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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80% of the global cases are concentrated, S. mansoni is the major species that is transmitted.2 Despite efforts to control this disease, which are based on treatment of infected people and elimination of snails, schistosomiasis remains a major health problem that results in significant financial burdens.2 The Kato-Katz parasitologic technique3 is currently the recommended method for diagnosing this disease1 because it is quantitative, relatively inexpensive, and simple. However, the sensitivity of this technique decreases when the prevalence and intensity of infection are low.4–6 Thus, this method is less efficient in areas of low endemicity, in post-treatment situations, and in the control of transmission.
Other diagnostic alternatives include immunologic methods, such as detection of parasite-specific antibodies and circulating antigens. Antibody detection assays have been shown to be more sensitive than the parasitologic examination,7 but generally lack specificity.8 The detection of circulating antigens is a highly specific assay,7,9 but has not been shown to be more sensitive than the detection of eggs in areas of low endemicity.7,10,11
Despite remarkable advances achieved over the last decade in this field, including the development of a polymerase chain reaction (PCR),12 molecular techniques have not been extensively used in the diagnosis of human infections with Schistosoma. We have recently published preliminary results demonstrating that the PCR is a useful technique for the detection of S. mansoni in human feces and serum.13 The amplification reaction is capable of detecting as little as 1 fg of a highly repetitive Schistosoma mansoni DNA sequence, showed no cross-reactivity with other related helminths, and was 10 times more sensitive than the Kato-Katz technique.
This report presents the results of a more extensive comparison between the PCR assay and the parasitologic Kato-Katz technique, in which fecal samples of individuals from a area of Brazilian endemic for S. mansoni infection were analyzed by both procedures.
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MATERIALS AND METHODS |
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Fecal samples. Samples were collected and stored at -70°C until use, except during transportation to the research laboratory, through which they were kept on ice. The collections were done on three consecutive days for all patients. Fecal samples were analyzed by both a PCR (one sample) and Kato-Katz stool examination (three samples). The collection of new fecal samples, specifically conducted for the investigation of discordant results between the two methods, was performed on at least four different days and the samples were reanalyzed by both procedures.
Forty-one patients with a positive stool examination result by the Kato-Katz diagnostic assay were treated with a single oral dose of praziquantel (50 or 60 mg/kg for adults and children, respectively). New fecal collections from these patients were done on three consecutive days two months after treatment. These samples were then analyzed for cure evaluation by the PCR technique (one sample) and the Kato-Katz stool examination method (three samples).
Twenty fecal samples of non-exposed persons from Belo Horizonte, Minas Gerais, Brazil with negative Kato-Katz stool examination results were used to evaluate the specificity of the PCR assay.
Stool examination by the Kato-Katz method. Fecal samples were evaluated for the presence of S. mansoni eggs by the quantitative Kato-Katz parasitologic method.3 Two glass slides (43 mg of feces per slide) were prepared for analyzing each sample, and the arithmetical mean number of eggs per gram of feces was the final result.
Stool examination by the PCR method. Sample preparation. Prior to extraction, approximately 0.5 g of feces were mixed with 1.0 mL of distilled water in a 1.5-mL microcen-trifuge tube, vigorously agitated, and centrifuged at 500 x g for two minutes. The supernatant was discarded and the pellet was washed by the same procedure, except that the sample was gently agitated for 10 minutes to favor the rupture of S. mansoni eggs.14,15 The final pellet was used for isolation of parasite DNA.
Isolation of DNA. The DNA from fecal samples was extracted following a modification of the rapid one-step extraction (ROSE) method.16 Briefly, 0.7 mL of ROSE buffer (10 mM Tris, pH 8.0, 300 mM EDTA, pH 8.0, 1% sodium lauryl sulfate, 1% polyvinylpolypyrrolidone) were mixed with the washed fecal pellet and heated to 95°C for 20 minutes, with the sample being agitated vigorously briefly after the first 10 minutes of heating. The sample was then centrifuged at 14,000 x g for 10 minutes at room temperature to precipitate proteins and fecal debris. Five hundred microliters of the supernatant was then collected in a separate tube, mixed with 50 µL of cold (4°C) 3 M sodium acetate, pH 5.3, and 1 mL of cold (-20°C) ethanol, and immediately centrifuged at 14,000 x g for 15 minutes. The supernatant was then discarded, and the pellet was washed once with 500 µL of 70% ethanol (-20°C) and centrifuged at 14,000 x g for 10 minutes. The supernatant was discarded, and the pellet was dried at 37°C for 15 minutes and resuspended in 100 µL of cold (4°C) buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0).
