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Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples
Tropical Medicine & International Health
Abstract
Until recently infection of humans with Oesophagostomum bifurcum was regarded as a rare zoonosis. But in northern Togo and Ghana its prevalence is 50% or more in certain villages. Diagnosis is hampered by the fact that the eggs of O. bifurcum are morphologically identical to those of the hookworm Necator americanus. Stools have to be cultured for 7 days to allow eggs to hatch to the characteristic third-stage (L3) larvae. We evaluated the applicability of specific polymerase chain reactions (PCRs) to amplify DNA from faecal samples as an alternative method for the differential diagnosis of the two infections. Oesophagostomum bifurcum-PCR was positive in 57 of 61 faecal samples known to contain O. bifurcum L3 larvae in coproculture. Necator americanus PCR was positive in 137 of 146 faecal samples known to contain N. americanus L3 larvae in coproculture. PCR also detected 26 additional O. bifurcum cases in 72 samples from O. bifurcum endemic villages in which no O. bifurcum larvae were found and 45 N. americanus cases in 78 samples in which no N. americanus larvae were found in coproculture. No O. bifurcum DNA was detected in 91 stool samples from individuals from two non-endemic villages. These results prove the usefulness of specific PCR assays as epidemiological tools to estimate the prevalence of O. bifurcum and N. americanus infections in human populations.
... The extraction is carried out using a Qiagen extraction kit (QIAamp Tissue Kit after treatment with 4% PVPP (polyvinylpolypyrolidone) (Sigma, Steinheim, Germany) and a mixture of Sodium dodecyl Sulphate-Proteinase K [19]. It can be more specific than Kato-Katz because PCR amplifications can be carried out with a very small amount of DNA, but it is much more expensive and more difficult to use because it can only be performed by very competent individuals in the domain of molecular biology who are acquainted to the reagents and equipment. ...
... Stool samples should be frozen prior to usage. The protocol for DNA extraction uses 0.5 g of stool which is mixed with 1 ml of distil water [19]. DNA extraction is not carried out using a Qiagen kit but following the ROSE protocol [20]. ...
... It uses the same principle and the same steps defined in the protocol of Verweij et al. [19]. Here, the stool samples collected from subjects need to be processed by making a dilution of 0.25g/ ml using 70% ethanol within a 24hrs time frame and the PCR used is a real-time PCR [16]. ...
... Recently, polymerase chain reaction (PCR)-based methods for the laboratory diagnosis of giardiasis and cryptosporidiosis were developed and showed excellent specificity and sensitivity, compared with antigen detection, and microscopy (14,15). ...
... Fresh duodenal aspirates samples and faecal suspension (200 µl) were incubated for 2 h at 56 0 C in 200 µl of lysis buffer containing 100 mM Tris-HCl (pH 8.0), 100 mM EDTA (pH 8.0), 2% sodium dodecyl sulfate, 150 mM NaCl, and 200 mg of proteinase K per ml. DNA was isolated with QIAamp Tissue Kit spin columns (QIAGEN, Germany) (14,19). ...
... Once considered, commonly accomplished by the identification of the diagnostic parasitic stage in faeces, which is time-consuming and lack sensitivity. Recently, simple and effective methods for the isolation of parasitic DNA from faeces have been developed (14). Easier and faster isolation, amplification and detection of DNA open ways of using these techniques in routine parasitology laboratories (38). ...
Giardiasis and cryptosporidiosis presents to hepatic and gastroenterology units with chronic non-specific
symptoms that mimic and confusing with other gastrointestinal and/or biliary disorders. A cross-sectional study was
conducted to determine the molecular prevalence and the diagnostic yield of duodenal aspirate for giardiasis and
cryptosporidiosis in patients attending gastroenterology unit, King Fahad hospital in Al-Madinah during routine upper
endoscopy for the first time in Al-Madinah from September, 2009 to April, 2010. Duodenal aspirates and concomitant
faecal samples from 109 cases (66 Males, 43 Female, mean age 43.38 years, range 18-79 years) were analyzed
microscopically, using ELISA and PCR for detection of G. lamblia and Cryptosporidium. They were molecularly
diagnosed in 12 (11%) and 7 (6.4%) of duodenal aspirate samples, with 95.1% and 96.2% specificity, respectively. Their
antigens were detected using ELISA in 13 (11.9%) and 8 (7.3%) cases with 94.1% and 95.1% specificity, respectively.
While, microscopy exhibited the least specificity (57.1 and 60%, respectively)> All three diagnostic methods exhibited
100% sensitivity. Compared to faecal examination, duodenal aspirate examination during upper endoscopy is less
sensitive for the diagnosis of giardiasis and cryptosporidiosis and significantly inferior. Molecular diagnostic yield of
duodenal aspirate is sensitive, specific, quick and reliable method for detection of Giardia and Cryptosporidium.
However, duodenal aspirates for molecular diagnosis of Giardia and Cryptosporidium may be unnecessary if faecal DNA
testing is available.
... The molecular approaches include conventional polymerase chain reaction (PCR), real-time PCR, digital PCR, loop-mediated isothermal amplification assay (LAMP) and, more recently, cell-free DNA detection and quantitative paper-based DNA reader. Various molecular diagnostic tests for STH diagnosis are summarised in Table 2. Molecular-based methods can detect and identify intestinal parasites in faecal samples (Rogers et al., 2021) from appropriate DNA targets (Verweij et al., 2001). Advances in the bioinformatics sequencing of nematode genomes with the availability of nematode sequence data have enabled the development of assays which target species-specific genomic regions (Khurana et al., 2021;Grant et al., 2019). ...
... Polymerase chain reaction (PCR)-based methods possess high sensitivity as parasite detection is possible with low DNA concentrations (Manuel et al., 2021;Verweij et al., 2001) americanus, and T. trichiura detection was 87 %, with a specificity of 83 % where a single copy of DNA was detectable. Nonetheless, agarose gel electrophoresis is required to visualise the results of PCR. ...
World Health Organization (WHO) reported that over 1.5 billion people are infected by soil-transmitted helminths (STH) worldwide in sub-Saharan Africa, the United States of America, China, and East Asia. Heavy infections and polyparasitism are associated with higher morbidity rates, and the patients are exposed to increased vulnerability to other diseases. Therefore, accurate diagnosis followed by mass treatment for morbidity control is necessary.STH diagnosis commonly involves the microscopic observation of the presence of the STH eggs and larvae in the faecal samples. Furthermore, molecular approaches are increasingly utilised in monitoring and surveillance as they show higher sensitivity. Their capability to differentiate hookworm species is an advantage over the Kato-Katz technique. This review discusses the advantages and limitations of microscopy and various molecular tools used for STH detection.
... Additionally, the main diagnostic approaches (particularly the Kato-Katz method) are unable to differentiate infections caused by different genera and species of hookworm or some other non-hookworm nematode species. 3,4 Therefore, hookworms are usually grouped together in epidemiological surveys without any consideration of the diversity of hookworm species that might be present in a given community. ...
The global distribution and morbidity effects for each specific hookworm species is unknown, which prevents implementation of the optimum intervention for local hookworm control. We did two systematic reviews of studies on the proportion of hookworm isolates of each species and genus by region of the world and associations between hookworm species-specific infections and morbidity outcomes, particularly severe anaemia. Necator americanus and Ancylostoma spp were present in all regions of the world, although at different ratios. No clear evidence was found for the differential morbidity effects of different hookworm species. Diagnostic methods that differentiate between hookworm species, including molecular methods, need to be developed for widespread use in control programmes to elucidate key features of hookworm epidemiology and control.
... The percent of single, double and triple infection of STH based on multiplex PCR examination.The speedy, high sensitive of molecular techniques, predominantly multiplex PCR make it suitable for diagnose STH. Until now, molecular revealing of STH was mostly limited to the research and study, on other hand, there is agreement of adopting molecular tests in the World Health Organization STH elimination programs[20]. Thus, STH infections are important public health problems and should be correctly diagnosed and treated to reduce the mortality and morbidity signi cantly[21]. ...
Ascaris lumbricoides, Trichuris trichiura, and Ancylostoma duodenale are soil-transmitted helminths (STHs) and medically neglected in Iraq country in spite of their effect on the public health. A cross-sectional study was performed in the Maternity and Childhood Teaching Hospital and General Education Hospital in Al-Dewanyia province, included 850 tool samples collected from patients who attended to the O&P lab. General stool examination (GSE), Direct wet mount method DWMM and Kato-Katz were using for diagnosis of STH infections through detected the adult and the ovum of the helminthes. A conventional multiplex PCR assay was used for detection of STHs in fecal samples. Base on microscopic examination. The results showed that 275/ 850 range among triple, double and single infection on other hand was 365/ 850 range among triple, double and single infection. In conclusions the investigative sensitivity of DWMM is notable for STH, in exception, it is capable to identify patients with the intention of highest required of management, and therefore contributes to the universal target to reduce STH as a community healthiness trouble.
... 15,28 In cases in which parasites were obtained and identified by morphological features, PCR was conducted using NC1 and NC2 primer sets for rDNA of helminths. 33 Bidirectional sequencing with the original primers was conducted as mentioned earlier. ...
