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.

Management of paediatric soil-transmitted helminthiasis in a non-endemic area: experience in a reference international health unit.

Soil-transmitted helminth (STH) infections inflict disability worldwide, especially in the poorest communities. Current therapeutic options against STHs show limited efficacy, particularly against Trichuris trichiura. The empirical management of patients coming from high-prevalence areas has been suggested for non-endemic areas. This study aimed to describe the management of STH infections in a non-endemic setting using an individualised approach. We performed a retrospective, descriptive study of all patients up to 16 years of age with STH infections attended at an international health unit in a non-endemic area (2014-2018), including all T. trichiura, Necator americanus, Ancylostoma duodenale, and Ascaris lumbricoides infections diagnosed using a formol-ether concentration technique and direct visualisation. Patients were treated according to current international guidelines. Sixty-one stool samples from 48 patients testing positive for STHs were collected, with 96% (46/48) reporting a previous long-term stay in endemic areas. Cure rates with 3-day benzimidazole regimens were 72% for T. trichiura, 40% for hookworms, and 83% for A. lumbricoides. The results were not influenced by any reinfection risk due to the study being performed in a non-endemic area. Patients coming from STH-endemic areas should be evaluated with appropriate diagnostic tools and followed up until cure control results. Cure rates in our cohort were moderate to low, similar to those published in studies in endemic areas. The efficacy of current treatment options is insufficient to recommend a specific empirical approach in high-income countries' healthcare systems.

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.

Intestinal helminthic infections: a narrative review to guide the hepatogastroenterologist.

Intestinal helminthic infections are not uncommon in Western Europe, mainly due to modern travel, emigration and globalization. Moreover, some helminthic infections are endemic in Western Europe and are part of the everyday clinical practice. The hepatogastroenterologist should therefore recognize and manage these patients or at least refer them to appropriate reference centers. Signs and symptoms are often unspecific or even absent. Discerning the disease at an early stage avoids expensive diagnostic testing, life-threatening complications and in some cases even further spread of the disease. This review article aims to guide the hepatogastroenterologist when suspecting a helminthic infection by addressing the most prevalent symptoms, summarizing the most probable associated helminthic entities, highlighting practical steps in diagnosis and available treatments.

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.

A general framework to support cost-efficient survey design choices for the control of soil-transmitted helminths when deploying Kato-Katz thick smear.

BACKGROUND: To monitor and evaluate soil-transmitted helminth (STH) control programs, the World Health Organization (WHO) recommends screening stools from 250 children, deploying Kato-Katz thick smear (KK). However, it remains unclear whether these recommendations are sufficient to make adequate decisions about stopping preventive chemotherapy (PC) (prevalence of infection <2%) or declaring elimination of STHs as a public health problem (prevalence of moderate-to-heavy intensity (MHI) infections <2%). METHODOLOGY: We developed a simulation framework to determine the effectiveness and cost of survey designs for decision-making in STH control programs, capturing the operational resources to perform surveys, the variation in egg counts across STH species, across schools, between and within individuals, and between repeated smears. Using this framework and a lot quality assurance sampling approach, we determined the most cost-efficient survey designs (number of schools, subjects, stool samples per subject, and smears per stool sample) for decision-making. PRINCIPAL FINDINGS: For all species, employing duplicate KK (sampling 4 to 6 schools and 64 to 70 subjects per school) was the most cost-efficient survey design to assess whether prevalence of any infection intensity was above or under 2%. For prevalence of MHI infections, single KK was the most cost-efficient (sampling 11 to 25 schools and 52 to 84 children per school). CONCLUSIONS/SIGNIFICANCE: KK is valuable for monitoring and evaluation of STH control programs, though we recommend deploying a duplicate KK on a single stool sample to stop PC, and a single KK to declare the elimination of STHs as a public health problem.

A general framework to support cost-efficient fecal egg count methods and study design choices for large-scale STH deworming programs-monitoring of therapeutic drug efficacy as a case study.

BACKGROUND: Soil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool. METHODS: We performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species (Ascaris lumbricoides, Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select (SS); screen, select and retest (SSR) and no selection (NS)) and number of subjects enrolled (100-5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design. PRINCIPAL FINDINGS: Kato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAKG2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAKG2) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity. CONCLUSIONS/SIGNIFICANCE: We confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO (SS) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.

