Diagnosis of soil-transmitted helminth infections with digital mobile microscopy and artificial intelligence in a resource-limited setting.

BACKGROUND: Infections caused by soil-transmitted helminths (STHs) are the most prevalent neglected tropical diseases and result in a major disease burden in low- and middle-income countries, especially in school-aged children. Improved diagnostic methods, especially for light intensity infections, are needed for efficient, control and elimination of STHs as a public health problem, as well as STH management. Image-based artificial intelligence (AI) has shown promise for STH detection in digitized stool samples. However, the diagnostic accuracy of AI-based analysis of entire microscope slides, so called whole-slide images (WSI), has previously not been evaluated on a sample-level in primary healthcare settings in STH endemic countries. METHODOLOGY/PRINCIPAL FINDINGS: Stool samples (n = 1,335) were collected during 2020 from children attending primary schools in Kwale County, Kenya, prepared according to the Kato-Katz method at a local primary healthcare laboratory and digitized with a portable whole-slide microscopy scanner and uploaded via mobile networks to a cloud environment. The digital samples of adequate quality (n = 1,180) were split into a training (n = 388) and test set (n = 792) and a deep-learning system (DLS) developed for detection of STHs. The DLS findings were compared with expert manual microscopy and additional visual assessment of the digital samples in slides with discordant results between the methods. Manual microscopy detected 15 (1.9%) Ascaris lumbricoides, 172 (21.7%) Tricuris trichiura and 140 (17.7%) hookworm (Ancylostoma duodenale or Necator americanus) infections in the test set. Importantly, more than 90% of all STH positive cases represented light intensity infections. With manual microscopy as the reference standard, the sensitivity of the DLS as the index test for detection of A. lumbricoides, T. trichiura and hookworm was 80%, 92% and 76%, respectively. The corresponding specificity was 98%, 90% and 95%. Notably, in 79 samples (10%) classified as negative by manual microscopy for a specific species, STH eggs were detected by the DLS and confirmed correct by visual inspection of the digital samples. CONCLUSIONS/SIGNIFICANCE: Analysis of digitally scanned stool samples with the DLS provided high diagnostic accuracy for detection of STHs. Importantly, a substantial number of light intensity infections were missed by manual microscopy but detected by the DLS. Thus, analysis of WSIs with image-based AI may provide a future tool for improved detection of STHs in a primary healthcare setting, which in turn could facilitate monitoring and evaluation of control programs.

Serological diagnosis of soil-transmitted helminth (Ascaris, Trichuris and hookworm) infections: A scoping review.

BACKGROUND: The World Health Organization emphasizes the importance of integrated monitoring and evaluation in neglected tropical disease (NTD) control programs. Serological assays offer a potential solution for integrated diagnosis of NTDs, particularly for those requiring mass drug administration (MDA) as primary control and elimination strategy. This scoping review aims (i) to provide an overview of assays using serum or plasma to detect infections with soil-transmitted helminths (STHs) in both humans and animals, (ii) to examine the methodologies used in this research field and (iii) to discuss advancements in serological diagnosis of STHs to guide prevention and control programs in veterinary and human medicine. METHODOLOGY: We conducted a systematic search in the Ovid MEDLINE, Embase and Cochrane Library databases, supplemented by a Google search using predefined keywords to identify commercially available serological assays. Additionally, we performed a patent search through Espacenet. PRINCIPAL FINDINGS: We identified 85 relevant literature records spanning over 50 years, with a notable increased interest in serological assay development in recent years. Most of the research efforts concentrated on diagnosing Ascaris infections in both humans and pigs, primarily using ELISA and western blot technologies. Almost all records targeted antibodies as analytes, employing proteins and peptides as analyte detection agents. Approximately 60% of sample sets described pertained to human samples. No commercially available tests for Trichuris or hookworms were identified, while for Ascaris, there are at least seven different ELISAs on the market. CONCLUSIONS: While a substantial number of assays are employed in epidemiological research, the current state of serological diagnosis for guiding STH prevention and control programs is limited. Only two assays designed for pigs are used to inform efficient deworming practices in pig populations. Regarding human diagnosis, none of the existing assays has undergone extensive large-scale validation or integration into routine diagnostics for MDA programs.

Follicular larva migrans.

