Resurgence of visceral leishmaniasis in Bangladesh*

Bulletin of the World Health Organization, 61 (1): 113-116 (1983) K World Health Organization 1983 Resurgence of visceral leishmaniasis in Bangladesh* K. M. RAHMAN' & N. ISLAM2 Out of 218 suspected cases of kala-azarfrom different parts of Bangladesh, 59 could be confirmed; and out of4J patients with suspected skin lesions, 6 were confirmed as post- kala-azar dermal leishmaniasis. Confirmation of diagnosis was based on the demon- stration of L-D bodies in tissue specimens (spleen and liver (after puncture) or skin scrapings). The complement fixation test (CFT) and aldehyde test (A T) were positive in all but one case of kala-azar. Their usefulness in the diagnosis of the disease has been emphasized. The aldehyde test is very simple to perform and does not require any equipment or electricity; it can therefore be used as a screening test in rural areas. Failure of the organism to grow in NNN medium in 2 cases and failure to maintain organisms isolatedfrom some other casesfor more than 2-3 weeks in the culture medium indicate the possibility that different strains were involved. As the number of kala-azar cases appears to be increasing, the threat of an epidemic is real. Visceral leishmaniasis was thought to have been eradicated from Bangladesh as a collateral effect of the malaria eradication programme on the sandfly vector. In India also, the disease had essentially disappeared in the 1960s, but recently there have been reports of a resurgence of kala-azar following suppression of spraying for malaria control. Esti- mates of up to a million cases have been made in Bihar State (1) which adjoins Bangladesh. Cessation of spraying for malaria in Bangladesh has evidently also resulted in a resurgence of kala-azar, and we are informed that patients are reporting to the major medical centres of Bangladesh from their respective catchment areas. Increasing numbers of visceral and post-kala-azar dermal leishmaniasis (PKADL) cases are being diagnosed in our laboratory. The danger exists that these sporadic cases have reached, or soon may reach, a level which could provide a reservoir of sufficient magnitude to spark a major epidemic in this country. However, to our knowledge, there have been no published reports on visceral leishmaniasis in Bangladesh for over a decade. The purpose of this communication is to call attention to this threat of an epidemic, and to present information on the current situation. We also want to call attention to the diagnostic pitfalls of reliance upon currently available laboratory * From the Institute of Postgraduate Medicine and Research, Dhaka, Bangladesh. ' Professor and Head of the Department of Microbiology. 2 Director and Professor of Medicine. methods in Bangladesh, since quick and reliable diagnosis is a prerequisite to effective control measures. MATERIALS AND METHODS A total of 218 consecutive cases of suspected kala- azar, who had been seen in the periods 1968-70 and 1973-80, were available for the study. All of them presented with chronic enlargement of the spleen, most had an enlarged liver, and the majority had pyrexia. Forty-one patients with suspected post-kala- azar dermal leishmaniasis, who had been referred to our laboratory, constituted another group of patients. The criterion for confirmation of the diseases was demonstration of Leishman-Donovan (L-D) bodies in specimens of tissue from the patients. The complement fixation test (CFT) for kala-azar, using an extract of Mycobacterium phlei (2), and the aldehyde test (AT) of Napier (3) were performed in all the subjects. A positive CFT in 1:100 (or more) dilutions of serum was taken as strongly positive, in 1:50 dilutions as moderately positive, and in 1:25 dilutions as weakly positive. In the aldehyde test, a positive reaction within 20 minutes was taken as strongly positive, within 2 hours as moderately posi- tive, and within 24 hours as weakly positive. Microscopic examinations and cultures for L-D bodies from splenic puncture specimens were performed in all but 4 of the suspected cases of kala- azar. In the 4 cases, no spleen specimen was 4266 -113 K. M. RAHMAN & N. ISLAM available; instead, specimens from liver or bone- marrow puncture were examined. The tissue specimens were cultured at 22 °C in modified NNN medium containing rabbit's blood (4). Patients