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Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA and IFAT for the diagnosis of visceral leishmaniasis
Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 172—178
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Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA and IFAT for the diagnosis of visceral leishmaniasis Mariana Junqueira Pedras a, Luciana de Gouvˆ ea Viana a,b, Edward Jos´ e de Oliveira a, Ana Rabello a,∗ a
˜o Oswaldo Cruz (FIOCRUZ), Laboratory of Clinical Research, Centro de Pesquisas Ren´ e Rachou (CPqRR), Fundac¸a Avenue Augusto de Lima, 1715, 30190002 Belo Horizonte, MG, Brazil b Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. Prof. Alfredo Balena, 190, 30130-100 Belo Horizonte, MG, Brazil Received 28 June 2007; received in revised form 7 November 2007; accepted 7 November 2007
KEYWORDS Visceral leishmaniasis; Diagnosis; Serological tests; Direct agglutination test; rK39; Sensitivity and specificity
Summary Five serological tests for the diagnosis of visceral leishmaniasis (VL) were compared: a direct agglutination test (DAT) based on freeze-dried antigen (DAT-fd); a locally produced DAT (DAT-LPC); an IgG ELISA against rK39 (ELISA-rK39); an IgG ELISA for Leishmania chagasi (ELISA-L. chagasi); and an IgG IFAT against L. chagasi. Serum samples from 88 patients with VL, 20 non-infected individuals and 85 patients with others infectious diseases were evaluated. The sensitivity rates were: DAT-fd, 96.6%; DAT-LPC, 95.5%; ELISA-rK39, 88.6%; ELISA-L. chagasi, 89.8%; and IFAT, 92.0% (P > 0.05). The specificity for the control groups varied from 53.3% to 100%. DAT-fd had the highest efficiency (97.4%), followed by DAT-LPC (91.7%) and ELISA-rK39 (90.7%). Our data suggest that DAT-fd, DAT-LPC and ELISA-rK39 are useful tests for the diagnosis of VL and could replace IFAT as the routine diagnostic test in Brazil. © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is endemic in 62 countries, with a total of 200 million people at risk and an estimated 500 000 new cases each year worldwide (Desjeux,
∗
Corresponding author. Tel.: +55 31 33497700; fax: +55 31 3295 3115. E-mail address: ana@cpqrr.fiocruz.br (A. Rabello).
2004). In Brazil from 2001—2004, the mean number of reported VL cases was 3320 (Minist´ erio da Sa´ ude, 2006). The clinical symptoms of VL include prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anaemia, pancytopenia and hypergammaglobulinaemia. The clinical diagnosis is difficult because it shares features with other infectious and non-infectious diseases such as schistosomiasis mansoni, hepatitis, malaria, acute toxoplasmosis, and haematological and autoim-
0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.11.004
Serodiagnosis of visceral leishmaniasis mune diseases. Therefore, adequate laboratory diagnosis is essential both for individual patient care and for control strategies. The diagnostic gold standard for VL is to confirm the presence of Leishmania spp. either through cultures or microscopic visualisation of parasites from aspirate of bone marrow, spleen, lymph node or liver (Hailu et al., 2005). IFAT has been widely used for the diagnosis of leishmaniasis (Duxbury and Sadun, 1964) and remains the routine serological test used by the public health services, despite requiring fluorescence microscopes and relatively well equipped laboratories. The direct agglutination test (DAT) offers high sensitivity and specificity and may be performed in laboratories with limited infrastructure (Chappuis et al., 2006; El Harith et al., 1986; Schallig et al., 2002). ELISA is highly sensitive but its specificity varies widely depending on the antigen used. The most commonly used antigen is a crude soluble antigen extracted from the promastigotes, with 80—100% sensitivity (Sundar and Rai, 2002). A major advance has been the identification and production of the recombinant Leishmania chagasi antigen K39 (rK39), which when used in ELISA was shown to be more sensitive and specific for the diagnosis of active VL than soluble antigens (Badar´ o et al., 1996). High efficiency was also reported for the rK39 immunochromatographic strip test (Chappuis et al., 2003, 2006; Ritmeijer et al., 2006; Sundar et al., 1998; Zijlstra et al., 2001). Despite the above mentioned developments in laboratory testing, diagnosis of VL poses a challenge to physicians working in endemic areas. The scientific literature suggests that both DAT and the detection of anti-rK39 antibodies provide high efficiency and reliability for the diagnosis of VL and therefore could replace IFAT as the routine diagnostic method. According to the Brazilian guidelines, in a known endemic area a case of suspected VL is defined by the presence of fever and splenomegaly. Confirmation is based on parasitological diagnosis or on positive IFAT (≥1:80) if other diseases are excluded (Minist´ erio da Sa´ ude, 2006). In Brazil, the IFAT kit produced by Biomanguinhos/FIOCRUZ (Rio de Janeiro, Brazil) is freely provided by the Ministry of Health to public laboratories. However, in a country with such a continental dimension, a clinical sample may have to travel long distances to reach the nearest laboratory where trained personnel and fluorescent microscopy are available. Consequently, the need for transportation of serum samples may delay the patient’s diagnosis and subsequent adequate treatment for several weeks. Despite the large number of studies evaluating the rK39 strip test and DAT for the diagnosis of VL, a comparative study with the other most relevant methods for serodiagnosis of VL in Brazil is lacking. The rational for this study was to evaluate the possible laboratory options to replace IFAT, which is the routinely used method for serological diagnosis of VL in Brazil, focusing on a locally produced DAT. Herein we evaluated the performance of five serological tests for the diagnosis of VL: a DAT kit that supplies freeze-dried antigen (DAT-fd); a locally produced DAT using freshly prepared aqueous antigen (DAT-LPC); detection of anti-L. chagasi and anti-rK39 IgG antibodies using the ELISA platform (ELISA-L. chagasi and ELISA-rK39, respectively); and IFAT.
173
2. Patients and methods 2.1. Patients and controls The study was conducted during the period 2004 to 2006 at the Reference Center for Leishmaniasis of the ‘Centro de Pesquisas Ren´ e Rachou’ (CPqRR/FIOCRUZ), which provides laboratorial and an ambulatory care reference services. To evaluate the sensitivity of the serological diagnostic methods, five serological tests were evaluated on six batches of human serum samples: a visceral leishmaniasis group (Group VL, n = 88); a Chagas disease group (Group CD, n = 30); a malaria group (Group M, n = 20); a syphilis group (Group S, n = 20); a schistosomiasis mansoni group (Group SM, n = 15); and a non-infected group (Group NI, n = 20). The VL group comprised 88 patients with a clinically and parasitologically confirmed diagnosis of VL. The mean age of the patients was 14 years (range 1—59 years) and 59% of patients were male. In addition to clinical and epidemiological features, diagnosis of VL was further confirmed when amastigotes were seen in Giemsa-stained smears or when promastigote forms were isolated from culturing bone marrow aspirates. All patients were treated with meglumine antimoniate (81 mg/ml of Sbv (Aventis Pharma Ltda, S˜ ao Paulo, Brazil)) by intravenous administration of 20 mg Sbv /kg/day for 30 days, according to the recommendations of the Brazilian Ministry of Health. To evaluate the specificities of the serological methods, serum samples from individuals living in the Brazilian states of Mato Grosso or Minas Gerais, which are endemic for leishmaniasis, were retrieved from the serum bank of the Laboratory of Clinical Research (CPqRR/FIOCRUZ) and placed into groups. Group CD contained 30 serum samples from patients living in Bambu´ı, Minas Gerais state, a former area endemic for Chagas disease. These patients had been previously diagnosed with Chagas disease by immunofluorescence and ELISA for Chagas disease. Group M comprised 20 samples from patients from Apiac´ as, Mato Grosso state, with a clinical and parasitological diagnosis of malaria. Group S contained 20 samples from patients with a clinical and serological diagnosis of syphilis treated at the Municipal Syphilis Health Care Centre of Belo Horizonte, Minas Gerais state. Group SM contained 15 samples from patients from Minas Gerais state who had an acute clinical form and parasitological diagnosis of schistosomiasis. Group NI contained 20 samples from healthy persons from the laboratory staff, who live in Belo Horizonte where VL is endemic, but who had no history of clinical suspicion of VL. Peripheral blood samples were collected by venipuncture and the serum samples were separated and stored at —20 ◦ C until use.