Follicular larva migrans (FLM) is a rare and atypical clinical presentation of hookworm-related cutaneous larva migrans (HrCLM). FLM is characterized clinically by follicular, round, small, erythematous papules that are sometimes topped by vesicles or pustules. These lesions are usually located on the abdomen, back, buttocks and thighs and are accompanied by more or less severe pruritus. Some typical and/or short and fragmented tracks may also be visible. FLM is more resistant to anti-helminthic drugs than classical HrCLM: this is likely due to the deep location of larvae in hair follicles. We present two cases of FLM and a review of the literature.

Egg excretion patterns of soil-transmitted helminth infections in humans following albendazole-ivermectin and albendazole treatment.

BACKGROUND: Control efforts of soil-transmitted helminthiases rely primarily on large scale administration of anthelminthic drugs. The assessment of drug efficacies and understanding of drug behavior is pivotal to the evaluation of treatment successes, both in preventive chemo-therapy programs as well as in research of novel treatment options. The current WHO guidelines recommend an interval of 14-21 days between the treatment and follow-up, yet no in-depth analysis of egg excretion patterns of Trichuris trichiura after treatment has been conducted to date. METHODS: Within the framework of a multi-country trial to assess the efficacy and safety of albendazole-ivermectin combination therapy vs albendazole monotherapy against T. trichiura infections, we conducted a study collecting daily stool samples over the period of 28 days post-treatment in 87 participants in Pak Khan, Lao PDR. Egg counts were derived by duplicate Kato-Katz on-site for T. trichiura, hookworm and Ascaris lumbricoides and stool sample aliquots were subsequently analyzed by qPCR for the detection of T. trichiura infections. Sensitivity and specificity was calculated for each day separately using data derived by Kato-Katz to determine the optimal timepoint at which to assess drug efficacy. RESULTS: Egg excretion patterns varied across treatment arms. For T. trichiura, only the albendazole-ivermectin treatment led to a considerable reduction in mean egg counts, whereas both treatments reduced hookworm egg counts and A. lumbricoides were cleared in all participants after day 7. For T. trichiura, we found sensitivity to be highest at days 18 and 22 when using egg counts as outcome and days 19 and 24 when using qPCR. Specificity was high (>0.9) from day 14 onwards. For hookworm, the highest sensitivity and specificity were found at days 17 and 25, respectively. CONCLUSIONS: Based on our study, the ideal time period to assess drug efficacy for soil-transmitted helminth infections would be between day 18 and 24. The current WHO recommendation of 14 to 21 days is likely to yield acceptable outcome measures for soil-transmitted helminth infections. TRIAL REGISTRATION: NCT03527732.

Pyrantel resistance in canine hookworms in Queensland, Australia.

Hookworms are the most common intestinal nematode parasites of dogs in Australia. The control of these parasites relies mostly on regular deworming with anthelmintics, with pyrantel-based dewormers being a relatively low cost and readily-available option for dog owners. Pyrantel resistance in canine hookworms in Australia was first reported in 2007, however pyrantel-based dewormers are still used against hookworm infection in dogs across Australia. The present study was conducted to evaluate the efficacy of pyrantel against hookworms infecting dogs housed in a shelter facility in Southeast Queensland which receives rescued or surrendered animals from greyhound rescue centres and dog shelters across this region. A total of 10 dogs were examined using the faecal egg count reduction test (FECRT). There was no reduction in FEC in any of the dogs following pyrantel treatment, with drug efficacies ranging from -0.9% to -283.3%. Given that these dogs originated from various sites across Southeast Queensland, the present study suggests that pyrantel resistance is widespread in this region, and hence this anthelmintic may not be a useful option for treatment of hookworm infections in dogs.

Hookworm Anthelmintic Resistance: Novel Fecal Polymerase Chain Reaction Ancylostoma caninum Benzimidazole Resistance Marker Detection in a Dog.

