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Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow.

ABSTRACT: Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.

SUBMITTER: Azzopardi KI

PROVIDER: S-EPMC8483301 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe 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.ResultsThe 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.ConclusionsOur 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.

Project description:BackgroundDue to the recent increased use of the McMaster (MM) fecal egg counting method for assessing benzimidazole drug efficacy for treating soil-transmitted helminth (STH) infections, the aim of the current study was to determine the operational value of including the MM method alongside the Kato-Katz (KK) fecal thick smear to increase the diagnostic sensitivity when STHs are co-endemic with trematode helminths (e.g., Schistosoma mansoni).MethodsA cross-sectional study was conducted in school-aged children aged 4-18 years in the northeastern region of the State of Minas Gerais (Brazil), where Necator americanus, Ascaris lumbricoides, Trichuris trichiura, and S. mansoni are co-endemic. One fecal sample from each participant was collected and transported to the field laboratory for analysis. Coprological diagnosis was performed on each fecal sample by three different methods: Formalin-Ether Sedimentation (FES), KK and the MM technique. The diagnostic sensitivity and negative predictive value (NPV) of each technique was calculated using the combination of all three techniques as the composite standard. In order to determine the agreement between the three techniques Fleiss´ kappa was used. Both the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) were calculated using the two quantification techniques (i.e., the MM and KK).ResultsFecal samples from 1260 children were analyzed. The KK had higher diagnostic sensitivity than the MM for the detection of both A. lumbricoides (KK 97.3%, MM 69.5%) and hookworm (KK 95.1%, MM 80.8%). The CR of a single dose of mebendazole varied significantly between the KK and MM for both A. lumbricoides (p = 0.016) and hookworm (p = 0.000), with lower rates obtained with the KK. On the other hand, the FECR was very similar between both techniques for both A. lumbricoides and hookworm.ConclusionThe MM did not add any diagnostic value over the KK in areas where both STHs and trematodes were co-endemic. The lower sensitivity of the MM would have an important impact on the administration of selective school-based treatment in this area since if only the MM were used, 36 (13.9%) children diagnosed with A. lumbricoides would have gone untreated.

Project description:BackgroundMonitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.Methodology/principal findingsThe diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO3) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO3 flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).Conclusions/significanceThis study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.

Project description:Objective To evaluate efficacies of anthelmintic drugs against soil transmitted helminths in terms of cure rates and egg reduction rates.Design Systematic review and network meta-analysis.Data Sources PubMed, ISI Web of Science, Embase, ScienceDirect, the Cochrane Central Register of Clinical Trials, and the World Health Organization library database from 1960 until 31 December 2016.Study selection Randomised controlled trials evaluating the efficacy of a single dose regimen of albendazole, mebendazole, levamisole, and pyrantel pamoate against Ascaris lumbricoides, hookworm (Necator americanus and Ancylostoma duodenale) and Trichuris trichiura. The primary outcomes included cure rates analysed by network meta-analysis with mixed logistic regression models and egg reduction rates with mixed linear models.Results 55 and 46 randomised controlled trials were included in the analysis of cure rates and egg reduction rates, respectively. All drugs were highly efficacious against A lumbricoides Albendazole showed the highest efficacy against hookworm infections with a cure rate of 79.5% (95% confidence interval 71.5% to 85.6%) and an egg reduction rate of 89.6% (81.9% to 97.3%). All drugs had low efficacy against T trichiura, with mebendazole showing the highest cure rate of 42.1% (25.9% to 60.2%) and egg reduction rate of 66.0% (54.6% to 77.3%). Estimates for the years 1995 and 2015 showed significant reductions in efficacy of albendazole against T trichiura: by 2015 the egg reduction rates fell from 72.6% (53.7% to 91.5%) to 43.4% (23.5% to 63.3%; P=0.049) and the cure rates fell from 38.6% (26.2% to 52.7%) to 16.4 (7.7% to 31.3%; P=0.027).Conclusions All four currently recommended drugs show limitations in their efficacy profile. While only albendazole showed good efficacy against hookworm infection, all drugs had low efficacy against T trichiura The decrease in efficacy of albendazole against T trichiura over the past two decades is of concern. The findings indicate the need for strengthening efforts to develop new drug treatments, with a particular focus on drugs against T trichiura.

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Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow.

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