Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of ...providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making.
Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease ...mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.
Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.
The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The capacity of vector insect surveillance to provide estimates of pathogen prevalence and transmission potential has long been recognized within the global communities tasked with eliminating ...lymphatic filariasis (LF), the underlying cause of elephantiasis and hydrocele, and onchocerciasis (river blindness). Initially restricted to the practice of dissection, the potential of vector monitoring has grown due to the advent of molecular methods capable of increasing the sensitivity and throughput of testing. However, despite such advancement, operational research gaps remain. If insufficiently addressed, these gaps will reduce the utility of molecular xenomonitoring (MX) for onchocerciasis as elimination efforts expand into Africa. Similarly, such shortcomings will limit the programmatic usefulness of MX for LF, resulting in this technique’s significant underutilization.
Preliminary recommendations now exist for the programmatic use of molecular xenomonitoring for lymphatic filariasis in Culex-vectored regions.
Novel methodologies for molecular xenomonitoring, such as excreta/feces testing, have the potential to increase the feasibility of mosquito-based monitoring efforts for lymphatic filariasis.
The Esperanza Window Trap has enabled the collection of black flies for the molecular xenomonitoring of onchocerciasis in the Americas and Uganda without the use of human landing collectors.
The high-throughput screening of onchocerciasis vectors enables the practical use of molecular xenomonitoring as a component of onchocerciasis verification of elimination efforts.
Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for ...parasite infections than mass drug administration, while providing other quality of life benefits.
We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides, Trichuris trichiura, hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A. lumbricoides, 1% with T. trichiura, 2% with hookworm, and 39% with G. duodenalis. The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, <10% had electricity access, and >90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio PR: 0.82 95% CI 0.67, 1.00, p = 0.056), the WSH arm (PR: 0.78 95% CI 0.63, 0.96, p = 0.021), and the WSHN arm (PR: 0.78 95% CI 0.64, 0.96, p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 95% CI 0.73, 1.08, p = 0.228), handwashing (PR: 0.89 95% CI 0.73, 1.09, p = 0.277), or nutrition (PR: 86 95% CI 0.71, 1.05, p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia. Reanalysis of stool samples by quantitative polymerase chain reaction confirmed the reductions in Ascaris infections measured by microscopy in the WSH and WSHN groups. Trial limitations included imperfect uptake of targeted intervention behaviors, limited power to detect effects on rare parasite infections, and that it was not feasible to blind participants and sample collectors to treatment status. However, lab technicians and data analysts were blinded to treatment status. The trial was funded by the Bill & Melinda Gates Foundation and the United States Agency for International Development.
Integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies for Ascaris infections, particularly in similar settings with recent or ongoing deworming programs. Combining nutrition with WSH did not provide further benefits, and water treatment alone was similarly effective to integrated WSH. Our findings provide new evidence that drinking water should be given increased attention as a transmission pathway for Ascaris.
ClinicalTrials.gov NCT01704105.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Molecular-based surveys have indicated that Ancylostoma ceylanicum, a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in Asia. Most current PCR-based ...diagnostic options for the detection of Ancylostoma species target the Internal Transcribed Spacer (ITS) regions of the ribosomal gene cluster. These regions possess a considerable degree of conservation among the species of this genus and this conservation can lead to the misidentification of infecting species or require additional labor for accurate species-level determination. We have developed a novel, real-time PCR-based assay for the sensitive and species-specific detection of A. ceylanicum that targets a non-coding, highly repetitive genomic DNA element. Comparative testing of this PCR assay with an assay that targets ITS sequences was conducted on field-collected samples from Argentina and Timor-Leste to provide further evidence of the sensitivity and species-specificity of this assay.