Canine parvovirus type 2 (CPV-2) is among the most important and highly contagious pathogens that cause enteric or systemic infections in domestic and nondomestic carnivores. However, the spillover of CPV-2 to noncarnivores is rarely mentioned. Taiwanese pangolins ( Manis pentadactyla pentadactyla) are threatened due to habitat fragmentation and prevalent animal trafficking. Interactions between Taiwanese pangolins, humans, and domestic animals have become more frequent in recent years. However, information about the susceptibility of pangolins to common infectious agents of domestic animals has been lacking. From October 2017 to June 2019, 4 pangolins that were rescued and treated in wildlife rescue centers in central and northern Taiwan presented with gastrointestinal signs. Gross and histopathological examination revealed the main pathologic changes to be necrotic enteritis with involvement of the crypts in all intestinal segments in 2 pangolins. By immunohistochemistry for CPV-2, there was positive labeling of cryptal epithelium throughout the intestine, and immunolabeling was also present in epidermal cells adjacent to a surgical amputation site, and in mononuclear cells in lymphoid tissue. The other 2 pangolins had mild enteritis without crypt involvement, and no immunolabeling was detected. The nucleic acid sequences of polymerase chain reaction (PCR) amplicons from these 4 pangolins were identical to a Chinese CPV-2c strain from domestic dogs. Quantitative PCR revealed a higher ratio of CPV-2 nucleic acid to internal control gene in the 2 pangolins with severe intestinal lesions and positive immunoreactivity. Herein, we present evidence of CPV-2 infections in pangolins.
... In the case of the second assumption, PCR false negative results may arise because the DNA yields from feces remain poor [82,83]. Giardia cysts wall is difficult to disrupt and this may lead to insufficient DNA yields, whereas at the same time stool specimens commonly contain compounds such as DNases, proteases, bile salts, and polysaccharides that might cause DNA degradation and inhibition of enzymatic reactions [56,84]. ...
Background
Infections by protozoans of the genus Giardia are a common cause of diarrhea in dogs. Canine giardiosis constitutes a disease with a zoonotic potential; however, it is often underestimated due to its challenging diagnosis. The objective of the study was to assess the diagnostic performance of an immunochromatographic strip test (SpeedTMGiardia, Virbac, France) comparing it with microscopy (zinc sulfate flotation) by utilizing the combination of an enzyme immunoassay (ProSpecTTMGiardia EZ Microplate Assay, Oxoid Ltd., UK) and the PCR as the gold standard. A positive result in both ELISA and PCR was set as the gold standard.
Methods
Initially, fecal samples from dogs with clinical signs compatible with giardiosis were tested with the SpeedTMGiardia test and separated into two groups of 50 samples each: group A (positive) and group B (negative). Thereafter, all samples were examined by zinc sulfate centrifugal flotation technique and assayed by the ProSpecTTMGiardia Microplate Assay and PCR. The performance of the SpeedTMGiardia and zinc sulfate centrifugal flotation tests were calculated estimating sensitivity, specificity, and positive and negative likelihood ratio; the chi-square and McNemar tests were used for the comparison of the two methods.
Results
Giardia cysts were not detected by microscopy in 16 out of the 50 samples (32%) of group A and in none of group B samples. Eight out of 50 samples in group B (16%) were tested positive both with the ProSpecTTMGiardia Microplate Assay and PCR. Fecal examination with the SpeedTMGiardia test was more sensitive (86.2%) than the parasitological method (58.6%, P < 0.001) while the specificity of both methods was 100%.
Conclusions
The SpeedTMGiardia test is an easy-to-perform diagnostic method for the detection of Giardia spp., which can increase laboratory efficiency by reducing time and cost and decrease underdiagnosis of Giardia spp. infections. This immunochromatographic strip test may be routinely exploited when a rapid and reliable diagnosis is required, other diagnostic techniques are unavailable and microscopy expertise is inefficient. In negative dogs with compatible clinical signs of giardiosis, it is recommended either to repeat the exam or proceed with further ELISA and PCR testing.
... The low diversity of strongylids in humans and particularly the absence of genera such as Oesophagostomum and Ternidens is somehow surprising given that they have been reported in humans before and are considered neglected human parasites (Gasser, de Gruijter, & Polderman, 2006;Verweij et al., 2001). Interestingly, the other orally transmitted strongylids, Strongylid nematodes are listed as important pathogens of livestock and humans and further studies on their communities in wild animals populations exposed to increasing contact with humans are crucial, not only for understanding risks of possible parasite spillover to humans, but also to endangered mammalian fauna. ...
The close phylogenetic relationship between humans and non‐human primates (NHPs) can result in a high potential for pathogen exchange. In recent decades, NHP and human interactions have become more frequent due to increasing habitat encroachment and ecotourism. Strongylid communities, which include members of several genera, are typically found in NHPs. Using optimized high‐throughput sequencing for strain‐level identification of primate strongylids, we studied the structure of strongylid communities in NHPs and humans co‐habiting a tropical forest ecosystem in the Central African Republic. General taxonomic assignment of 85 ITS‐2 haplotypes indicated that the studied primates harbor at least nine genera of strongylid nematodes, with Oesophagostomum and Necator being the most prevalent. We detected both host‐specific and shared strongylid haplotypes. Skin‐penetrating Necator gorillae haplotypes were shared between humans and gorillas but N. americanus were much more restricted to humans. Strongylid communities of local hunter‐gatherers employed as trackers were more similar to those of gorillas compared to their relatives, who spent more time in villages. This was due to lower abundance of human‐origin N. americanus in both gorillas and trackers. Habituated gorillas or those under habituation did not show larger overlap of strongylids with humans compared to unhabituated. We concluded that the occurrence of the human‐specific strongylids in gorillas does not increase with direct contact between gorillas and humans due to the habituation. Overall, our results indicate that the degree of habitat sharing between hosts, together with mode of parasite transmission, are important factors for parasite spillover among primates.
... Although, coproscopy was regarded as the "gold standard" for traditional diagnosis of intestinal parasites, however, it is of low sensitivity and hindered by the nonspecific clinical presentation [13,14,15]. PCR-based methods are used effectively for reliable detection of intestinal protozoa, with high sensitivity and specificity, a fact that has led many authors to consider PCR the "gold standard" method that will take over current conventional techniques [11,16,17,18,19]. ...
Background:
Children with underlying malignancies and those on chemotherapy are at risk for having intestinal parasitic infections, which can lead to a severe course and death. This cross-sectional study was done to assess the copro-parasitological and copro-molecular prevalence of entero-parasites in children with malignancies and those on chemotherapy.
Procedure:
Stool samples were collected from 137 Egyptian hospitalized cancerous children with different malignancies in the National Cancer Institute, and receiving chemotherapy.Faecal samples were examined microscopically. Genomic copro-DNA was extracted from fecal samples and amplified by 3 separate nPCR assays targeting Cryptosporidium, G. intestinalis and Entamoeba histolytica complex.
Result:
The overall prevalence of enteroparasites was 6.6 % (9 cases). Only Giardia copro-DNA was encountered in 2 (1.4%) faecal samples of patients. Coproscopy detected parasites in 7 cases: Blastocystis spp. in 5 cases (3.6%), Hymenolepis nana in 1 case (0.7%) and Ascaris lumbericoides in 1 case (0.7%).
Conclusion:
Low prevalence may be due to patient's use of prophylactic anti-parasitic and anti-fungal drugs, a standard protocol, basic hygienic practices and good nursing all of which are preventive against enteroparasites transmission. Among studied variables only diarrhoeic individuals who had a solid tumor, and soft/liquid stool with mucus and blood were predictors of intestinal parasitism.
... Conversely, failure to detect A. abstrusus DNA in one Baermann and one pharyngeal sample, coming from 1 of the 30 animals with a history of aelurostrongylosis, might be due to the concentration or the quality of parasitic DNA. The value of diagnostic sensitivity (i.e., ϳ97%) is in accordance with those previously recorded for other parasites by using two-step PCR approaches on fecal or pharyngeal samples (27,28,30), and it is higher than that recorded by employing classical diagnostic methodologies. The higher percentage of sensitivity shown by the two-step PCR over the conventional diagnostic methods is likely due to the specific amplification of the parasites' watersoluble DNA molecules present in feces and in pharyngeal swabs. ...
Peritoneal larval cestodiasis caused by Mesocestoides spp. is a rare infection in dogs. A 6-year-old female dog was presented for veterinary care with urinary incontinence which started 1 year earlier. After performing hematology, ultrasound, and computerized tomography, an exploratory laparotomy revealed canine peritoneal larval cestodiasis (CPLC) with the presence of Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia confirmed by morphological identification and PCR and DNA sequencing. Parasitic cysts were found around the urinary bladder and appeared to inhibit its normal function. An initial treatment with 5 mg/kg praziquantel subcutaneously every 2 weeks for four treatments failed to alleviate the clinical signs, and only treatment with fenbendazole at 100 mg/kg P.O. twice daily for 28 days was associated with the disappearance of ascites and regaining of urinary control. This is the first report of CPLC associated with urinary incontinence in dogs and the first description of this cyclophyllidean cestode in dogs in Israel.
... Human infections with other zoonotic parasites of the Strongylida order, such as Trichostrongylus sp. and Oesophagostomum sp. have also been described (Blotkamp et al., 1993;Gasser et al., 2006;Goldsmid, 1968;Lattes et al., 2011;Polderman and Blotkamp, 1995;Sato et al., 2011). Eggs from these parasites are not easily distinguished from hookworm eggs through light microscopy, and molecular approaches have demonstrated that misidentifications may be frequent (Verweij et al., 2001;Yong et al., 2007). ...
Hookworm infection persists focally in rural communities in Brazil. In this study, we analyze the mitochondrial nucleotide sequences obtained from hookworms infecting humans in order to characterize species composition and assess their genetic diversity and phylogenetic relationships. Field expeditions and cross-sectional surveys were carried out in three Brazilian municipalities from 2013 to 2017: Nossa Senhora de Nazaré (n = 605) and Teresina (n = 297), in the state of Piauí and Russas (n = 213) in the State of Ceará. Parasitological methods were used to evaluate fecal samples. Hookworm-positive samples had a partial mtDNA cox1 amplified and sequenced. Maximum-likelihood and Bayesian analysis demonstrated two strongly-supported clades, including Group A, corresponding to Necator americanus, and Groups B and C, corresponding to Necator sp. Group A was divided into three main clusters: A1 grouped with Asian sequences, A2 grouped with African sequences, and A3 had only Asian sequences. Group B was closely related to Necator sp., showing a sequence similarity of 98%–99% with African samples circulating zoonotically among humans and non-human primates. Twenty three N. americanus haplotypes were identified. N. americanus Median-Joining network revealed three distinct groups, designated again as A1, A2, and A3. Group A1 presented a star-like shape, with one dominant haplotype. The molecular dating suggested that the two clades dividing N. americanus and Necator sp. began to diverge during the middle Pleistocene. The most recent common ancestor among N. americanus groups was dated to the late Pleistocene. Hookworms circulating in the studied communities are structured in well-defined subpopulations presenting both Asian and African genetic backgrounds. This reveals a double origin for hookworms in northeastern Brazil and opens up new possibilities in phylogeographic, evolutionary, and molecular epidemiological studies in regions where hookworms persists focally, despite control efforts. The presence of potentially zoonotic species and the specific identification of Necator sp. should be further investigated.
