Composition useful for the early diagnosis of visceral...

Details Composition useful for the early diagnosis of visceral... Composition useful for the early diagnosis of visceral...

1998-10-26

2002-07-23

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C435S325000, C435S374000

Reexamination Certificate

active

06423529

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a composition containing Leishmania promastigotes and a protein stabilizing solute useful for the early diagnosis of visceral leishmaniasis. Particularly, this invention relates to a composition containing
Leishmania donovani
promastigotes and a protein stabilising solute useful for the early diagnosis of visceral leishmaniasis or kala-azar. More particularly, the invention is useful for the early diagnosis of visceral leishmaniasis or Kala-azar in field conditions using Direct Agglutination Test (DAT) based on the composition of as disclosed herein.
BACKGROUND Of THE INVENTION
Visceral Leishmaniasis or Kala-azar, caused by an intracellular protozoan parasite Leishmania donovani, is transmitted from man to man through
Phlebotomus argentipes
(sand fly) of the phlebotomidae family.
The disease is geographically distributed in tropics and substropics of the world extending through most of the central and South East Asia, India, China, the Mediterranean region and Africa 90% of all the cases occur in Bangladesh, Brazil, India and sudan.
Visceral leishmaniasis is caused by
Leishmania donovani
in the Indian subcontinent and in East Africa, by
Leishmania infantum
in the mediterranean region and by
Leishmania chagasi
in the New World, mainly in Brazil, Peru and Paraguay (J. D. Berman; Clin. Infect. Dis., 24, 684, 1997).
It has been estimated that about 15 million people carry Leishmania infection (R. W. Ashford, P. Deszeux, P. deRadt; Parasitol. Today, 8, 104, 1992) and over 400,000 new cases appear each year. The number of people at risk is estimated to be 350 million in about 88 countires (Anon.; Tropical Disease, Leishmaniasis, 14, 1991 and F. Modabber; Tropical Disease Research Programme, Eleventh Programme Report, page 77, 1992). In India, in 1991 the disease had posed a great threat involving 38 out of the 42 districts of Bihar state, 8 districts of West Bengal and 2 districts of Eastern Uttar Pradesh (TDR News Letter, Published by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases No. 36, 1991 and V. Srivastava; Ph.D. Thesis; Chemotherapy and Immunodiagnostic Investigations on Visceral Leishmaniasis, Agra University, Agra, India, 1992). From Bihar alone in 1991 the number of cases recorded were 2,50,000 with 2 to 3 thousand deaths and more than 4.5 lakh are directly exposed to the parasite (C. P. Thakur; Status Paper on Kala-azar in Bihar, Core group discussion on Kala-azar control in India, 24-25 September 1993). Sporadic cases have also been reported from Gujrat (F. B. W. Gajwani, A. Mehta, B. A. Sayed, N. N. Patel and R. S. Pandey; Ind. Med. Assoc., 49, 216, 1967), Kashmir (V.P. Jacob and S. L. Kalra; Ind. J. Med. Res., 39, 329, 1951), Himachal Pradesh (S. Gupta and R. S. Bhatia; Ind. Practitioner, 28, 609, 1975), Delhi (S. R. Naik, P. M. Rao, D. V. Dutta, S. Mehta, and P. N. Chuttani; Trans. R. Soc. Trop. Med. Hyg., 33, 61, 1979) and Uttar Pradesh (V. Srivastava; Ph.D. Thesis; Chemotherapy and Immunodiagnostic Investigations on Visceral Leishmaniasis, Agra University, Agra, India, 1992 and S. Gupta, S. Tiwari, A. K. Bagchi and J. C. Katiyar; Current Science 73,456, 1997)
The disease is characterized by irregular bouts of fever, often with loss of weight and appetite, hepatosplenomegaly, leucopenia, cachexia etc. If left untreated the fatality rate can be as high as 100%. In early stages the disease can be effectively treated with proper course of sodium stibogluconate (SSG) as there is little or no morbidity and no immunosuppression. At later stages disease is assoicated with many problems like greater morbidity, immunosuppression, secondary infection and drug resistance etc. and at this stage the patients respond poorly to various combinations of chemotherapy.
Keeping in view of these limitations, the essential component in the management. of the disease is the early detection and proper treatment with available chemotherapeutic agents.