with suspected PKADL were subjected to skin scrapings for microscopy and culture. RESULTS Of the 218 suspected cases, 59 were found to have kala-azar on the basis of demonstration of L-D bodies in the tissue specimens. Their ages ranged from 6 to 60 years (mean: 26 ± 11.5 years) and 46 of them were males. The places of residence of 34 of them had been recorded and these are shown in Fig. 1. None of these patients had been out of Bangladesh during any period of their lives. The results of the complement fixation test and aldehyde test on these 218 cases are shown in Table 1. Out of 41 suspected PKADL cases, 6 were found to have L-D bodies in the skin scrapings. L-D bodies were demonstrable by both direct microscopy and culture. They were all negative for CFT or AT, except in one case where the CFT was weakly positive. None of these patients was found to have an enlarged spleen or liver on clinical examination. Some interesting observations regarding culture of these organisms were noted. Several isolates were able to be maintained by subculture for several months in modified NNN medium containing rabbit's blood. Others could not be maintained for more than 2-3 weeks in the same medium. Leishman-Donovan bodies were demonstrated by direct microscopic examination of specimens after splenic puncture from one patient and liver puncture from another, but culture of both specimens was negative for L-D bodies. Fig. 1. Distribution of kala-azar cases in different areas of Bangladesh. Figures within a circle show the number of cases (the place of residence could be determined in only 34 out of the 59 cases). In two cases where L-D bodies were demonstrated in the splenic puncture specimens, there were dermal lesions as well and L-D bodies were also demon- strated from skin scrapings by direct microscopy and culture. About 20 years ago, these same two patients had been diagnosed as having kala-azar by the demonstration of L-D bodies in the bone marrow; the skin lesions developed subsequently. Inciden- tally, one of them also had pulmonary tuberculosis in addition to kala-azar. Table 1. Results of the complement fixation test (CFT) and the aldehyde test (AT) on 218 suspected cases of kala- azar CFTa ATa + + + + + + Total Neg. ++ + + + Total Neg. positive positive Patients with kala-azar 47 1 1 58 1 45 8 5 58 1(n = 59) Patients without kala-azar 1 6 9 1 6 143 1 4 9 14 145 In= 159) a +++ = strongly positive; + + = moderately positive; + = weakly positive; Neg. = negative. 114 4 0 VISCERAL LEISHMANIASIS IN BANGLADESH DISCUSSION The present study shows that both kala-azar and PKADL are prevalent in Bangladesh since subjects were found who came from almost all parts of the country. As the study was carried out in Dhaka, the number of cases from different areas reflects the available communications and their utilization. It is reported that kala-azar patients are also presenting themselves at other medical centres in the country from their respective catchment areas. Recently Khan (5) reported about 10 cases of PKADL from different areas of Bangladesh; these cases are epidemiologically important because the diagnosis is usually made long after the appearance of the lesion. In the past, kala-azar had been endemic in Bangladesh and the neighbouring parts of India. Epidemics used to occur almost regularly every 15-20 years (3). The disease was then virtually eradi- cated (through control of the vector) as a result of DDT spraying during the malaria eradication cam- paigns. The present study shows that kala-azar is now being encountered with increasing frequency; this seems to have followed the stopping of insecti- cide spraying, and makes us believe that the threat of an epidemic of kala-azar in the future is real. A similar situation exists in Bihar, India, adjoining Bangladesh, from where recently an epidemic has been reported (1, 6). All the subjects in the present study were adults, which is in contrast to the findings of Aikat from Bihar (personal communication, 1978), where the disease affected mostly children. All our patients were referred to us by physicians and it is possible that there was selection in favour of adult subjects. More than 701Vo of our patients were males, which may be due to the fact that males present themselves for treatment more frequently than females. African kala-azar has, however, been