Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting

Abstract

BackgroundSchistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE.MethodsA cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum).ResultsWe obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement.ConclusionThe positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0558-4) contains supplementary material, which is available to authorized users.

Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in Stool? A Comparison with Microscopy in Senegal and Kenya

“…PCRs based on the 121-bp tandem-repeat sequence showed more promising results with 7 to 28% higher percentages of S . mansoni infections detected than standard microscopy [ 10 , 13 , 17 , 33 – 37 ]. In contrast to the ITS2-based real-time PCR used in the present study however [ 29 ], this PCR cannot quantify DNA loads.…”

Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay

“…Some immunological tests developed in China have acceptable performance characteristics and some serological methods are used largely as a screening tool in China, such as indirect hemagglutination assay (IHA) [ 27 , 28 ]. Molecular methods, mainly DNA amplification techniques by PCR, based on the genomic DNA of schistosomes are able to detect DNA from all phases of the life-cycle of this parasite [ 29 , 30 ]. Although the adequacy and sensitivity of the PCR methods have been proven in schistosome infections in water buffaloes [ 31 ] and in other helminth and protozoan infections in humans [ 32 , 33 ], the sensitivity of molecular methods can be affected by the degree of infection and methods have poor agreement [ 34 ].…”

Comparative Study of the Accuracy of Different Techniques for the Laboratory Diagnosis of Schistosomiasis Mansoni in Areas of Low Endemicity in Barra Mansa City, Rio de Janeiro State, Brazil

“…This mixture was homogenized for 5 min using a vortex mixer, followed by centrifugation for 8 min at 16.863 ×g at 4°C. An aliquot of 400 μ L of the supernatant was mixed with 100 µ L of Rapid One-Step Extraction (ROSE) solution: 10 mM Tris-hydroxymethyl amino methane HCl, pH 8; 300 mM EDTA, pH 8.0; 1% sodium lauroyl sarcosinate (Sarkosyl); and 1% polyvinylpolypyrrolidone (PVPP) [ 38 ]. Then, 30 μ L of Proteinase K (Life Technologies, Carlsbad, CA, USA) was added and homogenized using a vortex mixer.…”

“…The undetermined qPCR and qPCR IPC samples were tested in triplicate. Duplicate amplifications of the samples obtained by qPCR were included in the positive results [ 38 ].…”