Mobammad Abdur Rab  ( National Institute of Health, Islamabad. )
Jamshaid Iqbal  ( National Institute of Health, Islamabad. )
Farrukh Hameed Azmi  ( National Institute of Health, Islamabad. )
Mohammad Arif Munir  ( National Institute of Health, Islamabad. )
Mohammad Saleem  ( Armed Forces Institute of Pathology, Rawalpindi. )

Between January’85 and August’87, 22 cases of VL were seen at National Institute of Health, Islamabad. Three (14.6%) came from the previously known endemic region of Gilgit, 15 (68.1%) from different localities in Azad Jammu and Kashmir (AJ&K), and 4(1 7.3%) from neighbouring foci in NWFP and Punjab. Mean age of the patients was 4.2 years, (Range, 10 months to 57 years) median 2.5 years and mode 2 years. High levels of Leishmania antibodies were detected by Indirect Immunofluorescent Antibody Technique (IFAT) in all cases. Leishmania were isolated from bone marrow aspirates of 2 patients and isoenzyme characterization performed in one of these, the organism was typed as Leishmania infantum sensu stricto. Sera from 289 children residing in 5 endemic localities in AJ&K was tested for Leishmania specific antibodies by IFAT and low levels of these antibodies were detected in 15.4% of the cases (JPMA 39: 225, 1989).

Human visceral leishmaniasis was first re­ported in Pakistan from the northern areas in the late fifties and early sixties^1,2, the disease was thou­ght to be restricted to foci in Gilgit and Skurdu. Since the advent of this decade, sporadic cases began to occur not only in different foci of AJ&K but also in some extreme north eastern areas of Punjab as well as nearby localities of NWFP³. The disease is acquired through the bite of infected phlebotomine sand flies. Amastigotes mu­ltiply within mononuclear phagocytes throughout the reticuloendothelial system including spleen liver, lymphnodes and bone marrow. Clinically, the disease is marked by intermittent fever, anaemia, lymphadenopathy and hepatosplenomegaly. Pro­gressive weight loss, cachexia and malaise, followed by bacterial superinfections and can sometimes result in death. In general once hepatosplenomega­ly occurs host defence mechanisms are not able to prevent further parasitic multiplication⁴. Patients. with VL during the acute stage have high levels of globulins and produce Leishmania-specific antibo­dies. Following successful treatment, these ant tibody levels decline and a gradual, cell mediated, delayed type hypersensitivity response to leishma nial antigen is elicited which manifests over several months, and resistance to infection usualiy deve­lops⁵. Bone marrow aspiration has been used ex­tensively for the diagnosis of VL. This is an invasive procedure and may lead to serious complications if performed by individuals not fully trained in this technique. IFAT, a relatively more sensitive proce­dure, is now widely used for the diagnosis and seroepidemiological studies of VL⁶. This pilot study was undertaken to detect leishmania anti­body levels in patients and determine baseline anti­body levels in endemic populations in AJ&K using IFAT.

