Performance of microscopy compared to conventional PCR in identification of soil-transmitted helminth infections among antenatal women in a low-prevalence setting.

PURPOSE: Traditional microscopy-based methods may provide inaccurate estimates of Soil transmitted helminth (STH) infections in mild intensity of infection. Therefore, we aimed to determine the prevalence of STH infections using molecular diagnostic methods and compare the diagnostic performance of microscopy with polymerase chain reaction (PCR) in stool samples collected from pregnant women in primary care settings in Puducherry, India. METHODOLOGY: A singleplex PCR assay was developed to detect three species of STHs, namely Ascaris lumbricoides, Necator americanus, and Ancylostoma duodenale, by targeting the internal transcribed spacer regions (ITS1 and ITS2) of 5.8S rRNA. The PCR generated 420, 662, and 515 base pairs of DNA for the respective organisms. In addition to singleplex PCR, wet and concentration microscopy techniques were used. The results were expressed as percentages with 95% confidence intervals, and the diagnostic performance of microscopy was compared with PCR in terms of sensitivity, specificity, and positive, negative predictive values and kappa statistics. RESULTS: Among the 650 pregnant women included, 48.8% were aged 25 years or less, 59% were primigravida, and half were from rural areas. The overall prevalence of any STH infection was higher in PCR compared to microscopy (8.9% vs. 7.2%). The prevalence of Ascaris lumbricoides was higher by microscopy (5.4% vs 2.6%), while the prevalence of Necator americanus was higher by PCR (6.3%) than by microscopy (1.8%). No species of Ancylostoma duodenale was detected. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of microscopy for detecting any STH infection was 22.4%, 94.3%, 27.7%, and 92.5%, respectively. The agreement between microscopy and PCR for the identification is as follows: for any STH infection, k â€‹= â€‹0.12, Ascaris k â€‹= â€‹0.16, and Necator k â€‹= â€‹0.20, respectively. CONCLUSION: The prevalence of any STH infection identified by PCR was higher than microscopy, and the agreement between the two methods was poor.

First molecular data on the human roundworm Ascaris lumbricoides species complex from the Bronze and Iron Age in Hallstatt, Austria.

Palaeoparasitological studies can provide valuable information on the emergence, distribution, and elimination of parasites during a particular time in the past. In the prehistoric salt mines of Hallstatt, located in the Austrian Alps, human faeces have been conserved in salt. The aim of this study was to recover ancient DNA of intestinal parasites from these coprolites. Altogether, 35 coprolites from the Hallstatt salt mines, dating back to the Bronze Age mining phase (1158-1063 BCE) and the Iron Age mining phase (750-662 BCE), respectively, were analysed by microscopy and molecular methods. In 91% of the coprolite samples, eggs of soil-transmitted helminths (STH), namely of Trichuris and/or Ascaris were detected by light microscopy. The Ascaris eggs were exceptionally well preserved. For further analysis, DNA was extracted from the palaeofaecal samples and species-specific primers targeting different genes were designed. While amplification of Trichuris DNA remained unsuccessful, sequence data of A. lumbricoides species complex were successfully obtained from 16 coprolites from three different genes, the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), the mitochondrial cytochrome B gene (cytB) and the mitochondrial NADH dehydrogenase subunit 1 gene (nadh1). Importantly, these included two Ascaris sequences from a coprolite from the Bronze Age, which to the best of our knowledge are the first molecular data of this genus from this period.

Transcriptome profiling of male and female Ascaris lumbricoides reproductive tissues.