... In the last 20 yr, many DNAbased methods have been developed for the detection of viral, bacterial, and parasitic DNA (Perandin et al., 2018). Throughout the years, those methods have enhanced their speed and accuracy (Verweij et al., 2001). Recently, we published a paper where it has been demonstrated how molecular biology can change the classical laboratory approach for intestinal protozoan infections, including Giardia, improving the sensitivity and specificity of the diagnosis (Formenti et al., 2017). ...
Giardia intestinalis is a parasite that commonly causes diarrheal disease throughout the world. An accurate and rapid diagnosis is essential to reduce the infection. Classically, diagnosis of giardiasis is performed by microscopic examination of stool samples, but in the recent years many DNA-based methods have been developed. In this preliminary observational study we compared the results of the commercial BD Max Enteric Parasite Panel (EPP) with an in-house real-time PCR for Giardia intestinalis. The study population was composed by 73 samples; of these, 27 resulted positive at both techniques and 39 negative. Seven samples were positive at the in-house real-time PCR and negative at the BD Max EPP. The Cohen's Kappa resulted 0.805 (95% CI 0.670-0.940). In conclusion, these preliminary results suggest that the Rt-PCR could possibly demonstrate higher sensitivity for the diagnosis of Giardia intestinalis than BD Max EPP, that tended to miss infection of low intensity.
... DNA isolation of all samples was performed using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions with slight modification of the final step for both taeniid-like and ascarid eggs [17,30] and increase of incubation time at 56°C to at least 16 h for ascarid eggs until eggs were completely lysed [31]. Before extraction, five cycles of freezing in liquid nitrogen 5 min and boiling at 100°C 7 min were conducted to disrupt the parasite egg wall. ...
Multiple zoonotic helminth infections in domestic dogs
... DNA isolation of all samples was performed using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions with slight modification of the final step for both taeniid-like and ascarid eggs [17,30] and increase of incubation time at 56°C to at least 16 h for ascarid eggs until eggs were completely lysed [31]. Before extraction, five cycles of freezing in liquid nitrogen 5 min and boiling at 100°C 7 min were conducted to disrupt the parasite egg wall. ...
Background:
Echinococcosis and toxocarosis caused by the genus of Echinococcus and Toxocara spp. are among important helminthic diseases worldwide. Limited data on the prevalence of these parasites persuaded us to determine the prevalence of E. granulosus, E. multilocularis, and T. canis infections in domestic dogs in rural areas of Ahvaz, southwestern Iran. Fecal samples from 167 domestic dogs were examined using both microscopy and PCR techniques. Multiplex PCR was performed for the presence of Echinococcus, and Taenia spp. and single PCR for detection of T. canis and Toxascaris leonina.
Results:
The total occurrence of identified parasites was 65 (38.9%). The microscopic examinations showed that 40 (24%), 18 (10.8%), and four (2.4%) of dogs were infected with taeniid-like, ascarid, and both genera eggs, respectively. Echinococcus granulosus was identified in seven (4.2%), Taenia spp. in 29 (17.4%), and mixed infection with both in 11 (6.6%) samples. Sequencing of PCR-positive samples identified E. granulosus s.s. (G1), 18 T. hydatigena (10.8%), five T. multiceps (3%), three T. serialis (1.8%), one T. ovis (0.6%), one Spirometra erinaceieuropaei voucher (0.6%), and two Mesocestoides corti (1.2%). This is the first report of S. erinaceieuropaei voucher and M. corti in dogs in Iran. Nine (5.4%) and 16 (9.6%) dogs showed infection with T. canis and T. leonina, respectively. Two samples showed coinfection with both ascarids.
Conclusions:
Several studies have reported echinococcosis and toxocarosis in intermediate hosts from the southwest of Iran; however, this study is the first molecular research on E. granulosus and T. canis in domestic dogs in a rural area of southwestern Iran. Furthermore, issues of soil contamination with dogs' feces and recent dust storms in Khuzestan may have a role in the spreading of these zoonotic infections to other provinces close to it, and neighboring countries such as Iraq.
... Conventional PCR and real-time PCR (RT-PCR) have achieved critical advances in the detection of several parasitic infections. Several studies have also applied PCR-based assays for sensitive and specific detection of STH DNA in human faecal samples [22,23]. ...
Background
Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Molecular diagnostics are powerful tools to identify closely related species, but the requirement for costly equipment makes their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for accurate estimation of STH infection intensity in MDA programmes in which the goal is to reduce morbidity following repeated rounds of chemotherapy.
Results
In this study, colourimetric isothermal assays were developed using SmartAmp2 primer sets and reagents in loop-mediated amplification (LAMP) assays. Species-specific primer sets, designed on a specific target sequence in the β-tubulin gene, were used to identify Necator americanus, Trichuris trichiura and Ascaris lumbricoides. After initial optimization on control plasmids and genomic DNA from adult worms, assays were evaluated on field samples. Assays showed high sensitivity and demonstrated high tolerance to inhibitors in spiked faecal samples. Rapid and sensitive colourimetric assays were successfully developed to identify the STHs in field samples using hydroxy napthol blue (HNB) dye.
Conclusions
Rapid and simple colourimetric diagnostic assays, using the SmartAmp2 method, were developed, with the potential to be applied in the field for detection of STH infections and the estimation of response to treatment. However, further validation on large numbers of field samples is needed.
Electronic supplementary material
The online version of this article (10.1186/s13071-017-2420-1) contains supplementary material, which is available to authorized users.
... The genomic DNA from adult C. sinensis and other parasites was extracted using QIAamp Tissue Kit (QIAgen) following the manufacturer's instruction. DNA from stool samples was extracted using the procedures previously described [30]. Briefly, 100 mg stool was washed twice with 1 ml PBS and centrifuged at 8000 rpm for 5 min. ...
Background:
Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited.
Methodology/principle findings:
The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2) of sensitivity and 100% (95% CI, 92.9-100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%.
Conclusions:
To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.
... Several researchers have demonstrated infection by parasites such as Echinococcus spp (19), Toxocara spp (20), F. hepatica (1,21), Ostertagia ostertagi (22), Oesophagostomum bifurcum and Necator americanus (23), and Opisthorchis viverrini (24) using PCR assay of feces. There is no evidence regarding molecular discrimination of Fasciola species agents in fecal specimens from living ruminants in Iran, though there are many studies that deal with the genetic characterization of adult forms of Fasciola species from slaughtered animals in different regions of Iran (4,(14)(15)(16). ...
Abstract
Background: Fasciola species are the main causes for fascioliasis with great financial losses and are among the most important food/water-borne parasites worldwide. The basic proceedings such as epidemiology and effective control of fascioliasis rely mainly on precise identification of Fasciola species. The present study was
conducted to determine the Fasciola species in ruminant fecal samples from East
Azerbaijan Province in Iran.
Methods: Overall, 2012 fecal samples were collected and processed initially for
microscopic examination of Fasciola eggs in 2014-15. Then, recovered eggs were
subjected to molecular identification. A fragment of 618 bp of the 28S rRNA gene
pertaining to Fasciola genus was amplified under PCR. The amplified fragment was
restricted by fast digest Ava II enzyme in order to a Restriction Fragment Length
Polymorphism.
Results: Based on microscopic examination, 72 samples were infected, from
which, 10 and 62 cases pertained to cattle and sheep samples respectively. Based on
RFLP, the PCR products restricted by the Ava II restriction enzyme produced 529
bp fragments only. According to the positive controls, all restriction patterns were
related to Fasciola hepatica, while no restriction patterns were linked to F. gigantica.
Conclusion: Based on PCR-RFLP, F. hepatica was dominant species in animals of
the studied areas and no evidence of F. gigantica was observed. Therefore, further
field studies to verify these results are suggested.
... Samples showing successful PCR amplification based on agarose gel electrophoresis were subjected to a second PCR. The second PCR included two forward primers, NA (5′-ATG TGC ACG TTA TTC ACT −3′) (for N. americanus) (Verweij et al., 2001), AD1 (5′-CGA CTT TAG AAC GTT TCG GC −3′) (for Ancylostoma spp.) (de Gruijter et al., 2005) and NC2 as a common reverse primer. All 3 primers were included in a single reaction for simultaneous amplification of N. americanus and Ancylostoma spp. ...
... Parasitic nematodes of Oesophagostomum spp., commonly known as "nodular worms", are one of the most widely distributed and prevalent emerging nematode zoonoses of ruminants, pigs and primates (Legesse and Erko 2004). Oesophagostomum genus members are the pathogen agents of Oesophagostomiasis (Zhao et al. 2013), and some previous researches had reported some serious clinical diseases on animals, which may emerge to be serious public health concerns in humans (McCarthy and Moore 2000; Verweij et al. 2001;Legesse and Erko 2004;Krief et al. 2010). Previously, oesophagostomiasis was reported to be a serious public health problem in human in northern Togo and Ghana, with an estimation of more than a quarter of a total million population infection oesophagostomiasis (Polderman and Blotkamp 1995;Polderman et al. 1999;Storey et al. 2000). ...
