identification of factors associated with kala-azar in
TRANSCRIPT
IDENTIFICATION OF FACTORS ASSOCIATED WITH KALA-AZAR IN HUMAN AT FULBARIA UPAZILA,
MYMENSINGH
A Thesis
Submitted to Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science in
Pathology
By
Md. Forrukh Hasan Khan
Roll No.: 10Vet Path JJ 07 M Registration No.: 31067, Session: 2004-05
Department of Pathology
Bangladesh Agricultural University Mymensingh
May, 2011
IDENTIFICATION OF FACTORS ASSOCIATED WITH KALA-AZAR IN HUMAN AT FULBARIA UPAZILA,
MYMENSINGH
A Thesis
Submitted to Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science in
Pathology
By
Md. Forrukh Hasan Khan
Approved as to style and contents by
Prof. Dr. Farida Yeasmin Bari Prof. Dr. A. S. Mahfuzul Bari Co-Supervisor Supervisor
Prof. Dr. Md. Rafiqul Islam Chairman, BOS & Head Department of Pathology
May, 2011
Dedicated to my beloved Elder uncle (mother side)
Md. Motaher Khaled
ex. Head department of Language,
Bangladesh Agricultural University,Mymensingh.
Who died at Canada during my MS study period.
iv
ACKNOWLEDGEMENTS
All the praises are due to the Almighty Allah, the creator and supreme authority of the universe, who enabled the author to successfully complete the research work and to build up this thesis.
The author expresses the deepest sense of gratitude and heartfelt indebtedness to his respected teacher and supervisor Professor Dr. A. S. Mahfuzul Bari , Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, for his scholastic guidance, cordial consistence, sympathetic supervision, valuable advice, constant inspiration, affectionate feeling, radical investigation and constructive criticism in all phases of research work and preparing this thesis.
The author is ever grateful and immensely indebted to his honourable teacher and co-supervisor Professor Dr.Farida Yeasmin Bari, Department of Surgery & Obstetrics, Bangladesh Agricultural University, Mymensingh, for her scholastic guidance, sympathetic supervision, valuable advice and encouragement throughout the period of this study.
The author would like to acknowledge his gratitude and sincere appreciation to his respected teachers Prof. Dr. Md. Abdul Baki, Prof. Dr. Md. Iqbal Hossain, Prof. Dr. Md. Habibur Rahman, Prof. Dr. Priya Mohan Das, Prof. Dr. Md. Rafiqul Islam, Prof. Dr. Mokbul Hossain, Prof. Dr. Emdadul Haque Chowdhury, and Lecturer Rokshana Parvin, Department of Pathology, Bangladesh Agricultural University, Mymensingh, for their encouragement, constructive and informative suggestions in course of the research work period. The author expresses his cordial thanks to Md. Enamul Haque, Md. Ataur Rahman Bhyian, Md. Abu Sufian, Md. Zakir Hossain, Umme Kulsum Rima, Md. Mushfiqur Rahman Ph.D students, Dept. of Pathology, Bangladesh Agricultural University, Mymensingh, for their valuable suggestions, practical help and co-operation in every step of the research work and in writing the thesis.
The author expresses his thanks to laboratory technicians and office staffs of the Department of Pathology, Bangladesh Agricultural University, Mymensingh, for their kind co-operation towards the completion of the research work.
The author extends his profound thanks to all of his friends especially Mohammad Faruk Hossain, Mohammad Mahabubul Alam, Md. Tusar Chowdhury, Md. Nuruzzaman, Urmi Roy, Fouzia Naznin , Mehidi Hasan for their friendly help, inspiration and encouragement during the study period.
The author acknowledges his deepest sense of gratitude and heartfelt thanks to his beloved father (K.M. Fakhrul Hasan Khan), mother (Manjurara Hasan Khan), who sweated much to bring him up today’s position, brothers and sister for their wishes, constant inspiration and great sacrifices throughout the entire period of the study.
The Author
May, 2011
v
CONTENTS
CHAPTER TITLE PAGE ACKNOWLEDGMENT iv
ABSTRACT v
LIST OF CONTENTS vi
LIST OF FIGURES viii
LIST OF ABBREVIATIONS AND SYMBOLS ix
I INTRODUCTION 1
II REVIEW OF LITERATURE 3
2.1 Life cycle of of Visceral leishmaniasis 3
2.2 Relationship of Kala-azar with age & sex 5
2.3 Living & breeding sites of sand fly 8
2.4 Host preference of sand fly & blood meal from
different host
9
2.5 Risk factors associated with kala-azar 10
2.6 Visceral leishmaniasis in animal 18
2.7 Post-kala-azar dermal leishmaniasis 21
III MATERIALS AND METHODS 23
3.1 Study area & source of data collection 23
3.2 Time of study 23
3.3
The questionnaires used for collection of information
from patient
24
vi
CONTENTS (Contd.)
CHAPTER TITLE PAGE 3.4
3.4.1
Methodology
Collection of data
25
25
3.4.2 Fulbaria 25
3.4.3 UHC Fulbaria , Mymensingh 27
IV RESULTS 28
4.1 Kala-azar & PKDL in different age group 28
4.2 Kala-azar patients in different sex group 30
4.3 Animal shed, mud house & knowledge about
sand fly
31
4.4 Use of mosquito net, sleeping pattern &
other factors
33
V
VI
DISCUSSION
SUMMARY AND CONCLUSION
38
43 REFERENCES
APPENDICES
45
57
vii
LIST OF FIGURES
FIGURE TITLE PAGE
1
Life cycle of visceral leishmaniasis.
4
2 Sand fly which is responsible for transmission of kala-azar. 4
3 Map of Fulbaria upazila. 26
4 Kala-azar patients in age wise catagories. 29
5 PKDL patients in age wise catagories. 29
6 Incidence of kala-azar & PKDL 30
7 Kala-azar in male & female. 30
8 Housing system of kala-azar patients. 32
9 Animal shed in kala-azar patients house. 32
10 Knowledge of rural people about sand fly. 33
11 Use of mosquito curtain by kala-azar patients. 34
12 Sleeping pattern of kala-azar patients. 34
13(a&b) PKDL patient with specific lesion at skin. 35
14(a&b) Kala-azar patints standing infront of their mud house 35
15(a&b) Two kala-azar patient in the same family having teen shed
house but earthen floor.
36
16(a&b) Cracked wall with many small hole in mud house &
dampess around the house.
36
17 Presenc of cattle in cattle shed of kala-azar patient house. 37
18 Animal shed in kala-azar patient house with cracked wall.
37
viii
LIST OF ABBREVIATIONS AND SYMBOLS
CFR
CI
DAT
F
Fig.
E
Km
M
m
N
N
P
PKDL
RR
OR
UHC
VL
Y
Y
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Case fatality rate
Confidence interval
Direct agglutination test
Female
Figure
East
Kilometer
Male
miter North
No
Probability
Post Kala-azar Dermal Leishmaniasis
Rate ratio
Odd ratio
Upazila health complex
Visceral leishmaniasis
Yes
Year
ix
ABSTRACT
Visceral leishmaniasis (VL), also known as kala-azar, is a fatal vector-borne parasitic disease.
Worldwide incidence is 500,000 cases per year. The true incidence of kala-azar in Bangladesh
is close to 40 000–45 000 per year. The objective of this present study was to identify the
factors associated with kala-azar and Post Kala-azar Dermal Leishmaniasis (PKDL) in human
in Fulbaria upazila of Mymensingh district. A total of 359 cases were recorded from the rural
area by door to door survey and the data from the Upazial Health Complex , Fulbaria. The
incidence of kala-azar is higher in male (58.22%) than female (41.78.%). Children less than 10
years of age were affected most frequently (40.11%). The incidence was found to decrease with
the increase of age (11-20 years =19.50 % cases, 21-30 years =19.22%, 31-40 years =10.58%,
41-50 = 6.40% and above 50 years = 4.17 % cases). Among the Kala-azar infected patients,
only 14.48% cases developed PKDL. Incidence of PKDL also decreased with age. The rural
area of Fulbaria is well known for mud house for living. Forty seven (47) percent of kala-azar
infected patient were found to living in a mud house and rest 53% have tin shed house but with
earthen floor in most of the cases. Among the kala-azar infected patients 66% people have been
found rearing domestic animal either cattle, goats or dogs. These animal sheds were made by
clay with cracked wall & wet area, which are favorable for sand fly. However the relationship
of domestic animals with spreading of the kala-azar is not established. Most of the people
(72%) were found having no idea about sand fly. Only 28 % people knew about the sand fly.
Vegetations around the house, living in proximity to prior case, cracked mud house walls with
humid area, poor economical condition, nutritional deprivation, illiterate people, lack of proper
treatment and diagnostic facilities, incomplete treatment, lack of awareness and proper
knowledge about the diseases were other risk factors found behind the increasing incidence of
kala-azar in Fulbaria. Attentions need to be paid considering the severity and effects of kala-
azar in Bangladesh.
