Real-time PCR for sexing Schistosoma mansoni cercariae.
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ABSTRACT:
SUBMITTER: Chevalier FD
PROVIDER: S-EPMC4841722 | biostudies-literature | 2016 Jan-Feb
REPOSITORIES: biostudies-literature
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Real-time PCR for sexing Schistosoma mansoni cercariae.
Chevalier Frédéric D FD Le Clec'h Winka W Alves de Mattos Ana Carolina AC LoVerde Philip T PT Anderson Timothy J C TJ
Molecular and biochemical parasitology 20160101 1-2
The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker un ...[more]
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Project description:As a unique feature among otherwise hermaphroditic trematodes, Schistosoma species are gonochoric parasites whose sex is genetically determined (ZZ for males and ZW for females). However, schistosome larvae are morphologically identical, and sex can only be discriminated by molecular methods. Here, we integrated published Schistosoma. japonicum transcriptome and genome data to identify W chromosome-specific genes as sex biomarkers. Three W chromosome-specific genes of S. japonicum were identified as sex biomarkers from a panel of 12 genes expressed only in females. An efficient duplex real-time PCR (qPCR) method for sexing cercariae was developed which could identify the sex of cercariae within 2 h without DNA extraction. Moreover, this method can be used to identify not only single-sex but also mixed-sex schistosome-infected snails. We observed a nearly equal proportion of single-male, single-female, and mixed-sex schistosome infections in artificially infected snails. Sex-known schistosome-infected snail models can be efficiently constructed with the aid of duplex qPCR. A field study revealed that single-sex schistosome infections were predominant among naturally infected snails. Finally, a schistosomiasis mouse model based on sex-known cercariae infection was shown to be more reliable than a model based on sex-unknown cercariae infection. The developed duplex qPCR method for sexing S. japonicum cercariae can be widely used for schistosomiasis modeling, genetic experiments, and field-based molecular epidemiological studies.
| S-EPMC7467314 | biostudies-literature
Project description:In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R(2) = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas.
| S-EPMC2580822 | biostudies-literature
Project description:BackgroundSchistosomiasis is a water-based disease acquired through contact with cercaria-infested water. Communities living in endemic regions often rely on parasite-contaminated freshwater bodies for their daily water contact activities, resulting in recurring schistosomiasis infection. In such instances, water treatment can provide safe water on a household or community scale. However, to-date there are no water treatment guidelines that provide information on how to treat water containing schistosome cercariae. Here, we rigorously test the effectiveness of chlorine against Schistosoma mansoni cercariae.MethodS. mansoni cercariae were chlorinated using sodium hypochlorite under lab and field condition. The water pH was controlled at 6.5, 7.0 or 7.5, the water temperature at 20°C or 27°C, and the chlorine dose at 1, 2 or 3 mg/l. Experiments were conducted up to contact times of 45 minutes. 100 cercariae were used per experiment, thereby achieving up to 2-log10 inactivations of cercariae. Experiments were replicated under field conditions at Lake Victoria, Tanzania.ConclusionA CT (residual chlorine concentration x chlorine contact time) value of 26±4 mg·min/l is required to achieve a 2-log10 inactivation of S. mansoni cercariae under the most conservative condition tested (pH 7.5, 20°C). Field and lab-cultivated cercariae show similar chlorine sensitivities. A CT value of 30 mg·min/l is therefore recommended to disinfect cercaria-infested water, though safety factors may be required, depending on water quality and operating conditions. This CT value can be achieved with a chlorine residual of 1 mg/l after a contact time of 30 minutes, for example. This recommendation can be used to provide safe water for household and recreational water activities in communities that lack safe alternative water sources.
| S-EPMC7467251 | biostudies-literature
Project description:The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.
| S-EPMC4519940 | biostudies-literature
Project description:BackgroundSchistosomiasis is a parasitic disease that is transmitted by skin contact with waterborne schistosome cercariae. Mass drug administration with praziquantel is an effective control method, but it cannot prevent reinfection if contact with cercariae infested water continues. Providing safe water for contact activities such as laundry and bathing can help to reduce transmission. In this study we examine the direct effect of UV light on Schistosoma mansoni cercariae using ultraviolet light-emitting diodes (UV LEDs) and a low-pressure (LP) mercury arc discharge lamp.MethodologyS. mansoni cercariae were exposed to UV light at four peak wavelengths: 255 nm, 265 nm, 285 nm (UV LEDs), and 253.7 nm (LP lamp) using bench scale collimated beam apparatus. The UV fluence ranged from 0-300 mJ/cm2 at each wavelength. Cercariae were studied under a stereo-microscope at 0, 60, and 180 minutes post-exposure and the viability of cercariae was determined by assessing their motility and morphology.ConclusionVery high UV fluences were required to kill S. mansoni cercariae, when compared to most other waterborne pathogens. At 265 nm a fluence of 247 mJ/cm2 (95% confidence interval (CI): 234-261 mJ/cm2) was required to achieve a 1-log10 reduction at 0 minutes post-exposure. Cercariae were visibly damaged at lower fluences, and the log reduction increased with time post-exposure at all wavelengths. Fluences of 127 mJ/cm2 (95% CI: 111-146 mJ/cm2) and 99 mJ/cm2 (95% CI: 85-113 mJ/cm2) were required to achieve a 1-log10 reduction at 60 and 180 minutes post-exposure at 265 nm. At 0 minutes post-exposure 285 nm was slightly less effective, but there was no statistical difference between 265 nm and 285 nm after 60 minutes. The least effective wavelengths were 255 nm and 253.7 nm. Due to the high fluences required, UV disinfection is unlikely to be an energy- or cost-efficient water treatment method against schistosome cercariae when compared to other methods such as chlorination, unless it can be demonstrated that UV-damaged cercariae are non-infective using alternative assay methods or there are improvements in UV LED technology.
| S-EPMC8284627 | biostudies-literature
Project description:BACKGROUND:To detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment. METHODS:The study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70?years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (?) was used to estimate the agreement between these diagnostic tests compared to a combined "gold standard" of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall's Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points. RESULTS:By using the combined "gold standard" of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time (which were determined by the serum-based real-time PCR) showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later. CONCLUSIONS:The data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.
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Project description:BACKGROUND: Schistosomiasis 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. METHODS: A 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). RESULTS: We 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. CONCLUSION: The 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.
| S-EPMC4210485 | biostudies-literature
Project description:BackgroundIn Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study.MethodologyStool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months.Principal findingsOut of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100).ConclusionsThe use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.
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Project description:Singular use of activity assays or staining dyes to assess pathogen agrochemical tolerance can underestimate tolerance if pesticides cause sublethal effects. We exposed Schistosoma mansoni cercariae, the aquatic life stage of this trematode that infects humans, to 4 insecticides at 5 concentrations using a 24-h time-to-death assay. We used Trypan blue dye, which stains dead tissue, and activity assays simultaneously to discriminate dead from live but paralyzed individuals. Whereas cypermethrin, deltamethrin, and dimethoate exposure did not affect cercariae at any ecologically relevant concentrations, methamidophos exposure increased survival of cercariae compared with those in the controls. This was because methamidophos-induced paralysis reduced cercarial activity and thus energy expenditures, extending the lifespan of this short-lived parasite that causes human schistosomiasis. These findings highlight that sublethal effects should be considered when pesticide effects on disease are under investigation. Environ Toxicol Chem 2020;39:1421-1428. © 2020 SETAC.
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