Polymerase chain reaction assay. The PCR primers were designed to amplify a highly repeated 121-basepair sequence of S. mansoni reported by Hamburger and others17 that comprises about 10% of the parasite genome (600,000 copies per cell). For the amplification of DNA extracted from stool specimens, samples were diluted 1:100 and 1 µL was used as template in a 10-µL final volume mixture containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl; 1.5 mM MgCl2; 0.5 µM of each primer; 200 µM of the four dNTPs, and 0.75 units of Taq DNA polymerase. The amplification reaction was carried out for 35 cycles, with each cycle consisting of a denaturation step at 95°C for 40 seconds and an annealing step at 65°C for 30 seconds. The first cycle had an extended denaturation step for five minutes and the reaction was ended with an extension step at 72°C for five minutes.13
To prevent carryover contamination, extraction and amplification of DNA and the preparation of the PCR mixture were conducted in separate facilities with total separation of materials. Aerosol barrier tips were used in all steps, including sample collection. Contamination was monitored by the inclusion of four negative controls containing water instead of extracted DNA in the amplification reaction, two of which came from the DNA extraction step. The PCR products were subjected to electrophoresis on 6% polyacrylamide gels and analyzed after silver staining.18
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RESULTS |
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. The prevalence observed using the PCR technique was approximately 13% higher (
2 = 26, P < 0.0001) than that determined with the Kato-Katz stool examination.
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. Statistical analysis showed a kappa index of 0.8, indicating good agreement between the two methods. Analysis of discordant results showed that 16 samples were positive only by the PCR and two positive samples were positive only by the Kato-Katz method. These two patients had very low egg outputs, with 12 eggs per gram of feces in the first sample and no eggs in the other two samples.
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Diagnostic parameters were calculated by two different approaches: 1) taking the Kato-Katz results alone as the reference for comparison or 2) considering the specificity of both the Kato-Katz and PCR methods as being 100%, and assuming as infected person anyone with positive result by either method. The values estimated by each approach are shown in Table 2.
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Parasitive-positive patients were treated and new fecal samples were collected two months after treatment and analyzed by both methods for cure evaluation. Table 3 shows that the PCR technique detected more positive cases than the Kato-Katz method. The parasitologic results showed that two months after treatment, 87.8% of the infected individuals had been cured, a value that decreased to 75.6% (2 = 17.6, P < 0.0001) when the PCR results were used.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Table 1, a single PCR survey detected more cases of infection with S. mansoni than three Kato-Katz stool examinations. There are two possible explanations for this result: 1) the PCR could be more sensitive than the Kato-Katz technique, in which case those results would actually correspond to S. mansoni-infected samples, or 2) those results could, in reality, be false-positive results. To better discern between these two possibilities, we conducted a re-investigation among 12 of the 16 patients with positive PCR and negative stool examinations results by performing at least four additional parasitologic examinations on each fecal sample. The other four discordant results could not be further scrutinized since their donors could not be located. The re-investigation of these discordant results showed that five (41.6%) of the 12 patients were infected with S. mansoni, demonstrating the higher sensitivity of the PCR. Similar results were obtained in a preliminary experimental comparison between the two methods.13 Seven samples remained negative even after more than four additional Kato-Katz examinations. It is possible that having passed through such extensive survey these samples represent truly negative samples, a fact that would reduce the overall efficiency of the PCR technique. However, it is also possible that those individuals had egg outputs less than the 20 eggs/gram detection limit of the Kato-Katz examination,3 in which case the infection would had been detected only by the PCR survey. This possibility is supported by the higher sensitivity of the PCR, and is consistent with a previously reported underestimation of the prevalence by the Kato-Katz method, even after four additional investigations.19 False-positive results could have been due to either a lack of specificity by the PCR assay or carry-over contamination. Cross-reactivity with other helminth infections can be ruled out since only two of these patients were infected with other helminths and the DNA from these worms is not amplified by the S. mansoni-specific primers.13 Carryover contamination is often difficult to exclude as a cause of false-positive results in PCR-based diagnostic assays due to the exquisite sensitivity of the technique. However, strict measures to avoid it were taken during the entire handling and processing steps of the assay (see Materials and methods).
The PCR failed to detect two samples positive by the Kato-Katz technique. There is no reason to believe that these cases correspond to false-positive parasitologic results since this method has a specificity of 100%. They were certainly misdiagnosed by the DNA amplification assay, a fact that could be due to many factors, such as inhibition of the amplification reaction by feces compounds and/or DNA degradation during transportation from the field. The fecal DNA dilution was shown to be sufficient in earlier tests to overcome inhibition of the PCR inhibition. However, such inhibition cannot be ruled out for each individual sample unless internal controls are used. In this case, controls were not used to decrease costs. Another possibility that cannot be excluded is the effect that a variation in egg output and uneven distribution in feces20,21 could have on PCR detection of S. mansoni DNA, especially for these two PCR-negative, Kato-Katz positive samples, which contained only 24 eggs per gram of feces.
