Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Espírito-Santo, Maria Cristina Carvalho do; Alvarado-Mora, Mónica Viviana; Pintó, Pedro; Carrilho, Flair José; Pinho, João Renato Rebello; Gryschek, Ronaldo César Borges

doi:10.1590/s0036-46652012000500002

Cited by 9 publications

(7 citation statements)

References 14 publications

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“…Given prior research on the need to blend stool samples sufficiently [35], and the importance of STH egg disruption by utilizing a high-speed bead-based homogenizer [36][37][38] we acknowledged that any method developed to form pools would be critical, and the subsequent accurate detection of the evenly distributed targets upon dilution in the pool, would be challenging.…”

Section: Methods Developmentmentioning

confidence: 99%

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

et al. 2019

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Background: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low-and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a 'pooling approach' can yield a low frequency of 'missed' infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in 'pools-of-five'. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.

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Section: Methods Developmentmentioning

confidence: 99%

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

et al. 2019

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“…[ 19 ]. DNA extraction from serum, stool, and saline solutions with eggs and adult worms were performed at the Schistosomiasis Laboratory and at the Gastroenterology Laboratory, both at USP Medical School, in São Paulo, Brazil, as previously described [ 20 ],[ 21 ]. DNA from unrelated parasites ( Ascaris lumbricoides and Strongyloides stercoralis ) was used to evaluate the specificity of the assay.…”

Section: Methodsmentioning

confidence: 99%

“…Sensitivity was determined using serial dilutions of a DNA sample extracted from 200 eggs, including 10-fold dilutions ranging from 2 to 0.002 eggs (1:10 to 1:100,000). PCR amplification efficiency was calculated to be of 103.8%, with a slope of 3.44 [ 21 ]-[ 23 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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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.

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“…Para a extração do DNA das amostras de fezes ou soluções salinas de ovos e verme adulto, foi utilizada a metodologia do isotiocianato de guanidina (GT), associado às pérolas de vidro segundo Espírito-Santo et al (2012b).…”

Section: Extração Do Dna Das Amostrasunclassified

“…Uma vez realizada a extração, o DNA extraído foi estocado a -20 °C (Chomczynski, Sacchi, 1987;Espírito-Santo et al, 2012b).…”

Section: Extração Do Dna Das Amostras De Solução Salina De Ovos E Verunclassified

Estudo comparativo da acurácia de diferentes técnicas para o diagnóstico laboratorial da esquistossomose mansoni em áreas de baixa endemicidade

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Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Cited by 9 publications

References 14 publications

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

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

Estudo comparativo da acurácia de diferentes técnicas para o diagnóstico laboratorial da esquistossomose mansoni em áreas de baixa endemicidade

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