Objective: Immuno-epidemiology of kala-azar in terms of Leishmanial infection rates among specified age groups in tribal community, Utility of some diagnostic tools in eliciting evidence of recent and past infection. Materials and Methods: A cross sectional study among tribal communities, using Leishmanin skin test (LST) and direct agglutination test (DAT). Results: The prevalence leishmanial infection was found to be 44.4% past infection (LST positivity) and the prevalence of recent infection 44% (DAT positivity), respectively. The annual rate of infection in age groups up to 40 years was 3-5%. Statistical association was significant only for the effect of age on LST c 2 = 16.83 (P<.05) OR = .14 [95% CI= .05 to .42]. Associations with other important variables like sex, fever, spleen, family size were insignificant for both LST and DAT. Conclusion: Kala-azar infection rates are high in the tribal communities of Jharkhand. There was no association found between LST and DAT results. The DAT seropositivity to leishmanial infection in any age group is an indication of individual’s experience with the leishmanial infection. One can’t conclude for the active disease based on the seropositivity since the antibody levels (IgG) remain high even in a disease-free state.
Keywords: ARI, direct agglutination test, leishmanin skin test, visceral leismanaiasis
Kala-azar is one of the major public health problems in India. Bihar accounts for the major share with nearly 75% of the total cases reported from India. Tribals in Bihar are worst affected with the disease.
In the present study, we attempted to assess the magnitude of the Leishmanial infection in the tribal area by using two screening tests, namely direct agglutination test (DAT) and Leishmanin skin test (LST). The LST has been around since 1940s. The DAT is relatively a newer test since last 15 years.
The leishmanin test is specific for leishmaniasis but is not species specific. Leishmanin positivity in endemic kala-azar areas varies inversely with incidence of kala-azar indicating population immunity. 
Spontaneous cure as wee as “premunition” ore “sterile” resistance due to sub-clinical infection in endemic zones is more frequently encountered in India kala-azar. It appears that in kala-azar, there is exaggerated stimulation to the production of immunoglobulins some protective and other non-specific. On the other hand, it fails to stimulate requisite CMI necessary for spontaneous cure and sub-clinical infection suggests that the human host can mount up sufficient immunological reactions both humoral and cell medicated, to protect the individual. CMI probably plays the crucial role. 
The study was carried out in 2000-01 and helps in understanding of the epidemiology of kala-azar in terms of Leishmanial infection rates among specified age groups in a tribal community, utility of some diagnostic tools in eliciting evidence of recent and past infections, and studying susceptibility status and development of immunity against Leishmanial infection.
Litipara block (Jharkhand)
Litipara block is the most predominant tribal block in the Pakur district of Jharkhand state with 25% Pahadias and 50% Santhal tribes.  It is one among the six blocks of the Pakur district of newly created Jharkhand State in India. Jharkhand literally means Jhar (cluster of thick forest) and khand (tract of road).  Tribal population in this area is mainly dependent on the forest products, which they either sell in the market or exchange for the food grains. Out of four villages chosen for the study, Santhal tribe inhabits one and the Pahadia tribe inhabited the rest three villages. Pahadias have a Dravidian background, speak Malto language who migrated from south India.  The total number of this tribe is less than a lakh; there is concern that they might become extinct. Government has banned family planning programs in this community.
DAT – The DAT was done according to the methods described by Addy and Nandy 1995.  After cleaning the thumb, the skin was punctured with a sterile lancet; a large drop of blood was allowed to fill the circles of 12.7 mm diameter on the DAT Whatman filter paper No.3 printed strips. It was allowed to air dry, sealed in a selfsealing polythene bag, and dispatched to the laboratory.
DAT antigen preparation
The Antigen was prepared according to the earlier methods  of Harith et al. (1988) and Addy et al. (1989) with minor modification. Promastigotes of Donovani were harvested after 48-72 h of growth on modified Ray’s medium (Nandy et al. 1987). Harvested parasites were washed three to four times in a Locke solution. The parasites were then washed at 4°C in 2% (wt/vol) formaldehyde in the Locke solution. The parasites were then washed in 0.15 M NaCl and 0.056 M sodium citrate solutions (pH 7.4) three times and were stained with 0.1% Coomassie Brilliant Blue (Sigma Cat No. B 0149) in citrate-saline solution and resuspended in a 0.43% (wt/vol) formaldehyde-citrate-saline solution. The suspension was then filtered through a nylon mesh and the parasite count was adjusted at 6.5΄ 107 to 7.5΄ 107 per ml. This parasite suspension was preserved at 4°C in the formalinized citrate-saline solution till use.