1
CHAPTER I
INTRODUCTION
Visceral leishmaniasis (VL), also known as kala-azar, black fever, and Dumdum fever, is a
disease caused by an intracellular protozoan parasites of the genus Leishmania and
transmitted by the sand fly of the genus Phlebotomus. It is the second-largest parasitic
disease killer in the world (after malaria), responsible for an estimated 500,000 cases each
year worldwide (Desjeux, 2001). The parasite migrates to the internal organs such as liver,
spleen (hence 'visceral)' and bone marrow and if left untreated will almost always result in
the death of the host. Signs and symptoms include fever, weight loss, mucosal ulcers,
fatigue, anemia and substantial swelling of the liver and spleen.
In Bangladesh, sporadic kala-azar cases were reported in the 1970, and an outbreak
occurred in Pabna district in 1980 (Elias et al., 1989). This is one of the major health
problem because 34 of total 64 districts are at high risk of Visceral Leishmaniasis infection
where 80% patients die within five years of affliction due to lack of diagnostic and
treatment facilities in rural areas (Hasib, 2004). The true incidence of kala-azar in
Bangladesh is close to 40 000–45 000 per year (Bern & Chowdhury, 2006).
Ninety percent of total cases in Bangladesh reported from 10 districts during a past study of
11 years (1994-2004) and from 2000-2004, more than 50% of total cases reported from
Mymensingh District. Out of 12 upazila (sub district) of Mymensingh, five upazila were
confirmedly reported of kala-azar patients. Among these five, Fulbaria upazilla showed the
highest incidence becoming epidemic in this area. Using the population of the respective
thana as the denominator, the incidence of kala-azar in Fulbaria thana ranged from 30 to
33/10,000/year (Bern and Chowdhury, 2006).
2
Post-kala-azar dermal leishmaniasis (PKDL) is usually a sequel to kala-azar (Das et al.,
2010). PKDL is also an important health hazard with skin lesions ranging from
hypopigmented macules, erythematous patches and papulonodular lesions either singly or in
combination in absence of systemic involvement. No actual statistical data for PKDL is
available in Bangladesh but some sporadic cases are reported (Hassan and Hossain, 2000).
The incidence of kala-azar is higher in male than female. Children are most vulnerable. The
incidence of this disease is decreasing with age (Palumbo, 2010; Singh et al., 2010 a & b;
Joshi, 2009).
Important risk factors for spreading Kala-azar in Fulbaria upaziala are poor economic
condition, living in mud house with cracks & crevices in their house floor, lack of knowledge
about transmission and prevention of disease, having cattle shed (Mamoon et al., 2006).
In many of the endemic areas, dogs are considered the major reservoir for human disease
while in other regions people are the principal reservoir for further human spread. Nine
Isolated foci of infections have been found in Texas affecting both humans and dogs,
however apparently healthy cases or Leishmania spp. have now been found in dogs in 21
states, including Oklahoma, Ohio, Texas, Michigan, NewYork & Alabama and two
Canadian provinces (Eddlestone, 2000).
The objectives of the present study were to:
• Identify factors (housing system, animal rearing, lack of knowledge about sand fly,
living with previously affected kala-azar patients etc) associated with this disease.
• Identify the ages and sex wise incidence of kala-azar.
3
CHAPTER II
REVIEW OF LITERATURE
The parasitic disease kala-azar (visceral leishmaniasis) was first described in 1824, in
Jessore district, Bengal in what is now Bangladesh. Historical records describe the classical
picture of kala-azar prolonged irregular fever, progressive emaciation, and enlargement of
the liver and spleen (Sengupta, 1947). Kala-azar appeared to have spread along the courses
of the Ganges and Brahmaputra rivers, the major transport routes (Sanyal, 1985). Visceral
leishmaniasis (VL) or Kala-azar is a severe and often fatal infection of human caused by
protozoal parasite of leishmania species (Leishmania donovani Complex, Leishmania chagasi,
Leishmania. infantum). Visceral leishmaniasis (VL) is transmitted by bite of a sand fly of the
genus phlebotomus mainly Phlebotomus argentipes and it occurs in all continents except
Australia (Quinnell & Courtenay 2009, Jeronimo et al., 2006, Melby, 2000).
2.1 Life cycle of of Visceral leishmaniasis
The promastigote form of L. donovani is transmitted into the skin by female phlebotomine
sandflies (Fig-2). Once transmitted, the parasites are internalized by dendritic cells and
macrophages in the dermis where they lose their flagella, transforming into the amastigote
form. The amastigotes multiply, destroy the host cell and infect other phagocytic cells. The
amastigotes disseminate through the lymphatic and vascular systems, eventually infiltrating
the bone marrow, liver and spleen (Fig-1) (Varki et al., 2009; Chappuis et al., 2007).
4
Fig-1: Life cycle of visceral leishmaniasis.
Fig-2: Sand fly which is responsible for transmission of kala-azar.
5
2.2 Relationship of Kala-azar with age & sex Leishmaniasis is distributed worldwide and 13 million people are estimated to be infected,
with about 1.8 million new cases each year. Approximately 50% of these patients are
children (Palumbo , 2010).
Visceral leishmaniasis (VL) is endemic in parts of Islamic Republic of Iran. There was no
significant difference in seropositivity between the sexes (2.8% males and 3.3% females at
Booyerahmad district in the south-west of the country. The highest rate of infection (5.2%)
was in the age group 10 years (Sarkari et al., 2010).
Muzaffarpur district in Bihar proportion of kala-azar increased with age. Women had a
significantly lower prevalence than men >14 years old. Owning domestic animals (cows,
buffaloes or goats) was associated with a higher risk of being direct agglutination test (DAT
)positive [OR 1.16 (95% CI 1.01–1.32)], but socio-economic status was not (Singh et al.,
2010).
In Nepal majority (70.9%) of persons affected by VL were aged 15 years and above,
followed by 10-14 years (13.9%), 5-9 years (11.9%) and 1-4 years (3.3%). VL cases
recorded from different district of Nepal for the year 2004, 2005, 2006 and 2007 are
recorded. case fatality rate (CFR) for the year 2004, 2005, 2006 and 2007 were 3.2%, 3.7%,
16.67% and 11.42% respectively (Joshi, 2009).
In Fortaleza, Ceará, Brazil. there were a marked increase and an elevated incidence of cases
of VL in urban areas. Children and young people were the most affected group.
(Albuquerque et al., 2009).
6
In Al-Nassiriya Pediatric Hospital during the year 2002; it was found that the majority of
cases (94.64%) were below the age of 3 years, 51.8% were males and 48.2% were females.
A high percentage of cases 85% were from rural areas and only 15% were from city center
(Raddam et al., 2007).
There were 62% males and 38% females. Their ages ranged from 3.5 months to 10 years.
At a hospital in the southwestern part of Iran the disease was most common in those
younger than 2 years of age. The majority presented in late winter (March and April,
40.2%), followed by spring (23.5%) and a few were seen in the summer (11.3%) and
autumn (18%) About 80% of the children were from rural areas and nomads (Rahim and
Ashkan, 2007).
Kala-azar outbreak in Libo Kemkem, Ethiopia for 492 kala-azar patients diagnosed at
AZHC between May 10 and October 18, 2005. Of these, 344 (74%) were men and 121
(26%) women (data missing for 27). The mean age was 17.4 years (median, 16 years; range,
0.7–60 years); females patients were younger than men (mean, 14.5 versus 18.6 years for
men; P < 0.001) (Alvar et al., 2007).
Data collected from eight zonal hospital in Terai region of Nepal. A total 25890 cases with
599 deaths reported during the year 1980-2006. Majority of persons affected by VL were
aged 15 years & above (70.9%), followed by 10-14years (13.9%), 5-9 years (11.9%) , 1-4
years (3.3%) (Joshi et al., 2006).
In Fulbaria upazila under Mymensingh district of Bangladesh total 218 respondents were
studied from 46 households. All the members of the households were interviewed and
7
their blood samples were taken .The highest percentage (40.7%) of them were in the age
group of < 10 years and male and female ratio was 1.7:1 (Mamoon et al., 2005).
Males in Venezuela were more frequently affected (59.5%) than were females (40.5%). In
terms of age, 67.7% of the VL patients were </= 4 years of age, and 80.6% were younger
than 15 years. The mortality rate among the persons with VL was 7.85% during the 1995-
2000 period. Serological screening with rK39 antigen of 1217 dogs from Margarita Island
found a 28.5% positivity rate. In contrast, the rate was 2.8% in the 1 808 samples from dogs
from 12 states on the mainland ( Zerpa et al., 2003).
