A FIELD STUDY TO DETERMINE THE USE OF GLUTARALDEHYDE FOR SCREENING KALA-AZAR CASES IN SIRAHA DISTRICT, NEPAL. Dr.

Uma Nath Devkota, MBBS, MSc. (Med.Epid.) Epidemiologist, Primary Health Care Project GTZ Dr. J. P. Steinmann, MD, MPH, Team Leader, Primary Health Care Project GTZ

Primary Health Care Project GTZ Department of Health Services Teku, Kathmandu, Nepal P O Box 1457, Tel:261404 Fax:977-1-261079

ABSTRACT A case control study was performed in Siraha district, Nepal, to determine the use of glutaraldehyde for screening kala-azar cases. The study was performed by combining a questionnaire/clinical examination with a blood test by mixing equal parts of 2.5% glutaraldehyde solution and EDTAblood. Glutaraldehyde test (GA-test) was found positive in 82.2% of the clinically diagnosed kala-azar cases at 240 seconds glutaraldehyde cut off point against 12.8% in the control group. Whereas, the aldehyde test was positive in 36.8% cases only. Similarly, 82.3% of the kala-azar cases were found positive for the presence of LD body in the bone marrow smear examination. Among the GA test positive kala-azar cases, 76.4% had shown LD body in the bone marrow examination. In a country like Nepal, where sophisticated laboratory tests to screen kala-azar are not possible at the peripheral health institutions, results of the study have shown clear advantage of glutaraldehyde over the aldehyde test. It is suggested that kala-azar case finding with glutaraldehyde combined with a simple questionnaire/clinical examination is very useful in screening suspected kala-azar cases at peripheral health institutions. The glutaraldehyde test could easily be done at peripheral health institution at a low cost and suspected cases could be referred earlier to the hospitals for the confirmation and initiation of the treatment.

INTRODUCTION: Kala-azar has been recognised as a serious public health problem in Nepal. Since 1980, a rise in kala-azar incidence is reported every year from 11 districts in the eastern and central terai region of the country. Available information suggests that as many as 11, 216 cases of kala-azar with 300 deaths have been reported since 1980 in Nepal. Siraha, one of the endemic districts for kala-azar is situated in the eastern development region and every year hundreds of kala-azar cases are reported. In 1984 only 3 cases of kala-azar were reported which in 1988 increased to 178. In 1996, 83 of 110 village Development Committees (VDCs) with a total population of 388, 484 were at risk of the disease. A total of 2335 cases of kala-azar with 45 deaths have been reported from the district for the year 1985-96. Siraha is spread over 1188 sq. km. with an estimated total population of 522,878 (1997). There are two district hospitals, 2 PHC, 12 health posts and 97 sub-health posts. In the district it is difficult to diagnose kala-azar due to the scarcity of laboratory facilities and trained personnel at peripheral level. Currently, Aldehyde test is the main screening tool used in the hospitals. At the peripheral level however, such a test is time consuming and needs serum, whereas, the Glutaraldehyde test could be carried out using patients blood directly. The gelification time of the glutaraldehyde test for kala-azar screening has been claimed to be less than 2 minutes, showing clear advantages of this text over the aldehyde test, So, it has been suggested that glutaraldehyde tests could be as well used as an alternative tools for kala-azar screening at the district and peripheral health institutions. OBJECTIVE OF THE STUDY:

The overall objective of the study was to determine the advantages of the glutaraldehyde test (GA-test) over the aldehyde and to determine whether the aldehyde test could be replaced by the glutaraldehyde test to screen suspected cases of kala-azar at the district and peripheral health institutions. The specific objectives of the study were to determine the gelification time (cut off point) of the glutaraldehyde for kala-azar screening at the same time to determine the frequency of bone marrow positive for LD body among the glutaraldehyde positive and negative cases. MATERIALS AND METHODS: A hospital based case control study was conducted at the Lahan and Siraha hospitals in the Siraha district. The study started from January 1996 and ended in October 1997. Cases A total of 62 kala-azar patients admitted or receiving ambulatory anti kalaazar treatment in the Lahan and Siraha hospitals were included in the study as cases. A case for this study was defined as a patient diagnosed clinically as kala-azar having either/or a combination fever, enlarged spleen and receiving anti kala-azar treatment at the Lahan and Siraha hospitals. Controls Similarly, a total of 125 normal healthy persons not suffering or suffered from chronic diseases, such as tuberculosis, polyarthritis, leprosy, chronic bronchitis, carcinomas etc. at the time of blood sampling, were included as the controls. The controls included the students, staffs and their family members from the Public Technical Institute, Siraha. Pregnant women were not included in the study.