Feasibility of Training Community Health Workers to Use Smartphone-Attached Microscopy for Point-of-Care Visualization of Soil-Transmitted Helminths in the Peruvian Amazon.

The prevalence of soil-transmitted helminths (STH) is high in communities within the Peruvian Amazon despite repeated mass-drug administration, demanding alternative strategies of control. Smartphone-attached microscopy (SAM) permits visualization of STH from a small portable microscope through a smartphone screen, potentially providing an inexpensive and rapid method of STH visualization in communities where diagnostic laboratories with microscopes are inaccessible. In this study, a total of 45 community health workers who work within the health systems of Loreto, Peru, attended a 1-day training session with lectures and practicums on STH and SAM. Participants received a pre- and post-intervention questionnaire. Post-intervention, participants were significantly more confident using SAM and identifying parasite images, symptoms, transmission, and treatment (P ≤ 0.0045). Post-intervention, participants correctly labeled a median of five of seven SAM apparatus components and five of eight steps of Kato-Katz technique, were less likely to choose taking medicine to prevent parasite infection (P = 0.0075), and were more likely to select Kato-Katz technique as a type of diagnostic test (P < 0.0001). Most participants felt ready to use SAM in their communities and stated that it could help rural communities far from health centers or laboratories (24%); provide faster identification, results, diagnosis (19%); permit at-home or on-the-spot visualization (14%); and save money (14%). Results show that community health workers show a high level of willingness and competency to learn about both STH and SAM and may be a yet-unexplored practical method of augmenting STH visualization, bringing healthcare to communities in Loreto with poor access to diagnostic laboratories and clinics.

Intestinal helminths-an outlander in a case of protein losing enteropathy.

Malabsorption is the major disease burden in tropical countries. Both primary and secondary forms exist and a secondary form overshadows the primary category. Intestinal parasitic infections lead to secondary form of tropical malabsorption in both native and travelers and presentation varies from mild glossitis to severe protein losing enteropathy. The underlying condition is often masked unless an endoscopic biopsy is performed. This is followed by a histopathological examination which unravels the etiology.

A review on innovative optical devices for the diagnosis of human soil-transmitted helminthiasis and schistosomiasis: from research and development to commercialization.

Diagnosis of soil-transmitted helminth (STH) and schistosome infections relies largely on conventional microscopy which has limited sensitivity, requires highly trained personnel and is error-prone. Rapid advances in miniaturization of optical systems, sensors and processors have enhanced research and development of digital and automated microscopes suitable for the detection of these diseases in resource-limited settings. While some studies have reported proof-of-principle results, others have evaluated the performance of working prototypes in field settings. The extensive commercialization of these innovative devices has, however, not yet been achieved. This review provides an overview of recent publications (2010­2022) on innovative field applicable optical devices which can be used for the diagnosis of STH and schistosome infections. Using an adapted technology readiness level (TRL) scale taking into account the WHO target product profile (TPP) for these diseases, the developmental stages of the devices were ranked to determine the readiness for practical applications in field settings. From the reviewed 18 articles, 19 innovative optical devices were identified and ranked. Almost all of the devices (85%) were ranked with a TRL score below 8 indicating that, most of the devices are not ready for commercialization and field use. The potential limitations of these innovative devices were discussed. We believe that the outcome of this review can guide the end-to-end development of automated digital microscopes aligned with the WHO TPP for the diagnosis of STH and schistosome infections in resource-limited settings.

A Pilot Comparison of Fixatives for Hookworm Real-time Polymerase Chain Reaction.

Polymerase chain reaction (PCR) is increasingly used in the diagnosis of soil-transmitted helminth infections. Despite this, few studies have evaluated the impact of different fecal fixatives on the outcome of fecal helminth qPCR analysis, and none have evaluated the effect of commercial parasitology fixatives commonly used in diagnostic laboratories. We fixed dog feces containing Ancylostoma spp. hookworm eggs in zinc polyvinyl alcohol (Zn-PVA) and Total-Fix, and with 70% ethanol (EtOH) as a control. DNA was extracted at timepoints 11, 33, 64, and 94 days and subjected to Ancylostoma spp. quantitative PCR (qPCR). A linear regression model was created to assess the effect of preservative types on the temporal change of qPCR quantification cycle number (Cq) values, accounting for variances among individual animals. Fixation in 70% EtOH least affected Cq values over 94 days. Total-Fix preservation yielded a higher Cq overall, but there was no significant difference compared with 70% EtOH fixation. Fixation in Zn-PVA resulted in significantly (P < 0.001) higher Cq values than 70% EtOH after only 33 days and loss of amplification at 64 days. Consistent with other helminth fixation studies, 70% EtOH performed well in preserving hookworm DNA over 94 days. Total-Fix provided a comparable alternative for qPCR analysis for hookworm. Fixation in Zn-PVA resulted in loss of detectable hookworm DNA at 64 days, as determined by qPCR.