Survey of visceral leishmaniasis from 13 villages and from nomadic tribes in Bushehr
province shows that almost all cases (94.1%) were in children under 10 years old
(Mohebali et al., 2001).
The average incidence rate of infection since 1985 was 2.8%/year with all ages equally at
risk in north-west Iran. One in 13 infections in children led to visceral leishmaniasis (VL),
and this ratio decreased significantly with age. Seroprevalence also dropped rapidly with
age, suggesting that the same process may affect both clinical outcome and the humoral
immune responses. Cell-mediated immunity was associated with a reduction in the
seroconversion rate and an increase in the serorecovery rate. Even amongst people with no
detectable cell-mediated immunity to Leishmania, the seroconversion rate decreased and the
serorecovery rate increased with age. All current VL patients had a negative leishmanin skin
test response. Hence, adults may develop protection against Leishmania infantum through 2
processes, 1 dependent and 1 independent of acquired cell-mediated immunity (Davies et
al., 1999).
8
In Meshkin-Shahr, Iran about 90% of kala-azar infected cases were under 5 years of age and
15% under 1 year. Very few cases occurred among older children and adults; 55% of
patients were males and 45% females (Soleimanzadeh et al., 1993).
2.3 Living & breeding sites of sand fly
Breeding of sand flies was detected in 46% of the villages and 7.3% of the soil samples
examined from 50 villages of 5 visceral leishmaniasis (VL) endemic districts of Bihar state.
Intra-domestic soil was found to be infested with 2 species of sand flies, Phlebotomus
argentipes Annandale and Brunetti and Phlebotomus papatasi (Scopoli). In comparison
with human houses, Phlebotomus argentipes was found to show greater propensity to breed
in cattle sheds; breeding prevalences in villages and soil samples, for the species, were
significantly higher in cattle sheds than in human houses. The preference for breeding sites
by the sand flies appeared to be associated with the pH of the soil. Phlebotomus argentipes
thus preferred to breed in the alkaline soil of cattle shed, and Phlebotomus papatasi in the
soil with neutral pH, of human houses. The emergence of adult Phlebotomus argentipes
from soil samples was mainly observed from April to October (Sing et al., 2008).
Of the 15 houses selected for the study 10 in India, including the cattle sheds and latrines,
were plastered with a mixture of mud and lime, up to a height of 1.22 m taking care to seal
all cracks and crevices. The remaining five houses were left unplastered and were
considered as control areas. The pre-treatment and post-treatment resting densities of the
sand fly were monitored both in treated and untreated houses. A sudden drop in the sand fly
density was noticed in the treated houses, whereas there was no significant reduction in the
check houses, suggesting an effective control (Kumar et al., 1995).
9
2.4 Host preference of sand fly & blood meal from different host
Blood meals of 304 Phlebotomus argentipes and 206 Phlebotomus papatasi, collected from
different biotopes from two Kala-azar affected districts in West Bengal, were tested against
seven different antisera by modified Ouchterlony gel diffusion techniques. It appeared that
host preference of Phlebotomus argentipes varied widely in different biotopes, which is
mainly zoophilic (62.80%), preferring to feed on man as the second choice (24.92%);
however it is also a “chance feeder” according to biotopes. Multiple blood meals are also
prevalent in Phlebotomus argentipes at a much higher rate than that of Phlebotomus
papatasi. The implication in relation to epidemiological significance has been discussed
(Palit et al., 2005).
In Bahapur, Patna district cattle appeared to be better as bait, since more than five females
P. argentipes were caught on them for each one caught on the human bait. Overall, although
P. argentipes were caught during each collection hour from 18.00--06.00 hours, the
numbers caught landing/biting peaked at 23.00--24.00 hours. There were, however, slight
seasonal variations in the timing of this peak in activity. The numbers of Phlebotomus
argentipes caught/collection night also varied with season, being significantly higher during
the summer than during the rainy season or winter (P < 0.01 for each), and apparently
inversely correlated with rainfall. Males Phlebotomus argentipes were caught in much
higher numbers than the females of this species, with female male ratio of 1:8 for the flies
caught on human bait and of 1:13 for those collected from cattle. If the probability of a
females Phlebotomus argentipes being infected with Leishmania donovani does not vary
with season, peak transmission of this parasite to humans probably occurs between
February and May, at the middle of night (Dinesh et al., 2001).
10
From eight districts of West Bengal (India) blood meal indices were found different in the
two biotopes, human dwellings and cowsheds. A total of 395 blood meals were analyzed.
The blood meal indices in the human dwellings were human 68.8 per cent, cow 38.9 per
cent, both human and cow 10.2 per cent, others 2.5 per cent and in the cowsheds: human
19.7 per cent, cow 91.6 per cent, human and cow 13.9 per cent and others 2.5 per cent.
These indicated that Phlebotomus argentipes did not show preference to any blood meal
(Ghosh et al., 1990).
2.5 Risk factors associated with kala-azar
A systematic review of risk factor analyses for South Asian Visceral Leishmaniasis shows
that within villages, areas become saturated over a period of several years; kala-azar
incidence then decreases while neighboring areas see increases. More recently, post kala-
azar dermal leishmaniasis (PKDL) cases have followed kala-azar peaks. Mud walls,
palpable dampness in houses, and peridomestic vegetation may increase infection risk
through enhanced density and prolonged survival of the sand fly vector. Bed net use,
sleeping on a cot and indoor residual spraying are generally associated with decreased risk.
Poor micronutrient status increases the risk of progression to kala-azar. The presence of
cattle is associated with increased risk in some studies and decreased risk in others,
reflecting the complexity of the effect of bovines on sand fly abundance, aggregation,
feeding behavior and leishmanial infection rates. Poverty is an overarching theme,
interacting with individual risk factors on multiple levels (Bern et al., 2010).
No significant associations between VL and keeping domestic animals inside the house (OR
of 0.88 for bovines and 1.00 for ‘any animal’) or ownership of domestic animals (OR of
0.97 for bovines and 1.02 for ‘any animal’). VL was associated with housing conditions.
Living in a thatched house (OR 2.60, 95% CI 1.50–4.48) or in a house with damp floors
11
(OR 2.60, 95% CI 1.25–5.41) were risk factors, independently from socio economic status
(Singh et al., 2010 a).
In Nepal the sero-survey (n = 5397) showed an infection prevalence rate of 9%, (range 5–
15% per cluster) with higher prevalence in men (9.9%) than in women (8.3%) (P = 0.049).
Male gender, increasing age and poverty were significant risk factors (Rijal et al., 2010).
The poorest of the poor: a poverty appraisal of households affected by visceral
leishmaniasis in Bihar, India showed that Visceral leishmanisis clearly affects the poorest of
the poor. They were found most vulnerable, as this vector-born disease is linked to poor
housing and unhealthy habitats. 83% of households were in communities with high VL
attack rates belonged to the two lowest quintiles of the Bihar wealth distribution (Boelaert
et al., 2009).
In India, Bangladesh, and Nepal screening for VL was done in 18,933 households (106,425
inhabitants). The estimated annual incidence of VL in the endemic districts was on average
22 times higher than the elimination target of less than one case per 10,000 inhabitants in
2015. The percentage of newly detected cases through the household screening was high in
the districts least covered by health-care services and much lower in districts with greater
availability of VL care (Mondal et al., 2009).
Ninety per cent of the 500,000 annual new cases of visceral leishmaniasis (VL) occur in
India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80-90% of human infections are
sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity.
Understanding the environmental and genetic risk factors that determine why two people
with the same exposure to infection differ in susceptibility could provide important leads for
12
improved therapies. Recent research using candidate gene association analysis and genome-
wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have
identified a number of genes/regions related both to environmental risk factors (e.g. iron), as
well as genes that determine type 1 vs. type 2 cellular immune responses (Blackwell et al.,
2009).
In Chatrakhali, West Bengal, India factors associated with infection included residing in
homes with mud walls (RR 4.3), dampness in the home (RR 2.5), proximity to bodies of
water (RR 2.5) and livestock ownership (RR 2.4). Sleeping dressed (RR 0.4), or under a bed
net (RR 0.5) or in a cot (RR 0.6) were associated with a lower risk. High rates of infection
indicated that transmission persisted in this community. Poor housing conditions were
associated with a higher risk, while personal protection measures against vectors were
effective (Saha, 2009).
In Bihar, India Kala-azar occurred more among persons living in the same household (24 of
25). However, there was no significant difference in disease conversion among children (5-
15 years of age) and adults (> 15 years of age) (Gidwani et al., 2009).
