Welcome to the ICOPA 2022 Online Program

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Next-generation sequencing (NGS) is a powerful tool to detect and characterize parasites in various sample types, including human clinical specimens for diagnostic purposes. In contrast to most other molecular diagnostic methods, NGS is target agnostic and has the potential to detect novel or poorly characterized parasites. However, one challenge is the low amount of parasites in comparison to human (and sometimes other eukaryotic) cells present in typical clinical specimens. Shotgun sequencing methods tend to produce an overabundance of human reads that may prevent parasites from being detected. Nonetheless, there are several examples of successful detection of parasites in human clinical specimens by shotgun NGS, including Balamuthia mandrillaris, Toxoplasma gondii and Taenia solium in cerebrospinal fluid, Plasmodium species and Fasciola hepatica in blood, and Leishmania infantum in bone marrow.

An alternative to shotgun sequencing is to amplify by PCR a region of interest and sequence the amplicon using NGS. This targeted amplicon deep sequencing offers better sensitivity and, depending on PCR primer design, better specificity, with the possibility to examine genetically complex specimen types such as feces. The 18S rRNA gene is the most commonly used marker, especially variable regions V4 and V9. However, due to the great sequence diversity even in conserved regions of the ribosomal genes, it has proven difficult to design PCR primers that will avoid amplifying human DNA and still capture all groups of parasites. Another approach is to use broadly reactive eukaryotic PCR primers together with a strategy to reduce the number of human reads being sequenced.

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Parasitic neglected tropical diseases (NTDs) affect over one billion people worldwide, with individuals from communities in low-socioeconomic areas being most at risk and suffering the most. Disease management programs are hindered by the lack of infrastructure and resources for clinical sample collection, storage, and transport and a dearth of sensitive diagnostic methods that are inexpensive as well as accurate. Many diagnostic tests and tools have been developed for the parasitic NTDs, but the collection and storage of clinical samples for molecular and immunological diagnosis can be expensive due to storage, transport, and reagent costs, making these procedures untenable in many areas of endemicity. The application of membrane technology, which involves the use of specific membranes for either sample collection and storage or diagnostic procedures, can streamline this process, allowing for long-term sample storage at room temperature. Membrane technology can be used in serology-based diagnostic assays and for nucleic acid purification prior to molecular analysis. This facilitates the development of relatively simple and rapid procedures, although some of these methods, mainly due to costs, lack accessibility in low-socioeconomic regions of endemicity. New immunological procedures and nucleic acid storage, purification, and diagnostics protocols that are simple, rapid, accurate, and cost-effective must be developed as countries progress control efforts toward the elimination of the parasitic NTDs.

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Following endorsement of the road map for neglected tropical diseases in 2020, the global community is now faced with the challenges of implementing programs and policies that can achieve the targets and milestones laid out in these commitments. As countries prepare to pursue elimination of these diseases as public health problems, or in some cases, seek to pursue elimination of transmission, additional tools will be required to verify progress and validate the achievement of the ambitious targets articulated in the road map.

Unfortunately, the global NTD community has long focused on outdated classical diagnostic tools and technologies and have not kept pace with rapid advancements in technology that allow far more sensitive, specific and efficient diagnostic performance than traditional microscopy-based assays. Existing diagnostics for many NTDs are limited by performance characteristics that make them unsuitable to support these programmatic needs. For example. despite a growing body of evidence suggesting significant sensitivity and specificity inadequacies, World Health Organization guidelines for the detection and quantification of soil-transmitted helminths (STH) remain reliant upon microscopy-based techniques (Kato-Katz). This reliance results less from the merits of the recommended coproscopic techniques than it does from the perceived difficulties in implementing newer, more sensitive DNA-based methods. Despite the availability of such diagnostic assays, demonstration of the utility of these assays, at scale within endemic country settings, has yet to be achieved.

In recent years, efforts aimed at demonstrating the use of real-time PCR (qPCR) at scale have intensified. The DeWorm3 project, a Bill & Melinda Gates Foundation funded initiative, has developed and validated a high-throughput assay for the diagnosis of soil-transmitted helminth infection. We have established operating laboratory capacity in West Africa (Benin), South Asia (India) and the United States and have validated the performance of this assay with a number of external advisory partners. The assay has now been utilized to successfully test tens of thousands of stool samples from Benin, India and Malawi. These efforts, spearheaded by collaborations between partner institutions in DeWorm3, have allowed for the development of well-controlled, semi-automated, multiplexed platforms capable of standardized, high-throughput testing. Utilizing carefully chosen highly-repetitive DNA sequences as targets, the highly sensitive nature of these assays helps to buffer their performance against minor platform inconsistences and PCR-inhibition from human stool. This platform offers the opportunity to accurately and effectively monitor, support and validate progress towards the targets and milestones at the heart of the WHO commitments described in the road map for neglected tropical diseases.

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Digital PCR (dPCR) has been used for pathogen DNA detection for diagnostic purposes, as well as for environmental and food monitoring. Although the application of dPCR in the field of medical parasitology is relatively less advanced than the recent and rapid progression observed for other infections, this approach can play an important role for the diagnosis and control of parasitic infections. Indeed, accurate diagnostic tools are needed in support of parasite control programmes in endemic regions, and for diagnosis in non-endemic areas. The principle of this technique is to amplify a single DNA template from maximally diluted samples, therefore generating amplicons that are exclusively derived from one template, based on Poisson statistics. Differently from real-time PCR, which produces an exponential signal, dPCR generates linear, digital signals, allowing quantitative analysis of the PCR product, detecting very rare mutations with high precision and sensitivity. In parasitology, dPCR can be very useful in detecting low parasitic loads and for differential analysis. Here, the applications of dPCR are reported for parasitology, highlighting the advantages, the potential issues and the perspectives for a clinical laboratory.