Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

Retrospective analysis of canine fecal flotation and coproantigen immunoassay hookworm positive results in Greyhounds and other dog breeds.

Recent studies demonstrated that Greyhounds are commonly infected with Ancylostoma caninum and these infections have been shown to be resistant to anthelmintics. This study evaluated samples submitted to a commercial reference laboratory (IDEXX Laboratories) for canine fecal flotation zinc sulfate centrifugation and coproantigen immunoassay between January 1, 2019, and July 30, 2023 for evidence that Greyhounds were more often positive for Ancylostoma spp. (hookworms) compared to other breeds. The purpose of the study was to determine if Greyhounds were more likely to be hookworm-positive compared to other breeds, if Greyhounds on preventives with efficacy against hookworm infections are more likely to test positive than other breeds, if their infections take longer to resolve, to estimate how long this takes and to assess whether the proportion of hookworm positive tests for all breeds is increasing over time. Records of 25,440,055 fecal results were obtained representing 17,671,724 unique dogs. Of these, 49,795 (∼0.3%) were Greyhounds. The overall odds ratio (OR) of 15.3 (p < 0.001) suggests that Greyhounds are at significantly higher risk than other breeds for hookworm positive float findings, and the OR of 14.3 (p < 0.001) suggests significantly higher risk for hookworm antigen positive results. The median time to negative testing event from the Turnbull distribution estimate was in the interval of 1-2 days for other breeds and 71-72 days for Greyhounds. These results provide evidence that anthelmintic resistant A. caninum strains may be having population-level impacts on the frequency and duration of infections in Greyhounds. The findings have broader health implications beyond Greyhounds as MADR A. caninum strains could spread to other breeds and even pet owners.

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.

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.

[Interpretation of Detection and identification standard of hookworm-Hook-worm larvae coproculture techniques (WS/T 791-2021)].

Detection and identification standard of hookworm-Hookworm larvae coproculture techniques (WS/T 791-2021) is the first recommended technical standard for hookworm detection and species identification using the hookworm larvae coproculture technique in China. This standard was issued on November 23, 2021, and had been in effect since May 1, 2022. This article provides a detailed interpretation pertaining to the background, drafting process, main contents, and dos and don'ts for better understanding and application of this standard among professionals working in disease control and prevention institutions and medical institutions.

Chronic Hemorrhagic Anemia Caused by Hookworm Infection: A Case Report.

PURPOSE: Hookworm infection is one of the causes of long-term chronic hemorrhagic anemia in patients. This article reports a case of chronic severe anemia caused by hookworm infection. METHODS: The capsule endoscopy showed that there were a large number of hookworms in the small intestine of a patient. At the same time, using the technique of saturated brine floatation and the automatic stool analyzer, hookworm eggs were detected. RESULTS: The patient's anemia was caused by hookworm infection and was significantly improved after anti-hookworm treatment. CONCLUSION: Hookworm infection cannot be ignored in the differential diagnosis of patients with chronic anemia. Capsule endoscopy combined with stool detection haves an important clinical value for the diagnosis of hookworm disease.

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.

Modeling the effect of different drugs and treatment regimen for hookworm on cure and egg reduction rates taking into account diagnostic error.

BACKGROUND: Hookworm infections, caused by Ancylostoma duodenale and Necator americanus, are of considerable public health importance. The World Health Organization recommends preventive chemotherapy as the key strategy for morbidity control. Meta-analyses have been conducted to estimate treatment efficacy of available drugs and drug combinations. However, in most studies, the relation between the diagnostic error and infection intensity have not been considered, resulting in an overestimation of cure rates (CRs). METHODOLOGY: A Bayesian model was developed to compare the 'true' CR and egg reduction rate of different treatment regimens for hookworm infections taking into account the error of the recommended Kato-Katz thick smear diagnostic technique. It was fitted to the observed egg count data which was linked to the distribution of worms, considered the day-to-day variation of hookworm egg excretion and estimated the infection intensity-dependent sensitivity. The CR was obtained by defining the prevalence of infection at follow-up as the probability of having at least one fertilized female worm. The model was applied to individual-level egg count data available from 17 treatments and six clinical trials. PRINCIPAL FINDINGS: Taking the diagnostic error into account resulted in considerably lower CRs than previously reported. Overall, of all treatments analyzed, mebendazole administered in six dosages of 100 mg each was the most efficacious treatment with a CR of 88% (95% Bayesian credible interval: 79-95%). Furthermore, diagnostic sensitivity varied with the infection intensity and sampling effort. For an infection intensity of 50 eggs per gram of stool, the sensitivity is close to 60%; for two Kato-Katz thick smears it increased to approximately 76%. CONCLUSIONS/SIGNIFICANCE: Our model-based estimates provide the true efficacy of different treatment regimens against hookworm infection taking into account the diagnostic error of the Kato-Katz method. Estimates of the diagnostic sensitivity for different number of stool samples and thick smears are obtained. To accurately assess efficacy in clinical trials with the Kato-Katz method, at least two stool samples on consecutive days should be collected.