Assessment of cetyl-trimethyl-ammonium bromide (CTAB) based method for the extraction of soil-transmitted helminth DNAs from stools for molecular dagnostic of soil-transmitted helminthiasis.

Although several protocols were developed to extract DNA for soil-transmitted helminthiasis diagnostic, amplifying these extracts remains challenging due to DNA polymerase inhibitors. This study aimed to assess a DNA extraction method for efficient detection of soil-transmitted helminth species by determining stool mass and the type of DNA polymerase that can be used for this extraction method. For this study, 141 stool samples harbouring soil-transmitted eggs and 50 samples without egg were obtained from school-aged children of Makenene in the Centre region of Cameroon. DNA was extracted from 10, 20, 40 and 80 mg of stool using commercial kit and/or cetyltrimethylammonium bromide (CTAB)-based method. The amount of stool for molecular diagnostic of soil-transmitted helminthiasis was determined by amplifying Ascaris lumbricoides DNA. The performances of three DNA polymerases and CTAB-based method were assessed by amplifying DNA of different soil-transmitted helminth species. For this study, 94 stools with A. lumbricoides eggs, 39 with Trichuris trichuria and 15 with hookworm were analyzed. DNA of A. lumbricoides, T. trichuria, Necator americanus and Ancylostoma duodenale were detected in 97.9% of extracts from stools harbouring soil-transmitted helminth eggs. Soil-transmitted helminth DNAs were significantly (X2 = 17.66; df = 3; p ã€ˆ00001) more amplified in extracts from 10 and 20 mg than those from 40 and 80 mg. The amplification rate with "Q5 high fidelity DNA polymerase" was significantly (X2 = 30.54; df = 2; p < 0.00001) higher than that of other DNA polymerases. Multiplex-PCR confirmed co-infections of A. lumbricoides with either T. trichuria or N. americanus. The extraction cost for the CTAB-based method was $1.45. This method appearedis reliable and 3 times cost effective than commercial kit. Its combination with the "Q5 high fidelity DNA polymerase" may improve soil-transmitted helminthiasis diagnostic.

Comparison of coproprevalence and seroprevalence to guide decision-making in national soil-transmitted helminthiasis control programs: Ethiopia as a case study.

BACKGROUND: WHO recommends periodical assessment of the prevalence of any soil-transmitted helminth (STH) infections to adapt the frequency of mass drug administration targeting STHs. Today, detection of eggs in stool smears (Kato-Katz thick smear) remains the diagnostic standard. However, stool examination (coprology) has important operational drawbacks and impedes integrated surveys of multiple neglected tropical diseases. Therefore, the aim of the present study was to assess the potential of applying serology instead of coprology in STH control program decision-making. METHODOLOGY: An antibody-ELISA based on extract of Ascaris lung stage larvae (AsLungL3-ELISA) was applied in ongoing monitoring activities of the Ethiopian national control program against schistosomiasis and soil-transmitted helminthiasis. Blood and stool samples were collected from over 6,700 students (median age: 11) from 63 schools in 33 woredas (districts) across the country. Stool samples of two consecutive days were analyzed applying duplicate Kato-Katz thick smear. PRINCIPAL FINDINGS: On woreda level, qualitative (seroprevalence) and quantitative (mean optical density ratio) serology results were highly correlated, and hence seroprevalence was chosen as parameter. For 85% of the woredas, prevalence based on serology was higher than those based on coprology. The results suggested cross-reactivity of the AsLungL3-ELISA with Trichuris. When extrapolating the WHO coproprevalence thresholds, there was a moderate agreement (weighted κ = 0.43) in program decision-making. Using the same threshold values would predominantly lead to a higher frequency of drug administration. SIGNIFICANCE: This is the first time that serology for soil-transmitted helminthiasis is applied on such large scale, thereby embedded in a control program context. The results underscore that serology holds promise as a tool to monitor STH control programs. Further research should focus on the optimization of the diagnostic assay and the refinement of serology-specific program decision-making thresholds.