2.2. Serological tests All serological tests were performed by two laboratory technicians with vast experience in the different assays, who were not aware of the results of the other tests.
174 2.2.1. DAT produced at the Laboratory of Clinical Research of CPqRR (DAT-LPC) Promastigotes of L. chagasi (MHOM/BR/2002/LPC-RPV) were grown in 0.5 l flasks at 26 ◦ C in NNN/LIT medium containing 20% heat inactivated fetal bovine serum (FBS) (Gibco/Invitrogen, New York, NY, USA). Log phase promastigotes were harvested and centrifuged for 15 min at 3200 × g at 4 ◦ C. The pellet was washed five times with 45 ml of cold Locke’s solution (0.9% NaCl, 0.25% glucose, 0.04% KCl, 0.02% CaCl2 , 0.02% NaHCO3 ). The pellet was treated with 20 equal volumes of 1.2% 2-mercaptoethanol (2-ME) (Sigma Chemical Co., St Louis, MO, USA) in Locke’s solution for 45 min at 37 ◦ C with agitation. After washing, the promastigotes were fixed by treatment with 2% (w/v) formaldehyde in cold Locke’s solution for 20 h at 4 ◦ C. Following the washes in cold saline-citrate solution (SCS), the fixed promastigotes were stained in SCS containing 0.02% Coomassie brilliant blue (R-250 Nuclear, Diadema, SP, Brazil) for 90 min using a magnetic stirrer at a moderate speed at room temperature. The stained promastigotes were then washed several times with cold SCS. Finally, the pellet was re-suspended (5 × 107 /ml) in cold 1% formaldehyde-SCS (w/v). The test was performed essentially as described by El Harith et al. (1988) and Garcez et al. (1996). Sera were diluted in 0.9% NaCl containing 1% heat inactivated FBS and 0.15 M 2-ME solution and a two-fold dilution series was made from 1:100 to 1:102 400. Then, 50 l of DAT-LPC antigen solution (concentration 5 × 107 parasites/ml) was added to each well of a V-shaped microtitre plate (Nunc-Immuno Plate Brand Products, Roskilde, Denmark) containing 50 l of diluted serum. After 18 h of incubation at room temperature, the end titre was read as the dilution immediately before the well with a clear sharp-edged blue spot identical in size to the negative control. Appropriate control samples with known DAT titres were included as controls. The cut-off value was determined by analysis of a receiveroperating characteristic (ROC) curve. Based on this analysis, 1:1600 was the best titre for interpretation of positive and negative results without compromising sensitivity and specificity. 2.2.2. Direct agglutination test (DAT-fd) The freeze-dried antigen from L. donovani promastigotes was supplied by KIT Biomedical Research (Amsterdam, The Netherlands). All procedures followed the manufacturer’s instructions. A two-fold dilution series of the serum samples was made following the same procedure described for DAT-LPC. 2.2.3. Anti-Leishmania chagasi and anti-rK39 ELISA Leishmania-reactive IgG antibody levels were measured by ELISA using crude water-soluble antigen preparations from L. chagasi (MHOM/BR/74/PP75). Promastigotes were cultivated in NNN/LIT medium for 7 days. The antigen was prepared according to (Ho et al., 1983). The dissolvable extract of L. chagasi antigen was prepared from 9 × 107 promastigotes/ml and sonicated six times for 20 s on ice. The suspension was centrifuged at 10 000 × g for 30 min at 4 ◦ C and stored in aliquots at −70 ◦ C until use. Protein concentration was determined according to the Lowry method (Lowry et al., 1951). The optimum working concentrations of
M.J. Pedras et al. antigens, sera and anti-human serum conjugates were determined with the checkerboard titration method. Polystyrene plates were coated with 100 l of 3 g/ml antigen diluted in carbonate—bicarbonate buffer (pH 9.6) per well. The plates were incubated overnight at 4 ◦ C and then washed five times with distilled water. Free binding sites were blocked with 150 l of 2% bovine serum albumin (BSA) in PBS (pH 7.4) buffer with 0.05% Tween-20 (PBS-BSA-T20) for 1 h at 37 ◦ C. The plates were then washed five times. Sera were diluted to 1:1000 in PBS-T20 buffer and 100 l aliquots were added as duplicates for each sample. Wells containing positive and negative control sera were included. Plates were incubated for 1 h at 37 ◦ C and then washed five times. Peroxidase conjugated anti-human IgG (Sigma no. A.6029) diluted 1:1000 was added (100 l/well). The plates were incubated for 1 h at 37 ◦ C and then washed five times. After washing, 100 l of substrate solution (ABTS) was added to each well. After 20 min, the plate was read at 405 nm in a microplate reader (Model 550; Bio-Rad Laboratories, Tokyo, Japan). Anti-rK39 IgG antibodies were determined by ELISA employing the same protocol as ELISA-L. chagasi with the following changes: the plates were coated with 50 ng/well of rK39 (a kind gift from S.G. Reed, Infectious Disease Research Institute, Seattle, WA, USA) and the serum samples were diluted to 1:100. The cut-off was determined as the mean + 2 SD of the absorbance readings of control sera (n = 20). Each serum was assayed in duplicate, taking the mean as the final result. Negative and positive controls were included in each plate. 2.2.4. IFAT The L. chagasi antigen was produced at the Laboratory of Clinical Research of CPqRR, according to the following procedure: promastigotes (MHOM/BR/2002/LPC-RPV) were centrifuged at 1300 × g at 4 ◦ C for 10 min. The suspension was washed three times with PBS containing 2% BSA (PBS-BSA). The parasites were fixed with 4% paraformaldehyde—PBS solution (w/v) for 30 min at room temperature. The suspension was again washed three times with PBS-BSA. The sediment was re-suspended in 10 ml of PBS and the number of parasites was adjusted to 3—4 × 106 promastigotes/ml. For IFAT, slides were coated with 10 l of antigen at room temperature for 12 h. For each reaction, 10 l of a two-fold serum dilution (1:40 to 1:640) in PBS were added over the slide holes. After incubation in a humid stove at 37 ◦ C for 30 min, the slides were washed for 3 min in PBS plus 3 min in distilled water. The conjugate was diluted to 1:100 in PBS with 0.025% Evan’s Blue and then 15 l of this solution was placed over the slide holes. The incubation and washing steps were repeated once, as outlined above. Slides were mounted with buffered glycerin, covered with a cover slip and read under an Olympus BX-FLA fluorescent microscope equipped with a 100 W mercury lamp with 400× magnifying power. The samples were defined as positive at titres >1:40.