BACKGROUND: Ascaris lumbricoides causes human ascariasis, the most prevalent helminth disease, infecting approximately 1 billion individuals globally. In 2019 the global disease burden was estimated to be 754,000 DALYs and resulted in 2090 deaths. In the absence of a vaccination strategy, treatment of ascariasis has relied on anthelminthic chemotherapy, but drug resistance is a concern. The propensity for reinfection is also a major challenge to disease control; female worms lay up to 200,000 eggs daily, which contaminate surrounding environments and remain viable for years, resulting in high transmission rates. Understanding the molecular mechanisms of reproductive processes, including control of egg production, spermatogenesis, oogenesis and embryogenesis, will drive the development of new drugs and/or vaccine targets for future ascariasis control. METHODS: Transcriptome profiles of discrete reproductive and somatic tissue samples were generated from adult male and female worms using Illumina HiSeq with 2 × 150 bp paired-end sequencing. Male tissues included: testis germinal zone, testis part of vas deferens, seminal vesicle and somatic tissue. Female tissues included: ovary germinal zone, ovary part of the oviduct, uterus and somatic tissue. Differentially expressed genes (DEGs) were identified from the fragments per kilobases per million reads (FPKM) profiles. Hierarchical analysis was performed to identify tissue-specific genes. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to identify significant terms and pathways for the DEGs. RESULTS: DEGs involved in protein phosphorylation and adhesion molecules were indicated to play a crucial role in spermatogenesis and fertilization, respectively. Those genes associated with the G-protein-coupled receptor (GPCR) signaling pathway and small GTPase-mediated signal transduction pathway play an essential role in cytoskeleton organization during oogenesis. Additionally, DEGs associated with the SMA genes and TGF-ß signaling pathway are crucial in adult female embryogenesis. Some genes associated with particular biological processes and pathways that were identified in this study have been linked to defects in germline development, embryogenesis and reproductive behavior. In the enriched KEGG pathway analysis, Hippo signaling, oxytocin signaling and tight junction pathways were identified to play a role in Ascaris male and female reproductive systems. CONCLUSIONS: This study has provided comprehensive transcriptome profiles of discrete A. lumbricoides reproductive tissue samples, revealing the molecular basis of these functionally important tissues. The data generated from this study will provide fundamental knowledge on the reproductive biology of Ascaris and will inform future target identification for anti-ascariasis drugs and/or vaccines.

Genetic diversity and identity of Ascaris worms from human and pig hosts in Thailand.

Ascaris roundworms are of public health and socio-economic importance worldwide. They are conventionally attributed to two taxa - A. lumbricoides infecting principally human and A. suum infecting principally pig. Phylogenomic analysis has revealed that Ascaris worms from both human and pig are represented in Clades A and B. A recent study indicates that the Ascaris worms from human and pig in Thailand belong to Clade A. We examined adult Ascaris worms from human and pig in Thailand by means of the partial sequences of three mitochondrial genes (cox1, cox2 and nad1) and concatenation of these genes. Phylogenomic analysis indicates that two isolates (H1,H2) of A. lumbricoides from human belonged to Clade B; one isolate (H3) belonged to Clade A (based on cox1, cox2 and concatenated sequences) or as an outlier to Clades A and B (based on nad1 sequences). All the eight isolates of A. suum from pig clustered in Clade A. The partial nad1 and the concatenated sequences revealed two lineages of A. suum isolates which were distinct from the two A. lumbricoides isolates of Clade B. It is evident that greater genetic diversity, and a more robust phylogeny, could be uncovered by the application of multiple genes. In sum, the present study reveals the presence in Thailand of A. lumbricoides from human in Clades A and B which necessitates appropriate treatment and control measures; Clades A and B have been reported to contain haplotypes of Ascaris worms from both human and pig in other parts of the world. A country wide study is needed to elucidate the identity, distribution, prevalence, cross transmission, genetic diversity and phylogeny of the Ascaris worms in Thailand.

Development of allele-specific PCR methodology (AS-PCR) to screening A. lumbricoides and A. suum.