Parasitic nematodes of Oesophagostomum spp., commonly known as ‘nodular worms’ are one of the most widely distributed
and prevalent emerging zoonotic nematodes. However, little is known about the prevalence and gene characteristics of those
parasites in Tibetan pigs. Therefore, a study was carried out to investigate the prevalence, isolation and identification of Oesophagostomum
spp from Tibetan pigs by genetic markers of nad1, cox3 and ITS1 for the first time. The results revealed that
the infection rate of O. dentatum and O. quadrispinulatum by genetic markers of nad1 was 81.13%; 35 (66.04%); the O. dentatum
and O. quadrispinulatum by genetic markers cox3 was 66.04%, and O. dentatum and O. stephanostomum by genetic
markers ITS1 was found to be 77.36%. Interestingly, the O. stephanostomum specie was identified and isolated from 90.48%
stomach and 69.23% colon samples by genetic markers of ITS1. The present study, for the first time has described the presence
and genetic characterization of Oesophagostomun spp of O. dentatum, O. quadrispinulatum and especially O. stephanostomum
in Tibetan pigs from the high and remote Tibetan plateau. A public concern should be raised in terms of economical losses
and severe public health problem.
... After sodiumdodecyl sulfate-proteinase K treatment (2h at 55°C), DNA was isolated using QIAamp DNA-easy 96-well plates (QIAgen, Limburg, the Netherlands). In each sample, 10 3 PFU/mL Phocin Herpes Virus 1 (PhHV-1) was included within the isolation lysis buffer [27,28]. ...
The current reference test for the detection of S. mansoni in endemic areas is stool microscopy based on one or more Kato-Katz stool smears. However, stool microscopy has several shortcomings that greatly affect the efficacy of current schistosomiasis control programs. A highly specific multiplex real-time polymerase chain reaction (PCR) targeting the Schistosoma internal transcriber-spacer-2 sequence (ITS2) was developed by our group a few years ago, but so far this PCR has been applied mostly on urine samples. Here, we performed more in-depth evaluation of the ITS2 PCR as an alternative method to standard microscopy for the detection and quantification of Schistosoma spp. in stool samples.
... As reduced sensitivity due to insuffi cient DNA release from Ascaris and hookworm eggs had to be expected, additional positive microscopic results were added to the positive PCR results for these organisms in this study. The time-consuming (several hours) and laborious nature of the procedure described [26,32] for DNA extraction from Ascaris eggs and hookworm eggs currently limits the diagnostic use of PCR for helminth detection in stool samples. Specially adapted and evaluated DNA extraction procedures for each parasite might be desirable for use in fi eld studies targeting single pathogens but these are hardly realistic for routine diagnosis. ...
Molecular methods, in particular PCR, are increasingly used for the diagnosis of enteric pathogens in stool samples. In high-endemicity settings, however, asymptomatic carriage or residual DNA from previous infections will hamper the interpretation of positive test results. We assessed the quantitative dimension of this problem in schoolchildren in the rural highlands of Madagascar.
Stool samples were collected from 410 apparently healthy Madagascan schoolchildren and analysed by multiplex real-time PCR for enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Campylobacter jejuni, Yersinia spp.), enteric protozoa (Entamoebea histolytica, Giardia duodenalis, Cryptosporidium spp., Cyclospora spp.), and helminths (Ascaris lumbricoides, Ancylostoma spp., Necator americanus, Strongyloides stercoralis). Symptoms of gastrointestinal disease were assessed.
Among the 410 samples, we detected Giardia duodenalis in 195, Campylobacter jejuni in 91, Ascaris lumbricoides in 72, Cyclospora cayetanenesis in 68, Shigella spp./EIEC in 56, and Strongyloides stercoralis and Cryptosporum spp. in 1 case each. Salmonella spp., Yersinia spp. and hookworms were not observed. Relative risk assessment suggested few and incoherent associations with pathogen detections, indicating asymptomatic carriage or DNA residuals. Only 26.1% of the schoolchildren were tested negative for all analysed pathogens.
The very high risk of detecting traces of asymptomatic carriage or residual DNA from previous infections limits the value of highly sensitive PCR for the causal attribution of detected enteric pathogens from stool samples to an infectious gastrointestinal disease in the high-endemicity setting. Evaluated standards for the interpretation of such results are needed both for the diagnostic routine and for epidemiological assessments.
... The advent of in vitro amplification techniques of nucleic acids has improved the diagnosis of parasites. Furthermore, the isolation of parasitic DNA from fecal samples has been improved and simplified (Verweij et al. 2001). ...
Abstract
Giardia lamblia and Cryptosporidium parvum are recognized as the most common protozoan infections in Saudi Arabia. Microscopic examination of stool samples, either direct or concentrated, for the recovery of G. lamblia cysts and trophozoites and C. parvum oocysts is still the most commonly used for the diagnosis of both parasites. We compared the conventional parasitological techniques of iodine-stained wet mount for G. lamblia and Kinyoun's acid-fast for C. parvum against ImmunoCard STAT® Cryptosporidium/Giardia and real-time polymerase chain reaction (PCR) detecting the 18S rRNA gene of G. lamblia and conventional PCR detecting the same gene of C. parvum at a tertiary hospital in Dhahran, Saudi Arabia. Out of 148 stool samples, 19 and 12 true positives were identified for G. lamblia and C. parvum, respectively, using a composite reference standard. In this case, true positives and negatives were considered as those with at least two positive or negative results out of the three tests. Both ImmunoCard STAT! and PCR methods were more sensitive than the microscopic tests of a single stool specimen of 85.7 % (CI = 62.6–96.2 %) and 85.7 % (CI = 56.2–97.5 %) for G. lamblia and C. parvum, respectively. However, specificity of microscopic tests was higher than other techniques for both parasites. Although PCR seems to be most sensitive for both G. lamblia and C. parvum, its low specificity may render its superiority over other techniques. When a single stool sample is used for detection of G. lamblia and C. parvum, better results can be obtained when coupled with serological testing. Although PCR is the most sensitive method for the detection of both G. lamblia and C. parvum, its use requires attention in relation to the increased possible false positives.
... Ethanol in the saliva and urine samples was removed by centrifuging at 18 500 g for 3 min, washing the sediment three times with sterile PBS and suspending in 200 ml PBS. DNA from saliva samples was extracted using the QIAamp DNA Mini Kit (QIAGEN Ltd) using the protocol of Verweij et al. 18 while DNA from urine samples was extracted using the QIAamp viral RNA kit (QIAGEN Ltd) to provide a higher DNA yield. 19 Positive and negative controls Positive and negative controls were included in all PCR reactions. ...
Malaria can be diagnosed in saliva and urine using mitochondrial PCR detection of Plasmodium DNA.
Blood, saliva and urine were collected from 99 febrile patients referred to health centers in Sistan and Baluchestan Province, southeastern Iran, from May to November 2011. The mitochondrial cytochrome b gene of Plasmodium falciparum and Plasmodium vivax was targeted in saliva, urine and blood samples using nested PCR.
Nested PCR proved to be more sensitive than microscopy for the diagnosis of sub-microscopic and mixed-species infections. The results of nested PCR amplifications of saliva and urine samples showed the same specificity of 97% and sensitivity of 91% and 70%, respectively. Nested PCR amplifications of saliva samples and microscopy showed the greatest area under the receiver operating characteristic (ROC) curve and were more accurate than nested PCR amplifications of urine samples.
Nested PCR amplification of saliva samples showed good levels of detection of mitochondrial Plasmodium DNA as compared to nested PCR of blood (к=0.84; AUC=0.94), which was used as a reference standard. Based on the results of nested PCR as well as the advantages of saliva sampling, we suggest that saliva could be an alternative to blood, in malaria diagnosis, in cases where repeat sampling is required. Further studies are needed to validate these findings.
... Before submitting the ethanol-preserved saliva and urine samples to DNA extraction, the samples were centrifuged for 3 min at 18,500 × g, the supernatant was discarded, and the sediment was washed three times with sterile PBS buffer. The pellets of the saliva samples were suspended in 200 l PBS with 2% polyvinylpolypyrolidone and held overnight at −20 • C. Following Verweij's procedure (Verweij et al., 2001), the frozen sample was heated to 100 • C for 10 min before DNA extraction using the QIAamp DNA Mini Kit (QIAGEN, Germany). The urine sample pellet was suspended in 200 l PBS, and DNA was extracted with the QIAmp viral RNA kit (QIAGEN, Germany) to provide a better yield of DNA than would be obtained with the Mini-kit (Bista et al., 2007). ...
This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23 respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.
... It is not hard to imagine that the latter is especially important in the isolation of parasite DNA from a complex matrix such as feces (11). Heating of the stool specimen and the addition of absorbent substances such as polyvinyl polypyrrolidone during the DNA isolation procedure or the addition of inhibition factorbinding substances, such as bovine serum albumin (BSA), or inhibitor-resistant DNA polymerases in the PCR mixture can be used to prevent inhibition of the amplification reaction (12)(13)(14)(15). ...
SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.
... For the DNA isolation, we followed the procedure described by Verweij and others. 39 All DNA samples were stored at −20 C and transferred on ice to the NIMR-MMRC, where PCR amplification and detection were conducted in June and November of 2012. ...
Sensitive diagnostic tools are crucial for an accurate assessment of helminth infections in low-endemicity areas. We examined stool samples from Tanzanian individuals and compared the diagnostic accuracy of a real-time polymerase chain reaction (PCR) with FLOTAC and the Kato-Katz method for hookworm and the Baermann method for Strongyloides stercoralis detection. Only FLOTAC had a higher sensitivity than the Kato-Katz method for hookworm diagnosis; the sensitivities of PCR and the Kato-Katz method were equal. PCR had a very low sensitivity for S. stercoralis detection. The cycle threshold values of the PCR were negatively correlated with the logarithm of hookworm egg and S. stercoralis larvae counts. The median larvae count was significantly lower in PCR false negatives than true positives. All methods failed to detect very low-intensity infections. New diagnostic approaches are needed for monitoring of progressing helminth control programs, confirmation of elimination, or surveillance of disease recrudescence.