2.3. Statistical analysis Data were processed with SPSS for Windows version 10.0.5 (SPSS Inc., Chicago, IL, USA). All variables were individually assessed with the W-test of normality. For comparisons of
Not infected (n = 20) Schistosomiasis (n = 15) Syphilis (n = 20) Malaria (n = 20) Chagas disease (n = 30) Total group (n = 105)
DAT-fd 85 (96.6) (89.7—99.1) 103 (98.1) (92.6—99.7) 28 (93.3) (76.5—98.8) 20 (100) (80—100) 20 (100) (80—100) 15 (100) (74.7—100) 20 (100) (80—100) 188 (97.4) (93.7—99.0) DAT-LPC 84 (95.5) (88.1—98.5) 93 (88.6) (80.5—93.7) 26 (86.7) (68.4—95.6) 18 (90.0) (66.9—98.3) 17 (85.0) (61.1—96.0) 12 (80.0) (51.4—94.7) 20 (100) (80—100) 177 (91.7) (86.7—95.0) ELISA-rK39 78 (88.6) (79.7—94.1) 97 (92.4) (85.1—96.4) 25 (83.3) (64.5—93.7) 17 (85.0) (61.1—96.0) 20 (100) (80—100) 15 (100) (74.7—100) 20 (100) (80—100) 175 (90.7) (85.4—94.2) ELISA-L. 79 (89.8) (83.8—96.5) 85 (81.0) (71.9—87.7) 16 (53.3) (34.6—71.2) 15 (75.0) (50.6—90.4) 19 (95.0) (73.1—99.7) 15 (100) (74.7—100) 20 (100) (80—100) 164 (85.0) (79.0—89.5) chagasi IFAT 81 (92.0) (83.8—96.5) 88 (83.8) (75.1—90.0) 20 (66.7) (47.1—82.1) 14 (70.0) (45.7—87.2) 20 (100) (80—100) 14 (93.3) (66.0—99.7) 20 (100) (80—100) 169 (87.6) (81.9—91.7) All other differences were non-significant (P ≥ 0.05). a Difference between specificity rates: Total group: DAT-fd × DAT-LPC, P < 0.001; DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P < 0.001. Chagas disease group: DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P = 0.01; DAT-LPC × ELISA-L. chagasi, P < 0.001; ELISA-rK39 × ELISA-L. chagasi, P = 0.012. Malaria group: DAT-fd × IFAT, P = 0.026. b Difference between efficiencies: DAT-fd × DAT-LPC, P = 0.01; DAT-fd × ELISA-rK39, P = 0.005; DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P < 0.001.
Diagnostic efficiency (n = 193), n (%) (95% CI)b Specificity in control groups, n (%) (95% CI)a
Table 1 shows the sensitivity and specificity rates obtained with the five serological tests. The sensitivity rates varied from 88.6% to 96.6% (non-significant difference among all five tests). The two DAT preparations presented similar values, with sensitivities of 95.5% (95% CI 88.1—98.5%) for DAT-LPC and 96.6% (95% CI 89.7—99.1%) for DAT-fd. Four serum samples presented false-negative reactions with DATLPC and three with DAT-fd. The sensitivity rates obtained with anti-rK39 and anti-L. chagasi ELISA were also very similar: 88.6% (95% CI 79.7—94.1%) and 89.8% (95% CI 83.8—96.5%), respectively. Using IFAT, seven sera presented false-negative results, conferring a sensitivity of 92.0% (95% CI 83.8—96.5%). Specificity rates were determined for the total group of non-leishmaniasis patients and for each group separately (Table 1). The specificity values observed for the total control group were 88.6% (95% CI 80.5—93.7%) for DATLPC, 98.1% (95% CI 92.6—99.7%) for DAT-fd, 92.4% (95% CI 85.1—96.4%) for ELISA-rK39, 81.0% (95% CI 71.9—87.7) for ELISA-L. chagasi and 83.8% (95% CI 75.1—90.0) for IFAT. However, the specificity observed for each group of related diseases varied widely: 53.3—93.3% for Chagas disease; 70—100% for malaria; 85—100% for syphilis; and 80—100% for schistosomiasis mansoni. The higher frequency of crossreactivity that reduced the specificity of the tests occurred with IFAT and ELISA-L. chagasi mainly with sera from patients with Chagas disease, followed by sera from patients with malaria. With DAT-fd, only 2 of the 30 serum samples from the Chagas disease group had any cross-reactivity. In the group of non-infected individuals, all serological tests had 100% specificity (Table 1). The serological tests that achieved the highest diagnostic efficiencies were DAT-fd (97.4%, 95% CI 93.7—99.0%), followed by DAT-LPC (91.7%, 95% CI 86.7—95.0%), ELISA-rK39 (90.7%, 95% CI 85.4—94.2%), ELISA-L. chagasi (85.0%, 95% CI 79.0—89.5%) and IFAT (87.6%, 95% CI 81.9—91.7%). Statistical differences were observed between the results presented by DAT-fd and all the other serological tests (P < 0.05) (Table 1). A good agreement beyond chance ( index) ranging from 0.60 to 0.80 was obtained when all the results from all five methods were cross-tabulated, including 193 serum samples from VL patients and the control groups (Table 2).
Sensitivity in VL group (n = 88), n (%) (95% CI)
3. Results
Serological test
quantitative results among the different groups, the titres and absorbance readings were transformed to log10 [result+1] and evaluated by the non-parametric Spearman’s correlation, considering significance at the 0.01 level (two-tailed). The equation TP+TN/TP+TN+FP+FN (where TP = true positive, TN = true negative and FP = false positive) was used to determine diagnostic efficiency (Galen and Gambino, 1975). The 2 test was employed for comparison of sensitivities, specificities and diagnostic efficiency rates. Agreement beyond chance was assessed using the Cohen coefficient (Cohen, 1968) and interpreted according to the scale of Landis and Koch (1977): 1.00—0.81, excellent; 0.80—0.61, good; 0.60—0.41, moderate; 0.40—0.21, weak; and 0.20—0.0 negligible. The significance level was set at <5% probability of ␣ error (Bhattacharyya and Johnson, 1977).
175 Table 1 Comparison of sensitivity in patients with visceral leishmaniasis (VL), specificity rates in control groups and diagnostic efficiency in VL patients plus control groups of the five serological tests
Serodiagnosis of visceral leishmaniasis
M.J. Pedras et al.
—– —– —– —–
—– —– —– —–
96 97 193
P N 82 5 14 92 96 97 0.80 (0.66—0.94) 0.79 (< 0.001) —– —– —– —–
—–
—– —– —–
98 95 193
99 94 193
87 106 193
96 97 193
index (95% CI) Spearman’s coefficient (P-value) DAT-LPC P N T index (95% CI) Spearman’s coefficient (P-value) IFAT P N T index (95% CI) Spearman’s coefficient (P-value) ELISA-L. chagasi P N T index Spearman’s coefficient (P-value)
P N T DAT-fd
P: positive; N: negative; T: total.
96 97 193
P N 81 6 17 89 98 95 0.76 (0.67—0.85) 0.81 (< 0.001) 79 17 19 78 98 95 0.63 (0.49—0.77) 0.75 (<0.001) —– —– —– —–
ELISA-L. chagasi
P N 78 9 21 85 99 94 0.69 (0.56—0.83) 0.65 (< 0.001) 78 18 21 76 99 94 0.60 (0.46—0.74) 0.66 (<0.001) 78 20 8 87 86 107 0.71 (0.57—0.90) 0.69 (<0.001) —– —– —– —– T 87 106 193
P N 76 11 10 96 86 107 0.78 (0.64—0.92) 0.79 (< 0.001) 79 17 7 90 86 107 0.75 (0.61—0.89) 0.75 (<0.001) 81 6 17 89 98 95 0.76 (0.67—0.85) 0.80 (<0.001) 73 26 13 81 86 107 0.60 (0.56—0.74) 0.70 (<0.001)
T 87 106 193
IFAT
T 87 106 193
DAT-LPC
T 87 106 193
The Spearman’s correlation coefficients between the quantitative values of each test are shown in Table 2. The log-transformed titres of both DAT tests, the IFAT and the absorbance readings from both ELISA tests obtained with the 193 serum samples from the VL patients and controls were analysed. All the paired tests present positive and significant correlations. Interestingly, the highest correlation coefficients were observed between IFAT and DAT-fd (0.81) and IFAT and ELISA-rK39 (0.80). Figure 1 shows the individual agglutination results obtained with the two DAT preparations. DAT-LPC agglutination was observed at the lowest serum dilution of the control samples. None the less, positive and significant correlation (0.79) was observed between the titres obtained with the two preparations (Table 2).