The diagnostic value of the PCR technique was estimated by two different approaches. In a traditional approach, the Kato-Katz assay was taken as the gold standard and all parameters were calculated using it as the reference. In another approach, both methods were considered to have specificities of 100%; thus, a true positive result was considered as one detected by either method. The validity of this approach would depend solely on the true efficiency of the PCR since the quality of the Kato-Katz technique has been established. Several facts support the assumption of this efficiency. With respect to specificity, no cross-reactivity has been observed when DNA from related helminths was used as template,13 and the PCR showed a specificity of 100% when used with 20 fecal samples of non-infected persons. With regard to sensitivity, retesting with the parasitologic method showed that 41% of the discordant results analyzed had been correctly diagnosed by the DNA amplification technique. In addition, a previous report has shown greater sensitivity for the PCR assay.13 With respect to the overall efficiency of the PCR technique, the statistical agreement found between the two methods was high (kappa value = 0.8).
Sensitivity values of the PCR technique were high by both approaches (>96%). A specificity value calculated by the first approach was lower (88% versus 100%), but still satisfactory if one considers that it was based on results obtained from an endemic area, and that the high sensitivity of the technique would lead to higher detection rates. The same reasoning is valid for positive predictive values obtained with the PCR technique (78.4% versus 100%). Negative predictive values were high and identical (98.3%) irrespective of the adopted gold-standard. Since all of these parameters are highly dependent on the infection prevalence among the population, test performance under other conditions could be different.
Cure evaluation by both methods also showed a higher detection capacity of the DNA amplification reaction. Among the 60 patients positive by the stool examination, 41 were treated and provided three fecal samples two months after treatment. The other 19 patients could not be treated, either because they could no longer be located or they had a restrictive medical condition. Since it detected more positive cases, the PCR provides a lower estimation of the cure rate than the parasitologic technique (75.6% versus 87.8%). The cure rate for S. mansoni infection in Brazil, as estimated by the Kato-Katz method, ranges from 80% to 90% after two months of treatment.22,23 If one considers the high negative predictive value found for the PCR technique (98%), the real cure rate is unlikely to be very different from what was calculated from the data obtained with both methods.
The data presented in this study show the results of a stringent evaluation of the PCR technique as a diagnostic tool for schistosomiasis in patients in an area of medium intensity of infection. In the analysis of this specific population, a single PCR survey has shown a high statistical correlation with three parasitologic examinations (kappa value = 0.8). However, it also had a higher sensitivity (97% versus 79%). Thus, this technique might prove to be especially useful in circumstances of lower intensity or prevalence of infection, a condition for which the parasitologic examination shows a well-documented limitation of its sensitivity.4–6
Received October 26, 2002. Accepted for publication February 3, 2003.
Acknowledgment: We thank Aureo Almeida for excellent technical assistance.
Financial support: This work was partially supported by the Conselho Nacional de Pesquisa (CNPq-Brazil) and the PAPES/Fundação Oswaldo Cruz. Emmanuel Dias-Neto thanks the Associação Beneficente Alzira Denise Hertzog Silva for their support.
Authors’ addresses: Luis A. Pontes, Maria C. Oliveira, and Ana Rabello, Laboratory of Clinical Research, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz-Fiocruz, Av. Augusto de Lima, 1715, 30190-002, Barro Preto, Belo Horizonte, Minas Gerais, Brazil, Telephone: 55-31-3295-3566, E-mail: luis{at}cpqrr.fiocruz.br. Naftale Katz, Laboratory of Schistosomiasis, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz-Fiocruz, Av. Augusto de Lima, 1715, 30190-002, Barro Preto, Belo Horizonte, Minas Gerais, Brazil. Emmanuel Dias-Neto, Laboratory of Neurosciences, Instituto de Psiquiatria, Faculdade de Medicina, Universidade de Sao Paulo. R. Dr. Ovidio de Campos, s/n Consolacao, 05403-010, São Paulo, SP, Brazil.
Reprints requests: Luís A. Pontes, Laboratory of Clinical Research, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz-Fiocruz, Av. Augusto de Lima, 1715, 30190-002, Barro Preto, Belo Horizonte, Minas Gerais, Brazil.
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  Y.-B. ZHOU, M.-X. YANG, Q.-Z. WANG, G.-M. ZHAO, J.-G. WEI, W.-X. PENG, and Q.-W. JIANG FIELD COMPARISON OF IMMUNODIAGNOSTIC AND PARASITOLOGICAL TECHNIQUES FOR THE DETECTION OF SCHISTOSOMIASIS JAPONICA IN THE PEOPLE'S REPUBLIC OF CHINA Am J Trop Med Hyg, June 1, 2007; 76(6): 1138 - 1143. [Abstract] [Full Text] [PDF]
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