ANDHYGIENE(1992) 86,259-262

Detection of high titres of Toxoplasma leprosy in Pakistan


in sera of patients


R. Hussain’, S. Jarnil’, H. M. Dockrel12, T. J. Chiang3 and R. Hasan 1 ‘Department of Microbiology, Aga Khan University, P.O. Karachi, Pakistan; 2Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London, UK; 3Marie Adelaide Leprosy Centre, Mariam Manzil, Saddar, Karachi, Pakistan

Abstract Untreated and treated leprosy patients and their household contacts were screened for antibody to Toxoplasma gondii using antigen-coated latex particles. A significantly high level of seroprevalence(29.6%) was observed in the untreated leprosy patients compared to endemic controls (P100000






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high titres of antibodies to T. go&ii. Sera were graded from 1 to 5 according to the intensity of agglutination, 5 being the highest. Agglutination was carried out in the presenceof either soluble T. gondii or M. leprae sonicate (250 ng) or buffer alone as control. Agglutination was assessedin both the presence and absence of each of the antigens. A single dilution of each serum was used corresponding to the mid range of the activity curve. The intensity of agglutination was graded from l-5 and expressed as an agglutination index, with 1 being the weakestagglutination. Statistical analysis Student’s t test and x2 analysis were used to determine

the significance of differences between groups of patients, using the Statview@package on an Apple Macintosh@microprocessor. Results Distribution of T. gondii antibodies in leprosy sera The distribution of anti-T. gondii antibodies in the

various groups of patients and controls are shown in Fig. 1. A surprisingly high level of T. gondii antibodies was observed in both the lepromatous and tuberculoid type of leprosy patients, compared to the control groups. The seroprevalence in the different groups was examined using a cut-off titre of 11512(Table). Even with this relatively high cut-off value, seropositivity in 98 untreated leprosy patients was 29.6%, compared to 7.8% in the control population (n=5 1). Since the employees and students of Aga Khan University may belong to slightly higher socioeconomic strata, household contacts of leprosy patients were also screened. The household contacts showed similar seropositivity (6.1%; n=50) to the Aga Khan University employee/student group, indicating that socioeconomic factors did not play a significant role in seropositivity to T. gondii. The difference in mean antibody titres of the seropositive patients was even more dramatic (Table). The interesting observation was that

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Fig. 1. Latex agglutinating (LA) antibody titre to T. gondii antigen in untreated (LS) and treated (LEP) leprosy patients with either tuberculoid type (BTITT) (open circles) or lepromatous type (LLIBL) of leprosy (closed circles), household contacts of leprosy patients (LCS), and endemic controls (NS). The number of individuals tested (N) is indicated Table. Seroprevalence patients

of Toxoplasma



Antibody titre

Percentage positi&


51 50 74 98

512iO 1024f102 1792k183 20007~413580

7.8 6.1 13.5 29.6

GS NS -1:512. ‘Probability determined by x2 test; NS=not


in leprosy

ldI.5 80 Fig. 2. Specificity of agglutination of T. gondii coated latex particles. Sera from five untreated leprosy patients (LS) were incubated with latex particles coated with either T. gondii antigen (panel A) or M. leprae antigen (panel B) in the presence of soluble T. go&i antigen (250 ngiml), M. leprae soluble antigens (CD75; 250 ngiml), or no antigen (buffer alone). The dilutions used for each serum corresponded fo the mid range of its activity curve. Latex agglutinating activity is expressed on a scale of 1 to 5 depending on the intensity of agglutination (S=maximum).

261 both the seropositivity rate and the mean antibody titres were much lower in treated leprosy patients (13.5%; n=74). Since both M. Zepraeand T. go&ii are susceptible to the sulphone drugs (dapsone), it is possible that the drop in antibody titres may be related to antigen clearance. These observations do not rule out the possibility of cross reactivity between T. gondii and M. leprae antigens being responsible for the apparent presence of T. gondii antibody in leprosy sera. In order to rule out cross reactivity of T. gondii antigens with M. leprae, cross inhibition experiments were set up with soluble M. leprae and T. gondii antigens. The results with 5 leprosy sera containing high titres of antibody to T. gondii are shown in Fig. 2. While both antigens were able to inhibit agglutination of the homologous antigen-coated latex, cross inhibition was not seen with either antigen. T. gondii antigen was able to inhibit the agglutinating activity completely in 4 of 5 positive sera and caused a 75% reduction of titre in the remaining serum, while addition of soluble M. Zeprae sonicate resulted in no inhibition and gave results comparable to the control (buffer alone; Fig. 2A). Conversely, soluble M. IO4 2

IO -' I - "'-t ""*'=I """.I ' "**'s, "'S'., """7 "a-1 10-l 100 IO' IO2 IO3 IO4 105 106 LA TITER (T. gondii)