A previously described platform for the design of primers/probe targeting non-coding highly repetitive regions was used for the development of this novel assay. The assay's limits of detection (sensitivity) and cross-reactivity with other soil-transmitted helminth species (specificity) were assessed with real-time PCR experiments. The assay was successfully used to identify infections caused by A. ceylanicum that were previously only identified to the genus level as Ancylostoma spp. when analyzed using other published primer-probe pairings. Further proof of sensitive, species-specific detection was provided using a published, semi-nested restriction fragment length polymorphism-PCR assay that differentiates between Ancylostoma species.
Due to the close proximity of people and domestic/wild animals in many regions of the world, the potential for zoonotic infections is substantial. Sensitive tools enabling the screening for different soil-transmitted helminth infections are essential to the success of mass deworming efforts and facilitate the appropriate interpretation of data. This study describes a novel, species-specific, real-time PCR-based assay for the detection of A. ceylanicum that will help to address the need for such tools in integrated STH deworming programs.
ANZCTR.org.au ACTRN12614000680662.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Proper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined ...various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time.
Quantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against "no preservative" controls and the "gold standard" of rapidly freezing samples at -20°C. The preservation methods were compared at both 4°C and at simulated tropical ambient temperature (32°C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA.
At 4°C there were no significant differences in DNA amplification efficiency (as measured by Cq values) regardless of the preservation method utilized over the 60-day period. At 32°C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cq value increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4°C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon ...assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions.
Utilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as "negative" using the less sensitive detection method.
Through the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Community presence of loiasis must be determined before mass drug administration programmes for lymphatic filariasis and onchocerciasis can be implemented. However, taking human blood ...samples for loiasis surveillance is invasive and operationally challenging. A xenosurveillance approach based on the molecular screening of mosquitoes and their excreta/feces (E/F) for Loa loa DNA may provide a non-invasive method for detecting the community presence of loiasis. Methods We collected 770 wild mosquitoes during a pilot study in a known loiasis transmission area in Mbalmayo, Cameroon. Of these, 376 were preserved immediately while 394 were kept in pools to collect 36-hour E/F samples before processing. Carcasses and E/F were screened for L. loa DNA. To demonstrate this method's potential for integrated disease surveillance, the samples were further tested for Wuchereria bancrofti, Mansonella perstans, and Plasmodium falciparum. Results Despite limited sample numbers, L. loa DNA was detected in eight immediately-stored mosquitoes (2.13%; 95% CI 1.08 to 4.14), one carcass stored after providing E/F (0.25%; 95% CI 0.04 to 1.42), and three E/F samples (estimated prevalence 0.77%; 95% CI 0.15 to 2.23%). M. perstans and P. falciparum DNA were also detected in carcasses and E/F samples, while W. bancrofti DNA was detected in E/F. None of the carcasses positive for filarial worm DNA came from pools that provided a positive E/F sample, supporting the theory that, in incompetent vectors, ingested parasites undergo a rapid, complete expulsion in E/F. Conclusions Mosquito xenosurveillance may provide a useful tool for the surveillance of loiasis alongside other parasitic diseases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The balance of expense and ease of use vs. specificity and sensitivity in diagnostic assays for helminth disease is an important consideration, with expense and ease often winning out in endemic ...areas where funds and sophisticated equipment may be scarce. In this review, we argue that molecular diagnostics, specifically new assays that have been developed with the aid of next-generation sequence data and robust bioinformatic tools, more than make up for their expense with the benefit of a clear and precise assessment of the situation on the ground. Elimination efforts associated with the London Declaration and the World Health Organization (WHO) 2020 Roadmap have resulted in areas of low disease incidence and reduced infection burdens. An accurate assessment of infection levels is critical for determining where and when the programs can be successfully ended. Thus, more sensitive assays are needed in locations where elimination efforts are approaching a successful conclusion. Although microscopy or more general PCR targets have a role to play, they can mislead and cause study results to be confounded. Hyper-specific qPCR assays enable a more definitive assessment of the situation in the field, as well as of shifting dynamics and emerging diseases.
Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both ...next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent.
Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing.
Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study's objectives.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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