Ascaris lumbricoides and Ascaris suum are described as helminths that infect humans and pigs, respectively. It is estimated that infection by A. lumbricoides affects about 447 million individuals living in tropical regions of developing countries. However, there is an increasing number of cases of human ascariasis in countries with no recent history of autochthonous infection by A. lumbricoides. In these places, pigs have been incriminated as the main source of human infection. Conventional parasitological diagnosis does not allow species-specific identification, and the real epidemiological scenario of human and swine ascariasis is still uncertain. Therefore, this work presents the application of a species-specific molecular diagnosis, based on the allele-specific PCR methodology (AS-PCR), using the Internal Transcript Space 1 (ITS-1) of the ribosomal DNA, as a target for differentiating between the two species, using DNA obtained from eggs. To validate the methodology, stool samples positive for Ascaris spp, were obtained from 68 humans from seven Brazilian states and from six pigs from the state of Minas Gerais. All samples obtained from humans were genotyped as A. lumbricoides and all samples obtained from swine were genotyped as A. suum. These results are in agreement with the literature, which demonstrates that in most endemic regions, transmission cycles are separate. Therefore, the execution of this work allowed the availability of a useful methodology for the differential diagnosis of the species, which may contribute to the characterization of the real epidemiological profile of human and swine ascariasis, and to the implementation of future control strategies.

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation.

Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/ µl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Molecular epidemiology of Ascaris species recovered from humans and pigs in Cameroon.

BACKGROUND: In Cameroon, considerable research has been conducted on human ascariasis, but no studies have been undertaken to determine whether pigs contribute to the persistence of the infection in children or to unravel the evolutionary relationship between human-derived and pig-derived Ascaris. METHODS: DNA was extracted from adult Ascaris worms collected from humans and pigs. Segments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were sequenced and analysed for 83 worms to dissect the local transmission dynamics of Ascaris in Cameroon. RESULTS: The data showed high genetic diversity and revealed demographically expanding populations in the human and pig Ascaris samples. A restricted gene flow between Ascaris lumbricoides and Ascaris suum populations correlating with the preference for humans and pigs, respectively, as hosts was evident. Phylogenetic analyses and haplotype networks split the haplotypes into two major clusters, A and B. However, support for cross-transmission between hosts and hybridization were revealed through shared haplotypes among worms from both hosts. CONCLUSIONS: This study provides useful baseline information for future studies of the genetics of Ascaris in Cameroon and suggests that effective and sustainable control of human ascariasis should target both human and pig hosts.

Characterization of the ß-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance.

BACKGROUND: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding ß-tubulin proteins, the principal targets of BZ drugs. METHODOLOGY AND PRINCIPAL FINDINGS: First, the ß-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different ß-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. SIGNIFICANCE: This study demonstrated the presence of at least seven ß-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the ß-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of ß-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Genotyping of Ascaris spp. infecting humans and pigs in Italy, Slovakia and Colombia.

BACKGROUND: The systematics and taxonomy of Ascaris lumbricoides and Ascaris suum, two of the world's most widespread nematodes, still represent a highly debated scientific issue. Two different transmission scenarios have been described according to endemicity: separated host-specific transmission cycles in endemic regions, and a single pool of infection shared by humans and pigs in non-endemic regions. The swine roundworm A. suum is now recognized as an important cause of human ascariasis also in endemic areas such as China, where cross-infections and hybridization have also been reported, as well as in non-endemic regions like Italy. This study aimed to investigate the molecular epidemiology of human and pig ascariasis in three countries representing different epidemiological scenarios: Italy as a non-endemic country, Colombia as an endemic country, and Slovakia as a non-endemic country, but with a poor socio-economic context linked to some focal populations of Roma settlements. MATERIALS AND METHODS: A total of 237 nematodes were analysed: 46 from Colombia (13 from humans, 33 from pigs), 114 from Slovakia (20 from humans, 94 from pigs) and 77 from Italy (17 from humans and 60 from pigs). Genotyping by PCR-RFLP of nuclear (ITS) and sequencing of mitochondrial (cox1) target regions were performed. ITS genotypes were used to estimate the Hardy-Weinberg (HW) equilibrium according to hosts and country of origin. The partial cox1 sequences were used to analyse genetic polymorphisms according to hosts and country of origin, as well as to infer the network of haplotypes, their evolutionary relationships and geographical distribution. RESULTS: 110 quality cox1 sequences were obtained. Haplotype network revealed three main groups corresponding to clade A, B and C. Clade C included most of the human cases from Italy, while those from Slovakia and Colombia were grouped in clade B. Ascaris from Italian and Colombian pigs showed HW equilibrium at the ITS marker, while disequilibrium was found in A. lumbricoides from Slovak pigs, which suggest a high unexpected amount of roundworms of human origin circulating also in pigs. CONCLUSIONS: This study updates and extends the current understanding of Ascaris species and genotypes circulating in different epidemiological scenarios, with particular attention to the inclusion of human-derived Ascaris in the phylogenetic cluster C. Despite the evidence of HW equilibrium in the ITS in pig-derived Italian samples, the amount of genetic variation seems to support the existence of two closely related species.