Methods A structured questionnaire was administered with the case and control groups to collect necessary information. The attending physicians were requested to fill and sign the questionnaires for the cases. Ready-made Glutaraldehyde tubes containing 1 ml of the reagent, manufactured by the ANDA Biologicals, France, was used in the study. From every subject, including the healthy volunteers, blood was collected in EDTA-tubes. One ml. of this blood was mixed with equal parts of glutaraldehyde and gently mixed, and the gelification time was observed. For cases, aldehyde test and hemoglobin percentage was performed from rest of the blood. The aldehyde test was performed by mixing 0.5 ml of the patients serum with 2-3 drips of 40% formaline in a test tube. The test was considered as positive when a white coagulation developed in less than 20 minutes. As the study was designed to confirm the diagnosis of kala-azar as well, bone marrow aspiration of the cases was performed to demonstrate the presence of the LD bodies. Out of 62 kala-azar patients bone marrow aspiration was performed only in 34 suspected cases of kala-azar through a puncture in the anterior superior iliac crest using aseptic precautions by a medical officer. Further staining and microscopic examination of the slide was done by an experienced laboratory technician working in Siraha hospital. Limitations Due to some operational difficulties, the planned enrollment of 100 cases of kala-azar in the study could not be done and only 62 kala-azar cases were enrolled in the study. So is the case with not performing LD body examination of all the enrolled cases of kala-azar.

RESULTS: Among the 62 clinically diagnosed kala-azar cases, 48.3% (n=30) were male and 51.7% (n=32) female. The mean age of the case was 29 years with a range of 3-80 years. Similarly, among the 125 healthy controls, 77.6% (n=97) were male and 22.4% (n-28) female. The mean age of the controls was 21 years with a range of 8-50 years (Table 1). Analysis of the clinical features of the kala-azar cases revealed that fever was present in all the cases (100%) (Table 2). Weight loss was present in 90% (n=56) of cases. it was found that enlarged spleen showed in 65% (n=40) cases, whereas the enlargement of liver was confirmed to only 11% (n=7) cases, which is surprisingly low compared to the findings where 100% spleenomegaly and 75% hepatomegaly have been reported. Among the spleenomegaly cases 50% (n=20) has enlarged spleen up to 3 cm and 28% (n=11) had 5 cm. The average spleenomegaly found was 4 cm with a range of 1.5-12 cm. Anemia was found in 76% of the cases. Among the anemic cases 68.8% had hemoglobin less than 10 gm.% with a mean of 7 gm.%. In the study, the GA-test was performed among the cases and controls to determine the cut off point for glutaraldehyde gelification for screening suspected kala-azar cases. Among the 62 clinically diagnosed kala-azar cases, 35.4% (n=22) had glutaraldehyde gelification time within 60 seconds as compared to zero for the control groups. Similarly, when 0-120 seconds gelification time period was taken into consideration, 56.4% (n-35) among the cases and 0.8% (n=1) for the control groups were found positive. It was found that when the gelification time period increased from 120 to 180 seconds 70.9% (n=44) of the cases were found positive. For the same time period 2.4% (n=4) were also found positive among the control group. Similarly, when the gelification time period was further 6