A 4 yr old castrated male greyhound presented with a history of chronic (>3 wk) intermittent diarrhea. Initial fecal analysis identified infection with Ancylostoma caninum. Despite treatment with routine anthelmintics, the dog remained persistently A caninum positive for several months. A novel fecal gastrointestinal real-time polymerase chain reaction (qPCR) parasite panel detected A caninum and the genetic benzimidazole (BZ) F167Y resistance marker in multiple samplings over 48 hr. This finding, together with the dog's clinical signs (diarrhea) and lack of response to routine anthelmintics, prompted treatment with cyclooctadepsipeptide emodepside, a drug currently not registered for dogs in the United States. The dog's clinical signs resolved and post-treatment fecal qPCR testing was negative. However, 5 mo later, retesting with fecal qPCR detected A caninum and concurrent BZ resistance marker, as well as Giardia. A presumptive diagnosis of re-infection was made and the emodepside treatment was continued. The dog again reverted to undetected (A caninum and the 167 resistance marker) on reassessment fecal qPCR. This case report describes the use of a novel fecal qPCR panel for gastrointestinal parasites, persistent hookworm and BZ F167Y resistance marker detection in a dog, and highlights the importance of a stepwise approach to clinical management, treatment, and retesting.

CANINE HOOKWORM PRESENCE IN TONGATAPU, TONGA.

Dogs are common in Tonga, where they exist in a close relationship with humans as pets and guard dogs and often are free-roaming due to limited property fencing and the need to scavenge for food. Little is known about the health status of these dogs and the type and level of pathogens carried by them. In this study, blood and fecal samples were analyzed to identify the presence of endoparasites and determine packed cell volume (PCV) values for dogs in Tonga. The current data deficit in this area made it necessary to develop a methodology and collect preliminary data to assist with future research in Tonga. Fecal analysis revealed hookworm in 73% of dogs, primarily those 4-12 mo old. Hematologic evaluation revealed low to borderline low PCV in 87% of dogs. The zoonotic nature of some of the pathogens identified poses a public health risk, with a particular risk for children.

Altered larval activation response associated with multidrug resistance in the canine hookworm Ancylostoma caninum.

Parasitic gastrointestinal nematodes pose significant health risks to humans, livestock, and companion animals, and their control relies heavily on the use of anthelmintic drugs. Overuse of these drugs has led to the emergence of resistant nematode populations. Herein, a naturally occurring isolate (referred to as BCR) of the dog hookworm, Ancylostoma caninum, that is resistant to 3 major classes of anthelmintics is characterized. Various drug assays were used to determine the resistance of BCR to thiabendazole, ivermectin, moxidectin and pyrantel pamoate. When compared to a drug-susceptible isolate of A. caninum, BCR was shown to be significantly resistant to all 4 of the drugs tested. Multiple single nucleotide polymorphisms have been shown to impart benzimidazole resistance, including the F167Y mutation in the ß-tubulin isotype 1 gene, which was confirmed to be present in BCR through molecular analysis. The frequency of the resistant allele in BCR was 76.3% following its first passage in the lab, which represented an increase from approximately 50% in the founding hookworm population. A second, recently described mutation in codon 134 (Q134H) was also detected at lower frequency in the BCR population. Additionally, BCR exhibits an altered larval activation phenotype compared to the susceptible isolate, suggesting differences in the signalling pathways involved in the activation process which may be associated with resistance. Further characterization of this isolate will provide insights into the mechanisms of resistance to macrocyclic lactones and tetrahydropyrimidine anthelmintics.

Nationwide USA re-analysis of amplicon metabarcoding targeting ß-tubulin isoform-1 reveals absence of benzimidazole resistant SNPs in Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala.

Nationwide sampling by Venkatesan and colleagues (2023) described the resistance status of the canine hookworm, Ancylostoma caninum, to benzimidazoles across the USA via ß-tubulin isotype-1 amplicon metabarcoding. In this study, we aimed to use the existing public amplicon metabarcoding data and mine it for the presence of ß-tubulin isotype-1 sequences that belong to hookworm species other than A. caninum. Through bioinformatics analysis we assigned species to A. caninum, Ancylostoma braziliense, Ancylostoma tubaeforme and Uncinaria stenocephala. All non-A. caninum sequences contained only the benzimidazole susceptible residues of ß-tubulin isotype-1. Using two ß-tubulin isotype-1 metabarcoding sequence data (assay targeting 134 and 167 codons, and assay targeting 198 and 200 codons), 2.0% (6/307) and 2.9% (9/310) individual samples had hookworms other than A. caninum (A. braziliense n = 5, A. tubaeforme n = 4 and U. stenocephala n = 2), respectively. We identified one sample containing A. braziliense in each of the Northeastern region and Midwestern region, and in three samples from the Southern region. Presence of A. tubaeforme in dog faeces is considered as pseudoparasitism. There were no statistically significant regional differences for the distribution of each species, for either of the two assays independently or combined (χ2 tests, P > 0.05). Our work demonstrates the utility of the amplicon metabarcoding for the identification of species through antemortem assays, thus resolving the dilemma of assigning hookworm species based on either post-mortem or egg sizes for the identification of hookworms.