Limitations of PCR detection of filarial DNA in human stools from subjects non-infected with soil-transmitted helminths.

The standard techniques for diagnosis of human filariasis are the microscopic examination of blood smears or skin biopsies, which are relatively invasive and poorly sensitive at low levels of infection. Recently, filarial DNA has been detected in fecal samples from non-human primates in Central Africa. The aim of this study was to demonstrate proof-of-concept of a non-invasive molecular diagnosis technique for human filariasis by targeting fragments of 12S rDNA, Cox1, ITS1 and LL20-15kDa ladder antigen-gene by conventional PCR in DNA extracted from stool samples of 52 people infected with Mansonella perstans and/or Loa loa. Of these, 10 patients were infected with soil-transmitted helminths (Trichuris trichiura and/or Ascaris lumbricoides), and none were positive for Necator americanus. Interestingly, no filarial gene fragments were detected in the stools of any of the 52 patients. Future studies should evaluate whether a co-infection with soil-transmitted helminths causing gastrointestinal bleeding and likely allowing (micro)filaria exit into the digestive tract, may facilitate the molecular detection of filarial DNA fragments in stool samples.

TITLE: Limites de la détection par PCR d'ADN de filaires dans les selles humaines de sujets non-infectés par les géohelminthes. ABSTRACT: Les techniques standards de diagnostic des filarioses humaines (examen microscopique de gouttes épaisses ou de biopsies cutanées) sont relativement invasives et peu sensibles à de faibles niveaux d'infection. De l'ADN de filaires a été récemment détecté dans des échantillons de fèces de primates non-humains en Afrique centrale. L'objectif de cette étude était de démontrer la preuve de concept d'un diagnostic moléculaire non invasif des filarioses chez l'homme en ciblant des fragments d'ADNr 12S, Cox1, ITS1 et l'antigène LL20-15kDa par PCR classique. L'ADN a été extrait d'échantillons de selles de 52 personnes infectées par Mansonella perstans et/ou Loa loa. Parmi ces patients, dix étaient infectés par des géohelminthes (Trichuris trichiura et/ou Ascaris lumbricoides) et aucun n'était positif pour Necator americanus. De manière intéressante, aucun fragment de gène de filaires n'a été détecté dans les selles des 52 patients. Des études futures devraient être menées pour évaluer si une coinfection avec des géohelminthes (provoquant des hémorragies gastro-intestinales et permettant probablement l'effraction de (micro)filaires dans le tube digestif) facilite la détection moléculaire de fragments d'ADN de filaires dans les selles.

Expression of Ascaris lumbricoides putative virulence-associated genes when infecting a human host.