Performance of Mini Parasep® SF stool concentrator kit, Kato-Katz, and formalin-ethyl acetate concentration methods for diagnosis of opisthorchiasis in Northeast Thailand.

BACKGROUND: Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections. METHODS: A cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases. RESULTS: The initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05). CONCLUSIONS: As the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis.

Affordable artificial intelligence-based digital pathology for neglected tropical diseases: A proof-of-concept for the detection of soil-transmitted helminths and Schistosoma mansoni eggs in Kato-Katz stool thick smears.

BACKGROUND: With the World Health Organization's (WHO) publication of the 2021-2030 neglected tropical diseases (NTDs) roadmap, the current gap in global diagnostics became painfully apparent. Improving existing diagnostic standards with state-of-the-art technology and artificial intelligence has the potential to close this gap. METHODOLOGY/PRINCIPAL FINDINGS: We prototyped an artificial intelligence-based digital pathology (AI-DP) device to explore automated scanning and detection of helminth eggs in stool prepared with the Kato-Katz (KK) technique, the current diagnostic standard for diagnosing soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworms) and Schistosoma mansoni (SCH) infections. First, we embedded a prototype whole slide imaging scanner into field studies in Cambodia, Ethiopia, Kenya and Tanzania. With the scanner, over 300 KK stool thick smears were scanned, resulting in total of 7,780 field-of-view (FOV) images containing 16,990 annotated helminth eggs (Ascaris: 8,600; Trichuris: 4,083; hookworms: 3,623; SCH: 684). Around 90% of the annotated eggs were used to train a deep learning-based object detection model. From an unseen test set of 752 FOV images containing 1,671 manually verified STH and SCH eggs (the remaining 10% of annotated eggs), our trained object detection model extracted and classified helminth eggs from co-infected FOV images in KK stool thick smears, achieving a weighted average precision (± standard deviation) of 94.9% ± 0.8% and a weighted average recall of 96.1% ± 2.1% across all four helminth egg species. CONCLUSIONS/SIGNIFICANCE: We present a proof-of-concept for an AI-DP device for automated scanning and detection of helminth eggs in KK stool thick smears. We identified obstacles that need to be addressed before the diagnostic performance can be evaluated against the target product profiles for both STH and SCH. Given that these obstacles are primarily associated with the required hardware and scanning methodology, opposed to the feasibility of AI-based results, we are hopeful that this research can support the 2030 NTDs road map and eventually other poverty-related diseases for which microscopy is the diagnostic standard.

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.

Spurious infection by Calodium hepaticum (Bancroft, 1983) Moravec, 1982 and intestinal parasites in forest reserve dwellers in Western Brazilian Amazon.

Subsistence hunting is the main source of protein for forest reserve dwellers, contributing to the development of spurious infections by Calodium hepaticum, frequently associated with the consumption of the liver from wild mammals. The prevalence of infections by soil-transmitted helminths (STHs) and intestinal protozoa is considered an indicator of the social vulnerability of a country, besides providing information on habits, customs and quality of life of a given population. Intestinal parasites mostly affect poor rural communities with limited access to clean water and adequate sanitation. This study reports the results of a parasitological survey carried out in 2017 and 2019, in two municipalities (Xapuri and Sena Madureira) in Acre State. Stool samples were collected from 276 inhabitants. Upon receipt, each sample was divided into two aliquots. Fresh samples without preservative were processed and examined by the Kato-Katz technique. Samples fixed in 10% formalin were processed by the spontaneous sedimentation and the centrifugal sedimentation techniques. Calodium hepaticum eggs were found in three stool samples. The overall STH prevalence was 44.9%. The hookworm prevalence (19.2%) was higher than that of Ascaris lumbricoides (2.5%) and Trichuris trichiura (0.7%), an unexpected finding for municipalities belonging to the Western Brazilian Amazon. When considering parasites transmitted via the fecal-oral route, Endolimax nana and Entamoeba coli showed the highest positivity rates, of 13% and 10.9%, respectively. This study is the first report of spurious infection by C. hepaticum among forest reserve dwellers that consume undercooked liver of lowland pacas. Additionally, this is the first report of Blastocystis sp. in Acre State.