increased to 240 seconds 82.2% (n=51) cases were found positive against 12.8% (n=16) in control group. In the analysis, it was found that when the gelification time period was even further increased to 300 seconds 990.3% (n=56) of the case were found positive for GA-test. But, at the same time 25.6% (n=32) among the control group were also found positive for glutaraldehyde test (Table 3). Thus, increasing the exposure time entails the possibility of increasing false positive cases. Taking this into consideration the optimal cut off point for gelification of glutaraldehyde for kala-azar screening was taken as 240 seconds. The GA-test positive cases, within the cut of time of 240 seconds, were compared with the bone marrow positively in the same patients to validate the GA-test. Out of 62 kala-azar cases, 34 cases only were subjected to bone marrow smear examination and 28 were found positive for LD body. Among these 28 LD body positive cases, 76.4% (n=26) were also positive for GA test at 240 seconds cut off time whereas 5.8% (n=2) turned negative. on the other had, among the 6 bone marrow negative cases, 11.7% (n=4) were GA-test positive and 5.8% (n=2) GA test negative. Similarly, comparison of aldehyde test and bone marrow examination revealed that among the 28 LD body positive cases 35.2% (n=12) only were positive for the aldehyde test. Thus, the GA-test was found positive in 82.2% (n=51) of the cases at 240 seconds cut off points whereas the aldehyde test in 36.8% (n=21) cases only. Similarly, among the total cases bone marrow smear examination for the presence of LD body was found positive in 82.3% cases (n=28) (Table 5). DISCUSSION: Over the last two decades kala-azar has emerged as an urgent public health problem in Nepal. Approximately 5.5 million populations are

estimated to be at risk of kala-azar and a total of 11,216 cases with 300 deaths were reported during 1980-1996. The available data reflects only the tip of the iceberg of the problem because case finding at peripheral levels of the health sector in Nepal is not properly made. A large number of unidentified kala-azar cases die before they are diagnosed or seek any medical help. This is all because currently there is no appropriate tool for kala-azar screening at the periphery. The introduction of ELISA-test, DAT-test and finding the leishmania parasite in the peripheral blood for kala-azar diagnosis require well trained staff and well equipped laboratories. They are also far too expensive to use in less developed countries like Nepal. Preliminary studies on the inexpensive and easily made method based on gelification by glutaraldehyde, as a screening tool, have given encouraging results to identify kala-azar and at the same time inspired to conduct this study. In Nepal, this kind of study is the first attempt to use glutaraldehyde for kalaazar screening and at the same time fixing the cut off point for gelification. The results clearly indicate that if the cut off point for glutaraldehyde gelification for kala-azar screening is taken as 240 seconds then 82.2% of the suspected kala-azar cases are found positive against 12.8% in the control group. But, when the cut off time period is increased to 300 seconds then the positivity rate among the cases increases to 90.3%. But, at the same time 25.6% of the non Kala-azar cases are also found positive. So, to avoid increasing false positive cases, it is recommended that the glutaraldehyde gelification cut off point be at 240 seconds. Since at this time period not only the 82.2% of kala-azar cases are found positive but 76.4% of kala-azar cases are also found positive for LD body in bone marrow smear examination. When the aldehyde test, glutaraldehyde test and bone marrow smear were compared, it was found that the positive rates of the glutaraldehyde and

bone marrow smear were almost similar (82.2% and 82.3% respectively) whereas the aldehyde test had positive rate of only 36.8%. It clearly suggests that the performance of the glutaraldehyde test is definitely superior to the aldehyde test. The reason why the GA gelification takes place with whole blood within few minutes in kala-azar patients is based on the chemical reactivity of GA. In dilute solutions the substance forms intramolecular bridge with heavy albumin, globulin and fibrinogen in suitable concentrations. Studies in Spain have shown that the GA-test correlates very well with the presently recommended diagnostic tools including the A 60 ELISA test. In low concentration, 1.25% GA in saline for cattle, and in 2.5% in humans, GA will polymerise the most basic blood proteins. Of course, elevated amount of various albumin fractions, globulin and fibrinogen can be found inpatients other than kala-azar. It seems, however, that with the accompanied questionnaire/examination chart the GA-screening tool is the most reliable and cheapest test for kala-azar finding in rural areas in poor countries. CONCLUSION: In a country like Nepal where the use of other sophisticate tests are not practically or economically viable at the peripheral level, the glutaraldehyde test, would fit in well as a simple tool to screen for suspected cases of kala-azar and form the basic for referral to the hospital. The glutaraldehyde test is cheap, simple and does not need any special training or equipment. This test could be performed by any peripheral health worker such as health assistant, auxiliary health worker and auxiliary nurse midwife after providing a one day orientation. Since, the results are available in 4 minutes, a large population could be screened in a short period during epidemics. A trail of this test on a bigger scale could