Fig. 3. Correlation scattergram of latex agglutinating (LA) antibodies to T. go&ii and M. &we in sera of untreated leprosy patients with either tuberculoid type (closed circles) or lepromatous type (open circles) of le-

leprae sonicate was able to inhibit agglutination of latex beads coated with the homologous antigen but not with soluble T. gondii antigen (Fig. 2B), showing the high degree of antigenic specificity within the system. If T. gondii antibody were related to a common crossreactive epitope with M. Zeprae, there should also have been a relation between antibodies to the 2 antigens in these patients. Fig. 3 is a scattergram of the distribution of both antibodies in lepromatous and tuberculoid leprosy patients. No significant relationship was observed between the levels of T. gondii and M. leprae specific antibodies! indicating that the response of T. gondii antibody was Independent of the M. leprae specific response in these patients. This was further indicated by the fact that a high proportion of lepromatous leprosy patients with extremely high levels of M. Zeprae specific antibodies showed a complete absence of T. gondii antibodies, while some of the tuberculoid leprosy patients with low to undetectable levels of antibodies to M. leprae showed high levels of antibodies to T. gondii. Polyclonal activation of B cells, which is a feature of many protozoa1 infections, is also known to occur in lepromatous leprosy and may in some cases result in unrelated antibody responses. To investigate whether this might have accounted for our observation, total IgG levels were determined in both the lepromatous and tuberculoid leprosy patients (Fig. 4). As expected, the leprosy patients showed significantly higher levels of total IgG in the lepromatous than in the tuberculoid leprosy group (PthoZogy.of Protozoal and Helminthic Diseases, Martial-Rojas, R. A. (editor). Baltimore: Williams and Wilkins Company, p. 254. Frenkel, J. K. & Ruiz, A. (1980). Human toxoplasmosis and cat contact in Costa Rica. American Journal of Tropical Medicine and Hygiene, 29, 1167-1180. Godal, T., Myklestad, B., Samuel, D. R. & Myrvang, B. (1971). Characterization of the cellular immune defect in lepromatous leprosy: a specific lack of circulating Mycobacterium leprae-reactive lymphocytes. Clinical and Experimental Immunology, 9,82 l-83 1. Harboe,, M. (1985). The immunology of leprosy. In: Leprosy, Hastings, R. C. (editor). London; Churchill Livingstone, pp. 53-87. Hasan, R., Dockrell, H. M., Chiang, T. & Hussain, R. (1989). Quantitative antibody ELBA for leprosy. InternationalJournal of Leprosy, 57,766-776. Hussain, R., Jamil, S., Kifayet, A., Firdausi, F., Dockrell, H. M., Lucas, S. & Hasan, R. (1990). Quantitation of IgM antibodies to the M. Zeprae svnthetic disaccharide can predict early bacterial multi&cation in leprosy. Intemational~oumal of Leprosy, 58,491-502. Jones, T. C., Yeh, S. & Hirsch, J. G. (1972). The interaction between Toxoplasma gondii and mammalian cells. Journal of ExperimentalMedicine, 136, 1157-1172. Luft, B. J., Brooks, R. G., Conley, F. K., McCabe, R. E. & Remington, I. S. (1984). Toxoplasmic encephalitis m patients with aiquired immune deficiency syndrome. Journ& of the AmericanMedicalAssociation,252,913-917. McLeod, R. & Remington, J. S. (1979). A method to evaluate the capacity of monocytes and macrophages to inhibit multiolication of an intracellular uathoaen. Ioumal of Zmmunolok&Methods, 27, 19-29. A G * Myrvang, B., Godal, T., Ridley, D. S., Froland, S. S. & Song, Y. K. (1973). Immune responsiveness to Mycobacterium leprae and other mycobacterial antigens throughout the clinical and histopathological spectrum ofleprosy. C%nical and Experimental Immunology, 14,541-553. Rao, K. N., Saha, K. & Bhatia, V. N. (1989). Toxoplusmagondii antibody in patients of lepromatous leprosy.JupaneseJournal of Medical Sciences and Biology, 42, 163-168. Schmitt, E., Meuret, G. & Stix, L. (1977). Monocyte recruitment in tuberculosis and sarcoidosis. British .youmal of Haematologv, 35, 11-17. Sibley, L. D. & Krahenbuhl, J. L. (1987). Mycobacterium leprue-burdened macrophages are refractory to activation by gamma interferon. Infection and Immunity, 55,446-450. Tweardy, D. J., Schacter,, B. Z. & Ellner, J. J. (1984). Association of altered dynamics of monocyte surface expression in tuberculosisJournal of Infectious Diseases? 149,31-37. Velimirovic, B. (1984). Toxoplasmosis in lmmunosuppression and AIDS. Infection, 12,315-317. Received 3 September 1991; revised 10 October accepted for publication 10 October 1991


Detection of high titres of Toxoplasma gondii antibodies in sera of patients with leprosy in Pakistan.

Untreated and treated leprosy patients and their household contacts were screened for antibody to Toxoplasma gondii using antigen-coated latex particl...
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