a) Selection of Localities:
Five villages in AJ&K (Total population, 2600) were selected for seroepidemiological sur­vey. From these villages atleast 7 active cases had been reported in the past two year period. These villages are located in the vicinity of 3 major towns in AJ&K i.e. Rawalakot, Dirkot and Chikar. The houses in these villages are generally scattered along the hills in this region.
b) Collection of Sera for IFAT:
Sera was obtained from 22 patients admitted in different hospitals of Rawalpindi and Islamabad. .Sera samples were also collected from 289 children (under 12 years) residing in 5 localitjes mentioned above. Of these 289, venous blood was drawn from 90. From the remaining 189 children, 100 microliter blood samples were drawn by finger pricks and dried on No. 4 Whatman filter paper. These were appropriately numbered and stored at -20C Se­rum was eluted in 1 ml Phosphate buffered saline (PBS, pH 7.4) to give a starting dilution of 1 in 20.
c) Preparation of Antigen:
Leishmania was isolated from a case of VL and an invitro culture was maintainS in NNN medium. Promastigotes were harvested from this culture and after three washings in PBS, stored overnight at 4°C in 1% formol PBS. This was wash­ed three times again with PBS and diluted to con­tain 30 to 40 parasites per high power field. One drop of this antigen was placed on each spot of a 12 well multispot slide (flow Laboratories). The antigen slides were air dried at room temperature, properly wrapped in aluminum foil and placed in self sealed polytbene bags, with a few crystals of silica gel. The slides were stored at -70°C till use.
d) Conjugate:
Commercially available antihuman IgG fluo­rescence isothiocyanate (FITC) conjugate obtain­ed from Sigma, was used at a working dilution of 1:20 in PBS. At this dilution no fluorescence was observed in the negative control.
e) Test Procedure:
The antigen slides were brought to room temperature before use. 25 micro liter of test serum was applied to each antigen spot and allowed to react for 30 minutes at room temperature inamoist chamber. The slides were washed 3 times in PBS through serial changes of 10 minutes each. An­tihuman IgG FITC conjugate was then applied and incubated for 30 minutes at room temperature in moist chamber. Washing procedure was repeated. The slides were mounted in a preparation of 90% glycerol in 03M Carbonate/Bicarbonate buffer (pH 9.0) and covered with coverslips. Both positive and negative control sera were used each time the test was performed.
f) Fluorescence Microscopy
Slides were examined with an Olympus fluo­rescent BH2 microscope using a dark field con­denser and 400 X magnification. Light source was 12 volts, 50-60HZ halogen lamp.
g) Isoenzyme Characterization:
Culture on NNN medium was obtained from bone marrow biopsy of a two year old male patient admitted in a local hospital. Isoenzyme charac­terization on this sample was performed at aWHO recognized center, Instituto Superiore Di Santa in Rome, Italy.

Serology of Patients
All confirmed cases of VL demonstrated very high titers of Leishmania antibodies (Figure 1).

9.1% were positive at titers of 1:400, 27.2% at 1:800, 182% at 1:1600 and 36.4% had titers of 1:3200 or more. Serology of Children in Endemic Areas Of the 289 children examined (Figure 2),

none showed clinical evidence of disease. IFAT results did not reveal detectable antibodies to Lei­shmania in 84.6%. 14.1% were positive at serum dilutions of 20, and 13% were positive at dilutions of 40 (Figure 3).

Sera from 40 control children living in nonendemic regions were all negative for leishmania antibodies.
Isoenzyme Characterization
Strain characterization on one isolate, iden­tified the parasite strain as Leishmania infantum sensu stricto. Reference stocks used for identifica­tion included L. infantum s. st., L.infantum NH variant Linfantum NH MDH ASAT variant and L.donovani. Enzyme patterns were examined for enzymes such as PGM, PGJ, GOT, G6PD, P6GD, ME, MDH, LDH, MPI and NH.