... The liver puree and PBS buffer were transferred to a 15 ml falcon tube, centrifuged at 8006g for 10 min, and 400 ml of the cell suspension in PBS buffer, equal to approximately 25 mg liver tissue, was transferred to 2 ml tubes. DNA was extracted using the QIAamp DNA Mini kit (QIAGEN, Germany) tissue protocol, according to manufacturer's instructions, and the Verweij et al. [25] protocol with slight modification as described [19]. DNA was stored at 220uC until molecular analysis. ...
Alveolar echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus multilocularis. Many species of small mammals, including arvicolid rodents or Ochotona spp., are natural intermediate hosts of the cestode. The main aim of this study was to identify natural intermediate hosts of E. multilocularis in Chenaran County, Razavi Khorasan Province, northeastern Iran, where the prevalence of infected wild and domestic carnivores is high.
A program of trapping was carried out in five villages in which this cestode was reported in carnivores. The livers of 85 small mammals were investigated for the presence of E. multilocularis infection using multiplex PCR of mitochondrial genes. Infections were identified in 30 specimens: 23 Microtus transcaspicus, three Ochotona rufescens, two Mus musculus, one Crocidura gmelini, and one Apodemus witherbyi.
A range of small mammals therefore act as natural intermediate hosts for the transmission of E. multilocularis in Chenaran County, and the prevalence suggested that E. multilocularis infection is endemic in this region. The existence of the life cycle of this potentially lethal cestode in the vicinity of human habitats provides a significant risk of human infection.
... Extending logically from a range of studies (Romstad et al., 1997a,b;Verweij et al., 2001Verweij et al., , 2007 which critically assessed the suitability of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as a 116 A.M. Polderman et al. genetic marker for the specific diagnosis of O. bifurcum infection and its differentiation from hookworm infection, a real-time PCR (rt-PCR) was developed (Verweij et al., 2007) for the simultaneous, semi-quantitative and specific detection of DNA from O. bifurcum, A. duodenale and N. americanus in alcohol-preserved faecal samples. An analysis of samples from villages endemic for Oesophagostomum in northern Ghana showed that 71 of 83 larval cultures containing O. bifurcum were also test-positive using the rt-PCR assay; in addition, 32 of 256 culture-negative samples gave positive PCR results. ...
Until recently, the infections of humans with representatives of the genus Oesophagostomum were thought to be rare and of zoonotic origin. In the 1980s, it was recognised that intense transmission associated with the disease was taking place in northern Togo and Ghana. Pathology can be severe and two clinical presentations, called ‘Dapaong Tumour’ and ‘multinodular disease’, have been described. Lesions can now be efficiently and specifically visualised by ultrasound. The prevalence of infection appeared to be high in many villages, although its distribution was limited and focal. Parasitological diagnosis has been based on the demonstration of third-stage larvae in stool cultures and more recently on PCR. Molecular and epidemiological evidence supports the proposal that Oesophagostomum bifurcum infections of humans in Ghana and Togo represent a distinct genotype from that of non-human primates in the area. Mass treatment, using repeated doses of albendazole rapidly resulted in the effective elimination of human oesophagostomiasis in the affected areas.This review takes a historic perspective on oesophagostomiasis of humans. This chapter discusses how the unexpected success of mass treatment together with aberrant pathogenesis, the severity of pathology in humans and the limited geographic distribution of the parasite all reflect a poor adaptation of the parasite to the human host.
... After centrifugation the pellet was resuspended into 200 ll of 2% polyvinylpolypyrolidone (PVPP) (Sigma) suspension and heated for 10 min at 100°C. After sodium-dodecyl sulphate-proteinase K treatment (2 h at 55°C), DNA was isolated using spin columns (QIAamp Tissue Kit, Qiagen, Germany) (Verweij et al. 2001). In each sample, 10 3 plaque-forming units/ml phocin herpes virus 1 (PhHV-1) was added as an internal control (Niesters 2002). ...
Since the redescription of the potentially invasive Entamoeba histolytica, separating it from the morphologically identical non-invasive Entamoeba dispar, there is a need for the reassessment of epidemiological data on amoebiasis. In this context we conducted a descriptive survey on the presence of E. histolytica and E. dispar in a rural area in northern Ghana. We found a high prevalence (39.8%) of the E. histolytica/E. dispar complex with microscopy, but E. histolytica and E. dispar-specific DNA amplification using real-time polymerase chain reaction identified only one E. histolytica case and revealed a considerably higher prevalence of E. dispar (82.8%).
... After sodiumdodecyl sulfate-proteinase K treatment (2 h at 558C), DNA was isolated using QIAamp DNA-easy 96-well plates (QIAgen). In each sample, 10 3 PFU/mL Phocin Herpes Virus 1 (PhHV-1) was included within the isolation lysis buffer (Verweij et al. 2001, Niesters 2002. ...
Ngamba Island Chimpanzee Sanctuary (NICS) in Lake Victoria, Uganda is currently home to 44 wild-borne, semi-captive chimpanzees. Despite regular veterinary health checks, it only came to light recently that many animals, and sanctuary staff, were naturally infected with Schistosoma mansoni. Indeed, local schistosome transmission appears firmly engrained for intermediate snail hosts can be found along almost the entirety of Ngamba's shoreline. Here, the epidemiology of infection is a dynamic interplay between human and chimpanzee populations, as revealed by genetic analyses of S. mansoni. In this review, our present understanding of this complex and evolving situation is discussed, alongside general disease control activities in Uganda, to highlight future interventions towards stopping schistosome morbidity and transmission within this conservation sanctuary setting.
... DNA was isolated using feces suspensions of 200 ml (60.5 gram of stool per ml PBS containing 2% poly-vinyl-poly-pyrolidone (Sigma, Steinheim, Germany)) and heated for 10 minutes at 100uC. After sodium-dodecyl-sulphate-proteinase K treatment (overnight at 55uC), DNA was isolated with QIAampTissue Kit spin columns (QIAgen, Hilden, Germany) [32]. Extracted DNA samples were transported for multiplex real-time PCR assessment in the Netherlands. ...
Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi.A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p
Trong nghiên cứu này, nhóm tác giả đã ứng dụng kỹ thuật PCR tổ (nested PCR) để xác định giun móc Ancylostoma spp. và giun mỏ Necator americanus trong phân người nhiễm bệnh ở cộng đồng dân cư xã Đức Lập Thượng, huyện Đức Hoà, tỉnh Long An. 80 mẫu ADN tách chiết từ 80 mẫu ấu trùng giun từ phân người bệnh được phân lập bằng phương pháp Sasa đã cho kết quả dương tính. Kết quả PCR tổ đặc hiệu vùng gen 28S rRNA-ITS2 thu được tỷ lệ nhiễm Necator americanus có 66 mẫu (82,5%), Ancylostoma spp. có 5 mẫu (6,25%) và đồng nhiễm cả 2 loài có 9 mẫu (11,25%). Từ kết quả nghiên cứu cho thấy, kỹ thuật này là một công cụ bổ sung có giá trị trong chẩn đoán và kiểm soát nhiễm bệnh ký sinh trùng ở các tỉnh/thành phố của Việt Nam.