Geographic Distribution of Human Infections with Zoonotic Ancylostoma ceylanicum and Anthropophilic Hookworms in Ecuador: A Retrospective Analysis of Archived Stool Samples.

Zoonotic human infections with Ancylostoma ceylanicum have recently been reported in the Americas. We used archived human stool samples to study the geographic distribution of human infections with A. ceylanicum and anthropophilic hookworms in different geoclimatic regions (coastal, Andean, and Amazon) of Ecuador. We analyzed retrospectively archived human stool samples from five studies previously screened for hookworm infection by microscopy, of which four included hookworm-positive samples only and one involved hookworm-negative samples to increase geographic distribution of sampling. Stools were analyzed using multi-parallel quantitative polymerase chain reaction (qPCR) assays to detect Necator americanus, Ancylostoma duodenale, A. ceylanicum, Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. Sequencing was done for the A. ceylanicum cox1 gene. A total of 132 samples were analyzed, of which 69 (52.3%) were from hookworm-positive and 63 (47.7%) from hookworm-negative individuals by microscopy. Overall, 82.6% of microscopy-positive samples and 33.3% of microscopy-negative samples were positive for hookworm by qPCR. Of microscopy-positive samples, 36.2% were A. ceylanicum, 37.7% A. duodenale, and 33.3% N. americanus, whereas equivalent proportions for microscopy-negative samples were 1.6%, 31.7%, and 1.6%, respectively. Ancylostoma duodenale was the most widely dispersed geographically, followed by N. americanus. Ancylostoma ceylanicum was least dispersed but was detected in coastal and Amazon regions. In conclusion, human infections with A. ceylanicum, A. duodenale, and N. americanus were detected in different geoclimatic regions of Ecuador. Additional studies are required to further define the epidemiology of human A. ceylanicum infections, but the potentially widespread presence of this helminth in human populations in Ecuador has implications for hookworm control strategies.

Molecular identification and genetic diversity of zoonotic hookworm infections in domestic dogs from northeastern, Thailand.

Hookworm infections remain a significant public health concern in tropical and subtropical regions, including Thailand. This study investigated the species and genetic diversity of hookworm infections in domestic dogs from northeastern Thailand. The molecular analysis focused on amplifying and sequencing specific regions of ribosomal RNA genes (ITS1-5.8S-ITS2 region) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene in hookworm larvae recovered from 21 domestic dog stool samples. Among 21 larvae (one larva per infected dog) analyzed, 14 had sequences identical to Ancylostoma caninum, and 7 showed sequences almost identical to Ancylostoma ceylanicum. Phylogenetic analysis of cox1 sequences placed A. caninum and A. ceylanicum in separate clades. The median-joining network of A. caninum cox1 sequences from Thailand showed high haplotype diversity and belonged to the same cluster as sequences from Australia while forming separate clusters from those of A. caninum samples from the USA. The available published A. ceylanicum cox1 sequences (n = 33), in combination with seven sequences in the present study, represented 15 haplotypes distributed among three clusters. Interestingly, A. ceylanicum sequences from dogs and humans shared the same haplotypes. These findings are crucial for recognizing the potential for zoonotic transmission, highlighting the necessity for targeted control measures, and increasing awareness among pet owners and healthcare professionals to mitigate the risk of hookworm transmission to humans.

Gastrointestinal parasite infections in Nepalese Gurkha recruits arriving in the United Kingdom from 2012-2020.