Seventy-six cases of visceral leishmaniasis (VL) were reported from the Hebron district of
the West Bank, Palestine between 1993 and 2007. All cases were in children less than 9
years old (median age 2 years). The average number of cases was 5.06/year and the average
annual incidence was 3.02/100000 children. Seropositivity was highest for children in
households of previous VL cases [odds ratio (OR) 7.5; 95% CI 3.17-17.61; P<0.001] and
among people who had domestic dogs and/or other animals (OR 2.4; 95% CI 1.19-4.68;
P=0.017). No difference was seen between male and female (P=0.073). A preliminary
survey of sand fly distribution showed the abundance of two putative vector species:
Phlebotomus syriacus (45%) and Ph. tobbi (10%). The focus of VL in Hebron district was
13
shown to follow the epidemiological pattern of paediatric disease characteristic of the
Mediterranean region (Amro et al., 2009).
In Bihar of India the houses were made up of mud, soil and bamboo; some homes were
made with walls of bricks and no plastering. However, with few exceptions, kala-azar cases
also occurred in concrete houses in north and south Bihar. Both areas have some alluvial
soil and some soft stem vegetation, such as banana, creepers and peridomestic vegetation,
giving the terrain the same amount of risk factor for kala-azar transmission (Kumar et al.,
2009).
The exposure rate of VL is more in boys (70%) than in girls (30%) in paediatric wards of
Mymensingh Medical College Hospital. The frequency rate of VL was higher in children
>10 years of age group (66.6%) than in other age groups. Mud house dwelling is
significantly associated with the development of VL (81.6%) and carries 28.9 times greater
risk of developing VL, than those dwelling in bamboo house (13.3%). The difference of this
proportion is statistically significant (p<0.001, OR=28.9). The exposure rate of VL was
slightly higher (51.6%) in mosquito net non-users than the mosquito net users. Grade III
malnutrition was found significantly associated with (83.3%) VL (p<0.005). The frequency
rate of VL was higher in children sleeping on floor than in sleeping on Chowki or Khat
(OR=2.07). Rainy season is significantly associated with VL (60%) and carries 2.4 times
greater risk of developing VL than other seasons (p<0.05, OR=2.4). It can be concluded that
mud house dwelling, grade III malnutrition, sleeping on floor and seasonality (rainy season)
are the important risk factors of VL in children (Rukunuzzaman & Rahman, 2008).
In a Bangladeshi community kala-azar and seroconversion incidence were 15.6 and 63.1 per
1,000 person-years, respectively in two villages of Uttar Pradesh, India .Proximity to a
14
previous kala-azar case increased the likelihood of both kala-azar and asymptomatic
infection. Bed net use protected against kala-azar (rate ratio = 0.35, P < 0.01), but not
subclinical infection (rate ratio = 1.1, P = 0.82). Kala-azar patients were younger (P <
0.001) and reported lower red meat consumption (P < 0.01) than asymptomatic seropositive
individuals. Retinol and zinc levels were lower in current kala-azar patients and those who
later developed kala-azar compared with uninfected and asymptomatically infected subjects.
The CRP levels were higher in kala-azar patients compared with the other two groups. Low
red meat intake and poor zinc and retinol status may characterize a group at higher risk of
symptomatic disease (Bern et al., 2007).
Fulbaria upazila under Mymensingh district of Bangladesh area is endemic for kala-azar .
Among the important risk factors like economic condition, 81.5% sero-positive cases had
less than 4000 taka monthly family incomes. In addition, 81.5% of the seropositive kala-
azar cases lived in mud houses, 11.1% in tin houses and 48.1% of them had cracks and
crevices in their house floor. Furthermore, 63.0% of the patients did not use mosquito net
and 51.9% of them possessed cattle shade, which are also contributing factors for high rate
of kala-azar infection. 45.7% respondents did not know how the disease is transmitted. On
the other hand, those who knew, among them 36.9% of respondents answered that the
disease could be transmitted by food, 28.3% by close contacts with the patients and 21.7%
by using clothes of patients & mosquito borne both. Only 2.1% replied that it was
transmitted by bite of sand flies (Mamoon et al., 2006).
In southeastern Nepal independent risk factors for Leishmania infection were proximity of
the house to ponds [odds ratio (OR) 3.7, 95% CI: 1.6–8.5], family size (OR 4.4, 95% CI:
1.6–12.6), age 15 years (OR 5.5, 95% CI: 1.2–25.0) and house constructed in mud (OR 3.0,
95% CI: 1.1–7.6). Bed nets, not impregnated and in poor condition, were used by 95.2%
15
(95% CI: 92.3–97.0) of the population, but did not show any protective effect (Schenkel et
al., 2006).
VL seroprevalences were 52.5% in Parkarin and 16.9% in Loboi of Kenya. Significant
associations among seropositivity and house construction, age, and proximity to domestic
animal enclosures were found. A significant spatial cluster of VL was found in Loboi. The
spatial distribution of cases in the two villages was different with respect to risk factors,
such as presence of domestic animals. This study suggests that disease control efforts could
be focused on elimination of sand fly habitat, placement of domestic animal enclosures, and
targeted use of insecticides (Ryan et al., 2006).
Human visceral leishmaniasis (HVL), caused by Leishmania infantum is mainly observed
as sporadic cases in Turkey and dogs are considered as the main reservoir of the disease.
Household members of individuals with previously confirmed visceral leishmaniasis were
found to have higher frequency of the disease suggesting the household members should be
included in the risk group for visceral leishmaniasis and serological screening should be
performed for the detection of possible infection (Sakru et al., 2006).
In common with its neighbours India and Nepal, VL in Bangladesh is anthroponotic. Living
in proximity to a kala-azar case is the strongest risk factor for disease, while consistent use
of bed nets in the summer months and the presence of cattle are protective. Shortages of
first-line antileishmanial drugs and insecticide for indoor spraying programmes have
hindered VL treatment and vector control efforts. Effective control of VL will require
activities to improve availability and access to diagnostic testing and antileishmanial drugs,
enhanced surveillance for kala-azar, post-kala-azar dermal leishmaniasis and VL treatment
failures, and increased coverage and efficacy of vector control programmes (Bern et al.,
2006).
16
In Bangladeshi community risk was highest for persons 3-45 years of age, and no
significant difference by sex was seen. In age-adjusted multivariable models, 3 factors were
identified: proximity to a previous kala-azar patients (odds ratio [OR] 25.4, 95% confidence
interval [CI] 15-44 within household; OR 3.2 95% CI 1.7-6.1 within 50 m), bed net use in
summer (OR 0.7, 95% CI 0.53-0.93), and cattle per 1,000 m2 (OR 0.8, 95% CI 0.70-0.94]).
No difference was seen by income, education, or occupation; land ownership or other
assets; housing materials and condition; or keeping goats or chickens inside bedrooms. Our
data confirm strong clustering and suggest that insecticide-treated nets could be effective in
preventing kala-azar (Bern et al., 2005).
In two villages in Uttar Pradesh, India reported to have had a recent outbreak 245
households with 2,203 people. Risk factors included living in the same household as a VL
case (odds ratio [OR] _ 76, P < 0.0005 in one village and OR _ 22, P < 0.0005 in the other
village), sleeping downstairs and outside in the summer (OR _ 4.7, P _ 0.004), and an age _
15 years old (OR _ 2.9, P _ 0.024). Increasing cattle density was a risk factor in one village
but not the other. We were not able to determine the route by which VL entered the villages.
Our data demonstrate a new spread of VL in previously unaffected areas (Barnett et al,
2005).
There was a 69.5% predominance of males cases among those registered and a greater
(52.4%) occurrence of the disease in children from zero to ten years old among Indians in
the State of Roraima, Brazi. The rate of natural infection was 10.3% out of 3,773 dogs
examined in 74 different locations. Lutzomyia longipalpis was found in 31 areas with
greater prevalence of the disease. The human and animal cases as well as the vectors were
17
concentrated in areas where mountains and arable soil predominate, typical locations for the
occurrence of American visceral leishmaniasis (Guerra et al., 2004).
Parasitic diseases, including human visceral leishmaniasis, are multifactorial.. Some
individuals, however, fail to control parasite expansion and dissemination and develop a
visceral disease. Most of the 660 subjects (90%) living in the central district were exposed
to Leishmania and 20.9% (n = 138), mostly teenagers, developed VL. VL cases increased
markedly in adults late in the outbreak, suggesting some changes in adult resistance status
or in Leishmania "virulence" during the epidemic. Age and ethnic origin of the patients
were the most important critical risk factors to account for the distribution of the VL cases
that were recorded during the whole epidemic. This and the high frequency of VL in certain
families suggest that host genetic factors played an important role in shaping the outbreak in
this village. However, environmental factors (the presence of cows and neems in the
households) that increase/decrease exposure to the parasite had significant effects on the
distribution of VL cases in the village in the first phase of the outbreak (Bucheton et al.,
2002).