Khoa học Y-Dược /Y học dự phòng 23 65(12) 12.2023 1. Mở đầu Nhiều nghiên cứu chỉ ra nhiễm giun móc/mỏ ở người tại các tỉnh miền Đông Nam Bộ của Việt Nam chiếm tỷ lệ còn cao, dù rằng cả tỷ lệ và cường độ nhiễm đang giảm dần do áp lực của thuốc điều trị và các yếu tố xã hội khác tác động. Ở người Việt Nam, giun móc gây bệnh là A. duodenale, chiếm tỷ lệ thấp hơn so với loài giun mỏ N. americanus. Gần đây, một báo cáo đã tìm thấy dấu vết của loài giun móc A. ceylanicum bằng kỹ thuật phân tử và kết luận loài này có tỷ lệ nhiễm thấp. A. ceylanicum và A. duodenale được gọi chung là giun móc và do vòng đời phát triển khá giống với loài giun mỏ N. americanus nên 3 loài giun này thường được ghép chung với nhau bằng danh từ giun móc/mỏ [1-3]. Chẩn đoán nhiễm bệnh thường dựa vào hình thể trứng và ấu trùng của 3 loài giun từ bệnh phẩm phân. Điểm khác biệt về hình thể học giữa các loài là rất nhỏ, nên việc định danh còn nhiều điểm chưa rõ ràng. Vì vậy, hạn chế của chẩn đoán hình thể học khi áp dụng thực tế là chỉ dừng lại ở mức độ chẩn đoán chung chung, đó là có hay không nhiễm giun móc/mỏ. Với sự phát triển của kỹ thuật sinh học phân tử, chẩn đoán định loài đã chính xác và toàn diện. Kỹ thuật phân tử, đặc biệt là giải trình tự gen đã khắc phục được những nhược điểm tồn tại của các kỹ thuật chẩn đoán truyền thống. Ngoài ra, kỹ thuật này có khả năng xác định loài giun móc A. ceylanicum gây bệnh ở người Việt Nam có nguồn gốc từ động vật [4, 5]. Nghiên cứu này được tiến hành nhằm xác định chính xác thành phần từng loài giun móc, giun mỏ gây bệnh ở người dân bị nhiễm cư ngụ tại xã Thạnh Bình, huyện Tân Biên, tỉnh Tây Ninh. Từ đó nghiên cứu góp phần làm sáng tỏ hiện trạng nhiễm loài giun mỏ hay giun móc nào là phổ biến ở người Việt Nam. 2. Đối tượng và phương pháp nghiên cứu 2.1. Đối tượng Từ tháng 2/2022 đến 8/2022 tại xã Thạnh Bình, huyện Tân Biên, tỉnh Tây Ninh, 65 người dân sống tại đây được xác định nhiễm giun thông qua phương pháp nuôi cấy mẫu phân phối hợp với kỹ thuật soi trực tiếp. Ấu trùng giun móc/mỏ được thu thập từ các mẫu phân trên được sử dụng cho nghiên cứu. 2.2. Phương pháp nghiên cứu Hóa chất và môi trường cấy phân bằng phương pháp Sasa sử dụng trong nghiên cứu này được sản xuất từ Hãng Merck (Đức). Kít tách chiết DNA của Hãng ABT Biological Solution Company Limited, Việt Nam (Hà Đông, Hà Nội, Việt Nam). Các hoá chất thực hiện kỹ thuật PCR của Hãng Promega, Mỹ. Trình tự các đoạn mồi của các gen được tổng hợp bởi Công ty IDT, Singapore. Địa điểm nghiên cứu là Phòng Thí nghiệm của Bộ môn Ký sinh Y học, Khoa Khoa học Cơ bản-Y học Cơ sở, Trường Đại học Y khoa Phạm Ngọc Thạch và giải trình tự gen tại Công ty First BASE Laboratories-Axil Scientific, Singapore. Xác định hình thể ấu trùng và trứng của giun móc/mỏ bằng soi trực tiếp dưới kính hiển vi: Xét nghiệm tầm soát nhiễm giun cho người dân tình nguyện bằng phương pháp nuôi cấy Sasa và soi trực tiếp dưới kính hiển vi xác định được các trường hợp dương tính người dân nhiễm bệnh giun móc/mỏ. Tiến hành thu thập tất cả ấu trùng giun trong từng mẫu cấy bằng kỹ thuật ly tâm lắng cặn với tốc độ 3.000 vòng trong 5 phút. Phần cặn lắng sau ly tâm được soi dưới kính hiển vi kiểm tra sự hiện diện ấu trùng giai đoạn I, II Xác định thành phần loài giun móc (Ancylostoma duodenale, Ancylostoma ceylanicum) và giun mỏ Necator americanus gây bệnh cho người tại tỉnh Tây Ninh bằng kỹ thuật sinh học phân tưL Ngày nhận bài 22/3/2023; ngày chuyển phản biện 24/3/2023; ngày nhận phản biện 18/4/2023; ngày chấp nhận đăng 21/4/2023 Tóm tắt: Nhiễm giun móc/mỏ ở người Việt Nam là bệnh khá phổ biến. Tác nhân gây bệnh là Necator americanus và Ancylostoma duodenale. Một số nghiên cứu gần đây đã xác định thêm một tác nhân mới gây bệnh trên người-giun móc Ancylostoma ceylanicum, vốn thường ký sinh ở chó. Nghiên cứu sử dụng kỹ thuật PCR lồng (nested PCR) và giải trình tự gen nhằm xác định sự hiện diện và thành phần của các loài giun móc A. duodenale, A. ceylanicum và giun mỏ N. americanus. Các mẫu phân người dân nghi nhiễm sống tại xã Thạnh Bình, huyện Tân Biên, tỉnh Tây Ninh được tiến hành nuôi cấy, thu thập ấu trùng và xác định sơ bộ bằng hình thái học. 65 mẫu DNA được tách chiết từ các mẫu ấu trùng dùng cho các kỹ thuật sinh học phân tử. Kết quả cho thấy, 44 mẫu đơn nhiễm N. americanus (67,7%), 11 mẫu A. ceylanicum (16,9%) và 1 mẫu A. duodenale (1,6%). Các trường hợp đồng nhiễm (13,8%) là A. ceylanicum và N. americanus. N. americanus có nguồn gốc gần với loài được công bố tại Malaysia, A. ceylanicum có nguồn gốc gần với loài đã công bố tại Thái Lan. Kết luận, tại xã Thạnh Bình, loài mới A. ceylanicum phổ biến hơn loài truyền thống A. duodenale, chiếm 95,2 so với 4,8%. Từ khóa: Ancylostoma ceylanicum, giải trình tự gen, Necator americanus, PCR lồng, phân người, phương pháp Sasa. Chỉ số phân loại: 3.3
Hookworm infections are common in Vietnam and are caused by two known pathogens: Necator americanus and Ancylostoma duodenale. Recent studies have identified a new hookworm species - Ancylostoma ceylanicum, which typically parasitises in dogs. To determine the prevalence of these three hookworm species: A. duodenale, A. ceylanicum, and N. americanus, nested PCR and gene sequencing were employed in fecal samples from suspected individuals residing in Thanh Binh commune, Tan Bien district, Tay Ninh province. Larvae were collected and identified by morphology before 65 DNA samples were extracted and analysed using molecular biology techniques. Results showed that 67.7% (44/65) of the samples were infected with N. americanus, while 16.9% (11/65) and 1.6% (1/65) were infected with A. ceylanicumand A. duodenale, respectively. Co-infection was present in 13.8% of cases, with A. ceylanicum and N. americanus being the most common combination. The origin of the N. americanus species identified was found to be closely related to that reported in Malaysia, while A. ceylanicum was more closely related to the species reported in Thailand. In conclusion, the study found that the new hookworm species A. ceylanicum was more prevalent than the traditional species A. duodenale in Thanh Binh commune (95.2 vs 4.8% of hookworm infections).
The present review mainly highlighted on hookworm infections and the Ancylostoma species distribution, infection rate, epidemiology, prevalence, pathophysiology, diagnosis and treatments were analyzed. Hook Worms are located in the intestinal tract and/or tissues. Several investigations have reported the interesting phenomenon that the infective larvae of canine hookworm, Ancylostoma canium and other species. The immune response to worm infections also depends upon the location of infestation. Gastrointestinal nematode infections have always been a major animal health problem of domestic animals and/or ruminant livestock. Hosts with nematode infections present a series of pathological effects these changes include tissue damage, alterations in blood constituents, elevation or decrease of enzymatic levels. Hookworm diseases are most common in tropical and subtropical climatic conditions and the infections were observed in both animals and humans. As per the available information globally more than 740 million peoples are infected with hookworm. In sub-Saharan Africa, Asia, approximately 200 million people have been infected with hookworm, 90 million of them were children. The main objective of this review was to identify the prevalence, epidemiology and determinant factors of hookworm infection. Diagnostic methods that differentiate between hookworm species, including molecular methods, need to be developed for widespread use in control programmes to elucidate key features of hookworm epidemiology and control.
Parasites are important pathogens with significant global economic, public and animal health impacts. Successful control or elimination of many parasitic diseases, not least neglected tropical parasites, will require scalable, sensitive and cost-effective monitoring tools. Environmental DNA (eDNA) methods, used extensively in ecology for biomonitoring in natural ecosystems, offer promising advantages such reduced costs and labor requirements for species monitoring. Yet, the use of eDNA-based methods in parasitology and disease surveillance, has only recently begun to be explored. With this review, we wish to give an up-to-date overview of current uses and limitations of eDNA in human and veterinary parasitology, and how existing challenges can be overcome to fully utilize the potential of eDNA for monitoring and control of parasitic diseases. We begin by systematically searching published literature to identify studies that apply eDNA methods in parasitology and synthesize the main findings from these studies. We find that eDNA applications in parasitology only account for a small proportion (73/1960) of all eDNA publications up to now, and even fewer (27/73) studies, that apply eDNA methods specifically for parasites of human or veterinary importance. The majority of studies concern snail-borne trematodes and their intermediate host snails, while a few apply eDNA for mosquito vector species detection. A strong geographical bias, with only very few studies undertaken on the African continent, where parasites are of the biggest public health concern, is also noted. Current obstacles hindering further advances of eDNA methods in parasitology include incomplete reference databases, and challenges related to real-time monitoring in remote areas, and in certain LMIC settings. Finally, we point to future opportunities for eDNA-based research in parasitology and highlight recent innovations in eDNA research, which could further develop its application for monitoring and control of parasitic diseases and vectors in the future.
Giardia duodenalis is an intestinal protozoan parasite of humans and animal hosts and comprises eight microscopically indistinguishable molecularly-diverse lineages designated as assemblages A–H. Assemblages A and B are the primary sources of infections in humans and a wide range of mammals. Here, we identified assemblages, and inter-/intra-assemblage genetic diversity of human G. duodenalis isolates based on the multilocus sequence typing of the triosephosphate isomerase (tpi), β -giardin (bg), and glutamate dehydrogenase (gdh) loci. Multilocus sequence analysis of 62 microscopically-positive G. duodenalis fecal samples identified 26 (41.9%), 27 (43.5%), and nine (14.5%) isolates belonging to assemblages A, B, and discordant assemblages, respectively. The tpi locus assemblage-specific primers identified dual infections with A and B assemblages (45.2%). The sequence analysis of multiple alignments and phylogenetic analysis showed low genetic polymorphism in assemblage A isolates, classified as sub-assemblage AII at three loci, subtype A2 at tpi and gdh loci, and subtype A2 or A3 at bg locus. High genetic variations were found in assemblage B isolates with 14, 15, and 23 nucleotide patterns at tpi, bg, and gdh loci, respectively. Further concatenated sequence analysis revealed four multilocus genotypes (MLG) in 24 assemblages A isolates, two previously-identified (AII-1 and AII-5), with one novel multilocus genotype. However, the high genetic variations observed in assemblage B isolates among and within the three genetic loci prevented the definitive designation of specific MLGs for these isolates. Multilocus sequence typing may provide new insight into the genetic diversity of G. duodenalis isolates in Tehran, suggesting that humans are likely a potential source of G. duodenalis infection. Further host-specific experimental transmission studies are warranted to elucidate the modes of transmission within multiple host populations.
