Diagnostic performance indices for immunofluorescent tests and enzyme immunoassays of leishmaniasis sera from northern and north-eastern Brazil.

Diagnostic performance indices for immunofluorescent tests and enzyme immunoassays of leishmaniasis sera from northern and north-eastern Brazil M.C.S. Guimaraes,1 B.J. Celeste,2 & E.L. Franco3 A total of 341 sera were screened for anti-Leishmania IgA, IgG, and IgM antibodies by immunofluorescent (IF) tests and enzyme immunoassay (ELISA). Altogether, 292 of the sera originated from patients with clinically as well as parasitologically diagnosed (positive lesion imprint or the Montenegro skin test) cutaneous leishmaniasis; 49 of the sera were from controls from the same base population. In terms of diagnostic performance, the ELISAs for IgG and IgM yielded indices of diagnostic utility, and the positive predictive value for the JgG-ELISA was 94.6%. A remarkably high specificity (100%) was obtained with the IgA-lF test, but its sensitivity was very low. Introduction The leishmaniases are endemic in Latin America from northern Mexico and southern Texas to north- ern Argentina; in Brazil they occur mainly in the northern and north-eastern regions of the country. Over the period 1979-85, 40985 cases occurred in Brazil and in 1985 alone a total of 11 508 were reported (1,2). The Leishmania mexicana and L. braziliensis complexes are present in endemic areas in Brazil and usually cause a cutaneous leishmaniasis (CL) only, but if the disease is not treated, the oropharynx mucosa of patients infected with L. b. braziliensis can be invaded and destroyed. Leishmania spp. can be identified using monoclonal antibodies (3), isoenzyme methods (4), or DNA probes (5); however, such tests have yet to be standardized. For field work or routine diagnosis in in- or outpatient departments, mucocutaneous leishmaniasis (MCL) is diagnosed clinically followed by lesion imprint (6) or delayed-type skin tests (7). Labelled-antibody tech- niques, such as the immunofluorescent (IF) test (8) ' Associate Professor, Department of Preventive Medicine, Facul- dade de Medicina da Universidade de Sao Paulo and Chief, Seroepidemiology Laboratory, Instituto de Medicina Tropical de Sao Paulo, av. Dr Eneas de Carvalho Aguiar, 470, 05403, Sao Paulo, Brazil. Requests for reprints should be sent to the latter address. 2 Technician, Seroepidemiology Laboratory, Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, Brazil. 3Associate Professor, Epidemiology and Preventive Medicine Research Center, Institut Armand Frappier, Laval, Quebec, Canada. Reprint No. 5043 and enzyme immunoassays (ELISAs) employing either soluble antigens (9, 10) or particulate antigens (Dot-ELISA) (11), have also been developed for the serodiagnosis of the leishmaniases. Diagnostic performance indices for the IF and ELISA methods have only recently been estimated for a group of leishmaniasis patients who exhibited long-standing or severe symptoms and for controls without any previous contact with the etiological agent (12). Although very useful for test standardiza- tion, such a series does not reflect the epidemiological pattern of the disease as found in endemic areas. In the present study, 341 sera obtained from 292 patients with CL and 49 controls from endemic areas in Brazil were screened for the presence of anti- Leishmania anti-IgG, anti-IgA, and anti-IgM anti- bodies using IF and ELISA methods. Subsequently, the diagnostic performance indices of sensitivity, specificity, positive predictive value, negative predic- tive value, and efficiency were calculated to determine whether such serological tests could be used to diag- nose the leishmaniases. Materials and methods Sera A total of 341 sera were collected at regional offices of the Superintendencia de Campanhas de Saiude Puiblica, Brazilian Ministry of Health (SUCAM), in northern and north-eastern Brazil. The lesion imprint (6) and Montenegro skin tests (7) were used to make a diagnosis of CL. Thirteen sera from patients who Bulletin of the World Health Organization, 68 (1): 39-43 (1990) © World Health Organization 1990 39 M.C.S. Gulmaries et al. had a clinical diagnosis of MCL were also screened, but the results obtained were used only to calculate the performance indices (Tables 1 and 2). Sera were collected from untreated cases and were shipped frozen to the Seroepidemiology Laboratory, Instituto de Medicina Tropical de Sao