BACKGROUND: Ascaris lumbricoides is the most common causative agent of soil-transmitted helminth infections worldwide, with an estimated 450 million people infected with this nematode globally. It is suggested that helminths are capable of evading and manipulating the host immune system through the release of a spectrum of worm proteins which underpins their long-term survival in the host. We hypothesise that the worm overexpresses these proteins when infecting adults compared to children to cirvumvent the more robust defence mechanisms of adults. However, little is known about the parasite's genes and encoded proteins involved during A. lumbricoides infection. Hence, this study was conducted to assess the expression profile of putative virulence-associated genes during an active infection of adults and children. METHODS: In this study, quantitative PCR was performed to evaluate the expression profile of putative virulence-associated genes in A. lumbricoides isolated from infected children and adults. The study was initiated by collecting adult worms expelled from adults and children following anthelminthic treatment. High-quality RNA was successfully extracted from each of six adult worms expelled by three adults and three children, respectively. Eleven putative homologues of helminth virulence-associated genes reported in previous studies were selected, primers were designed and specific amplicons of A. lumbricoides genes were noted. The expression profiles of these putative virulence-associated genes in A. lumbricoides from infected adults were compared to those in A. lumbricoides from infected children. RESULTS: The putative virulence-associated genes VENOM, CADHERIN and PEBP were significantly upregulated at 166-fold, 13-fold and fivefold, respectively, in adults compared to children. Conversely, the transcription of ABA-1 (fourfold), CATH-L (threefold) and INTEGRIN (twofold) was significantly suppressed in A. lumbricoides from infected adults. CONCLUSIONS: On the basis of the expression profile of the putative virulence-associated genes, we propose that the encoded proteins have potential roles in evasion mechanisms, which could guide the development of therapeutic interventions.

Molecular evidence of hybridization between pig and human Ascaris indicates an interbred species complex infecting humans.

Human ascariasis is a major neglected tropical disease caused by the nematode Ascaris lumbricoides. We report a 296 megabase (Mb) reference-quality genome comprised of 17,902 protein-coding genes derived from a single, representative Ascaris worm. An additional 68 worms were collected from 60 human hosts in Kenyan villages where pig husbandry is rare. Notably, the majority of these worms (63/68) possessed mitochondrial genomes that clustered closer to the pig parasite Ascaris suum than to A. lumbricoides. Comparative phylogenomic analyses identified over 11 million nuclear-encoded SNPs but just two distinct genetic types that had recombined across the genomes analyzed. The nuclear genomes had extensive heterozygosity, and all samples existed as genetic mosaics with either A. suum-like or A. lumbricoides-like inheritance patterns supporting a highly interbred Ascaris species genetic complex. As no barriers appear to exist for anthroponotic transmission of these 'hybrid' worms, a one-health approach to control the spread of human ascariasis will be necessary.

Detection of Ascaris lumbricoides infection by ABA-1 coproantigen ELISA.

Intestinal worms, or soil-transmitted helminths (STHs), affect hundreds of millions of people in all tropical and subtropical regions of the world. The most prevalent STH is Ascaris lumbricoides. Through large-scale deworming programs, World Health Organization aims to reduce morbidity, caused by moderate-to-heavy intensity infections, below 2%. In order to monitor these control programs, stool samples are examined microscopically for the presence of worm eggs. This procedure requires well-trained personnel and is known to show variability between different operators interpreting the slides. We have investigated whether ABA-1, one of the excretory-secretory products of A. lumbricoides can be used as a coproantigen marker for infection with this parasite. Polyclonal antibodies were generated and a coproantigen ELISA was developed. Using this ELISA, it was found that ABA-1 in stool detected Ascaris infection with a sensitivity of 91.5% and a specificity of 95.3%. Our results also demonstrate that there is a correlation between ABA-1 levels in stool and A. lumbricoides DNA detected in stool. Using a threshold of 18.2 ng/g stool the ABA-1 ELISA correctly assigned 68.4% of infected individuals to the moderate-to-heavy intensity infection group, with a specificity of 97.1%. Furthermore, the levels of ABA-1 in stool were shown to rapidly and strongly decrease upon administration of a standard anthelminthic treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that ABA-1 remained undetectable until day 28 and was detected at day 42 or 56, concurrent with the appearance of worm eggs in the stool. This report demonstrates that ABA-1 can be considered an Ascaris -specific coproantigen marker that can be used to monitor infection intensity. It also opens the path for development of point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site.

Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial.

BACKGROUND: Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. RESULTS: Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using "bead-beating" for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. CONCLUSIONS: In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the "bead-beating protocol" should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

Study on the population evolution of Ascaris lumbricoides and Ascaris suum based on whole genome resequencing.

Ascaris lumbricoides and Ascaris suum are parasitic nematodes that mainly parasitize the small intestines of people and pigs, respectively. Ascariasis seriously endangers human health and causes huge economic losses in the pig industry. A. lumbricoides and A. suum have similar morphologies and genetic structures, and occasionally these organisms cross-infect the alternate host. Therefore, their taxonomies are controversial. In this study, the whole genomes of A. lumbricoides (n = 6) and A. suum (n = 6) were resequenced using a HiSeq X Ten sequencing platform. Phylogenetic, principal component, and population structure analyses showed clear genetic differentiation between the two Ascaris populations. Linkage disequilibrium analysis indicated that the A. lumbricoides population was more primitive than the A. suum population. In the selective elimination analysis, 160 and 139 candidate regions were screened in A. lumbricoides and A. suum, respectively, and the selected regions were analyzed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The A. lumbricoides population had no significant enrichment in GO terms, but two KEGG pathways, the RNA degradation and tyrosine metabolism pathways, were significantly enriched. Five GO entries and one KEGG pathway, the alanine, aspartate, and glutamate metabolism signaling pathway, were significantly enriched in the A. suum population. An analysis of the demographic histories of Ascaris populations revealed that A. lumbricoides and A. suum had similar trends in effective population size in different historical periods. Ascaris populations peaked about 1 million years ago and then began to decline. In the last glacial period, they dropped to a historical low and continued at this level until the last glacial maximum. This phenomenon may be associated with the cold climate at that time. This study provides new information on the genetic differentiation, evolutionary relationships, gene functional enrichment, and population dynamics of Ascaris populations, with implications for host differences, evolution, and classification of A. lumbricoides and A. suum.

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan.

BACKGROUND: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. METHODS: Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. RESULTS: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. CONCLUSIONS: To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.

Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool.

BACKGROUND: A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. METHODOLOGY AND PRINCIPAL FINDINGS: In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. CONCLUSIONS: The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs.

First identification of the benzimidazole resistance-associated F200Y SNP in the beta-tubulin gene in Ascaris lumbricoides.

The main control strategy for Ascaris lumbricoides is mass drug administration (especially with benzimidazoles), which can select strains of parasites resistant to treatment. Mutations in the beta-tubulin isotype-1 gene at codons 167, 198 and 200 have been linked to benzimidazole resistance in several nematodes. The mutation in codon 200 is the most frequent in different species of parasites, as previously observed in Necator americanus and Trichuris trichiura; however, this mutation has never been found in populations of A. lumbricoides. This study aimed to screen for single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codon 200 in A. lumbricoides. We developed a technique based on an amplification refractory mutation system (ARMS-PCR) for the analysis of 854 single A. lumbricoides eggs collected from 68 human stool samples from seven Brazilian states. We detected the mutation in codon 200 at a frequency of 0.5% (4/854). This is the first report of this mutation in A. lumbricoides. Although the observed frequency is low, its presence indicates that these parasite populations have the potential to develop high levels of resistance in the future. The methodology proposed here provides a powerful tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations.

Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool.