reported to affect four times as many males as females (7). The results of laboratory investigations showed that both the complement fixation test and the aldehyde test are reliable. One false negative reaction was observed in our series and only a small percentage of cases showed a false positive reaction. These false positive cases were mostly weakly positive, a few were moderately positive, and only one was strongly positive. One limitation in the present study is due to the fact that no normal control was included. However, it can safely be concluded from our results that patients with kala-azar are unlikely to be negative in the two laboratory tests (CFT and AT). In our view, patients with pyrexia or splenomegaly (but with no demonstrable L-D bodies) would serve better as controls than the normal population because the former are often sent for confirmation of a tentative diagnosis of kala-azar. Of the two tests, AT is very simple to perform and does not require any instru- ment or electricity, so that it can be used as a screen- ing test in surveys. ELISA and immunofluorescence methods, using specific antigens, are being currently employed in many centres outside Bangladesh for diagnosis and in surveys of kala-azar. These methods need trained manpower and other facilities which at present are not easily available in Bangladesh. Complete failure of the organism to grow in modified NNN medium containing rabbit's blood in two cases, and also failure to maintain the organism (in some cases) in the culture medium for more than 2-3 weeks point to the possible existence of multiple strains in certain areas. The observation that patients with visceral leishmaniasis can develop a dermal lesion while the visceral lesion persists is of interest, because it is generally considered that PKADL develops several years after the cure of visceral leishmaniasis. Visceral cure is followed by dermal resistance to new infection. After a variable period, however, skin resistance is lost and a resurgence of the old infection leads to PKADL while the visceral organs remain unaffected (8). Simultaneous visceral and dermal lesions point to the possibility of involvement by multiple strains or an unusual immunological status of the individual. ;UME REAPPARITION DE LA LEISHMANIOSE VISCERALE AU BANGLADESH Sur 218 cas suspects de kala-azar survenus dans diffe- rentes regions du Bangladesh, 59 ont pu etre confirmes, et parmi 41 patients presentant des 1lsions cutanees soup- qonn6es d'etre imputables a cette parasitose, on a etabli dans 6 cas qu'il s'agissait effectivement d'une leishmaniose cutanee post-kala-azar. La confirmation du diagnostic a e faite par la mise en evidence de corps de Leishman-Donovan dans des echantillons de tissus (preleves par ponction sternale ou hepatique ou par grattage cutane). Dans tous les cas de kala-azar, sauf un, la reaction de fixa- 115 116 K. M. RAHMAN & N. ISLAM tion du complement et l'epreuve a l'aldehyde ont et posi- tives. Leur utilite pour le diagnostic de la maladie a et souligne. L'epreuve A l'aldehyde est d'execution tres simple et n'exige ni equipement, ni source d'electricite, de sorte qu'on peut l'employer pour le depistage en zone rurale. Vu qu'il a et& impossible, dans 2 cas, de faire pousser les leishmanies dans le milieu NNN et, dans quelques autres cas, d'entretenir les parasites isoles plus de 2-3 semaines dans le milieu de culture, il semble qu'on se soit trouve en presence de souches differentes. Devant la multiplication apparente des cas de kala-azar, la menace d'epidemie est tout a fait reelle. REFERENCES 1. THAKUR, C. P. ET AL. Journal of tropical medicine and hygiene, 84: 271-276 (1981). 2. RAHMAN, K. M. Bangladesh Medical Research Council Bulletin, 1: 32 (1975). 3. NAPIER, L. E. The principles and practice of tropical medicine. New York, The Macmillan Co., 1946, p. 134. 4. CRUICKSHANK, R. Medical microbiology, 11th ed. Edinburgh and London, Churchill Livingstone, 1965, pp. 777-778. 5. KHAN, H. M. Bangladesh Medical Research Council Bulletin, 3: 130 (1977). 6. INDIAN COUNCIL OF MEDICAL RESEARCH. Proceedings of the meeting of an Expert Group on Kala-Azar held in New Delhi on 9.9.1977. 7. WILCOCKS, C. & MANSON-BAHR, P. E. C. Manson's tropical diseases, 7th ed. Baltimore, Williams, 1972, pp. 123-130. 8. HEYNEMAN, D. Bulletin of the World Organization, 44: 499-514 (1971).