Elution of blood from filter paper strips
Filter paper blood sample of each patient was extracted from single corresponding blood spot, overnight at 4°C in 1 ml, 0.15 M saline to provide a dilution of 1:30.
Direct agglutinated test
Each eluted sample was diluted further to 1:200 in tubes and then over microtiter “V” plates (Greeiner, West Germany) doubly till 12,800. The diluents used was 0.2% gelatin (Difco) in a citrate-saline solution with 0.78% 2-Mercaptoethanol. The first well of each transverse row was used as antigen control without a blood sample. Equal volume (50 μl) of antigen suspension was added to each well including the controls, missed by gentle shaking over the table top and incubated at 22°C overnight. The last well showing definite agglutination of the parasite was considered as the end point.
Leishmanin skin test
After antiseptic measures, 0.1 ml of antigen is injected intradermally into the volar surface of the forearm. A palpable nodule, 5 mm or more in diameter, after 48-72 h is considered positive.  The Leishmanin skin antigen was sourced from Centre for tropical medicine and Parasitology, Calcutta.
Census of the entire population of the four tribal study villages was conducted for sampling purpose. Every individual in each family according to hierarchy of the family was enumerated. Population in the census record formed a sampling frame. Through systematic random sampling technique, every fourth individual was enlisted as the study sample. Voluntary informed consent was obtained before taking necessary clinical information and subjecting them to the screening tests. All the newly diagnosed cases in the study sample were treated with sodium stibogluconate, free of cost.
Annual rate of infection for Leishmania was computed as follows:
For annual rate of infection incidence was found applying
e -It = 1 – CI
The study population was dominated by young and middle age group [Table 1] which is expected in a tribal population due to lower life expectancy. The prevalence of LST positivity (indurations >5 mm) in the study area was of 44.4% (95%CI = 33-55%). The prevalence of DAT seropositivity (³ 1:800) among the tribal communities in the study area was 44% (95%CI = 35-53%).The interpretation of test results in combination helps us distinguishing between resent and past infections [Table 2].
Annual rate of infection (ARI) of Leishmanial infection was derived for various age groups [Table 3], ARI for visceral Leishmania was highest (4%) in the age group of 11-25 years and the least (0.8%) in the age group ≥ 40 years.
This could be derived here as age-specific point prevalence which is an indication of Cumulative Incidence in that specified age groups since the LST positivity is a lifetime characteristic
Association of LST and DAT and their association with gender, spleen size, family size none of which turned out to be non-significant [Table 4]. However, the effect age on LST was statistically significant with OR= 0.14 [95% CI= 0.05 to 0.42].
The prevalence of the LST positivity in our study area was 44.4% (CI= 33-55%). Our prevalence figure was similar to the one reported from the Mediterranean region being 44.2% (in vallyzena). From India, the prevalence figures are available from West Bengal (19.2%) reported by Nandy et al. 
Earlier studies have shown male preponderance in kala-azar. , A retrospective study conducted on the Bihar epidemics in 1970s by Thakur  showed that the male:female ratio was 5.5:1. In our community, such a phenomenon was not observed. Most of the retrospective studies are done based on hospital records which are biased samples.
The significance of the prevalence of this magnitude that we have got is in the fact that kala-azar epidemics do not occur in communities when more than 40% of the population is Leishmanin skin positive, i.e., immune to the infection. 
The prevalence of LST positivity depends on
Patients who have recovered from infections achieve lifelong immunity. No record of second attack of visceral leishmaniasis is there. A positive leishmanin test, acquired naturally from unapparent infections, also protects infection. 