In northwest Iran random sample of 3,872 children and 199 dogs in 38 villages was
surveyed by the direct agglutination test. Dog ownership details among these households
were collected by questionnaire. Parasites isolated from 16 patients and 12 dogs were
characterized as Leishmania infantum. Average seroprevalence in dogs (21.6%) was much
higher than in children (7%). Child seropositivity increased significantly with village dog
density in absolute terms (P < 0.001) and in relation to dog/human ratios (P = 0.028). Dog
ownership within villages also was a significant risk factor for child seropositivity (P =
0.003) (Gavgani et al., 2002).
18
Malnutrition is a risk factor for the development of visceral leishmaniasis. Malnutrition
causes a failure of lymph node barrier function after L. donovani infection, which may be
related to excessive production of PGE(2) and decreased levels of IL-10 and nitric oxide.
(Anstead et al. 2001).
Univariate analyses in Nepal among 84 cases and 105 controls, protective factors included
sleeping on a bed or cot (Odds ratio [OR] 0.44, P < 0.01) and sleeping under a bed-net
regularly (OR 0.23, P < 0.001) or in the warm months (OR 0.20, P < 0.001). The bed-nets in
use in this region were commercially available and untreated with insecticide. Ownership of
a cow or buffalo was protective (OR 0.34, P < 0.001), whereas dampness observed in the
mud floor of the house was a strong risk factor (OR 4.0, P < 0.001). In multivariable
models, bed-net usage, cow or buffalo ownership, and damp floors were significantly
associated with altered risk. A program to increase bed-net usage could therefore decrease
the incidence of VL in Nepal (Bern et al., 2000).
In Varanasi district of India the prevalence of kala-azar was lower among literate persons,
but the differences were not statistically significant .The prevalence of the disease was
highest among males whose primary occupation was in agriculture; a similar trend was
observed among women who worked in agriculture and weaving (Kumar et al., 1999).
2.6 Visceral leishmaniasis in animal
In Nepal Leishmania infections were determined by using PCR. Infections among persons
was (6.1%), cows (5%), buffaloes (4%), and goats (16%). Data were georeferenced and
entered into a geographic information system. The bivariate K-function results indicated
spatial clustering of Leishmania spp.–positive persons and domestic animals. Classification
tree analysis determined that among several possible risk factors for Leishmania infection
19
among persons, proximity of Leishmania spp.–positive goats ranked first (Bhattarai et al.,
2010).
Leishmaniases are an endemic zoonosis in the Mediterranean basin. Dogs are considered
the major host for these parasites, and the main reservoir for human visceral infection.
Parasites are transmitted by the bite of phlebotomine sand flies. The global climate changes
associated with a higher density and activity of sand flies during a larger period might
enhance the number of days favorable for transmission of parasites to humans and animals
with a concomitant increase of incidence (Campino & Maia, 2010).
Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is an important
disease of humans and dogs. ZVL is maintained by sand fly transmission, transmission may
also occur by non-sand fly routes, such as congenital and sexual transmission. Dogs are the
only confirmed primary reservoir of infection. Meta-analysis of dog studies confirms that
infectiousness is higher in symptomatic infection; infectiousness is also higher in European
than South American studies. A high prevalence of infection has been reported from an
increasing number of domestic and wild mammals; updated host ranges are provided. The
crab-eating fox Cerdocyon thous, opossums Didelphis spp., domestic cat Felis cattus, black
rat Rattus rattus and humans can infect sand flies, but confirmation of these hosts as
primary or secondary reservoirs requires further xenodiagnosis studies at the population
level. Thus the putative sylvatic reservoir(s) of ZVL remains unknown (Quinnell &
Courtenay, 2009).
Among the 468 dogs examined urban area of Cuiabá, Brazil , 16 presented an indirect
immunofluorescence reaction, thus resulting in an overall prevalence of 3.4%. There was no
predisposition towards canine leishmaniasis occurrences in relation to breed, sex or age.
The main risk factors for occurrences of canine infection identified in the city of Cuiabá
20
were the presence of dogs living in areas surrounding homes and the proximity of homes to
forests. Thus, there have been changes in leishmaniasis occurrences within the urban
environment (Almeida et al., 2009).
Dogs are the most important reservoir of visceral leishmaniasis (VL). A male child who
lives in Köseköy in Kocaeli was diagnosed with VL. Since this child had never been outside
Kocaeli, serum samples of 65 stray dogs were analyzed for canine VL using the indirect
fluorescence antibody test (IFAT) and ELISA. Two dogs (3.07%) tested positive with both
ELISA and IFAT. Leishmania amastigotes were observed in the lymph aspiration material
from one of them (Tamer et al. 2008).
Synanthropic and domestic animals can act as source of infection for phlebotomine sand fly
vectors. Dogs have long been implicated as the main domestic reservoirs of Leishmania
(Leishmania) infantum, the aetiological agent of zoonotic visceral leishmaniasis, and there
exists an increasing trend to regard dogs as the main domestic reservoirs of Leishmania
(Viannia) braziliensis, the most widespread aetiological agent of American tegumentary
leishmaniasis (Torres, 2007).
Foxhounds infected with Leishmania spp. were found in 18 states and 2 Canadian
provinces. No evidence of infection was found in humans. The infection in North America
appears to be widespread in foxhounds and limited to dog-to-dog mechanisms of
transmission; however, if the organism becomes adapted for vector transmission by
indigenous phlebotomines, the probability of human exposure will be greatly increased
(Dupery et al., 2006).
Using an indirect fluorescent antibody test, anti-Leishmania antibodies were detected in 130
out of 322 dogs in an urban area in the State of Pernambuco (Brazil) where human cases of
21
visceral leishmaniasis have sporadically been reported. The overall seroprevalence found
was 40.3%. Furthermore, it was observed that 85.3% of all seropositive dogs showed no
clinical signs of leishmaniasis. The results showed a high seroprevalence of anti-Leishmania
antibodies among dogs from an urban area of Pernambuco - with a large proportion of
asymptomatic seropositive dogs - indicating that the prevalence of Leishmania infection in
this area has been underestimated (Torres et al., 2006).
In endemic foci of Iran Anti-leishmanial antibodies were detected by the direct
agglutination test (DAT) from domestic and wild canines. Of the 1568 serum sampled
collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). Dogs
of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the
seropositive domestic dogs had clinical signs. Parasitology and serology tests that were
performed in 30 wild canines showed 10% these animals were infected by Leishmania
infantum (Mohebali et al., 2005)
2.7 Post-kala-azar dermal leishmaniasis
PKDL is reported mainly from two regions - Sudan in eastern Africa and the Indian
subcontinent, with incidences of 50-60% and 5-10%, respectively. Importantly, patients
with PKDL are considered as reservoirs of VL.The etiopathogenesis of PKDL is
presumably due to an immunological assault on latent dermal parasites (Ganguly et al.,
2010).
Post-kala-azar dermal leishmaniasis is usually a sequel to visceral leishmaniasis. A 25-year-
old woman presented with hypopigmented maculopapular lesions all over the body for the
past 4 years without any previous history of visceral leishmaniasis. She was on treatment
for leprosy and pulmonary tuberculosis for the past 2 months, but did not show any
22
improvement. Investigations confirmed that she had post-kala-azar dermal leishmaniasis
associated with pulmonary tuberculosis and HIV-1 infection (Das et al., 2010).
In a prospective study in a village in the endemic area for kala-azar in the Sudan, 105 of 183
(57%) kala-azar patients developed PKDL. There was a significantly higher PKDL rate
(69%) in those who received inadequate and irregular treatment of kala-azar than in those
who were treated with stibogluconate 20 mg kg-1 daily for 15 days (35%). The group of
patients who developed PKDL did not differ from those who did not develop PKDL with
regard to age and sex distribution, reduction in spleen size, and conversion in the leishmania
skin test (LST).Severe PKDL was more frequent in younger age groups (P < 0.001)
(Zijlstra et al., 2000 a).
PKDL is characterised by a macular, maculopapular, and nodular rash in a patients who has
recovered from VL and who is otherwise well. The rash usually starts around the mouth
from where it spreads to other parts of the body depending on severity. It is mainly seen in
Sudan and India where it follows treated VL in 50% and 5-10% of cases, respectively.
Thus, it is largely restricted to areas where Leishmania donovani is the causative parasite.
The interval at which PKDL follows VL is 0-6 months in Sudan and 2-3 years in India.
PKDL probably has an important role in interepidemic periods of VL, acting as a reservoir
for parasites. There is increasing evidence that the pathogenesis is largely immunologically
mediated; higconcentrations of interleukin 10 in the peripheral blood of VL patients predict
the development of PKDL (Zijlstra et al., 2000 b).
23
CHAPTER III
MATERIALS AND METHODS
This study was undertaken to identify the factors ( housing system, animal rearing, lack of
knowledge about sand fly, living with previously affected kala-azar patients etc) associated
with kala-azar. The study also deals with the identification of the age & sex wise incidence
of kala-azar & PKDL in different villages of Fulbaria upazila, Mymensingh. This was done
by the following approaches:
a. Collecting records of Kala-azar infected patients from Upazila health complex Fulbaria .
b. Surveillances in the villages of kala-azar infected areas through structured
questionnaires.