4. Discussion
ELISA-rK39
Serological test Serological test
Table 2 Agreement, index values and Spearman’s correlation coefficient between paired quantitative results of the five serological tests for the diagnosis of visceral leishmaniasis (n = 193)
176
In this study, we compared five different techniques for serological diagnosis of VL, three of which are locally prepared (DAT-LPC, IFAT and ELISA-L. chagasi) and two are dependent on imported antigen (ELISA-rK39 and DAT-fd). DAT is a well validated test for serodiagnosis of VL and has been used in Sudan for the last two decades (El Harith et al., 1986, 1987; Ritmeijer et al., 2006), combining high levels of intrinsic validity and ease of use (Chappuis et al., 2006). The antigen supplied by KIT Biomedical Research is prepared from cultured promastigotes of L. donovani, whereas human VL in Brazil is caused by L. chagasi. The heterologous antigen did not impair its high performance, according to Andrade et al. (1989). The freeze-dried DAT antigen is currently available from the Prince Leopold Institute (Antwerp, Belgium) and The Royal Tropical Institute (Amsterdam, The Netherlands). The cost of one test is approximately US$2.00 (H. Schallig, personal communication). Although similar sensitivities were obtained both for the locally produced and commercial DAT, a superior specificity was observed with the industrialised kit (DAT-fd). In the present study, a relatively high specificity (88.6%) for the DAT-LPC test was observed, which is comparable with previous studies where DAT-fd was also used (Meredith et al., 1995; Schoone et al., 2001). Other studies showed that the specificity of DAT depends on the chosen cut-off titre. A cut-off titre of higher serum dilutions generally yields higher specificities (Chappuis et al., 2003). The cut-off titre of the industrialised DAT with freeze-dried antigens was previously defined, whereas the definition of the best cut-off value for the laboratory-made DAT-LPC had to be determined using a ROC curve (data not shown). The cut-off titre for the DAT-LPC is low compared with those defined in other studies (Chappuis et al., 2003). It is possible that further standardisation steps would improve the DAT-LPC, which would result in better interpretation of positive/negative agglutination reactions. Nevertheless, the good performance of the locally prepared DAT, with a index of 0.80, and the high correlation between the agglutination titres of both DAT assays (0.79; P < 0.01), showed that national production is possible, as all the necessary biological and chemical reagents are nationally available. Although microplates and micropipettes may be an obstacle for widespread field use of the DAT in some countries, this should not be a major
Serodiagnosis of visceral leishmaniasis
177
Figure 1 Agglutination titres of VL patients and healthy and diseased controls tested with (A) a direct agglutination test (DAT) based on freeze-dried antigen (DAT-fd) and (B) a locally produced DAT using freshly prepared aqueous antigen (DAT-LPC). Results were considered positive with titres ≥1:100 and >1:1600 for DAT-fd and DAT-LPC, respectively. M: malaria; S: syphilis; SM: Schistosoma mansoni; NI: non-infected; CD: Chagas diseases; VL: visceral leishmaniasis.
concern in Brazil. Nevertheless, the laboratory infrastructure needed to carry out DAT is much simpler than either ELISA or IFAT, as no sophisticated equipment is required. The estimates of sensitivity and specificity should be carefully interpreted as the selection of samples may misrepresent the population exposed to the parasite in an endemic area. The higher sensitivity rate presented by ELISA-rK39 (88.6%) was not statistically different (P > 0.577) from the value obtained with the ELISA-L. chagasi with crude soluble antigens (89.8%). This result corroborated the findings of Braz et al. (2002), who evaluated ELISA-rK39 and ELISA-L chagasi in 120 serum samples from Brazilian patients with VL. However, the higher specificity of ELISA-rK39 was significantly different when testing serum from patients with Chagas disease (83.3% vs. 53.3%) and malaria patients (85.0% vs. 75.0%). The dipstick format to detect anti-rK39 antibodies has been validated in different regions and presents heterogeneous sensitivity and specificity and needs to be largely evaluated in Brazil (Chappuis et al., 2006). In conclusion, the overall performance of DAT-fd, DAT-LPC and ELISA-rK39 scored higher than IFAT and ELISA-L. chagasi. The results point to the possible role of the evaluated methods in the replacement of IFAT as the routine serological method for diagnosis of VL in Brazil. A large-scale prospective study to validate the tests in the target population needs to be carried out. This assessment and an analysis to estimate costs and sustainability of the different methods are needed before definite recommendations can be given to the Leishmaniasis Control National Programme. Authors’ contributions: MJP and LGV carried out the immunoassays; EJO and AR carried out analysis and interpretation of the data and drafted the manuscript. All authors read and approved the final manuscript. AR is guarantor of the paper. Acknowledgements: We thank Dr Henk Schallig from the Laboratory for Biomedical Research, The Royal Tropical Institute, Amsterdam, The Netherlands, for providing the Leish-KIT, DAT kits and for critical review of the manuscript.
We also thank Dr Steven Reed from the Infectious Disease Research Institute, Seattle, WA, USA, for providing the rK39 antigen. The Health Care Centre of Belo Horizonte is thanked ´rika Braga from the for providing the syphilis sera, and Dr E Federal University of Minas Gerais state for providing the malaria samples. Funding: CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnol´ ogico), Bras´ılia, DF, Brazil (grant ˜o de Amparo a ` Pesquisa 350594/1995-3); FAPEMIG (Fundac ¸a de Minas Gerais), Belo Horizonte, MG, Brazil (CDS: 55013/ 02); and FIOCRUZ Rio de Janeiro, RJ, Brazil (PDTIS RID06). Conflicts of interest: None declared. Ethical approval: The Ethical Review Board of Centro de Pesquisas Ren´ e Rachou (CPqRR)—FIOCRUZ, Belo Horizonte, MG, Brazil, approved the use of stored serum samples (CEPSH/CPqRR no. 05/2007), in agreement with Resolution 357/05 of the National Health Council of the Ministry of Health, which regulates research involving human subjects in Brazil.
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178 ing active from subclinical infection. Am. J. Trop. Med. Hyg. 67, 344—348. Chappuis, F., Rijal, S., Singh, R., Acharya, P., Karki, B.M., Das, M.L., Bovier, P.A., Desjeux, P., Le Ray, D., Koirala, S., Loutan, L., 2003. Prospective evaluation and comparison of the direct agglutination test and an rK39-antigen-based dipstick test for the diagnosis of suspected kala-azar in Nepal. Trop. Med. Int. Health 8, 277—285. Chappuis, F., Rijal, S., Soto, A., Menten, J., Boelaert, M., 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333, 723. Cohen, J., 1968. Weighted kappa: nominal scale agreement with provisions for scales disagreement of partial credit. Psychol. Bull. 70, 213—220. Desjeux, P., 2004. Leishmaniasis. Nat. Rev. Microbiol. 2, 692—693. Duxbury, R.E., Sadun, E.H., 1964. Fluorescent antibody test for the serodiagnosis of visceral leishmaniasis. Am. J. Trop. Med. Hyg. 13, 525—529. El Harith, A., Kolk, A.H., Kager, P.A., Leeuwenburg, J., Muigai, R., Kiugu, S., Laarman, J.J., 1986. A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 80, 583—587. El Harith, A., Kolk, A.H.J., Kager, P.A., Leeuwenburg, J., Faber, F.J., Muigai, R., Kiugu, S., Laarman, J.J., 1987. Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA. Trans. R. Soc. Trop. Med. Hyg. 81, 603—606. El Harith, A., Kolk, A.H., Leeuwenburg, J., Muigai, R., Huigen, E., Kager, P.A., 1988. Improvement of a direct agglutination test for field studies of visceral leishmaniasis. J. Clin. Microbiol. 26, 1321—1325. Galen, R.S., Gambino, S.R., 1975. Beyond Normality: the predictive value and efficiency of medical diagnosis, first ed. John Wiley & Sons Inc., New York, NY. Garcez, L.M., Shaw, J.J., Silveira, F.T., 1996. Direct agglutination tests in the serodiagnosis of visceral leishmaniasis in the state of Par´ a. Rev. Soc. Bras. Med. Trop. 29, 165—180 [in Portuguese]. Hailu, A., Musa, A.M., Royce, C., Wassuna, M., 2005. Visceral leishmaniasis: new health tools are needed. PLoS Med. 2, 590—594.