A case of severe hookworm infection. / 临床极重症钩虫感染1例.

With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.

Ancylostoma ceylanicum Infection in a Miniature Schnauzer Dog Breed.

PURPOSE: Canine hookworm disease is a global zoonotic parasitic disease caused by a variety of nematodes in families Ancylostomatidae, including Ancylostoma spp., Necator spp., and Uncinaria spp., in the small intestine (mainly the duodenum) of dogs. The disease is widely distributed in China. The purpose of this study is to systematically diagnose and treat canine hookworm disease through the case of miniaturization Schnauzer dog feed infected with A. ceylanicum, so as to provide experimental basis for subsequent prevention and control of canine hookworm disease. METHODS: In the current study, we isolated hookworm eggs from a diseased miniature schnauzer, then the polymerase chain reaction (PCR) was used to amplify the ITS1-5.8S-ITS2 gene sequence from genomic DNA extracted from hookworms. Phylogenetic analysis based on ITS1-5.8S-ITS2 gene sequence sequences was inferred using MEGA-X. After phylogenetic analysis, etiologic and symptomatic therapies were used to treat the canine hookworm disease. RESULTS: The sequencing results showed that the length of the ITS1-5.8S-ITS2 gene sequence was approximately 960 bp, and ITS1 and ITS2 were extracted to analyze similarity with other hookworms to build a phylogenetic tree. After phylogenetic analysis, the results showed that the diseased miniature schnauzer was infected by A. ceylanicum. Using etiologic and symptomatic therapies, the sick dog with an A. ceylanicum infection was also treated for 5 days. CONCLUSIONS: To our knowledge, this is the first report of diagnosis and treatment for canine hookworm disease in Guangzhou city. In addition, with the improvement of economic level, the scale of pet dog breeding is also increasing. The diagnostic methods and treatment schemes adopted in this report will help to standardize the prevention and control of canine hookworm disease.

[Proteinuria associated with hookworm infection: a case report].

As a common soil-borne nematode, hookworm is mainly parasitized in the intestine, and the clinical manifestations of hookworm infections mainly include gastrointestinal symptoms and iron-deficiency anemia. In addition, hookworm may be also parasitized in other organs in addition to gastrointestinal system, resulting in development of disorders in other systems. Proteinuria caused by hookworm infections is rare and easy to be misdiagnosed in clinical practices. Hereby, the diagnosis and treatment of a case of proteinuria associated with hookworm infections was reported, in order to increase the understanding of hookworm infection-associated proteinuria among clinicians.

A case of severe hookworm infection / 中南大学学报(医学版)

With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.

The Effect of Drug Pricing on Outpatient Payments and Treatment for Three Soil-Transmitted Helminth Infections in the United States, 2010-2017.

The price of certain antiparasitic drugs (e.g., albendazole and mebendazole) has dramatically increased since 2010. The effect of these rising prices on treatment costs and use of standard of care (SOC) drugs is unknown. To measure the impact of drug prices on overall outpatient cost and quality of care, we identified outpatient visits associated with ascariasis, hookworm, and trichuriasis infections from the 2010 to 2017 MarketScan Commercial Claims and Encounters and Multi-state Medicaid databases using Truven Health MarketScan Treatment Pathways. Evaluation was limited to members with continuous enrollment in non-capitated plans 30 days prior, and 90 days following, the first diagnosis. The utilization of SOC prescriptions was considered a marker for quality of care. The impact of drug price on the outpatient expenses was measured by comparing the changes in drug and nondrug outpatient payments per patient through Welch's two sample t-tests. The total outpatient payments per patient (drug and nondrug), for the three parasitic infections, increased between 2010 and 2017. The increase was driven primarily by prescription drug payments, which increased 20.6-137.0 times, as compared with nondrug outpatient payments, which increased 0.3-2.2 times. As prices of mebendazole and albendazole increased, a shift to alternative SOC and non-SOC drug utilization was observed. Using parasitic infection treatment as a model, increases in prescription drug prices can act as the primary driver of increasing outpatient care costs. Simultaneously, there was a shift to alternative SOC, but also to non-SOC drug treatment, suggesting a decrease in quality of care.

Hookworm infection in infants: a case report and review of literature.