Soil-transmitted helminths (STH) including the hookworms Necator americanus and Ancylostoma spp., Ascaris lumbricoides, and Trichuris trichiura affect over 1.5 billion people worldwide and are estimated to have caused 1.9 million disability-adjusted life years (DALYs). With the concerted effort in expanding and improving targeted mass drug administration (MDA) programs over the past decade, along with decreasing prevalence, infections in several endemic areas tend to be of low intensity. Conventional microscopy-based methods recommended for the detection of STH in parasitological surveys have been shown to be less sensitive in these low-intensity settings. As communities progress towards STH elimination through MDA and improved sanitation, there is a pressing need for highly sensitive techniques that detect the true prevalence of STH to evaluate the effectiveness of ongoing programs and interventions. Molecular methods that involve analysis of DNA rather than the morphology of the organism are highly sensitive and specific, allowing for both quantitation and species discrimination. The following review discusses different sample collection strategies, pre-processing steps, DNA extraction platforms, and nucleic acid detection methods available for diagnosis and surveillance of STH. We have contrasted the utility of these molecular tools against conventional microscopy-based methods currently used in most endemic settings. While the detection methods are primarily qPCR based, several newer technologies have also become available along with automation and increased throughput, making these molecular tools increasingly cost-effective and potentially amenable for use in low-resource settings.
Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it is necessary to use molecular methodologies, since the eggs are morphologically similar. Here, we performed the molecular identification of single hookworm eggs from six Brazilian states. Of the 634 eggs individually analyzed, 98.1% (622/634) represented Necator americanus, and surprisingly, 1.9% (12/634 eggs from the same patient) represented Ancylostoma caninum. DNA analysis of the A. caninum-positive stool sample revealed no contamination with animal feces. This is the first report of the presence of A. caninum eggs in human feces, which may have a direct implication for the epidemiology of hookworm infection caused by this species. This suggests the need for special attention regarding prophylaxis, as different reservoirs, previously not described, may have great relevance for the spread of A. caninum.
Appropriate diagnostic techniques are crucial to global soil-transmitted helminth (STH) control efforts. The recommended Kato-Katz method has low sensitivity in low-transmission settings. Quantitative polymerase chain reaction (qPCR) is a highly sensitive alternative diagnostic option. However, little is known about the variability in qPCR results, and there are few published comparisons between qPCR and other microscopy-based techniques such as sodium nitrate flotation (SNF). Using 865 stool samples collected from 571 individuals, we compared SNF and qPCR in terms of diagnostic sensitivity and infection intensity measurements. In addition, we conducted repeated examinations on a single Necator americanus-positive stool sample over a 6-month period. Results showed good diagnostic agreement between SNF and qPCR for Ascaris spp. (κ = 0.69, P < 0.001), and moderate agreement for hookworm (κ = 0.55, P < 0.001) and Trichuris spp. (κ = 0.50, P < 0.001). Quantitative polymerase chain reaction demonstrated higher sensitivity than SNF for Ascaris spp. (94.1% versus 68.1%) and hookworm (75.7% versus 66.9%) but not for Trichuris spp. (53.1% versus 81.3%), which had very low prevalence. Sodium nitrate flotation and qPCR infection intensity measurements were strongly correlated for Ascaris spp. (ρ = 0.82, P < 0.001) and moderately correlated for hookworm (ρ = 0.58, P < 0.001). Repeated examinations using qPCR showed that N. americanus cycle threshold values decreased significantly at 1 month and remained stable thereafter. Results confirm the high diagnostic sensitivity of qPCR for Ascaris spp. and hookworm, particularly for light-intensity infections, which is ideal for settings approaching transmission elimination. Results support the potential for qPCR to be used as a quantitative assay for STH. Further research is needed in settings where Trichuris trichiura is endemic.
Historically, the diagnosis of soil-transmitted helminths (STHs) (e.g., Strongyloides stercoralis, Trichuris trichiura, Ancylostoma duodenale, Necator americanus, and Ascaris lumbricoides) has relied on often-insensitive microscopy techniques. Over the past several years, there has been an effort to use molecular diagnostics, particularly quantitative polymerase chain reaction (qPCR), to detect intestinal pathogens. While some platforms have been approved by regulatory bodies (e.g., Food and Drug Administration) to detect intestinal bacteria, viruses, and protozoa, there are no approved tests currently available for STH. Although studies comparing qPCR to microscopy methods for STH are imperfect, due in large part to a lack of a sufficient gold standard, they do show a significant increase in sensitivity and specificity of qPCR compared with microscopic techniques. These studies, as well as the advantages and disadvantages of using qPCR for STH diagnosis, are discussed. Guidelines for those designing future studies utilizing qPCR are proposed for optimizing results, as is the proposition for using standardized molecular diagnostics routinely for STH in clinical laboratories and for field-based studies when possible.
The family Taeniidae is of great importance in the medical and veterinary fields, particularly in the tropics and subtropics. Identification of eggs of different Taenia spp. in the final host by morphological examination is difficult owing to their similarity. Therefore, a multiplex polymerase chain reaction (PCR) targeting a mitochondrial gene was applied to identify morphologically indistinguishable eggs. Fecal samples from 100 domestic dogs, from the Mazandaran province in Iran, were examined using the flotation/sieving method followed by multiplex PCR. Taeniid eggs were observed in 24 % samples, of which 12 %, 10 %, and 2 % were infected with Echinococcus granulosus, Taenia spp., and both E. granulosus and Taenia spp., respectively. E. multilocularis was absent in these samples. The prevalence of E. granulosus in the examined domestic dogs as definitive hosts in north of Iran was high (14 %). Therefore, people living in this region of Iran are in danger of acquiring hydatid cyst, which is a serious public health problem.
Data on the presence and distribution of horse habronemosis in alive animals are lacking due to the high difficulties both in the clinical diagnosis and in a reliable coprodiagnosis in vivo. To overcome such limitations, a two-step semi-nested PCR assay for the specific amplification of the ribosomal second Internal Transcribed Spacer (ITS-2) of Habronema microstoma and Habronema muscae from horse faecal samples has been established. Characterization of the ITS-2 revealed sequences of different length and GC contents (296 bp and 29.5% for H. microstoma, and 334 bp and 35.9% for H. muscae, respectively), while the two-step semi-nested PCR assay showed a sensitivity of 96.7% and a specificity of 100%. The PCR assay showed to be much more powerful and reliable than the classical coprological examination, Besides its importance in large scale epidemiological surveys on alive animals, this diagnostic tool could be applied, under a practical point of view, for the differential diagnosis of both gastric and cutaneous habronemosis, for the evaluation of the efficacy of compounds against Habronema species in live horses, for studying the vector/s.
Resumen L a teniasis e himenolepiasis son consideradas endé-micas en el Ecuador. Estas zoonosis son causadas por las formas adultas de cestodos del género Tae-nia e Hymenolepis. La infestación por Taenia spp. es una zoonosis cuyas tasas de prevalencia varían en función de diversos factores socio-económicos y culturales. El comportamiento humano,
Ascaris lumbricoides, Trichuris trichiura, and Necator americanus are medically important soil-transmitted helminths (STHs) occurring frequently worldwide including Thailand. Fecal examination using a microscope has been recommended as the gold standard for diagnosis of STH infections, but suffers from low sensitivity. Recently, highly sensitive and specific assays, such as multiplex quantitative PCR, has been established, but the high cost and need for special instruments are still barriers limiting their applications in routine diagnosis. Therefore, a conventional multiplex PCR assay, with its lower cost and greater simplicity, was developed, for the simultaneous detection of STHs in fecal samples. The multiplex PCR assay was species-specific to the three STHs, and could detect one copy of DNA target. Compared with microscopic examination of fecal samples, sensitivity and specificity of the multiplex PCR was 87% and 83%, respectively. This multiplex PCR assay provides an alternative method for routine diagnosis of STHs infection, and might be applied for epidemiological studies of STHs in endemic areas.
Prevalence and intensity of parasitic infections is often higher in male than in female vertebrates. This bias may represent either differences between host sex in exposure or susceptibility to parasites. The former may be due to sex-specific behaviour of the host, including differential habitat use or diet. Differences in susceptibility are often regarded as a negative effect of male sex-steroid hormones on the immune system. Host-parasite dynamics are of great interest in terms of reptile survival, ecology and conservation. We used, for the first time, molecular diagnostics to track nematode parasitism in wild populations of reptiles non-invasively. Using slow worms (Anguis fragilis) as a model species, we investigated the interacting effects of time of year, sex, length, weight and climatic variables on the prevalence of the gastroenterological parasitic nematode Neoxysomatium brevicaudatum. Faeces were collected from three sites over two years. There was an interaction between sex and time of year, with lower nematode prevalence in males than females in July or August (different between years) but a high prevalence in males in April. As the latter is during the slow worm breeding season, this may be the result of testosterone-induced immunosuppression. A second order interaction between slow worm length and weight was found to be significant, with a positive association between prevalence and body condition in young slow worms and a negative association in older slow worms. The convex pattern of nematode prevalence with age that emerged suggests an increase with age-related exposure and a decrease with age-related acquired immunity. This article is protected by copyright. All rights reserved.
Measuring genetic variation is important for studying the epidemiology, systematics and population genetics of parasitic nematodes
as well as for their diagnosis and control. Technological advances pave the way for rapid progress in gene discovery and analysis.
In particular, mutation scanning allows high-resolution and high-throughput analysis of sequence or allelic variation between
and within individual parasitic nematodes and their populations. This chapter has highlighted a range of applications of SSCP
(combined with selective DNA sequencing) to parasitic nematodes for the purposes of species identification or delineation,
detection of cryptic species and diagnosis of infections. Importantly, it proposes future applications of the approach to
population genetic and molecular evolutionary studies, and indicates its attributes for investigating the ecology and epidemiology
of intestinal nematodes of humans.