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Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA and IFAT for the diagnosis of visceral leishmaniasis Mariana Junqueira Pedras a, Luciana de Gouvˆ ea Viana a,b, Edward Jos´ e de Oliveira a, Ana Rabello a,∗ a
˜o Oswaldo Cruz (FIOCRUZ), Laboratory of Clinical Research, Centro de Pesquisas Ren´ e Rachou (CPqRR), Fundac¸a Avenue Augusto de Lima, 1715, 30190002 Belo Horizonte, MG, Brazil b Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. Prof. Alfredo Balena, 190, 30130-100 Belo Horizonte, MG, Brazil Received 28 June 2007; received in revised form 7 November 2007; accepted 7 November 2007
KEYWORDS Visceral leishmaniasis; Diagnosis; Serological tests; Direct agglutination test; rK39; Sensitivity and specificity
Summary Five serological tests for the diagnosis of visceral leishmaniasis (VL) were compared: a direct agglutination test (DAT) based on freeze-dried antigen (DAT-fd); a locally produced DAT (DAT-LPC); an IgG ELISA against rK39 (ELISA-rK39); an IgG ELISA for Leishmania chagasi (ELISA-L. chagasi); and an IgG IFAT against L. chagasi. Serum samples from 88 patients with VL, 20 non-infected individuals and 85 patients with others infectious diseases were evaluated. The sensitivity rates were: DAT-fd, 96.6%; DAT-LPC, 95.5%; ELISA-rK39, 88.6%; ELISA-L. chagasi, 89.8%; and IFAT, 92.0% (P > 0.05). The specificity for the control groups varied from 53.3% to 100%. DAT-fd had the highest efficiency (97.4%), followed by DAT-LPC (91.7%) and ELISA-rK39 (90.7%). Our data suggest that DAT-fd, DAT-LPC and ELISA-rK39 are useful tests for the diagnosis of VL and could replace IFAT as the routine diagnostic test in Brazil. © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is endemic in 62 countries, with a total of 200 million people at risk and an estimated 500 000 new cases each year worldwide (Desjeux,
∗
Corresponding author. Tel.: +55 31 33497700; fax: +55 31 3295 3115. E-mail address: ana@cpqrr.fiocruz.br (A. Rabello).
2004). In Brazil from 2001—2004, the mean number of reported VL cases was 3320 (Minist´ erio da Sa´ ude, 2006). The clinical symptoms of VL include prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anaemia, pancytopenia and hypergammaglobulinaemia. The clinical diagnosis is difficult because it shares features with other infectious and non-infectious diseases such as schistosomiasis mansoni, hepatitis, malaria, acute toxoplasmosis, and haematological and autoim-
0035-9203/$ — see front matter © 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.11.004
Serodiagnosis of visceral leishmaniasis mune diseases. Therefore, adequate laboratory diagnosis is essential both for individual patient care and for control strategies. The diagnostic gold standard for VL is to confirm the presence of Leishmania spp. either through cultures or microscopic visualisation of parasites from aspirate of bone marrow, spleen, lymph node or liver (Hailu et al., 2005). IFAT has been widely used for the diagnosis of leishmaniasis (Duxbury and Sadun, 1964) and remains the routine serological test used by the public health services, despite requiring fluorescence microscopes and relatively well equipped laboratories. The direct agglutination test (DAT) offers high sensitivity and specificity and may be performed in laboratories with limited infrastructure (Chappuis et al., 2006; El Harith et al., 1986; Schallig et al., 2002). ELISA is highly sensitive but its specificity varies widely depending on the antigen used. The most commonly used antigen is a crude soluble antigen extracted from the promastigotes, with 80—100% sensitivity (Sundar and Rai, 2002). A major advance has been the identification and production of the recombinant Leishmania chagasi antigen K39 (rK39), which when used in ELISA was shown to be more sensitive and specific for the diagnosis of active VL than soluble antigens (Badar´ o et al., 1996). High efficiency was also reported for the rK39 immunochromatographic strip test (Chappuis et al., 2003, 2006; Ritmeijer et al., 2006; Sundar et al., 1998; Zijlstra et al., 2001). Despite the above mentioned developments in laboratory testing, diagnosis of VL poses a challenge to physicians working in endemic areas. The scientific literature suggests that both DAT and the detection of anti-rK39 antibodies provide high efficiency and reliability for the diagnosis of VL and therefore could replace IFAT as the routine diagnostic method. According to the Brazilian guidelines, in a known endemic area a case of suspected VL is defined by the presence of fever and splenomegaly. Confirmation is based on parasitological diagnosis or on positive IFAT (≥1:80) if other diseases are excluded (Minist´ erio da Sa´ ude, 2006). In Brazil, the IFAT kit produced by Biomanguinhos/FIOCRUZ (Rio de Janeiro, Brazil) is freely provided by the Ministry of Health to public laboratories. However, in a country with such a continental dimension, a clinical sample may have to travel long distances to reach the nearest laboratory where trained personnel and fluorescent microscopy are available. Consequently, the need for transportation of serum samples may delay the patient’s diagnosis and subsequent adequate treatment for several weeks. Despite the large number of studies evaluating the rK39 strip test and DAT for the diagnosis of VL, a comparative study with the other most relevant methods for serodiagnosis of VL in Brazil is lacking. The rational for this study was to evaluate the possible laboratory options to replace IFAT, which is the routinely used method for serological diagnosis of VL in Brazil, focusing on a locally produced DAT. Herein we evaluated the performance of five serological tests for the diagnosis of VL: a DAT kit that supplies freeze-dried antigen (DAT-fd); a locally produced DAT using freshly prepared aqueous antigen (DAT-LPC); detection of anti-L. chagasi and anti-rK39 IgG antibodies using the ELISA platform (ELISA-L. chagasi and ELISA-rK39, respectively); and IFAT.
