Quantitative PCR-Based Diagnosis of Soil-Transmitted Helminth Infections: Faecal or Fickle?

doi:10.1016/j.pt.2019.04.006

Trends in Parasitology

Volume 35, Issue 7, July 2019, Pages 491-500

https://doi.org/10.1016/j.pt.2019.04.006 Get rights and content

Highlights

Programmes for treating or controlling infections or diseases caused by soil-transmitted helminths (STHs) require the use of sensitive, specific, and practical diagnostic tools to monitor their success.
Conventional coprological methods are often used for the microscopic detection of worm eggs, but there is a need for improved sensitivity and measures of infection intensity as programmes approach STH elimination.
‘Transmission breakpoint’ has promoted the adoption of qPCR as a diagnostic tool.
There can be challenges in relying on qPCR as a 'gold-standard' diagnostic tool for STH infections; a point in question would be copy number variation of the molecular target in the parasites.
Studies are required to accurately relate qPCR data (in relation to target copy number) to infection intensity.

Treatment and control programmes tackling soil-transmitted helminth (STH) infections require sensitive, reliable, and accurate diagnostic tools. There is a growing need for measures of infection intensity as programmes approach STH control. Quantitative real-time PCR (qPCR) is well suited to the detection of DNA targets present in stool, even in low-prevalence settings. Detecting low levels of infection becomes increasingly important when the breakpoint of transmission is approached, and is vital when monitoring for recrudescence once control, or possibly 'elimination', is achieved. We address key challenges and questions that remain as barriers to incorporating qPCR as a cornerstone diagnostic tool for STH infections.

Section snippets

A Timely Diagnosis

The rapid, sensitive, and accurate diagnosis of human helminth infections underpins efforts to control or possibly eliminate disease-causing parasitic worms. For many decades, microscopy has been the most widely used and mainstay tool for detecting, identifying, and enumerating parasites. Advances in molecular biology and genomics provides opportunities for better tests. Of considerable interest is the application of qPCR to diagnose STH infections by testing stool samples; qPCR and its

Cost of qPCR

qPCR is a fairly complex tool and a relatively expensive procedure, requiring established facilities, an adequate supply of reagents, sufficient training for protocol reproducibility or troubleshooting, and local (company, contract, country) support. Overall, cost is decreasing due to increased reproducibility of qPCR results, obviating the need for multiple sampling [5], and reductions in the costs of some reagents [1]. Importantly, evaluating cost as ‘price per test alone’ is misguided, since

Concluding Remarks

It is dispiriting that some significant species of intestinal helminths, known to infect hundreds of millions of people and cause destructive diseases, have not been able to ‘make the cut’ to the list of parasites to be controlled. Helminths such as Strongyloides stercoralis are not included in current intervention agendas because diagnosis has not been standardized; with no egg output, and with highly varied levels of larval output, the intensity of infection cannot be reliably measured [45].

Outstanding Questions

What is needed for qPCR to be a robust, accurate, and reliable method for STH diagnosis and as a measure for STH infection intensity? If all that is measured by qPCR is copies of the target DNA present, how can outputs (C_q-value) be related to intensity of infection?
Is there a need to demonstrate the presence and utility of repetitive elements identified from limited genomic data across species ranges and in perpetuity?
How can identification and choice of molecular markers be improved as

Glossary

Breakpoint of transmission: defined by mathematical modelling, the point at which parasite reproduction becomes unsustainable and continuation of the parasite life cycle (transmission) falls to such levels that do not allow recrudescence.
Inhibition: any factor than can stall a PCR reaction; matrices like blood, stool, and urine carry a plethora of inhibitors (bacterial debris, bile salts, haeme, humic acids, polysaccharides) that modern nucleic acid extraction kits remove or wash away by

