significantly lower thanthat of the other strains, and the number of trypomastigotes
released in group I was significantly higher than that ingroup IIb (p<0.05) (Table 2).
The digestive form of this disease is rare in Central
America but frequently observed in South America. It has
been suggested that the association of clinical symptoms
and severity of the disease with certain geographic regions
is related to genetic factors within the species [2-10]. The
fact that the isolates of group IIb showed low proliferative
activity of amastigotes in vitro might be directly or indirectly related to the pathogenicity of magacolon in SouthAmerica.

ACKNOWLEDGMENTS
We are grateful to Dr. Carlota Monroy and Vivian
Matta, San Carlos University, for their kind help in performing field work in Guatemala. We also thank Professor
F. Sendo, Yamagata University, for providing the local isolates from Paraguayan patients.
This work was supported in part by JICA, Japan International Cooperation Agency (JIET-141), by a Grant-in-Aid
for Scientific Research (B) from Ministry of Education,
Culture , Sports , Science and Technology Japan(No.15406016) and by a grant-in-aid for International
Health Cooperation Research from the Ministry of Healthand Welfare of Japan. J. Mu was supported by a research
fellowship from the Uehara Memorial Foundation.

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