173
2. Patients and methods 2.1. Patients and controls The study was conducted during the period 2004 to 2006 at the Reference Center for Leishmaniasis of the ‘Centro de Pesquisas Ren´ e Rachou’ (CPqRR/FIOCRUZ), which provides laboratorial and an ambulatory care reference services. To evaluate the sensitivity of the serological diagnostic methods, five serological tests were evaluated on six batches of human serum samples: a visceral leishmaniasis group (Group VL, n = 88); a Chagas disease group (Group CD, n = 30); a malaria group (Group M, n = 20); a syphilis group (Group S, n = 20); a schistosomiasis mansoni group (Group SM, n = 15); and a non-infected group (Group NI, n = 20). The VL group comprised 88 patients with a clinically and parasitologically confirmed diagnosis of VL. The mean age of the patients was 14 years (range 1—59 years) and 59% of patients were male. In addition to clinical and epidemiological features, diagnosis of VL was further confirmed when amastigotes were seen in Giemsa-stained smears or when promastigote forms were isolated from culturing bone marrow aspirates. All patients were treated with meglumine antimoniate (81 mg/ml of Sbv (Aventis Pharma Ltda, S˜ ao Paulo, Brazil)) by intravenous administration of 20 mg Sbv /kg/day for 30 days, according to the recommendations of the Brazilian Ministry of Health. To evaluate the specificities of the serological methods, serum samples from individuals living in the Brazilian states of Mato Grosso or Minas Gerais, which are endemic for leishmaniasis, were retrieved from the serum bank of the Laboratory of Clinical Research (CPqRR/FIOCRUZ) and placed into groups. Group CD contained 30 serum samples from patients living in Bambu´ı, Minas Gerais state, a former area endemic for Chagas disease. These patients had been previously diagnosed with Chagas disease by immunofluorescence and ELISA for Chagas disease. Group M comprised 20 samples from patients from Apiac´ as, Mato Grosso state, with a clinical and parasitological diagnosis of malaria. Group S contained 20 samples from patients with a clinical and serological diagnosis of syphilis treated at the Municipal Syphilis Health Care Centre of Belo Horizonte, Minas Gerais state. Group SM contained 15 samples from patients from Minas Gerais state who had an acute clinical form and parasitological diagnosis of schistosomiasis. Group NI contained 20 samples from healthy persons from the laboratory staff, who live in Belo Horizonte where VL is endemic, but who had no history of clinical suspicion of VL. Peripheral blood samples were collected by venipuncture and the serum samples were separated and stored at —20 ◦ C until use.
2.2. Serological tests All serological tests were performed by two laboratory technicians with vast experience in the different assays, who were not aware of the results of the other tests.
174 2.2.1. DAT produced at the Laboratory of Clinical Research of CPqRR (DAT-LPC) Promastigotes of L. chagasi (MHOM/BR/2002/LPC-RPV) were grown in 0.5 l flasks at 26 ◦ C in NNN/LIT medium containing 20% heat inactivated fetal bovine serum (FBS) (Gibco/Invitrogen, New York, NY, USA). Log phase promastigotes were harvested and centrifuged for 15 min at 3200 × g at 4 ◦ C. The pellet was washed five times with 45 ml of cold Locke’s solution (0.9% NaCl, 0.25% glucose, 0.04% KCl, 0.02% CaCl2 , 0.02% NaHCO3 ). The pellet was treated with 20 equal volumes of 1.2% 2-mercaptoethanol (2-ME) (Sigma Chemical Co., St Louis, MO, USA) in Locke’s solution for 45 min at 37 ◦ C with agitation. After washing, the promastigotes were fixed by treatment with 2% (w/v) formaldehyde in cold Locke’s solution for 20 h at 4 ◦ C. Following the washes in cold saline-citrate solution (SCS), the fixed promastigotes were stained in SCS containing 0.02% Coomassie brilliant blue (R-250 Nuclear, Diadema, SP, Brazil) for 90 min using a magnetic stirrer at a moderate speed at room temperature. The stained promastigotes were then washed several times with cold SCS. Finally, the pellet was re-suspended (5 × 107 /ml) in cold 1% formaldehyde-SCS (w/v). The test was performed essentially as described by El Harith et al. (1988) and Garcez et al. (1996). Sera were diluted in 0.9% NaCl containing 1% heat inactivated FBS and 0.15 M 2-ME solution and a two-fold dilution series was made from 1:100 to 1:102 400. Then, 50 l of DAT-LPC antigen solution (concentration 5 × 107 parasites/ml) was added to each well of a V-shaped microtitre plate (Nunc-Immuno Plate Brand Products, Roskilde, Denmark) containing 50 l of diluted serum. After 18 h of incubation at room temperature, the end titre was read as the dilution immediately before the well with a clear sharp-edged blue spot identical in size to the negative control. Appropriate control samples with known DAT titres were included as controls. The cut-off value was determined by analysis of a receiveroperating characteristic (ROC) curve. Based on this analysis, 1:1600 was the best titre for interpretation of positive and negative results without compromising sensitivity and specificity. 2.2.2. Direct agglutination test (DAT-fd) The freeze-dried antigen from L. donovani promastigotes was supplied by KIT Biomedical Research (Amsterdam, The Netherlands). All procedures followed the manufacturer’s instructions. A two-fold dilution series of the serum samples was made following the same procedure described for DAT-LPC. 2.2.3. Anti-Leishmania chagasi and anti-rK39 ELISA Leishmania-reactive IgG antibody levels were measured by ELISA using crude water-soluble antigen preparations from L. chagasi (MHOM/BR/74/PP75). Promastigotes were cultivated in NNN/LIT medium for 7 days. The antigen was prepared according to (Ho et al., 1983). The dissolvable extract of L. chagasi antigen was prepared from 9 × 107 promastigotes/ml and sonicated six times for 20 s on ice. The suspension was centrifuged at 10 000 × g for 30 min at 4 ◦ C and stored in aliquots at −70 ◦ C until use. Protein concentration was determined according to the Lowry method (Lowry et al., 1951). The optimum working concentrations of
M.J. Pedras et al. antigens, sera and anti-human serum conjugates were determined with the checkerboard titration method. Polystyrene plates were coated with 100 l of 3 g/ml antigen diluted in carbonate—bicarbonate buffer (pH 9.6) per well. The plates were incubated overnight at 4 ◦ C and then washed five times with distilled water. Free binding sites were blocked with 150 l of 2% bovine serum albumin (BSA) in PBS (pH 7.4) buffer with 0.05% Tween-20 (PBS-BSA-T20) for 1 h at 37 ◦ C. The plates were then washed five times. Sera were diluted to 1:1000 in PBS-T20 buffer and 100 l aliquots were added as duplicates for each sample. Wells containing positive and negative control sera were included. Plates were incubated for 1 h at 37 ◦ C and then washed five times. Peroxidase conjugated anti-human IgG (Sigma no. A.6029) diluted 1:1000 was added (100 l/well). The plates were incubated for 1 h at 37 ◦ C and then washed five times. After washing, 100 l of substrate solution (ABTS) was added to each well. After 20 min, the plate was read at 405 nm in a microplate reader (Model 550; Bio-Rad Laboratories, Tokyo, Japan). Anti-rK39 IgG antibodies were determined by ELISA employing the same protocol as ELISA-L. chagasi with the following changes: the plates were coated with 50 ng/well of rK39 (a kind gift from S.G. Reed, Infectious Disease Research Institute, Seattle, WA, USA) and the serum samples were diluted to 1:100. The cut-off was determined as the mean + 2 SD of the absorbance readings of control sera (n = 20). Each serum was assayed in duplicate, taking the mean as the final result. Negative and positive controls were included in each plate. 2.2.4. IFAT The L. chagasi antigen was produced at the Laboratory of Clinical Research of CPqRR, according to the following procedure: promastigotes (MHOM/BR/2002/LPC-RPV) were centrifuged at 1300 × g at 4 ◦ C for 10 min. The suspension was washed three times with PBS containing 2% BSA (PBS-BSA). The parasites were fixed with 4% paraformaldehyde—PBS solution (w/v) for 30 min at room temperature. The suspension was again washed three times with PBS-BSA. The sediment was re-suspended in 10 ml of PBS and the number of parasites was adjusted to 3—4 × 106 promastigotes/ml. For IFAT, slides were coated with 10 l of antigen at room temperature for 12 h. For each reaction, 10 l of a two-fold serum dilution (1:40 to 1:640) in PBS were added over the slide holes. After incubation in a humid stove at 37 ◦ C for 30 min, the slides were washed for 3 min in PBS plus 3 min in distilled water. The conjugate was diluted to 1:100 in PBS with 0.025% Evan’s Blue and then 15 l of this solution was placed over the slide holes. The incubation and washing steps were repeated once, as outlined above. Slides were mounted with buffered glycerin, covered with a cover slip and read under an Olympus BX-FLA fluorescent microscope equipped with a 100 W mercury lamp with 400× magnifying power. The samples were defined as positive at titres >1:40.