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Schistosomiasis is a life-threatening infectious disease categorized by the World Health Organization as a public health issue. New molecular diagnostic alternatives for intestinal schistosomiasis caused by Schistosoma mansoni, such as the loop-mediated isothermal amplification (LAMP), a fast and simple amplification technique, have been proposed for control of this NTD in low-endemicity locations. A LAMP assay was performed to detect the internal transcribed spacer 1 ribosomal gene of S. mansoni (SmITS1-LAMP) in 322 DNA extracted from stool samples from schistosomiasis endemic area in Brazil. Kato-Katz analysis of human stool samples was used as the gold standard test, detecting 144 positive samples. SmITS1-LAMP detection limit achieved a maximum analytical sensitivity of 10 fg/μL using S. mansoni genomic DNA, subsequently detecting 17/144 (11.8%) positive samples. SmITS1-LAMP sensitivity and specificity were 12% (95%CI: 7%–18%) and 93% (95%CI: 89%–96%), respectively. Positive predictive value (PPV) and negative predictive value (NPV) were 59% (95%CI: 39% - 76%); and 57% (95%CI: 51% - 62%), respectively. Most cases involved men (61.8%), predominantly young adults (20–39 years old) in cases diagnosed by Kato-Katz and adults (40–59 years old) in cases diagnosed by LAMP. The low number of eggs per gram of stool (1–99 EPG) was the most frequently identified by both Kato-Katz and LAMP. Further studies are needed to evaluate the applicability of SmMIT-LAMP on Schistosoma mansoni diagnosis and surveillance of schistosome infections.
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Schistosoma is a genus of trematodes causing schistosomiasis, a major neglected tropical disease infecting more than 240 million people and with 700 million people at the risk of infection in the tropical and subtropical regions of the world, especially low-income countries. For the elimination of the disease, accurate diagnostic tools are needed. Besides allowing early treatment, early detection prevents environmental contamination and in turn ensures safe water sources in the endemic areas. Cell-free DNA (cfDNA) biomarker detection is a relatively new tool, used for the diagnosis of schistosomiasis in the early stages of infection from non-invasive clinical or experimental samples. cfDNA can be detected in Schistosoma infected host body fluids such as urine, serum, saliva and tissues, mainly in blood offering significant benefits for accurate diagnosis. In the current review, we described different characteristics of cfDNA, evidencing and supporting its potential uses in Schistosoma diagnosis and the improvement of treatment effectiveness.
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Citation Excerpt :
Zhang et al. (2022) developed a triplex TaqMan-based real-time PCR to detect and characterize three E. multilocularis, E. granulosus sensu stricto, and E. shiquicus in canid fecal samples with sensitivity 10 copies plasmid/μl, which regarding the high possibility of contamination of vegetable farmlands with canid feces, could be employed to investigate food and water samples (Zhang et al., 2022). However, beside advantages of real-time PCR for detection of pathogens in different samples, need for expensive instruments, trained technicians, risk of contaminations, and non-specific amplification, particularly due to primer-dimer, have limited applications of this technique for detection of pathogens in food and water resources (Maurer, 2011; Papaiakovou et al., 2019). There are a lot of nucleic acid amplification (NAA) techniques that amplify target genes at isothermal conditions, such as nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR) (Fahy et al., 1991), strand displacement amplification (SDA) (Walker et al., 1992), multiple displacement amplification (MDA) (Huang et al., 2020), recombinase polymerase amplification (RPA) (Rostron et al., 2019), and LAMP (Wong et al., 2018).
Increasing demands for foods, lack of enough arable land, and limited water resources for irrigation are the important challenges of the humanity in the century ahead. Food-related diseases are dramatically increasing and are one of main cause of death with huge effects on global economy. Therefore, food safety is a necessity, which should be considered by health policy makers. Laboratory diagnosis of foodborne pathogens is being dramatically improved during recent decades to increase accuracy and sensitivity, and decrease time and facilities of diagnostic tests. Loop-isothermal mediated amplification (LAMP) is a simple sensitive rapid test, which decreases dependency to current facilities, that are needed for conventional molecular methods. More recently, an incorporation of laboratory sciences with engineering has been led to emerging a new approach, called lab-on-a-chip, which is known as point of care (POC) diagnostic method. Here, we provided a state-of-the-art review on available diagnostic techniques from conventional PCR to microfluidic LAMP for detection of foodborne parasites.

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Trends in Parasitology

OpinionQuantitative PCR-Based Diagnosis of Soil-Transmitted Helminth Infections: Faecal or Fickle?

Highlights

Section snippets

A Timely Diagnosis

Cost of qPCR

Concluding Remarks

Outstanding Questions

Glossary

J. Microbiol. Methods

J. Clin. Virol.

Trends Parasitol.

Acta Trop.

Dev. Cell

Int. J. Food Microbiol.

Clin. Microbiol. Infect.

Adv. Parasitol.

Clin. Microbiol. Infect.

Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming

Parasit. Vectors

Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR

Am. J. Trop. Med. Hyg.

Diagnostic accuracy of Kato–Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania

Am. J. Trop. Med. Hyg.

Quantification strategies in real-time polymerase chain reaction

Sources of variability in the measurement of Ascaris lumbricoides infection intensity by Kato-Katz and qPCR

Parasit. Vectors

Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance: a Starworms study protocol

PLoS Negl. Trop. Dis.

Nonhuman DNA Typing: Theory and Casework Applications

Molecular Diagnostics of Infectious Diseases

Real-Time PCR: Current Technology and Applications

Error estimation in environmental DNA targets quantification due to PCR efficiencies differences between real samples and standards

Folia Microbiol. (Praha)

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data

Nucleic Acids Res.

PCR inhibitors – occurrence, properties and removal

J. Appl. Microbiol.

Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test

J. Clin. Microbiol.

Pre-PCR processing of samples

Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples

PLoS ONE

Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA

Appl. Environ. Microbiol.

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

J. Appl. Microbiol.

Limit of blank, limit of detection and limit of quantitation

Clin. Biochem. Rev.

Testing for soil-transmitted helminth transmission elimination: analysing the impact of the sensitivity of different diagnostic tools

PLoS Negl. Trop. Dis.

Identifying optimal threshold statistics for elimination of hookworm using a stochastic simulation model

Parasit. Vectors

Opinion
Quantitative PCR-Based Diagnosis of Soil-Transmitted Helminth Infections: Faecal or Fickle?