3.1 Study area & Source of Data Collection
This investigation was carried out at the Fulbaria upazila of Mymensingh district. This area
is selected for this study as there is a high incidence of kala-azar patients in this this area(
Bern and Chowdhury, 2006). Data were recorded from the rural area by door to door survey
& from the Upazila Health Complex (UHC) Fulbaria, Mymensigh. In total eight villages of
Fulbaria upaziala were visited for the collection of data. These are Kaladah, Kalakanda
,Pachkohania, Kalanajani, Bakta , Andarpara, Chowda , Chankanda.
3.2 Time of Study
The study was conducted during June 2010 to December 2010.
24
3. 3 The questionnaires used for collection of information from patients:
Epidemiology of Kala-azar/ PKDL in selected villages of Fullbaria Upazila (Preformed Questionnaires)
1) Name of the patient Age.................. 2) Father / Husband Name 3) Sex M F
4) Name of the Village :
5) Total house hold number 1-5 Above 5
6) Number of infected persons in the house: 1-5 Above 5
7) Do they know about sand fly? Y N
8) Do they use Mosquito curtain? Y N
9) Where do they sleep? Floor Bed
10) House made of Clay Other (Tin shed)
11) Infection with Kala- azar Y N
12) Infection with PKDL Y N
13) How long ago Kalazor was diagnosed? 1-2 Y 2-5 Y
14) Do they have Cattle / Shed? Y N
15) How many cattle? 1-5 Above 5
16) How many goats? 1-5 Above 5
17) Do they have pet dog? Y N
Name of the Interviewer
Signature
Date
25
3.4 Methodology
3.4.1 Collection of data:
A total of 359 cases were recorded. Among these 259 were collected from Upazila health
complex, Fulabaria which include age, sex and PKDL related information. Other 100 data
were collected from eight villages of Fulbaria by the questionnaries which include all the
information mentioned in the questionnaires. The person who have been previously
diagnosed as kala-azar patient and have diagnostic report was considered as kala-azar
patients. In conducting interviews all possible measures were taken to ensure valid &
reliable data from the respondents. All the questions were carefully explained to the
respondents so that they could answer them easily & properly.
A questionnaire was developed for household–based individual interviews. Data were
collected with this preformed questionnaires which include details information about the
patients (Name, age , fathers / husband name , sex ,village name, total house hold number) ;
housing system, bedding system, knowledge about sand fly, number of infected person in
their family, about type of infection (kala-azar or PKDL), about animal rearing( cattle, goat
or dog). The disease was diagnosed in hospital by a strip test named as rk39 (Al-Nahhas et
al., 2003) as described by the doctors attending the hospital.
3.4.2 Fulbaria:
Fulbaria is located at 24°38′00″N 90°16′00″E / 24.6333°N 90.2667°E . It has 68469 units of
house hold and total area 402.41 km². Fulbaria Upazila (mymensingh district) with an area
of 402 km2, is bounded by muktagachha upazila on the north, bhaluka upazila on the south,
trishal and mymensingh sadar upazilas on the east, ghatail, madhupur and Muktagachha
upazilas on the west (Fig-3).
26
Fig-3: Map of Fulbaria upazila.
Main rivers are Khiru, Nageshwari, banar and Bajua; main beel is Bhawal. The upazila
consists of 13 union parishads, 104 mouzas and 137 villages. Fulbaria has an average
literacy rate of 20.6% (7+ years), and the national average of 32.4% literate. Maximum
people in this rural area live in mud house.
27
3.4.3 Upazila Health Complex (UHC) Fulbaria , Mymensingh:
It is the main source for the rural people of Fulbaria from where they can take treatment of
different kinds of diseases very chiefly. It is a 50 seated health complex. There is a separate
section in this health complex for kala-azar patients. The main duty of this section is to give
proper treatment and advice to kala-azar infected patients.
28
CHAPTER IV
RESULTS
A total of 359 cases of patients were recorded during this study from UHC of Fulbaria,
Mymensingh & its surrounding affected villages. Among them 259 were collected from the
UHC and 100 from eight villages of Fulbaria. The influence of age, sex, housing system,
presence of domestic animals & their shed & knowledge about sand fly, sleeping pattern,
use of mosquito curtain by kala-azar patients were considered during this investigation. The
result of this research has been described in the following paragraph.
4.1 Kala-azar & PKDL in different age group
Out of 359 cases it was found that children were more vulnerable to be affected by kala-azar
than adult. Children less than 10 years of age were found to be affected most frequently
(40.11%). The incidence were found to be decreased with the increase of age (11-20 years
=19.50 % cases, 21-30 years =19.22%, 31-40 years =10.58%, 41-50 = 6.40% & above 50
years = 4.17 % cases) (Fig-4; Appendix-2). Ratio of children <10 years group with 11-10
years group was 2.05 & with > 50 years group was 9.6(Appendix-2). PKDL is usually a
sequel to kala-azar that developed 6 months to 2 years after kala-azar ( Das et al., 2010).
Like other countries it is also recorded in Bangladesh (Bern and Chowdhury, 2006). Among
the kala-azar infected patients 14.48% cases were developed to PKDL (Fig-6; Appendix-2).
Kala-azar vs PKDL ratio was 6.90:1. Children less than 10 years of age were most
susceptible (4.46%) to develope PKDL. The incidence of PKDL were also decreasing with
age (11-20 years =3.34% cases, 21-30 years =2.23%, 31-40 years =1.95%, 41-50 = 1.95%
& above 50 years = 0.56% cases) (Fig-5; Appendix-2). Ratio of children with 11-10 years
group was 1.33 & with > 50 years group was 8 (Appendix-2).
29
30
Fig-5: PKDL patients in age wise categories.
4.2 Kala-azar in different sex group Out of 359 cases 209 were males & 150 were females. The incidence was higher in males
(58.22%) than females (41.78%) (Fig-7; Appendix-3). In all age group it was found that
males were affected in higher proportion than females (ratio 1.39) (Appendix-3).
31
4.3 Animal shed, Mud house & Knowledge about sand fly
Among the 8 villages total 100 kala-azar infected patients were interviewed. The rural area
of Fulbaria is well known for the presence of mud house. Forty seven (47) percent of kala-
azar infected patients were found to be lived in a house which are totally made by clay
(ratio 0.89) & rest 53% have tin shed house or house that made by straw but having earthen
floor in most of cases (Fig-8; Appendix-4). These mud house & earthen floor have several
cracked & small hole all over the place which are favorable breeding and hiding place for
the sand fly. Even during insecticide spraying if the chemical cannot reach upto the hole &
sand fly may remain intact long-term house-spraying campaigns against VL vectors may
also be difficult to sustain (Joshi et al., 2003). Sixty six (66) percent of these people have
been found rearing domestic animal (ratio 1.94) mainly cattle & goats within animal shed in
their house (Fig-9; Appendix-4). These animal sheds were also made by clay with earthen
floor, cracked wall & always remain wet. All this condition found in animal sheds are also
favorable for the sand fly. One of these two condition (mud house or animal shed) was
present in almost all cases. The village called kalakanda among 18 cases only 2 mud house
found but 13 animal sheds were present. Similarly in Chankanda out of 19 cases 10 mud
house & 16 animal shed were found. In pachkohania out of 13 cases 5 mud house & 9
animal sheds. In Andarpara out of 37 cases 23 mud house & 19 animal sheds were found.
Most of the people (72%) were found having no idea about sand fly. Only 28 % people
knows about the role of sand fly in the transmission of kala-azar(ratio 0.39). Moreover they
have no idea about the transmission & prevention of kala-azar.(Fig-10; Appendix-4)
32
33
4.4 Use of mosquito net, sleeping pattern & other factors Most of the kala-azar infected patients were found using mosquito curtain (98%) (Fig-11,
Appendix-4) and sleeping in bed rather than floor (90%) (Fig-12,Appendix-4). Only 2%
kala-azar infected patients did not use mosquito curtain during night ( ratio 0.02) & 10 %
were found sleeping in floor (ratio 0.11) ( Appendix:4). Almost in every family more than
one kala-azar patient was found & some other family where single kala-azar patients were
detected they usually have history of having kala-azar patients in past or living proximity to
other kala-azar infected house. They were mostly illiterate, having no knowledge about the
disease. There was also lack of proper treatment & diagnostic facility.
34
35
Fig- 13(a) Fig-13(b)
Fig-13(a&b): PKDL patients with specific lesion at skin. Fig-14(a) Fig-14(b)
Fig-14(a&b): Kala-azar patirnts standing infront of their mud house.
36
Fig-15(a) Fig-15(b) Fig-15(a&b): Two kala-azar patients in the same family(b) having teen shed house but
earthen floor(a).