Paulo, where they were assayed for anti-Leishmania IgG, IgM and IgA antibodies by IF and ELISA. Sera from a group of 49 controls, who were either in good health or had clinical and pathological diagnoses of skin diseases such as yaws, chromomycosis, eczema, varicose ulcers, or sporotrichosis, but negative parasitological tests for Leishmania spp., were also collected. Controls and cases originated from the same base population. Antigens IF antigens. The IF antigens were prepared as de- scribed by Guimaraes et al. (8) from 7-day-old Leish- mania major-like (MHOM/BR/71/49) promastigotes grown in liver infusion and tryptose (LIT) culture medium (13). ELISA antigens. The ELISA antigens were prepared as described by Guimaraes et al. (10) from 7-day-old, LIT-cultured L. major-like (MHOM/BR/71/49) pro- mastigotes. IF tests IF tests for the detection of the IgG and IgM antibodies were performed using the technique de- scribed by Guimaraes et al. (8), while the IgA antibodies were detected using the IF tests described by Shaw & Lainson (14); conjugates were heavy chain specific. The IgM antibodies were detected after absorption of sera by heat-aggregated immuno- globulin (15). The end-point titre was taken to be the final dilution to give a continuous, bright green membrane and flagellum fluorescence. A positive and a negative control serum were included in all tests. ELISA methods ELISAs were carried out using the procedure de- scribed by Guimaraes et al. (10) with an anti-human IgG (y-chain specific) or an anti-human IgM (ju-chain specific) conjugated to horseradish peroxidase (type VI),& according to the method described by Nakane & Kawaoi (16). The chromogen was 200 p1 of a 5.2-mmol/A solution of 5-aminosalicylic acid and 1.5 mmol/l hydrogen peroxide. In all assays the ' Sigma Chemical Co., St. Louis, MO, USA. following controls were included: a positive serum, three pools of negative serum containing 10 sera each (previously found to be negative for anti-Leishmania and anti-Trypanosoma antibodies by IF and ELISA tests), conjugate, and antigen. The end-point titration was taken to be the absorbance just greater than the mean + 2 standard deviations of negative serum pools at a 1:20 dilution. IgM antibodies were detected after absorption of sera by heat-aggregated immunoglobulin (15). Statistical analysis For a given test, the serological titre for each sample was used to construct 2 x 2 contingency tables to further classify sera according to disease attribute (CL or not; leishmaniases or not). The frequencies of true positives, true negatives, false positives, and false negatives with respect to disease attribute could then be determined. Diagnostic indices such as sensitivity, specificity, positive and negative predictive value, and efficiency were calculated using previously described formulae (17), while 95% confidence intervals were estimated by Wilson's method (18) using Diagval (E.L. Franco & R. Simons, unpublished software, 1985), a customized template for Lotus 123. Results Skin and lesion Imprint tests A total of 194 patients (47.9% of respondents) were evaluated using the Montenegro skin test and 276 (55.7% of respondents) by the lesion imprint test. Serologkal tests The proportion of sera with titres that were above the prescribed cut-off level for each test (> 1:40 in the IgG-ELISA, 1:10 in the IgA-IF, and > 1:20 for the IF tests and the IgM-ELISA) was used to compare the anti-Leishmania response for each antigen. Immunofluoreuent test. IgG-IF test-27.7% of CL sera had titres > 1:20. IgM-IF test-20.5% of sera had titres > 1:20. IgA-IF test-3.1% of CL sera (9 patients) exhibited a positive response. One of these patients had experienced symptoms for 2 months, while five had had symptoms for 3-6 months, indicating that the invasive stage of the disease may occur earlier than that esti- mated by Shaw & Lainson (14). Five of the sera were collected in the northern Amapa State and the remainder in north-eastern states of the country; two patients were 7 years old. Parasite findings for four patients were positive, while those for one patient were negative; also, five patients had a WHO Bulletin OMS. Vol 68 1990. Diagnostic performance Indices for IF tests and ELISAs of leishmaniasis sera positive Montenegro skin test. There was a positive association between the results of the IF-IgA test and serum origin (P = 0.0015). Enzyme Immunoassays. The distribution of