2.3. Statistical analysis Data were processed with SPSS for Windows version 10.0.5 (SPSS Inc., Chicago, IL, USA). All variables were individually assessed with the W-test of normality. For comparisons of
Not infected (n = 20) Schistosomiasis (n = 15) Syphilis (n = 20) Malaria (n = 20) Chagas disease (n = 30) Total group (n = 105)
DAT-fd 85 (96.6) (89.7—99.1) 103 (98.1) (92.6—99.7) 28 (93.3) (76.5—98.8) 20 (100) (80—100) 20 (100) (80—100) 15 (100) (74.7—100) 20 (100) (80—100) 188 (97.4) (93.7—99.0) DAT-LPC 84 (95.5) (88.1—98.5) 93 (88.6) (80.5—93.7) 26 (86.7) (68.4—95.6) 18 (90.0) (66.9—98.3) 17 (85.0) (61.1—96.0) 12 (80.0) (51.4—94.7) 20 (100) (80—100) 177 (91.7) (86.7—95.0) ELISA-rK39 78 (88.6) (79.7—94.1) 97 (92.4) (85.1—96.4) 25 (83.3) (64.5—93.7) 17 (85.0) (61.1—96.0) 20 (100) (80—100) 15 (100) (74.7—100) 20 (100) (80—100) 175 (90.7) (85.4—94.2) ELISA-L. 79 (89.8) (83.8—96.5) 85 (81.0) (71.9—87.7) 16 (53.3) (34.6—71.2) 15 (75.0) (50.6—90.4) 19 (95.0) (73.1—99.7) 15 (100) (74.7—100) 20 (100) (80—100) 164 (85.0) (79.0—89.5) chagasi IFAT 81 (92.0) (83.8—96.5) 88 (83.8) (75.1—90.0) 20 (66.7) (47.1—82.1) 14 (70.0) (45.7—87.2) 20 (100) (80—100) 14 (93.3) (66.0—99.7) 20 (100) (80—100) 169 (87.6) (81.9—91.7) All other differences were non-significant (P ≥ 0.05). a Difference between specificity rates: Total group: DAT-fd × DAT-LPC, P < 0.001; DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P < 0.001. Chagas disease group: DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P = 0.01; DAT-LPC × ELISA-L. chagasi, P < 0.001; ELISA-rK39 × ELISA-L. chagasi, P = 0.012. Malaria group: DAT-fd × IFAT, P = 0.026. b Difference between efficiencies: DAT-fd × DAT-LPC, P = 0.01; DAT-fd × ELISA-rK39, P = 0.005; DAT-fd × ELISA-L. chagasi, P < 0.001; DAT-fd × IFAT, P < 0.001.
Diagnostic efficiency (n = 193), n (%) (95% CI)b Specificity in control groups, n (%) (95% CI)a
Table 1 shows the sensitivity and specificity rates obtained with the five serological tests. The sensitivity rates varied from 88.6% to 96.6% (non-significant difference among all five tests). The two DAT preparations presented similar values, with sensitivities of 95.5% (95% CI 88.1—98.5%) for DAT-LPC and 96.6% (95% CI 89.7—99.1%) for DAT-fd. Four serum samples presented false-negative reactions with DATLPC and three with DAT-fd. The sensitivity rates obtained with anti-rK39 and anti-L. chagasi ELISA were also very similar: 88.6% (95% CI 79.7—94.1%) and 89.8% (95% CI 83.8—96.5%), respectively. Using IFAT, seven sera presented false-negative results, conferring a sensitivity of 92.0% (95% CI 83.8—96.5%). Specificity rates were determined for the total group of non-leishmaniasis patients and for each group separately (Table 1). The specificity values observed for the total control group were 88.6% (95% CI 80.5—93.7%) for DATLPC, 98.1% (95% CI 92.6—99.7%) for DAT-fd, 92.4% (95% CI 85.1—96.4%) for ELISA-rK39, 81.0% (95% CI 71.9—87.7) for ELISA-L. chagasi and 83.8% (95% CI 75.1—90.0) for IFAT. However, the specificity observed for each group of related diseases varied widely: 53.3—93.3% for Chagas disease; 70—100% for malaria; 85—100% for syphilis; and 80—100% for schistosomiasis mansoni. The higher frequency of crossreactivity that reduced the specificity of the tests occurred with IFAT and ELISA-L. chagasi mainly with sera from patients with Chagas disease, followed by sera from patients with malaria. With DAT-fd, only 2 of the 30 serum samples from the Chagas disease group had any cross-reactivity. In the group of non-infected individuals, all serological tests had 100% specificity (Table 1). The serological tests that achieved the highest diagnostic efficiencies were DAT-fd (97.4%, 95% CI 93.7—99.0%), followed by DAT-LPC (91.7%, 95% CI 86.7—95.0%), ELISA-rK39 (90.7%, 95% CI 85.4—94.2%), ELISA-L. chagasi (85.0%, 95% CI 79.0—89.5%) and IFAT (87.6%, 95% CI 81.9—91.7%). Statistical differences were observed between the results presented by DAT-fd and all the other serological tests (P < 0.05) (Table 1). A good agreement beyond chance ( index) ranging from 0.60 to 0.80 was obtained when all the results from all five methods were cross-tabulated, including 193 serum samples from VL patients and the control groups (Table 2).
Sensitivity in VL group (n = 88), n (%) (95% CI)
3. Results
Serological test
quantitative results among the different groups, the titres and absorbance readings were transformed to log10 [result+1] and evaluated by the non-parametric Spearman’s correlation, considering significance at the 0.01 level (two-tailed). The equation TP+TN/TP+TN+FP+FN (where TP = true positive, TN = true negative and FP = false positive) was used to determine diagnostic efficiency (Galen and Gambino, 1975). The 2 test was employed for comparison of sensitivities, specificities and diagnostic efficiency rates. Agreement beyond chance was assessed using the Cohen coefficient (Cohen, 1968) and interpreted according to the scale of Landis and Koch (1977): 1.00—0.81, excellent; 0.80—0.61, good; 0.60—0.41, moderate; 0.40—0.21, weak; and 0.20—0.0 negligible. The significance level was set at <5% probability of ␣ error (Bhattacharyya and Johnson, 1977).
175 Table 1 Comparison of sensitivity in patients with visceral leishmaniasis (VL), specificity rates in control groups and diagnostic efficiency in VL patients plus control groups of the five serological tests
Serodiagnosis of visceral leishmaniasis
M.J. Pedras et al.
—– —– —– —–
—– —– —– —–
96 97 193
P N 82 5 14 92 96 97 0.80 (0.66—0.94) 0.79 (< 0.001) —– —– —– —–
—–
—– —– —–
98 95 193
99 94 193
87 106 193
96 97 193
index (95% CI) Spearman’s coefficient (P-value) DAT-LPC P N T index (95% CI) Spearman’s coefficient (P-value) IFAT P N T index (95% CI) Spearman’s coefficient (P-value) ELISA-L. chagasi P N T index Spearman’s coefficient (P-value)
P N T DAT-fd
P: positive; N: negative; T: total.
