Real-time PCR for detection of low intensity Schistosoma japonicum infections in a pig model

Lier, Tore; Johansen, Maria Vang; Hjelmevoll, Stig Ove; Vennervald, Birgitte J.; Simonsen, Gunnar Skov

doi:10.1016/j.actatropica.2007.10.004

Cited by 73 publications

(97 citation statements)

References 29 publications

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“…Chemotherapy has been used on a large scale in countries where Schistosoma is endemic. This has led to a lower intensity of infections and consequently lower diagnostic values of commonly used diagnostic tests like serology and Kato-Katz stool smear (Lier et al 2006). The cell-free circulating schistosome DNA consists of the fragments of parasite-derived DNA that exist in the host's bodily fluids (Wichman et al 2009(Wichman et al , 2013.…”

Section: Resultsmentioning

confidence: 99%

Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study

Eraky

Aly

2014

J Parasit Dis

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Searching for a more sensitive and accurate marker for schistosomiasis diagnosis and treatment follow up is a potential necessity. Hereby, we evaluated usefulness of circulating free DNA as a marker for schistosomiasis diagnosis, assessing drug efficacy and monitoring the control interventions impact using SYBR green real-time PCR. A batch of mice were infected by 90 ± 10 Schistosoma mansoni cercariae. Starting from the 2nd day post infection (p.i.), groups of 2 or 3 mice were sacrificed every 3 days until 30 days p.i. The remaining animals were treated by a single dose of 400 mg/kg mefloquine and sacrificed in group at 5, 10, 21 days post treatment (35, 40, 51 days p.i.). Using SYBR green real time qPCR, pooled sera DNA were extracted and amplified. The results showed that, circulating free S. mansoni DNA was detected from the 2nd day post infection (p.i.) onwards with gradual decrease in the cycle threshold value C t which indicates the gradual elevation of the DNA level (Log quantity was 2.6-3.1 IU/ml), As the infection progressed, DNA quantity was increased(Log quantity was 6.29 IU/ml). Initial increase of circulating free DNA was observed 10 days post treatment (40 days p.i.) (Log quantity was 7.38 IU/ ml). That was followed by a progressive decrease in DNA level by the end of 21st day, post treatment (51 p.i.) (Log quantity 4.35 IU/ml). In conclusion, circulating free S. mansoni DNA is a reliable marker in the diagnosis of schistosomiasis and for assessing drug efficacy and monitoring the impact of control interventions.

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Section: Resultsmentioning

confidence: 99%

Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study

Eraky

Aly

2014

J Parasit Dis

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“…The specific and conserved mitochondrial NADH dehydrogenase 1 (nad1) and retrotransposon SjR2 genes were selected as target sequences, as these sequences are highly abundant in the S. japonicum genome (Lier et al, 2006 The length of the amplicon, analysed by gel electrophoresis, was checked for the expected band size. The amplification product was sequenced, and the resulting sequence compared to sequences in Genbank with the Basic Local Alignment Search Tool (BLAST) considering the identity and Expect (E) value parameters.…”

Section: Selection Of Target Genes and Primer Designmentioning

confidence: 99%

“…The assay detected target sequences in different sources of Schistosoma DNA isolated from adult worms, schistosomules and eggs; it is highly sensitive and can detect very low amounts of parasite DNA (Lier et al, 2006;Xia et al, 2009;Xu et al, 2010). The specificity of the assay is high, with no cross reactivity found when tested using DNA from other helminth parasites as template, nor was there any crossreactivity with host DNA.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Weerakoon

Gordon

Gobert

et al. 2016

Journal of Microbiological Methods

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Schistosomiasis is a chronically debilitating helminth infection with a significant socioeconomic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostics procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

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“…Similar to the previous method, SYBER Green dye was used as an intercalating dye into double-strand DNA to measure the change in fluorescence after each PCR cycle. In artificially spiked stool samples, this method demonstrated high sensitivity, even in samples containing a single egg 90 . The same research group used this test in a comparative study in China among 1,727 participants from an endemic area.…”

mentioning

confidence: 99%

Diagnosing schistosomiasis: where are we?

Gomes

Enk

Rabello

2014

Rev. Soc. Bras. Med. Trop.

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In light of the World Health Organization's initiative to extend schistosomiasis morbidity and mortality control programs by including a disease elimination strategy in low endemic settings, this paper reviews diagnostic tools described during the last decades and provide an overview of ongoing efforts in making an efficient diagnostic tool available worldwide. A literature search on PubMed using the search criteria schistosomiasis and diagnosis within the period from 1978 to 2013 was carried out. Articles with abstract in English and that used laboratory techniques specifically developed for the detection of schistosomiasis in humans were included. Publications were categorized according to the methodology applied (parasitological, immunological, or molecular) and stage of development (in house development, limited field, or large scale field testing). The initial research generated 4,535 publications, of which only 643 met the inclusion criteria. The vast majority (537) of the publications focused on immunological techniques; 81 focused on parasitological diagnosis, and 25 focused on molecular diagnostic methods. Regarding the stage of development, 307 papers referred to in-house development, 202 referred to limited field tests, and 134 referred to large scale field testing. The data obtained show that promising new diagnostic tools, especially for Schistosoma antigen and deoxyribonucleic acid (DNA) detection, which are characterized by high sensitivity and specificity, are being developed. In combination with international funding initiatives these tools may result in a significant step forward in successful disease elimination and surveillance, which is to make efficient tests accessible and its large use self-sustainable for control programs in endemic countries.

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Real-time PCR for detection of low intensity Schistosoma japonicum infections in a pig model

Cited by 73 publications

References 29 publications

Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study

Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Diagnosing schistosomiasis: where are we?

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