Species identification of human hookworm infections among eight communities in rural areas of Peninsular Malaysia was determined during 2009-2011. Fecal samples were examined by microscopy and subsequently, the internal transcribed spacer 2 and 28S ribosomal RNA region of Necator americanus and Ancylostoma spp. were sequenced. Overall, 9.1% (58 of 634) were identified positive by microscopy for hookworm infection, and 47 (81.0%) of 58 were successfully amplified and sequenced. Sequence comparison found that N. americanus (87.2%) was the most predominant hookworm identified, followed by Ancylostoma ceylanicum (23.4%). No A. duodenale infection was detected in this study. Detection of A. ceylanicum in humans highlighted the zoonotic transmission among humans living near dogs. Thus, implementation of effective control measures for hookworm infections in future should seriously consider this zoonotic implication.
PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.
Infection with Oesophagostomum sp. is normally considered a rare zoonosis and up to this time its diagnosis has been based on the demonstration of larvae and young adult worms in the typical nodules formed in the intestinal wall. Only in Dapaong, in North Togo, and Bawku, North Ghana, have larger series of clinical cases been described. In the rural areas around these towns, a survey was made in which stool samples were collected and cultured. Third-stage larvae of Oesophagostomum sp. could be found after 5-7 days of incubation at room temperature, and the prevalence of infection with this parasite was often high but varied from one village to another. It was over 30% in seven villages out of the 15 villages surveyed. Anthelmintic treatment resulted in the evacuation of adult males and females of O. bifurcum. It is concluded that O. bifurcum is a locally common parasite of humans, not requiring an animal reservoir for completion of its lifecycle.
Fifty one patients with a peculiar tumour-like abdominal condition, part of them with intestinal occlusion, were operated upon in a period of 45 months at the Centre Hospitalier Regional of Dapaong in northern Togo, near to the border with Ghana. Thirty patients were children, less than 10 years old. The M/F ratio was 1.1/1. No geographical, social, familial or ethnic clustering was observed. The surgical appearance was highly characteristic with multiple nodules with central necrosis studding part or the whole of the large intestine. Histological appearances were typical of helminthoma in 21 of 24 biopsied cases. Twenty seven worms were observed in- or collected from the lesions of 14 patients of all ages. All identifiable specimens are immature male and female nematodes belonging to a single species, tentatively identified as Oesophagostomum bifurcum. The clinical features of the disease are recognized in the area and surgical interventions concern only part of the cases, most of which cure spontaneously in a few months. The frequency of the disease in humans and the absence of an obvious animal reservoir suggest that man himself may be the source of infection. A similar situation was described in 1964 in North-Eastern Ghana, just over the border.
Recently, it has been established that human infection with Oesophagostomum bifurcum is common in northern Togo and northeastern Ghana. Two surveys were conducted in this area. In a regional survey, O. bifurcum infection appeared to occur in 38 of 43 villages. The highest prevalences (up to 59%) occurred mostly in small isolated villages and were usually associated with high hookworm infection rates. The infection was relatively rare in children under five years of age (7% infected). In older individuals, females showed higher prevalences than males (30% v. 24%).
In a second survey, the entire population of two high-prevalence villages was examined. Infection rates were low in children under three years of age, but rose quickly thereafter, suggesting intense transmission. A stable level of infection was reached by 10 years of age.
Oesophagostomum larvae were found more frequently in hookworm-positive than in hookworm-negative coprocultures, and possible explanations for the association between infection with Oesophagostomum and hookworm are discussed.
When cultured alone or concurrently with Trichostrongylus colubriformis in sheep faeces, Ostertagia circumcincta produced fewer infective larvae per 100 eggs than did T. colubriformis. Averaged over five trials 60% of T. colubriformis eggs were recovered as infective larvae while for O. circumcincta the figure was only 39%. This result was observed for two strains of O. circumcincta and was independent of when larvae were harvested from culture (days 6-10 at 25 degrees C). The mortalities of both species occurred at the first and second larval stages. These observations are of concern when using larval differentiation from faecal culture to make quantitative estimates of worm egg numbers for each species present. Species such as T. colubriformis which have a low mortality during culture are likely to have their egg numbers overestimated when cultured with a species, like O. circumcincta, that suffers high mortality in culture.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
A sensitive and specific enzyme-linked immunosorbent assay (elisa) is described to diagnose human infection with Oesophagostomum bifurcum. In an elisa using crude soluble antigen, prepared from adult O. bifurcum, many cross reactions occurred when measuring IgG titres in patients with other helminth infections. An elisa based on the detection of specific IgG4, however, had a specificity of over 95%. The sensitivity of the IgG4 elisa was difficult to assess because a reliable parasitological diagnosis is not available. The IgG4-elisa described seems to be a powerful new tool to study the distribution of this little known but locally very common nematode
parasite.
Infection with Oesophagostomum sp. appears to be extremely common in man in northern Togo and Ghana. Adult specimens were recovered from the intestinal lumen by treatment with pyrantel pamoate and the morphological characteristics of oesophagostomes of man could for the first time be compared with information available on the morphology of oesophagostomes of monkeys. The observations and measurements demonstrated that the species involved is Oesophagostomum bifurcum and that the eggs of this species cannot be differentiated from those of Necator americanus. Both infections occur simultaneously in the population involved. The L1 larvae, too, cannot be differentiated from hookworm L1 larvae. The L3 larvae, however, are characteristic. Diagnoses of human Oesophagostomum infections is based on the detection of these larvae in coprocultures. In the present paper, the eggs, the L1 and L3 larval stages and the adults, are carefully described and photos are given.
Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.
Oesophagostomum bifurcum, as well as hookworm infections are hyperendemic among humans in northern Togo and Ghana. For parasite-specific diagnosis a coproculture is obligatory, because only the infective larvae, and not the eggs, can be distinguished morphologically. The sensitivity of duplicate coprocultures from a single stool sample was found to be above 90% in comparison to a gold standard of 10 coprocultures made from a single stool specimen. Prevalence of infection with O. bifurcum and hookworm further increased with the number of coprocultures made from each individual stool. Notwithstanding the high sensitivity, intensity of infection per individual varied considerably from day-to-day and the number of larvae found in different samples out of 1 stool also varied highly, both showing a heterogeneous distribution. Surprisingly, daily fluctuation and within-specimen variation could not be differentiated from each other, probably because of the variation created by the coproculture technique. To estimate the intensity of infection, it is sufficient to make repeated coprocultures from only 1 individual stool sample. Laborious collection of stool samples on subsequent days does not give better estimates of the individual infection status.
DNA technology is having a major impact in many areas of veterinary parasitology. In particular, the polymerase chain reaction (PCR) has found broad applicability because its sensitivity permits enzymatic amplification of gene fragments from minute quantities of nucleic acids derived from limited amounts of parasite material. This paper discusses some recent applications of PCR-based methods to parasites and highlights their usefulness or potential for those of veterinary importance. The focus is on PCR tools for the accurate identification of parasites and their genetic characterisation, the diagnosis of infections, the isolation and characterisation of expressed genes, the detection of anthelmintic resistance, and mutation scanning approaches for the high resolution analysis of PCR products.
Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodule worm) is of major human health significance in northern Togo and Ghana where the human hookworm, Necator americanus, also exists at high prevalence. Accurate diagnosis of O. bifurcum infection in humans is central to studying the epidemiology and controlling the parasite. To overcome limitations of current copro-diagnostic methods, we have developed an alternative, molecular approach. Utilising genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, we have established a two-step, semi-nested PCR method for the specific amplification of minute amounts (fg) of O. bifurcum DNA from human faecal samples. Using a panel of 155 well-defined faecal and DNA samples, the assay achieved a sensitivity of 94.6% and a specificity of 100%. This PCR assay will be useful for the diagnosis of O. bifurcum infection and as a molecular tool for elucidating the epidemiology of human oesophagostomiasis.
The intestinal helminth Oesophagostomum bifurcum is highly and focally endemic in northern Ghana and Togo, and its juveniles produce a nodular inflammatory response as they develop in the intestinal wall. This pathology can produce clinical symptoms. We report on 156 cases of oesophagostomiasis presenting in 1996-98 to Nalerigu hospital in northern Ghana. The disease accounted for 0.2% of the out-patient department new presentations (about 1 patient per week), and 1% (16) of the major acute surgical cases. Children aged 5-9 years were most commonly affected. Multinodular disease (13% of the cases) results from hundreds of pea-sized nodules within the colon wall and other intra-abdominal structures, and presents with general abdominal pain, persistent diarrhoea and weight loss. Dapaong tumour (87%) presents as an abdominal inflammatory mass often associated with fever. The 3-6-cm tumour is painful, well-delineated, smooth, spherical, 'wooden', periumbilical, and adhered to the abdominal wall. Cases most commonly presented during the late rains and early dry season. Diagnosis by ultrasound has reduced the need for exploratory surgery, and the ability to sonographically evaluate conservative treatment with albendazole has curtailed management by colectomy or incision and drainage.
Oesophagostomum spp are normally found as nematode parasites of ruminants, pig and monkeys. Occasionally humans are involved. In the past decade it became clear that, in some parts of Africa, humans are adequate final hosts. In those areas, prevalences of infection are high and morbidity is significant. The presence of lumen-dwelling adult worms, which do not seem to cause a great deal of pathology, can be demonstrated through coproculture. The presence of immature worms, encapsulated in nodules and responsible for pathology, on the other hand, is more difficult to confirm. It is not known what factors limit the distribution of endemic human oesophagostomiasis to a small focus in West Africa. The relationship between the 'helminthomas' described a long time ago in Uganda and the human Oesophagostomum infections in West Africa is unclear and it remains a mystery how humans get infected so effectively by ingesting L3 larvae. In this overview, Ton Polderman and Coby Blotkamp give an account of what is known and what is still to be elucidated in human Oesophagostomum infections.
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