BACKGROUND: Gastrointestinal parasite (GIP) infections are a major cause of global morbidity, infecting hundreds of millions of people each year and potentially leading to lifelong infection and serious complications. Few data exist on screening for GIP infections in migrants entering the UK or on the current performance of different traditional diagnostic approaches. This study aimed to describe the prevalence of GIP infections in Nepalese Gurkha recruits screened on arrival in the UK. METHODOLOGY/PRINCIPAL FINDINGS: We present a retrospective analysis of data from screening male adults (18-21 years) who arrived in the UK from Nepal between 2012 and 2020. Three separate faecal samples were obtained from participants at weekly intervals and processed for formalin-ethyl acetate (FEA) concentration/light microscopy and charcoal culture. Serum samples were analysed for IgG antibodies to Strongyloides stercoralis by ELISA. Results were available from 2,263 participants, of whom 463 (20.5%, 95% CI 18.8%-22.2%) had a positive diagnostic test for at least one GIP infection. A total of 525 potential infections were identified. Giardia duodenalis was most common (231/2263, 10.2%), followed by S. stercoralis (102/2263, 4.5%), and hookworm species (86/2263, 3.8%). Analysis (microscopy and culture) of the initial stool sample diagnosed only 244/427 (57.1%) faecally identified pathogens, including 41/86 (47.7%) hookworm infections. The proportion of participants infected with any GIP showed a downward trend over the study period. Log-binomial regression showed risk of infection decreasing by 6.1% year-on-year (95% CI 3.2% - 9.0%). This was driven predominantly by a fall in hookworm, S. stercoralis and Trichuris trichiura prevalence. CONCLUSIONS/SIGNIFICANCE: The level of potentially pathogenic GIP infection in young Nepalese men migrating to the UK is high (20.5%) and requires a combined diagnostic approach including serology and analysis of multiple stool samples incorporating specialised parasitological methods. Advances in molecular approaches may optimise and simplify the intensive screening strategy required.

Benzimidazole F167Y polymorphism in the canine hookworm, Ancylostoma caninum: Widespread geographic, seasonal, age, and breed distribution in United States and Canada dogs.

Surveillance data for Ancylostoma spp. and the A. caninum benzimidazole treatment resistance associated F167Y polymorphism using molecular diagnostics was obtained in a large population of dogs from the United States and Canada. Real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting a single nucleotide polymorphism (SNP) F167Y was used in 262,872 canine stool samples collected between March and December of 2022. Ancylostoma spp. was found at an overall prevalence of 2.5% (6538/262,872), with the highest prevalence in the Southern US, 4.4% (4490/103,095), and the lowest prevalence in Canada 0.6% (101/15,829). The A. caninum F167Y polymorphism was found with the highest prevalence (13.4%, n = 46/343) in the Western US and the lowest in Canada at 4.1% (4/97). The F167Y polymorphism was detected every month over the 10-month collection period. Seasonal distribution showed a peak in June for both Ancylostoma spp. (3.08%, 547/17,775) and A. caninum F167Y (12.25%, 67/547). However, the A. caninum F167Y polymorphism prevalence was highest in September (13.9%, 119/856). Age analysis indicates a higher prevalence of both hookworm infections and occurrence of resistant isolates in puppies. The breeds with the highest F167Y polymorphism prevalence in Ancylostoma spp. detected samples were poodles (28.9%), followed by Bernese Mountain dogs (25%), Cocker spaniels (23.1%), and greyhounds (22.4%). Our data set describes widespread geographic distribution of the A. caninum benzimidazole resistance associated F167Y polymorphism in the United States and Canada, with no clear seasonality compared to the Ancylostoma spp. prevalence patterns. The F167 polymorphism was present in all geographic areas with detected hookworms, including Canada. Our study highlights that the F167Y polymorphism is represented in many dog breeds, including greyhounds.

Hookworm infestation as a rare cause of melaena in tropics: A case report.

Melaena due to hookworm infestation is a rare clinical presentation. It usually presents with symptoms of iron-deficiency anaemia owing to slow blood loss. Here we present a case of 45-year male who presented with a one-year history of intermittent melena requiring multiple blood transfusions. Preliminary endoscopies at different centres were normal. The presence of hookworm in the duodenum was only detected on repeat upper endoscopy as the cause of blood loss, and treatment with albendazole was successful in curing the patient.

Immunity mediates host specificity in the human hookworm Ancylostoma ceylanicum.