IFAT has been used to study leishmania an­tibodies in patients suffering from YL. All patients exhibited high levels of such antibodies, except two children, in whom the levels were rather low (Posi­tive at dilution of 200). Both these patients had re ceived a course of Pentavalent Antimony before their serum sample was tested. Successful treat­ment of VL with pentavalent antimonials results in a rapid decline in leishmania specific antibodies and this is used as an indicator to monitor and assess the response to treatment in patients^7,8. Titers over 1:256 are considered to be diagnostic for VL⁹, and in our study, 91% of the patients presenting with clinical disease had antibody levels higher than 1:400 (Figure 1). Of the 22 patients in the study, the disease was diagnosed both by a positive bone marrow aspiration and high levels of leishmania antibodies detected by tEAT in 18. IFAT alone was used successfully for diagnosis in the remaining four, and two of these patients had IFAT antibody levels of 1:800 while the other two had titers in excess of 1:3200. Clinical studies in this region show evidence that the disease is of the Mediterranean type, af­fecting most commonly young children. The dis­ease is manifested by prolonged fever, anaemia, hepatosplenomegaly, generalised lymphadenopa­thy, weakness, wasting and emaciation. Laboratory investigations in patients revealed pancytopenia and markedly increased levels of gammaglobulins. Leishmania isolate obtained from bone marrow of a two year old male child admitted in a local hospi­tal was typed as Leishmania infantum ss on isoen­zyme characterization. Seven cases occurred in five villages of AJ&K. A randomly selected sample of 289 child­ren under 12 years of age, from these villages revea­led low levels of Leishmania antibodies. Of these, 14.1% had antibody titers of 1:20 and only 13% had levels of 1:40, while 84.6% of the children were negative for leishmania specific antibodies. Sera obtained from 40 control children from non en­demic area were also negative. In one house hold where a six year old female patient had recently expired of VL, none of her family members includ­ing 3 sisters (age range 2-10 years) showed any clinical evidence nor had history suggestive of past disease. They were also negative on serology. In another village a 5 year old girl who had suffered from the disease two years before, and had since fully recovered demonstrated an antibody level of 1:20 in her serum. Careful foflowup of the children with low levels is therefore essential. Asymptomatic and subclinical forms of VL are known to occur. ¹⁰ In a prospective study in Brazil¹¹, of the 86 children with leishmania an­tibodies 23.3% remained asymptomatic, 17.4% de­veloped classical VL within weeks of index serol­ogy. The remaining 59.3% children showed sub­clinical disease manifested by mild constitutional symptoms malaise, diarrhoea, poor work-play tole­rance and intermittent hepatomegaly. Of those showing subclinical disease 25% progressed to cla­ssical VL, and the remaining resolved theft illness after a prolonged period. As Geimsa stained sme­ars of bone marrow aspirates are usually negative for leishmania in the absence of classic Kala-azar, infection rates in populations can be determined by sensitive serological techniques such as IFAT, EUSA, RIA¹² and by delayed hypersensitivity skin testing^13,14. Identification of vector in this region has not yet been achieved. Although a large variety of phle­botomine sandflies are found in Pakistan¹⁵, the Ones that have been identified in the northern areas include Ph.chinensis, Ph. sergenti, Ph.kendeliki bu­rnei and Ph.major. Promastigotes have not been found in any of the species so far. Dogs and other wild carnivores such as foxes, jackals and wolves are primarilybelieved to be the reservoir of disease in most endemic areas¹⁶. The prevalence of human infection cannot be directly correlated with that in dogs. A number of domestic dogs were examined for evidence of clinical disease such as cutaneous lesions, onychogyrophosis, keratoconjunctivitis, H­gidity of posterior limbs etc. None were found tobe affected. Sera from 4 dogs tested for leishmania antibodies by bemagglutination, yielded negative results. Recently however, seilsitive and more reli­able techniques such as IFAT¹⁷ have been success­fully employed in the detection of antibodies in the reservoir host. In conclusion, therefore it is observed that sporadic cases of VL are occurring in the northern parts of the country, most commonly affecting you­ng children. The disease was first reported from the northern areas about three decades ago and was thought to be restricted to foci in that region. How­ever, since the advent of this decade, cases of vis­ceral leishmaniasis began to be reported from dif­ferent foci in Azad Jammu and Kashmir. Even more alarming is the fact that the disease has also been reported from regions of the North West Frontier Province as well as Punjab that neighbour AJ&K. There is thus conclusive evidence that the disease is gradually spreading southwards in the country. In order to limit this disease the exact epidemiological pattern needs to be defmed and knowledge regarding both vector as well as the reservoir host is essential before comprehensive control efforts can be undertaken.

The authors are indebted to Drs. Gramiccia, M. and Gradoni,L. of the Instituto Superiore Di Santa, in Rome, Italy, for isoenzyme characteriza­tion of a Leishmania isolate. Support of Drs K Abbas and Z. Kundi from Polyclinic and Holy Family hospitals at Islamabad/Rawalpindi is also gratefully acknowledged. Thanks are also due to Mr. Manzar All, Technician, Immunology Depart­ment, NIH, for helping us in field studies. This study was sponsored by the WHO/TDR Special Program for Research and Training in Tropical Diseases.

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