BACKGROUND: With the expansion of soil transmitted helminth (STH) intervention efforts and the corresponding decline in infection prevalence, there is an increased need for sensitive and specific STH diagnostic assays. Previously, through next generation sequencing (NGS)-based identification and targeting of non-coding, high copy-number repetitive DNA sequences, we described the development of a panel of improved quantitative real-time PCR (qPCR)-based assays for the detection of Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Trichuris trichiura, and Strongyloides stercoralis. However, due to the phenomenon of chromosome diminution, a similar assay based on high copy-number repetitive DNA was not developed for the detection of Ascaris lumbricoides. Recently, the publication of a reference-level germline genome sequence for A. lumbricoides has facilitated our development of an improved assay for this human pathogen of vast global importance. METHODOLOGY/PRINCIPAL FINDINGS: Repurposing raw DNA sequence reads from a previously published Illumina-generated, NGS-based A. lumbricoides germline genome sequencing project, we performed a cluster-based repeat analysis utilizing RepeatExplorer2 software. This analysis identified the most prevalent repetitive DNA element of the A. lumbricoides germline genome (AGR, Ascaris germline repeat), which was then used to develop an improved qPCR assay. During experimental validation, this assay demonstrated a fold increase in sensitivity of ~3,100, as determined by relative Cq values, when compared with an assay utilizing a previously published, frequently employed, ribosomal internal transcribed spacer (ITS) DNA target. A comparative analysis of 2,784 field-collected samples was then performed, successfully verifying this improved sensitivity. CONCLUSIONS/SIGNIFICANCE: Through analysis of the germline genome sequence of A. lumbricoides, a vastly improved qPCR assay has been developed. This assay, utilizing a high copy-number repeat target found in eggs and embryos (the AGR repeat), will improve prevalence estimates that are fundamental to the programmatic decision-making process, while simultaneously strengthening mathematical models used to examine STH infection rates. Furthermore, through the identification of an optimal target for PCR, future assay development efforts will also benefit, as the identity of the optimized repeat DNA target is likely to remain unchanged despite continued improvement in PCR-based diagnostic technologies.

Genetic diversity of Ascaris spp. infecting humans and pigs in distinct Brazilian regions, as revealed by mitochondrial DNA.

In this study, we assessed the genetic diversity of Ascaris lumbricoides / Ascaris suum circulating in humans and pigs, exploring potential zoonotic cycles in endemic areas in Brazil. We carried out cross-sectional surveys in four municipalities: Santa Isabel do Rio Negro (SIRN-AM) (n = 328); Nossa Senhora de Nazaré (NSN-PI) and Teresina (TER-PI) (n = 605 and n = 297, respectively); and Cachoeiras de Macacu (CAM-RJ) (n = 543). We also studied 61 fecal samples/adult worms obtained from pigs (n = 53 in NSN-PI and n = 8 in TER-PI). A ~450 bp fragment of the Ascaris cytochrome c oxidase subunit 1 (cox1) and ~400 bp of the NADH dehydrogenase subunit 1 (nad1) were amplified and sequenced. Maximum-likelihood (ML) tree and Median-joining (MJ) haplotype network analyses were performed. We also performed scanning electron micrographs of adult specimens. Positivity rates were 93/328 (28.4%) in SIRN-AM, 6/297 (2.0%) in TER-PI, 0/605 (0%) in NSN-PI, and 6/543 (1.1%) in CAM-RJ. In NSN-PI it reached 11/53 (20.7%) in pigs. The MJ network based on cox1 locus (383 bp) revealed three main clusters, one centered around haplotypes H01/H28/H32 and the other around H07/H11. The cox1 haplotypes had a heterogeneous distribution, showing no pattern by geographic region, and high haplotype diversity. The ML trees based on cox1 and nad1 loci showed a similar topology with each other, and with the haplotype networks. Three distinct clusters were observed. Sequences of cox1 and nad1 from humans and animals were distributed throughout the tree and it was not possible to differentiate specimens of human and swine origin. Ascaris populations obtained from humans and swine in different Brazilian regions are not discriminable through the genetic markers used, which indicates the potential for zoonotic transmission and the need for better control of these infections in swine herds, mainly when created in a peridomestic environment.