Hookworm infection affects millions globally, leading to chronic conditions like malnutrition and anaemia. Among the hookworm species, Ancylostoma ceylanicum stands out as a generalist, capable of infecting various hosts, including humans, cats, dogs and hamsters. Surprisingly, it cannot establish in mice, despite their close phylogenetic relationship to hamsters. The present study investigated the development of A. ceylanicum in immunodeficient NSG mice to determine the contribution of the immune system to host restriction. The infections became patent on day 19 post-infection (PI) and exhibited elevated egg production which lasted for at least 160 days PI. Infective A. ceylanicum larvae reared from eggs released by infected NSG mice were infectious to hamsters and capable of reproduction, indicating that the adults in the NSG mice were producing viable offspring. In contrast, A. ceylanicum showed limited development in outbred Swiss Webster mice. Furthermore, the closely related canine hookworm Ancylostoma caninum was unable to infect and develop in NSG mice, indicating that different mechanisms may determine host specificity even in closely related species. This is the first report of any hookworm species completing its life cycle in a mouse and implicate the immune system in determining host specificity in A. ceylanicum.

Hookworms dynamically respond to loss of Type 2 immune pressure.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.

Anthelmintic treatment follow up in a rural community in Camamu, Bahia, Brazil.

Enteroparasites are an important public health problem and the treatment seeks to cure and reduce transmission. The aim of this study was to evaluate the therapeutic efficacy of anthelmintic treatment in individuals living in a rural community area in Camamu, Bahia, Brazil. The parasitological diagnosis was performed by spontaneous sedimentation, Baermann-Moraes and Agar Plate Culture methods. A total of 212 individuals were evaluated. The most frequent helminth was Trichuris trichiura, 24.5% (52/212), followed by Ascaris lumbricoides, 21.2% (45/212), hookworms, 16.5% (35/212), and S. stercoralis, 4.7% (10/212). In the anthelmintic treatment follow up, T. trichiura infection presented the lowest parasitological cure rate, only 60.6% (20/33). Hookworm, Ascaris lumbricoides and Strongyloides stercoralis infections demonstrated cure rates of 70.5 (12/17), 78.1 (25/32) and 100% (5/5), respectively. Individuals who remained infected underwent a new drug therapy. The second parasitological cure rate for T. trichiura was 38.5% (5/13), and 66.7% (2/3) and 75% (3/4) for hookworms and Ascaris lumbricoides, respectively. Trichuris trichiura infection presented the lowest parasitological cure rate at this second evaluation. This reinforces the need to perform a follow-up of all treated individuals. The possibility of drug resistance denotes the necessity for studies to clarify the mechanisms and to evaluate new therapeutic approaches.

Distribution and treatment needs of soil-transmitted helminthiasis in Bangladesh: A Bayesian geostatistical analysis of 2017-2020 national survey data.

BACKGROUND: In Bangladesh, preventive chemotherapy targeting soil-transmitted helminth (STH) infections in school-age children has been implemented since 2008. To evaluate the success of this strategy, surveys were conducted between 2017 and 2020 in 10 out of 64 districts. We estimate the geographic distribution of STH infections by species at high spatial resolution, identify risk factors, and estimate treatment needs at different population subgroups. METHODOLOGY: Bayesian geostatistical models were fitted to prevalence data of each STH species. Climatic, environmental, and socioeconomic predictors were extracted from satellite images, open-access, model-based databases, and demographic household surveys, and used to predict the prevalence of infection over a gridded surface at 1 x 1 km spatial resolution across the country, via Bayesian kriging. These estimates were combined with gridded population data to estimate the number of required treatments for different risk groups. PRINCIPAL FINDINGS: The population-adjusted prevalence of Ascaris lumbricoides, Trichuris trichiura, and hookworm across all ages is estimated at 9.9% (95% Bayesian credible interval: 8.0-13.0%), 4.3% (3.0-7.3%), and 0.6% (0.4-0.9%), respectively. There were 24 out of 64 districts with an estimated population-adjusted STH infection prevalence above 20%. The proportion of households with improved sanitation showed a statistically important, protective association for both, A. lumbricoides and T. trichiura prevalence. Precipitation in the driest month of the year was negatively associated with A. lumbricoides prevalence. High organic carbon concentration in the soil's fine earth fraction was related to a high hookworm prevalence. Furthermore, we estimated that 30.5 (27.2; 36.0) million dosages of anthelmintic treatments for school-age children were required per year in Bangladesh. CONCLUSIONS/SIGNIFICANCE: For each of the STH species, the prevalence was reduced by at least 80% since treatment was scaled up more than a decade ago. The current number of deworming dosages could be reduced by up to 61% if the treatment strategy was adapted to the local prevalence.