96 97 193
P N 81 6 17 89 98 95 0.76 (0.67—0.85) 0.81 (< 0.001) 79 17 19 78 98 95 0.63 (0.49—0.77) 0.75 (<0.001) —– —– —– —–
ELISA-L. chagasi
P N 78 9 21 85 99 94 0.69 (0.56—0.83) 0.65 (< 0.001) 78 18 21 76 99 94 0.60 (0.46—0.74) 0.66 (<0.001) 78 20 8 87 86 107 0.71 (0.57—0.90) 0.69 (<0.001) —– —– —– —– T 87 106 193
P N 76 11 10 96 86 107 0.78 (0.64—0.92) 0.79 (< 0.001) 79 17 7 90 86 107 0.75 (0.61—0.89) 0.75 (<0.001) 81 6 17 89 98 95 0.76 (0.67—0.85) 0.80 (<0.001) 73 26 13 81 86 107 0.60 (0.56—0.74) 0.70 (<0.001)
T 87 106 193
IFAT
T 87 106 193
DAT-LPC
T 87 106 193
The Spearman’s correlation coefficients between the quantitative values of each test are shown in Table 2. The log-transformed titres of both DAT tests, the IFAT and the absorbance readings from both ELISA tests obtained with the 193 serum samples from the VL patients and controls were analysed. All the paired tests present positive and significant correlations. Interestingly, the highest correlation coefficients were observed between IFAT and DAT-fd (0.81) and IFAT and ELISA-rK39 (0.80). Figure 1 shows the individual agglutination results obtained with the two DAT preparations. DAT-LPC agglutination was observed at the lowest serum dilution of the control samples. None the less, positive and significant correlation (0.79) was observed between the titres obtained with the two preparations (Table 2).
4. Discussion
ELISA-rK39
Serological test Serological test
Table 2 Agreement, index values and Spearman’s correlation coefficient between paired quantitative results of the five serological tests for the diagnosis of visceral leishmaniasis (n = 193)
176
In this study, we compared five different techniques for serological diagnosis of VL, three of which are locally prepared (DAT-LPC, IFAT and ELISA-L. chagasi) and two are dependent on imported antigen (ELISA-rK39 and DAT-fd). DAT is a well validated test for serodiagnosis of VL and has been used in Sudan for the last two decades (El Harith et al., 1986, 1987; Ritmeijer et al., 2006), combining high levels of intrinsic validity and ease of use (Chappuis et al., 2006). The antigen supplied by KIT Biomedical Research is prepared from cultured promastigotes of L. donovani, whereas human VL in Brazil is caused by L. chagasi. The heterologous antigen did not impair its high performance, according to Andrade et al. (1989). The freeze-dried DAT antigen is currently available from the Prince Leopold Institute (Antwerp, Belgium) and The Royal Tropical Institute (Amsterdam, The Netherlands). The cost of one test is approximately US$2.00 (H. Schallig, personal communication). Although similar sensitivities were obtained both for the locally produced and commercial DAT, a superior specificity was observed with the industrialised kit (DAT-fd). In the present study, a relatively high specificity (88.6%) for the DAT-LPC test was observed, which is comparable with previous studies where DAT-fd was also used (Meredith et al., 1995; Schoone et al., 2001). Other studies showed that the specificity of DAT depends on the chosen cut-off titre. A cut-off titre of higher serum dilutions generally yields higher specificities (Chappuis et al., 2003). The cut-off titre of the industrialised DAT with freeze-dried antigens was previously defined, whereas the definition of the best cut-off value for the laboratory-made DAT-LPC had to be determined using a ROC curve (data not shown). The cut-off titre for the DAT-LPC is low compared with those defined in other studies (Chappuis et al., 2003). It is possible that further standardisation steps would improve the DAT-LPC, which would result in better interpretation of positive/negative agglutination reactions. Nevertheless, the good performance of the locally prepared DAT, with a index of 0.80, and the high correlation between the agglutination titres of both DAT assays (0.79; P < 0.01), showed that national production is possible, as all the necessary biological and chemical reagents are nationally available. Although microplates and micropipettes may be an obstacle for widespread field use of the DAT in some countries, this should not be a major
Serodiagnosis of visceral leishmaniasis
177
Figure 1 Agglutination titres of VL patients and healthy and diseased controls tested with (A) a direct agglutination test (DAT) based on freeze-dried antigen (DAT-fd) and (B) a locally produced DAT using freshly prepared aqueous antigen (DAT-LPC). Results were considered positive with titres ≥1:100 and >1:1600 for DAT-fd and DAT-LPC, respectively. M: malaria; S: syphilis; SM: Schistosoma mansoni; NI: non-infected; CD: Chagas diseases; VL: visceral leishmaniasis.
concern in Brazil. Nevertheless, the laboratory infrastructure needed to carry out DAT is much simpler than either ELISA or IFAT, as no sophisticated equipment is required. The estimates of sensitivity and specificity should be carefully interpreted as the selection of samples may misrepresent the population exposed to the parasite in an endemic area. The higher sensitivity rate presented by ELISA-rK39 (88.6%) was not statistically different (P > 0.577) from the value obtained with the ELISA-L. chagasi with crude soluble antigens (89.8%). This result corroborated the findings of Braz et al. (2002), who evaluated ELISA-rK39 and ELISA-L chagasi in 120 serum samples from Brazilian patients with VL. However, the higher specificity of ELISA-rK39 was significantly different when testing serum from patients with Chagas disease (83.3% vs. 53.3%) and malaria patients (85.0% vs. 75.0%). The dipstick format to detect anti-rK39 antibodies has been validated in different regions and presents heterogeneous sensitivity and specificity and needs to be largely evaluated in Brazil (Chappuis et al., 2006). In conclusion, the overall performance of DAT-fd, DAT-LPC and ELISA-rK39 scored higher than IFAT and ELISA-L. chagasi. The results point to the possible role of the evaluated methods in the replacement of IFAT as the routine serological method for diagnosis of VL in Brazil. A large-scale prospective study to validate the tests in the target population needs to be carried out. This assessment and an analysis to estimate costs and sustainability of the different methods are needed before definite recommendations can be given to the Leishmaniasis Control National Programme. Authors’ contributions: MJP and LGV carried out the immunoassays; EJO and AR carried out analysis and interpretation of the data and drafted the manuscript. All authors read and approved the final manuscript. AR is guarantor of the paper. Acknowledgements: We thank Dr Henk Schallig from the Laboratory for Biomedical Research, The Royal Tropical Institute, Amsterdam, The Netherlands, for providing the Leish-KIT, DAT kits and for critical review of the manuscript.
We also thank Dr Steven Reed from the Infectious Disease Research Institute, Seattle, WA, USA, for providing the rK39 antigen. The Health Care Centre of Belo Horizonte is thanked ´rika Braga from the for providing the syphilis sera, and Dr E Federal University of Minas Gerais state for providing the malaria samples. Funding: CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnol´ ogico), Bras´ılia, DF, Brazil (grant ˜o de Amparo a ` Pesquisa 350594/1995-3); FAPEMIG (Fundac ¸a de Minas Gerais), Belo Horizonte, MG, Brazil (CDS: 55013/ 02); and FIOCRUZ Rio de Janeiro, RJ, Brazil (PDTIS RID06). Conflicts of interest: None declared. Ethical approval: The Ethical Review Board of Centro de Pesquisas Ren´ e Rachou (CPqRR)—FIOCRUZ, Belo Horizonte, MG, Brazil, approved the use of stored serum samples (CEPSH/CPqRR no. 05/2007), in agreement with Resolution 357/05 of the National Health Council of the Ministry of Health, which regulates research involving human subjects in Brazil.
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