be done in one of the kala-azar endemic districts in Nepal and based on the results, it could be replied in other district. ACKNOWLEDGEMENT: Sincere gratitude and appreciation goes to the Medical Superintendents of the Lahan and Siraha hospitals for their kind co-operation in data collection. We are also very much thankful to Mr. Styanarayan Pandit, the Laboratory technician of Siraha hospital for all the laboratory work performed by him. Special thanks also extended to the Chairman, students and all the staff of the Public Technical Institute, Siraha for being kind and helpful in blood sample collection necessary for the study. We are also very much thankful to the ANDA-Biologicals, France for being kind enough to provide us with GA-test kits and make this study a success. At the end, we would like to express our deepest appreciation and heartfelt thanks to Stig Larsson, Professor in Community & Tropical Medicine, Austria and Dr. M K Banerjee for their valuable technical inputs for the success of this study. REFERENCES: 1. Report of the Annual Internal Assessment of the Malaria and Kala-azar Control activities, Division of Epidemiology and Disease Control, Department of Health Services, Ministry of Health, Nepal, 1997. 2. Larsson et al., The glutaraldehyde test as a rapid screening method for pulmonary tuberculosis: a preliminary report. Ann. Trop. med. Parasitol., 84, 110, 1990. 3. Bryceson M, Leishmaniasis, Mansons tropical Diseases, 20th edition, W B Saunders, 24-28 Oval road, London, 65, 1232-1233, 1996.

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4. Sandholm MM, A preliminary report of a rapid method for the demonstration of abnormal gammaglobulin level in bovine whole blood. Res. Vet. Sci., 17, 32-35, 1974 5. Larsson et al., Further studies with the glutaraldehyde test as a screening tool for pulmonary tuberculosis, J Inst. ed. (Nepal), 12, 279288, 1990 6. Larsson S, A preliminary study on an easy and inexpensive method for the detection of tuberculosis in cattle using the glutaraldehyde test. J. Nep. Med. Assoc. 26, 9-17, 1998.

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Table I. General characteristics of the study population. Subject Mal s e Case Control 30 97 % 48. 3 77. 6 Femal e 32 28 % 51. 7 22. 4 Total 62 125 Mean age (years) 29 21

Table II. Clinical features of kala-azar cases. Features 1. 2. 3. 4. 5. 6. 7. 8. Fever Weakness Weight loss Anaemia Spleenomegaly Cough Pain abdomen Nausea & vomiting 9. Hepatomegaly 10. Diarrhoea 11. Malnutrition No. of cases 62 57 56 47 40 27 21 33 7 6 6 Percentage 100 92 90 76 65 44 34 53 11 10 10

Table III. Table showing glutaraldehyde gelification time among cases and controls Time in seconds 0-60 61-120 121-180 181-240 241-300 >300 No. of No. of +ve cases cumulative cases 22 (35.4%) 13 (20.9%) 9 (14.5%) 7 (11.2%) 5 (8%) 6 (9.6%) 22 (35.4%) 35 (56.4%) 44 (70.9%) 51 (82.2%) 56 (90.3%) 62 (100%) No. of +ve controls 0 1 (0.8%) 2 (1.6%) 13 (10.4%) 16 (12.8%) 93 (74.4%) No. of cumulati ve controls 0 1 (0.8%) 3 (2.4%) 16 (12.8%) 32 (25.6%) 125 (100%)

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Table IV. Glutaraldehyde gelification time and bone marrow positive among cases. Time in seconds 0-60 61-120 121-180 181-240 241-300 301-480 No. of marrow +ve cases Cumulative no. of marrow +ve cases 11 (32.3%) 11 (32.3%) 8 (23.5%) 19 (55.8%) 2 (5.8%) 21 (61.7%) 5 (14.7%) 26 (76.4%) 2 (5.8%) 28 (82.3%) 0 28 (82.3%) No. of Cumulative marrow ve no. of cases marrow ve cases 2 (5.8%) 2 (5.8%) 0 2 (5.8%) 2 (5.8%) 4 (11.7%) 0 4 (11.7%) 0 4 (11.7%) 2 (5.8%) 6 (17.6%)

Table V. Table showing Glutaraldehyde, aldehyde and bone marrow positive among kala-azar cases. Glutaraldehyde test (240 seconds cut off point) Aldehyde test Positive n=51 Negativ n=11 e Positive n=21 Negativ n=36 e Positive n=28 Negativ n=6 e 82.2% 17.8% 36.8% 63.2% 82.3% 17.7%

Bone marrow

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