Fig-16(a) Fig-16(b) Fig-16(a&b): Cracked wall with many small hole in mud house (a)& dampess around
the house(b).
37
Fig-17: Presenc of cattle in cattle shed of kala-azar patients house. Fig-18: Animal shed in kala-azar patients house with cracked wall.
38
CHAPTER V DISCUSSION
This study revealed higher incidence of kala-azar patients in males (58.22%) than females
(41.78%) in Fulbaria upazila under Mymensingh district. Male female ratio was 1.39:1.
This indicate that males were highly exposed to the risk of affected with the disease than
females. These results are consistent with other studies done in Bangladesh (Rukunuzzaman
& Rahman, 2008; Mamoon et al., 2005) and in other countries (Rijal et al., 2010; Singh et
al., 2010 a&b; Rahim and Ashkan, 2007; Alvar et al., 2007; Raddam et al., 2007; Guerra et
al., 2004; Zerpa et al., 2003; Soleimanzadeh et al., 1993). Although exact cause for the
higher incidence of kala-azar in male cannot be explained but several factors including
clothing pattern, sleeping habits & occupation may be associated with this (Bari, 2008).
But some other study showed that there was no significant difference between sexes among
kala-azar patients (Sarkari et al., 2010; Amro et al., 2009; Bern et al., 2005). This difference
in result may be due to the equal exposure to the sand fly by men & women.
It was found that the majority of cases (40.11%) were below the age of 10 years; this
suggests children are at higher risk. The incidence of kala-azar was decreasing with the
increase of age & adults are less affected than children. Similar result also found in
Bangladesh (Mamoon et al., 2005) & other countries (Palumbo, 2010; Sarkari et al., 2010;
Albuquerque et al. 2009; Raddam et al., 2007; Bern et al., 2007; Zerpa et al., 2003;
Mohebali et al., 2001; Davies et al., 1999; Soleimanzadeh et al., 1993).
This is probably because of low immunity & lower prevalence of protective delayed type
hepersensitivity (DTH) responses in children & young than adult (Bern et al., 2006). But
opposite result were also reported (Joshi et al., 2006). No significant difference in disease
39
conversion among children and adults reported by Gidwani et al. (2009). This difference
may be due to the lower level of protective immunity in the adult people from where data
were collected.
PKDL is a common complication following kala-azar. It is mostly developed in those who
received inadequate and irregular treatment of kala-azar (Zijlstra et al., 2000). PKDL is
reported mainly from two regions - Sudan in eastern Africa and the Indian subcontinent,
with incidences of 50-60% and 5-10%, respectively. Importantly, patients with PKDL are
considered as reservoirs of VL. The etiopathogenesis of PKDL is presumably due to an
immunological assault on latent dermal parasites (Ganguly et al., 2010).
The sduy indicate that PKDL patients are also present at Fulbaria upazila, Mymensingh.
Among the kala-azar infected patients 14.48% were developed to PKDL. The incidence of
PKDL was more frequent in younger age groups. Higher incidence of PKDL in younger age
groups also found by Zijlstra et al. (2000). More over PKDL may developed without any
previous history of visceral leishmaniasis reported by Das et al. (2010); Ramesh (1995).
In this study attempt were taken to find out the important factors associated with the
presence of higher amount of kala-azar patients in Fulbaria upazila, Mymensing. These
important factors are living in mud house or other types of house but having earthen floor,
presence of animal shed which also made by mud & lack of knowledge about the role of
sand fly for the transmission of disease, living in the same house hold with kala-azar
patients, poor economic condition etc.
Forty seven (47) percent of kala-azar infected patients were found to be lived in houses
which are totally made by clay (ratio 0.89) & rest 53% have tin shed house or house that
40
made by straw but having earthen floor in most of the cases. There were many cracks &
crevices in these mud houses, with dampness & earthen floor. These cracks & crevices in
mud house are favorable living & breeding place for the sand fly. Plastering of these cracks
& crevices in mud house showed sudden decrease of sand fly density than unplastered mud
house (Kumar et al., 1995).
Some other study also showed that living in mud house is a important factors associated
with kala-azar (Bern et al., 2010; Singh et al., 2010; Saha, 2009; Kumar et al., 2009;
Rukunuzzaman & Rahman, 2008; Mamoon et al., 2006; Schenkel et al., 2006; Bern et al.,
2000)
It was found that 66% of kala-azar patients rearing domestic animal & having animal shed
in their house (ratio 1.39). These cattle sheds and surroundings area acts as a breeding sites
of Phlebotomus argentipes (Singh et al., 2008; Kundu et al., 1995; Kesari et al., 1992;
Ghosh et al., 1991; Dhiman et al., 1983). Phlebotomus argentipes also bites cattle & goat
take blood meal from them (Palit et al., 2005; Basak et al., 1995; Ghosh et al., 1990). So
these domestic animals may serves as a reservoir of kala-azar. Some other study also
showed that rearing domestic animals (cattle, goat, dog) & having cattle shed are important
factors associated with kala-azar (Singh et al., 2010; Amro et al., 2009; Mamoon et al.,
2006; Gavgani et al., 2002). It was found that increasing cattle density & domestic animal
as a risk factor in one village but not the other (Barnett et al., 2005; Ryan et al., 2006).
But ownership of a cow or buffalo could be protective against kala-azar spreading (Bern et
al., 2007; Bern et al., 2006; Bern et al., 2005; Bern et al., 2000). As sand fly take blood
meal from domestic animal they may not willing to bite human being so these may be
protective. Although no significant associations between VL and keeping domestic animals
inside the house as reported by Singh et al.( 2010).
41
In this study it was found that only 28% kala-azar patients knew about the sand fly ( ratio
0.39). Rest 82% did not know anything about this. Lack of knowledge about sand fly
ultimately results as a failure to protect the kala-azar spreading. Some other study also
showed that lack of knowledge about sand fly as a important risk factors for the
transmission of kala-azar (Mamoon et al., 2006).
The use of mosquito curtain & sleeping pattern (in bed or floor) does not have any impact to
prevent the kala-azar. 98% of kala-azar patients have been found using mosquito curtain
during night & 90% sleeping in bed whether only 10% sleeping in floor (ratio 0.11). Sand
flies usually bite during dawn & dusk period (Bern et al., 2010) the time when rural people
usually do not sleep. Higher exposure rate of VL in mosquito net non-users than the
mosquito net users (Rukunuzzaman & Rahman, 2008; Mamoon et al., 2006; Schenkel et al.,
2006). This is may be due to the use of normal bed net which are not protective against sand
fly. So insecticide treated bed net should be used. Use of insecticide treated bed net is
protective against kala-azar (Picado et al., 2010; Bern et al., 2005). But some study showed
that use of normal bed net protective against kala-azar (Bern et al., 2010; Saha, 2009; Bern
et al., 2007; Bern et al., 2006). This variation may be due to the different life style of kala-
azar patients in different region from where data were collected. Higher frequency rate of
VL were found in persons sleeping on floor than in sleeping on Chowki or Khat
(Rukunuzzaman & Rahman, 2008). This may depend on sleeping pattern of kala-azar
patients from where data were collected.
In some other study odd ratio was calculated to evaluate the result (Singh et al., 2010). But
in this study all data were collected from positive cases so the odd ratio cannot be
calculated.
42
Maximum kala-azar patients were found with poor economical condition. They were most
vulnerable, as this vector-born disease is linked to poor housing and unhealthy habitats
(Boelaert et al., 2009; Saha, 2009). Poorest people are most frequently affected reported by
Rijal et al. (2010); Bern et al.(2010); Mamoon et al. (2006). But some other study showed
no relation of economical condition with the disease (Singh et al., 2010; Bern et al., 2005).
That may be due to living in unhealthy & poor housing system by the reach people in the
rural area as like as poor people.
In every family more than one kala-azar infected patients were found. That indicate kala-
azar occurred more among persons living in the same household. This findings is also
reported by reported by Gidwani et al. (2009); Amro et al. (2009); Bern et al. (2007);
Sakru et al. (2006); Bern et al. (2006); Barnett et al. (2005). In a study of one Bangladeshi
community showed that, people living within 50 meters had a 3-fold increase in kala-azar
risk and those with a previous case in the same household were 26 times more likely to
develop kala-azar, compared to individuals living more than 50 meters away (Bern et al.,
2005)
Vegetation around the house, illiterate people, lack of proper treatment & diagnostic
facility, incomplete treatment, lack of awareness & proper knowledge about the diseases
these are the other factors found by talking with kala-azar infected patients & observing
their living condition. Vegetation around the house is a factors associated with kala-azar
also reported by Bern et al. (2010); Kumar et al. (2009).