titres for IgG antibodies was as follows: 32.5% of sera had titres <1:40, while 67.5% had titres > 1:40. There was a positive association between the IgG-ELISA titre and serum origin (P = 0.0366) as well as the age (P = 0.0006) and sex (P = 0.0174) of patients. IgM-ELISA-the distribution of IgM titres was as follows: 44.5% were <1:20, while 55.5% were >1:20. There was a positive association between the IgM-ELISA titres and the serum origin (P = 0.0001) and age (P = 0.0126) of patients, since 72.0% of children's sera (18 out of 25 individuals aged 1-17 years) and 55.6% of adult's sera (148 out of 266 individuals aged 18-49 years) gave positive IgM- ELISA results. Diagnostic performance Indices By basing diagnosis of leishmaniasis on a positive lesion imprint test or a positive Montenegro skin test and the preselected cut-off titre for each test, it was possible to evaluate the relative clinical and epidemiological utility of the five anti-Leishmania serological tests investigated. Tables 1 and 2 show the results of such an analysis in the form of diagnostic performance indices and their respective 95% con- fidence intervals for all five tests for the disease groups CL and CL plus MCL. As indicated by the total number of correct diagnoses, both positive and negative (efficiency index), the best disease indicator was the IgG-ELISA followed by the IgM-ELISA for both groups. These were the only tests with sen- sitivity indices and predictive values (positive and negative) as well as specificity indices that had values significantly different from those expected by chance alone. Discussion Although the study was carried out using antigens prepared from L.major-like parasites, L.b.braziliensis and L.b.guyanensis are the prevalent organisms in the survey area. At present, parasites of the L.braziliensis complex cannot yet be cultured to yield the large mass of promastigotes required to perform the sero- logical tests described. As shown in Table 1, the efficiency indices of the ELISAs, which are a measure of a test's ability to detect diseased or non-diseased status, were twice those of IF tests, regardless of whether CL sera were used to calculate the index or whether sera from the 13 invasive MCL cases were also included. Also, the ELISAs had sensitivity and specificity indices and positive and negative predictive values for both CL and MCL sera that were significantly different from those that would have arisen by chance alone (Tables 1 and 2). The IF tests had significant specificity indices (87.8% for the IgM-IF and 100.0% for the IgA-IF); however, the IgG-IF specificity indices were lower than those of the IgG-ELISAs for CL or MCL sera. The significance of the specificity index is of no assistance in making a diagnosis by exclusion of false positive results, since the positive predictive value of Table 1: Performance Indices for Immunofluorescent (IF) tests and enzyme immunoassays (ELISAs) for antigens In sera from patients with ieishmaniasis In northern and north-eastern Brazil Performance index (%) Sensitivity Specificity Efficiency Cutaneous leishmaniasis versus controls IgG-IF 27.7 (22.9,3.31)"b 71.4 (57.6,82.1) 34.0 1gM-IF 20.5 (16.3,25.5)b 87.8 (75.8,94.3) 30.2 IgA-IF 3.1 (1.6,5.7)b 100.0 (0.927,1.00) 17.1 IgG-ELISA 66.9 (61.3,72.1) 77.5 (64.1,87.0) 68.4 IgM-ELISA 56.6 (50.8,62.3) 72.9 (59.0,83.4) 59.0 Leishmaniasest versus controls IgG-IF 28.2 (23.4,33.5)b 71.4 (57.6,82.1) 31.2 1gM-IF 21.0 (16.8,25.9)b 87.8 (75.8,94.3) 30.2 IgA-IF 4.3 (2.5,7.9)b 100.0 (92.7,100.0) 17.6 IgG-ELISA 66.3 (60.8,71.4) 77.5 (64.1,87.0) 67.9 IgM-ELISA 56.2 (50.5,61.8) 72.9 (59.0,83.4) 58.5 Figures in parentheses are 95% confidence intervals. The interval includes the value that would be obtained by chance alone.C Both cutaneous and mucocutaneous leishmaniasis. WHO Bulletin OMS. Vol 68 1990. M.C.S. Guimaraes et al. Table 2: Performance Indices for immunofluorescent (IF) tsts and enzyme Immunoassays (ELISAs) for antigens In sera from patients with leishmanlasis In northern and north-eastern Brazil Performance index (%) Positive predictive Negative predictive value value Cutaneous leishmaniasis versus controls IgG-IF 85.3 (76.8,91.0)ab 14.2 (10.4,19.1)b IgM-IF 90.9 (81.5,95.6)b 15.6 (11.8,20.4)b IgA-IF 100.0 (70.1,100.0)b 14.8 (11.4,19.0)b IgG-ELISA 94.6 (94.6,96.9) 28.6 (21.6,36.8) IgM-ELISA 92.6 (87.7,95.6) 22.0 (16.3,29.1) Leishmaniasesc versus controls IgG-IF 86.0 (77.9,91.5)b 13.8 (10.1,18.6)b 1gM-IF 91.4 (82.5,96.0)b 15.1 (11.4,19.8)b IgA-IF 100.0 (77.2,100.0)b 14.4 (11.1,18.5)b IgG-ELISA 94.8 (90.9,97.0) 27.3 (20.6,35.3) IgM-ELISA 92.8 (88.0,95.7) 21.2 (15.7,28.1) ' Figures in parentheses are 95% confidence intervals. bThe interval contains the value that would be obtained by chance alone. c Both cutaneous and mucocutaneous leishmaniasis. the three IF tests lacked significance (Tables 1 and 2). The performance of the IF and ELISA tests was also investigated for a series of sera from long-stand- ing cases of leishmaniasis (12), and, as expected, the indices differed from those reported above. Of note was the performance of the IgM-ELISA. For the sera from the long-standing cases the performance indices for this test were not significantly different from those that would have arisen by chance alone; however, in the present study, probably because the cases were of shorter standing, the IgM-ELISA indices were signi- cantly different from those that would have arisen by chance. In general, whenever the performance indices were significantly different from those that would have arisen by chance alone (both for the long-stand- ing cases and those in the present study), the values of the indices accrued in this study were the lowest; in contrast, the positive predictive value for the IgG- ELISA increased from 73.7% for the long-standing cases to 94.6% for those in the present study, thereby permitting the detection of true positives from among a greater number of suspected cases. When the sera used in the present study were screened by the Dot-ELISA test (Guimaraes et al., unpublished results, 1989) the specificity index increased to 91.4% and the positive predictive value to 98.8%. Our results for the ELISAs show therefore that serological tests for cutaneous leishmaniasis may be used for diagnosis, even with an L. major-like heterologous antigen. 42 The diagnostic ability of the IgA-IF test is still not clear; for example, although its sensitivity index was not significantly different from that which would have arisen by chance, nine cases that were diagnosed as CL only had positive IgA-IF tests. Since none of the controls had cross-reactive results, these patients represented true invasive cases of the disease (MCL), which were still in the cutaneous stage, and, unless given adequate therapy and followed up, invasion of the oropharynx mucosa could result. Acknowledgements We gratefully acknowledge the excellent technical assis- tance given by Mr N. Campos Filho and Mr P. de Oliveira. This paper was supported by the UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases (Grant No. 840453), Conselho Nacional do Desenvolvimento Cientifico e Tecnol6gico (CNPq)- PIDE VI (Grant No. 403608-84), and Laborat6rios de lnvestigacao M6dica (LIM-38). R6sum6 Indices de quallte du diagnostic pour les epreuves d'immunofluorescence et les 6preuves immuno-enzymatiques de s6rodia- gnostic de la leishmaniose dans 1e nord et le nord-est du Br6sil On a examine 341 echantillons de serum (292 de sujets atteints de leishmaniose cutan6e et 49 de sujets temoins) en provenance du nord et du nord- est du Bresil, par immunofluorescence (IF) et epreuves immuno-enzymatiques (ELISA) de recherche des anticorps IgG, IgA et IgM anti-Leish- mania. Le diagnostic a 6t6 confirm6 sur empreinte de la lesion et par cuti-reaction de Montenegro. Les epreuves s6rologiques presentaient dif- ferents degr6s d'association avec les marqueurs 6pid6miologiques. Par exemple, pour l'lgG-EL18A on observait une association positive entre les resultats et l'origine geographique du serum, de meme qu'avec le sexe et l'Age des malades; en revanche, pour l'lgM-ELISA on n'observait une association positive qu'avec l'origine g6ogra- phique du serum et avec I'Age des malades. Parmi les methodes etudiees, les meilleurs indicateurs de la maladie etaient les resultats de l'lgG-ELISA et de l'IgM-ELISA. La valeur pr6dictive positive de cette derni6re 6preuve etait de 94,6%, ce qui permet de deceler les sujets positifs vrais parmi un plus grand nombre de cas suspects qu'il n'6tait auparavant possible de le faire; les autres WHO Bulletin OMS. Vol 68 1990. Diagnostic performance Indices for IF tests and ELISAs of lelshmanlasls sera indices de qualite pour les epreuves ELISA dif- feraient significativement de ceux qui auraient ete obtenus par pur hasard. 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