BACKGROUND: Hookworm infections (Necator americanus, Ancylostoma duodenale) are common in rural areas of tropical and subtropical countries. Human acquisition results from direct percutaneous invasion of infective larvae from contaminated soil. Overall, almost 472 million people in developing rural countries are infected. According to simulation models, hookworm disease has a global financial impact of over US$100 billion a year. Hookworm infection in newborn or infancy is rare, and most of the cases reported in literature are from endemic countries. Here, we describe the case of an infant with an Ancylostoma duodenale infection and review the literature currently available on this topic. CASE PRESENTATION: An Italian 2-month-old infant presented with vomit and weight loss. Her blood exams showed anemia and eosinophilia and stool analysis resulted positive for hookworms' eggs, identified as Ancylostoma duodenale with real time-PCR. Parasite research on parents' stools resulted negative, and since the mother travelled to Vietnam and Thailand during pregnancy, we assumed a transplacental transmission of the infection. The patient was treated successfully with oral Mebendazole and discharged in good conditions. DISCUSSION: Hookworm helminthiasis is a major cause of morbidity in children in the tropics and subtropics, but rare in developed countries. Despite most of the patients is usually asymptomatic, children are highly exposed to negative sequelae such as malnutrition, retarded growth and impaired cognitive development. In infants and newborns, the mechanism of infection remains unclear. Although infrequent, vertical transmission of larvae can occur through breastfeeding and transplacentally. Hookworm infection should be taken into account in children with abdominal symptoms and unexplained persistent eosinophilia. The treatment of infants infected by hookworm has potential benefit, but further studies are needed to define the best clinical management of these cases.

Comparison of direct wet mount, Kato-Katz and formol ether sedimentation technique for the diagnosis of hookworm infection in Debre Elias Woreda, Northwest Ethiopia.

Hookworm infection is a neglected parasitic disease. Direct wet mount is the usual method for the diagnosis of hookworm in Ethiopia, but its sensitivity is unsatisfactory. A cross-sectional study was therefore conducted from January to February 2018 among 192 study participants in Debre Elias district. Stool samples were processed by three methods: the prevalence of hookworm was 77.6%, 68.2% and 49.0% by Kato-Katz, formol ether sedimentation technique and direct wet mount, respectively. Direct wet mount had relatively low sensitivity (61.4%) compared to formol ether sedimentation technique (85.6%) and Kato-Katz (97.4%). The latter two compared well with the gold standard method with kappa values of κ = 0.94, κ = 0.71 and κ = 0.39, respectively, and are therefore recommended over against the direct wet mount technique in detecting hookworm.

Evaluating the performance of diagnostic methods for soil transmitted helminths in the Amhara National Regional State, Northwest Ethiopia.

BACKGROUND: Soil-transmitted helminths are more prevalent in tropics and sub-tropics including Ethiopia. Despite their high prevalence, direct saline microscopy with its low sensitivity has been used as a diagnostic method in almost all health facilities in Ethiopia. Alternative diagnostic methods which have higher sensitivity are not yet implemented. Therefore, this study aimed to compare and evaluate the performance of diagnostic methods for soil transmitted helminths. METHODS: A cross-sectional study among 520 school children was conducted from October to December, 2019 in Amhara National Regional State. The study participants were selected using systematic random sampling technique. Stool samples were processed via formol ether concentration, Kato-Katz, spontaneous tube sedimentation and agar plate culture techniques. Data was entered into Epi-data version 3.1 and analysis was done using SPSS version 20.0. The sensitivity, specificity, and negative predictive value were calculated against the combined result. Strength of agreement of the diagnostic methods was determined by Kappa value. RESULTS: The Overall prevalence of soil transmitted helminths was 40.8% using combination of methods. The prevalence 24.4, 22.5, and 32.4%, respectively was recorded by using formol ether concentration, Kato-Katz and spontaneous tube sedimentation. The highest prevalence of hookworm (29.2%) was detected by the agar plate culture. The sensitivity and negative predictive value of formol ether concentration were 57.9 and 78.4%, for Kato-Katz thick smear 55.2 and 76.4%, for spontaneous tube sedimentation were 79.2 and 87.5% to soil transmitted helminths detection, respectively. The sensitivity and negative predictive value of agar plate culture to hookworm detection were 86.4 and 93.5%, respectively. CONCLUSION: Spontaneous tube sedimentation shows higher sensitivity in the detection of soil transmitted helminth infections. Agar plate culture method also indicated better performance for hookworm detection than other methods. Therefore, the employment of spontaneous tube sedimentation technique for routine laboratory and agar plate culture for research purposes will significantly aid in accurate diagnosis of parasitic infections.