Epidemiological and molecular updates on hookworm species in dogs from southern Italy.

BACKGROUND: The zoonotic hookworms Ancylostoma caninum and Uncinaria stenocephala are widespread soil-transmitted helminths in dogs in Europe. Given the veterinary and public health importance of hookworms in dogs and the recent changes in the molecular epidemiology of some species, there is a need to continuously monitor the epidemiological and molecular prevalence of these parasites also at the "local" level. The present study aimed to update the epidemiological scenario of hookworm infections in both owned and stray dogs in southern Italy and to discriminate between different hookworm species (A. caninum and U. stenocephala) through molecular analyses. For this purpose, a retrospective analysis was performed over 10 years (2011-2021), including a total of 7008 owned dogs and 5642 stray dogs referred to our laboratory for copromicroscopic examinations. Moreover, 72 faecal samples, from dogs naturally infected by hookworms, were used to discriminate between A. caninum and U. stenocephala using two PCR protocols. Prior to molecular analyses, a subsample of 40/72 positive faecal samples was used for morphometric investigations on hookworm eggs. RESULTS: The results of the ten-year retrospective analysis (2011-2021) showed an overall prevalence of hookworm infection of 9.16%, specifically 5.1% in owned dogs and 14.2% in stray dogs. Logistic regression showed a significant association between positivity to hookworms and the variable "puppies" both in stray (13.84%; OR = 2.4) and owned (7.07%; OR = 2.2) dogs. The results of molecular analyses showed that positivity was confirmed only in 21/72 samples, specifically, 6 samples using protocol A and 19 with protocol B. Sequencing revealed 15 samples positive to U. stenocephala and 6 to A. caninum. CONCLUSIONS: The findings of this study showed a high prevalence of hookworm infections in dogs in southern Italy, updating the epidemiological scenario of the last decade. Moreover, the results of the study revealed the first identification of hookworm species in dogs in Italy by molecular studies, highlighting that U. stenocephala is more prevalent than A. caninum.

Assessment of the capacity of Whatman filter papers as support to store stools for the molecular diagnostic testing of soil-transmitted helminthiasis.

Storage of stools for the detection of soil-transmitted helminths (STH) remains challenging for the molecular diagnostic testing of STH infections. This study aimed to overcome this challenge by assessing the capacity of Whatman filter papers to store stools for the molecular detection of STHs. Stool samples were collected from school-aged children of soil-transmitted helminthiasis endemic areas of Cameroon and then, analysed using Kato Katz technique. For this study, 128 and 40 stool samples respectively with and without STH eggs were analysed. From each sample, 10, 20, 40 and 80 mg of stool were weighted and spread on 6 grades of Whatman filter papers that were stored at room temperature from one to ten weeks. DNA was extracted from spread stool using CTAB based-method. The amount of stool to spread on filter papers and the grade of filter paper offering good storage were determined by amplifying specific DNA fragments of Ascaris lumbricoides. The capacity of filter papers to store stool samples for several weeks before the molecular detection of STH species was assessed by amplifying specific DNA fragments of different STHs. The amplification rates of A. lumbricoides were significantly higher (P < 0.0001) for 10 and 20 mg of stored stools. Stools spread on Whatman paper grade 2 yielded the highest amplification rate of 100% for A. lumbricoides, T. trichiura and hookworm. PCR revealed STH infections in all the 128 spread stools carrying STH eggs. It also revealed Necator americanus and Ancylostoma duodenale respectively in 10 and 13 of 15 spread stools contained hookworm eggs. PCR confirmed the co-infections of these hookworm species as well as that of A. lumbricoides and Trichuris trichiura in 7 spread stools. Out of 40 stools without STH eggs, PCR revealed that 5 (12.5%) and 9 (22.5%) had respectively A. lumbricoides and T. trichiura infections. The amplification rate of each STH species was 100% from one to 8 weeks and decreased to 86.7% after 10 weeks of storage. This study highlighted the capacity of filter papers to store stools for the molecular detection of STHs. Storing stools on these papers will enable to monitor and evaluate control programs and ensure post-elimination surveillance.