43
CHAPTER VI SUMMARY AND CONCLUSION
Kala-azar which is also known as visceral leishmaniasis, black fever or damdam fever is
one of the most threatening parasitic disease in the world. Worldwide incidence is 500,000
cases per year. In Bangladesh more than 50% of total cases has been reported from
Mymensingh District. People of Five upazila of Mymensingh district specially people of
Fulbaria upazila is in higher risk to be affected by this disease. In this study attempts were
taken to find out the important factors associated with the increasing incidence of kala-azar
in Fulbaria upazila, Mymensingh. To do this data were collected from Upazila Health
Complex , Fulbaria & from rural areas of Fulbaria by door to door visit through a preformed
questionnaire. Result from this study showed that children less than 10 years of age
(40.11%) and males (58.22%) are in higher risk than females (41.78%). The incidence of
the disease was found to be decreased with the increase of age (11-20 years =19.50 % cases,
21-30 years =19.22%, 31-40 years =10.58%, 41-50 = 6.40% & above 50 years = 4.17 %
cases). Post-kala-azar dermal leishmaniasis is usually a sequel to kala-azar. Like other kala-
azar infected countries it is also present in Bangladesh. Among kala-azar infected patients
14.48 % were found to developed PKDL. Children less than 10 years of age were most
susceptible (4.46%) to developed PKDL. The incidence of PKDL were also decreasing with
age (11-20 years =3.34% cases, 21-30 years =2.23%, 31-40 years =1.95%, 41-50 = 1.95%
& above 50 years = 0.56% cases).
47% kala-azar patients were found living in mud house with cracks, crevices & dampness
in the wall & floor which are favorable living place for sand fly, rest 53 % living in other
types of house but almost all cases having earthen floor. Among kala-azar infected patients
66% have been found rearing domestic animal & having animal shed in their house which
44
are also made by clay. Only 28% patients were found having knowledge about sand fly.
Most of these people were poor & living proximity to previously affected patients. The use
of mosquito curtain & sleeping pattern (in bed or floor) does not have any impact to prevent
the kala-azar. 98% of kala-azar patients have been found using mosquito curtain during
night & 90% sleeping in bed whether only 10% sleeping in floor.
Other factors that are likely to be associated with increasing incidence of kala-azar in
Fulbaria are vegetation around the house, illiteracy, lack of proper treatment & diagnostic
facility, incomplete treatment, lack of awareness & proper knowledge about the diseases
these are the other important factors.
However in this experiment data were collected only from eight villages & UHC of
Fulbaria. Therefore extensive work is needed to determine if there are any other factors
associated with this disease & to find out the actual role of domestic animal whether they
serves as a reservoir of Leishmania sp. to spread the disease into human or not.
As the disease become a sever health problem in Fulbaria upazila so immediate attempt
should be taken from government & non government organization to make people aware
about the disease and take proper steps for the prevention & treatment of the disease.
45
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APPENDICES
Appendix-1: rk39 test
It is a rapid dipstick test based on the recombinant K39 protein is available for rapid
diagnosis of kala-azar. K39 is an epitope apparently conserved on amastigotes of
Leishmania species that cause visceral infection; by use of laboratory ELISA testing,
circulating anti-K39, IgG is detectable in 95%- 100% of patients who have kala-azar,
irrespective of geographic region. Using K39 antigen-impregnated nitrocellulose strips
developed for field conditions, fingerstick-obtained blood and serum samples tested from
Indian subjects demonstrated a positive anti-K39 immunochromatographic reaction in 362
patients with aspirate-proven kala-azar; with an estimated sensitivity of 100% and a
specificity of 97%. The strip testing proved simple to perform and yielded results within ten
minutes.
Kit Contents : Kala-azar dipstick test strip.s is a membrane, pre-coated with a recombinant VL antigen on
the test line region and chicken anti-protein A on the control line region. The Kit contains
the following:
1. Twenty-five (25) individually pouched Test Strips or twenty-five (25) test strips in a vial
with desiccant in the cap.
2. One vial of Chase Buffer solution.
Principle: The kala-azar dipstick rapid test is a immunochromatographic assay for qualitative
detection of antibodies to L. donovani in human serum. The assay is for aid in the
presumptive diagnosis of VL. During testing the serum sample reacts with the dye
conjugate (Protein A Colloidal Conjugate, which has been pre-coated in the test device).
58
The mixture then migrates upwards on the membrane chromatographically by capillary
action to react with rK39 antigen on the membrane and generate a red line. Presence of this
red line indicates a positive result while its absence indicates a negative result. Regardless
of the presence of antibody to VL antigen, as the mixture continues to migrate across the
membrane to the immobilized chicken anti protein
A region, a red line at the control line will always appear. The presence of this red line
serves as a verification for sufficient sample volume and proper flow and is a control for the
reagents.
Test Procedure: 1. 1 or 2 drops of finger prick blood assayed for anti K39 IgG.
2. Kala-azar dipstick strip was removed from the pouch or vial
3. One drop of blood was placed on the absorbent pad on the strip bottom.
4. The test strip was placed into a test tube so that the end of the strip remain facing
downward.
5. The mixture was allowed to migrate upto the strip by capillary action.
6. 2-3 drops of the Buffer solution were added which provided with the test kit to the pad.
7. The results was read after 10 minutes. The background should be clear before reading
the test, especially when samples have low titer of anti-Leishmanial antibody, and only a
weak band appears in the test region (T). Results interpreted after 10 minutes can be
misleading.
Interpretation: A positive result: The test was positive when a control line and test line appear in the test area. A positive
result indicates that the Kala-azar dipstick detected antibodies to L.donovani. A faint line is
59
a positive result. The red color in the test region will vary depending on the concentration of
anti Leishmanial antibodies present. The test line for .weakly positive. sera samples may
show
results between a weak positive red line to a faintly red, almost white background. (.Weakly
positive. samples are those with low affinity or low titer antibodies against the recombinant
test antigen).
A Negative Result: The test was negative when only the control line appears. A negative result indicates that
the Kala-azar dipstick did not detect antibodies to L. donovani.
An Invalid Result: If no lines appear at either the control or test line areas the test is invalid. The test is also
invalid if no control line appears, even though a test line is seen. It is recommended to retest
using a fresh dipstick and fresh blood sample should be used in such a case.
Storage: The sealed pouch or vial containing the test strip along with the buffer vial is designed to be
stored at room temperature (200C - 280C) for the duration of its shelf life. Exposure to
temperatures over 300C can impact the performance of the test and should be minimized.
The strips should not be frozen. The test should be used within one hour after removal from
the pouch or vial to prevent exposure to humidity. The shelf life of dip-stick is quiet long so
the dip-stick can be easily stored in Primary Health Centre (PHC), in the endemic states
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Appendix-2: Kala-azar in sex wise categories
Age(years) Males Percentage(%) Females Percentage(%) Sex ratio
Up to 10 76 52.78 68 47.22
Males vs
Females
1.39
11-20 43 61.43 27 38.57
21-30 37 53.62 32 43.38
31-40 24 63.16 14 36.84
41-50 16 69.57 07 30.43
Above 50 13 86.67 02 13.33
Total 209 58.22 150 41.78
Appendix -3: Patients of different age group suffering from kala-azar & PKDL
Age
(years)
No. of Kala-azar cases
Percentage
(%)
Age ratio
No. of PKDL cases
Percentage
(%)
Age ratio
Kala-azar vs PKDL ratio =6.90
Up to 10
144 40.11 <10 vs 11-20 = 2.05
16 4.46 <10 vs 11-20 = 1.33
11-20 70 19.50 11-20 vs21-30 =1.01
12 3.34 11-20 vs 21-30 =1.5
21-30 69 19.22 21-30 vs 31-40 =1.81
08 2.23 21-30 vs 31-40 =1.14
31-40 38 10.58 31-40 vs 41-50 =1.65
07 1.95 31-40 vs 41-50 =1
41-50 23 6.40 41-50 vs > 50 =1.53
07 1.95 41-50 vs > 50 =3.5
Above 50
15 4.17 < 10 vs > 50 =9.6
02 0.56 < 10 vs > 50 =8
Total 359 100 - 52 14.48 - -
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Appendix -4: Number of kala-azar patients living in mud house , having animal shed, sleeping pattern, use of mosquito curtain & their knowledge about sand fly. Name of village
No. of kala-azar patients
No. of mud house
Ratio
No. of animal shed
Ratio
No. of patients knows about sand fly
Ratio
Sleeping in floor
Mosq-uito curta-in non user
Kaladah 04 03 0.89
03 1.94
03 0.39
10 (ratio 0.11)
02 (ratio 0.02)
Kalakanda 18 02 13 08
Kalanajani
04 03 04 02
Pachkohania 13 05 09 04
Andarpara 37 23 19 05
Bakta 02 00 01 00
Chowda
03 01 01 01
Chankanda
19 10 16 05
Total 100 47 - 66 - 28 - - -