Prior Art References
Till date, there is no suitable method for the diagnosis of Kala-azar particularly in early stages.
The definitive diagnosis of visceral leishmaniasis is based on demonstration of parasites in biopsies or aspirates of infected spleen, bone marrow and lymph nodes. Culture of aspirates from these locations is also sometimes successful. Both approaches suffer because of lack of adequate sensitivity when the load of organism is low. Spleen biopsy detects 90% active cases whereas bone marrow aspirate can detect 60-70% active cases. By lymph gland biopsy only 50-60% active cases can be detected. These procedures at times are hazardous, painful and labour extensive, require skilled hands, possible in hospitals only where results could be available at least 24 hours later, costly, fail to detect early and slight infective stage.
For Kala-azar, some of the non-specific tests which were in use in early days and being practiced even today at places include; Formal gel or Aldehyde test and Chopra's antimony test. They are also related to the level of IgG and positive results are also obtained when other causes of raised immunoglobulin levels are present whether of IgG or IgM classes. It is therefore a screening test, which, if positive, requires further investigations. The test is positive in about 85% of patients with VL. The test becomes positive about three months after the infection. So these tests may detect only symptomatic cases, they fail to detect early leishmaniasis.
Serological tests are a useful adjunct and are specially valuable in early or highly immune cases when amastigotes may be present in insufficient numbers to be seen easly. Besides, these tests have the advantages that the blood sampling is relatively easy and can be performed with little inconvenience to the patient and many samples may be processed simultaneously.
A battery of immunological procedures have been developed and they demonstrate antileishmanial antibodies for confirming clinical diagnosis. These include Indirect Immunofluorescent Antibody Test (C. L. Jaffe and D. McMahon Pratt.; Trans. R. Soc. Trop. Med. Hyg., 81, 587, 1987), Enzyme Linked Immunosborent Assay (A. Jahn and H. J. Diesfeld; Trans. R. Soc. Trop. Med. Hyg., 77, 451, 1983 and E. R. M. El Amin, E. P. Wright, P. A. Kager, J. J. Laarman and K. W. Pondman; Trans. R. Soc. Trop. Med. Hyg., 79, 344, 1985), Counter Current Immunoelectrophoresis (J. Kohanteb, S. M. Ardehali and M. R. Rijai; Trans. R. Soc. Trop. Med. Hyg., 81, 578, 1987) and Complement Fixation Test (M. G. Pappas, L. T. Canon, W. T. Hakmeyer and D. H. Smith; Ann. Trop. Med. Parasitol., 79, 147, 1985). However, some have low sensitivity, others are cross reactive and still others require elaborate laboratory facilities (IFA and RIA). Thus they do not meet the requirements of field test as mentioned below.
An ideal immunodiagnostic test should be simple, quick, specific and cheap, and be applicable in the fields even in the adverse conditions that prevail in many areas where leishmaniasis is endemic.
Direct Agglutination Test (A. E. Harith, A. H. Z. Kolk, P. A. Kager, J. Leeuwenburg, R. Muigai, S. Kingu and J. J. Laarman; Trans. R. Soc. Trop. Med. Hyg.; 80, 583, 1986 and A. E. Harith, A. H. Z. Kolk, J. Leeuwenburg, R. Muigai, E. Huigen, T. Jelsma and P. A. Kager; J. Clin. Microbiol.; 26, 1321, 1988) is promising in meeting these requirements. In DAT the antigen preparation consists of whole organism and the serological response to surface borne antigen is measured and the test could be performed on samples of whole blood, thus the difficulties of preparation and storage of serum, plasma and blood in filter paper are avoided. Allain and Kagan first described DAT (S. Allan and I. G. Kagan; Trop. Med. Hyg., 24, 332, 1975) for diagnosis of visceral leishmaniasis, Side 1986, DAT was modified and simplified to increase sensitivity and specificity and also comparisons were made with other serological tests. The test is also capable of detecting canine visceral leishmaniasis (A. E. Harith, R. J. Slappendel, I. Reiter, F. van Knappen, P. de

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