The trend of Leishmanial infection in a community can be assessed thorough computing annual rate infection. Age-specific data on the prevalence of Leishmanial infection could be used to estimate ARI.The knowledge of cumulative incidence for a given disease helps to compute the ARI for that disease in a given community. In our study area, the annual rate of leishmanial infection was almost same in all the age groups ranging from 3% to 5%. This was an interesting finding. It is especially so when annual rates of infection are compared for diseases like tuberculosis (TB) which is about 1% in India.
While Leishmanin test is a good indicator of the past infection in the community shown by hypersensitivity to the leishmanin antigen by cell-mediated response, it does not give any information about recent infection, which mainly elicits humoral response in the body. Since our objective was to study the prevalence of leishmanial infection in the community irrespective of whether it was past or present, we had to employ another serological test called DAT, which reacts with the circulating antibodies. LST in recent infection or active disease will be negative due to suppression of cell-mediated immunity thereby will not pick up active infections.
The middle age groups showed higher rates of LST positivity, an index of immunity, compared to the extremes on either side of age spectrum. This is attributed to high transmission of infection in kala-azar endemic areas, where the population gets infected early in childhood; consequently, by middle age, half of the population would have experienced leishmanial infection and hence be immune to disease.
We looked for any association between DAT seropositivity with fever and splenomegaly as surrogate variables for kala-azar, since the probability of the kala-azar is higher in cases with these signs in an endemic area, but no association could be found.
High titer of antibodies can be elicited in subclinical and asymptomatic cases although the mean level in such cases will always be lower than clinical cases. 
Proportion of leishmanin skin positives was higher in the afebrile group as compared to the febrile group. This was expected, since the probability of a febrile person being a kala-azar case is higher than an afebrile person. LST will never be positive in active disease. DAT seropositivity rate was higher in the afebrile group than the febrile group owing to the fact that in an kala-azar endemic area people with past infection continue to be seropositive for several years.
Correlation was not seen between seropositvity and LST. We were interested to know if seronegative groups have higher or lower LST positivity rate or the difference in sero status of a group differed in leishmanin-tested groups.
With Leishmanin picking up the past infection and DAT picking up the recent infection and active disease, we could have a better assessment disease burden in the community.
In our study area, the prevalence seropositivity (titer ≥ 1:800) to leishmanial infection was 44% (95% CI = 35-53). By employing the DAT we could capture extra 18 (23.1%) cases in the community in whom the LST was negative.
The absence of association between family size and kala-azar can be explained on the fact that overcrowding has no bearing on the transmission of leishmanial infection, unlike in diseases like TB or measles.
Correlation between leishmanial infection and splenomegaly was not significant in our study. This could be explained in part by the confounding effect by malaria.
Having both serological and leishmanin test result to our disposal, our interest was to look for any correlation between the two results and especially to see whether LST positivity was higher in seronegative as compared to the seropositive cases. No negative correlation was seen.
The DAT seropositivity to leishmanial infection in any age groups is an indication of individual’s experience with the leishmanial infection. One can’t conclude for the active disease based on the seropositivity since the antibody levels (IgG) remain high even in a disease-free state.
There are several hypotheses on why the antibodies linger very long after cure:
In kala-azar as in leprosy, there is deranged cell-mediated immunity with a normal or exaggerated humoral response. Indian kala-azar offers certain unique features which are not present in other viscerotropic leishmaniasis. There does not appear to be an effective local cellular response of lymphocytes and plasma cells, which are a common consequence of cutaneous, allergic form of leishmaniasis. The T cells are suppressed during active phase of the disease. 
The initial response to kala-azar seems to stimulate both specific and non-specific increase of immunoglobulins. The specific response to leishmania antigens is not perhaps protective. The non-specific increase of immunoglob sulins in kala-azar may be the result of deviation to systemic lymphoreticular system of antigens which are normally mopped up by the kupffer cells of liver. The non-specific antibodies may remain elevated up to 1 year even after the complete cure, hence serological tests may not be able to discriminate between current and past infections among the newly cured cases. 
Leishmania parasites are able to persist very successfully in immunocompetent individual and they are also able to suppress the immune response suggesting that relationship of the parasite with man is very old. Immunity acquired from infection is cellular. According to the degree of cellular immunity, the spectrum of infection varies from inapparent infection to overt disease. 
Source of Support: Community Health Cell Bangalore, Dr. Nalini Abraham, Delhi., Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]