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Journal of Microbiological Methods Dec 2020Microscopy is the gold standard for diagnosis of intestinal parasitic diseases in many countries, including Cuba, although molecular approaches often have higher...
Microscopy is the gold standard for diagnosis of intestinal parasitic diseases in many countries, including Cuba, although molecular approaches often have higher sensitivity as well as other advantages. Fecal samples from 133 patients were analyzed by light microscopy and also real-time multiplex qPCR targeting Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica, and, separately, Dientamoeba fragilis. Microscopy revealed G. duodenalis occurred most commonly (17 patients), followed by Blastocystis spp. (12 patients). In a few patients, Entamoeba histolytica/E. dispar, Cryptosporidium spp., and Cyclospora cayetanensis were identified. Molecular analysis identified 4 more G. duodenalis infections and 2 more Cryptosporidium spp. infections; concordance between microscopy and PCR showed almost perfect agreement for G. duodenalis (κ = 0.88) and substantial agreement for Cryptosporidium (κ = 0.74). PCR indicated that E. dispar, rather than E. histolytica, had been identified by microscopy. Additionally, 16 D. fragilis infections were detected using molecular methods. Although both microscopy and molecular techniques have a place in parasitology diagnostics, for parasites such as D. fragilis, where microscopy can underestimate occurrence, molecular techniques may be preferable, and also essential for distinguishing between morphologically similar microorganisms such as E. histolytica and E. dispar. Although in resource-constrained countries such as Cuba, microscopy is extremely important as a diagnostic tool for intestinal parasites, inclusion of molecular techniques could be invaluable for selected protozoa.
Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Cross-Sectional Studies; Cryptosporidiosis; Cryptosporidium; Cuba; Dientamoeba; Dientamoebiasis; Entamoeba histolytica; Entamoebiasis; Feces; Female; Giardia lamblia; Giardiasis; Humans; Intestinal Diseases, Parasitic; Male; Microscopy; Middle Aged; Molecular Diagnostic Techniques; Real-Time Polymerase Chain Reaction; Young Adult
PubMed: 33188802
DOI: 10.1016/j.mimet.2020.106102 -
Pathogens (Basel, Switzerland) Mar 2021, the brown or Norway rat, is the most abundant mammal after humans in urban areas, where they live in close proximity to people. Among rodent-borne diseases, the...
, the brown or Norway rat, is the most abundant mammal after humans in urban areas, where they live in close proximity to people. Among rodent-borne diseases, the reservoir role of Norway rats of zoonotic parasites in cities has practically been ignored. Considering the parasitic diseases in the One Health approach, we intended to identify and quantify the zoonotic intestinal protozoans (ZIP) in an urban population of in the city of Barcelona, Spain. We studied the presence of ZIP in 100 rats trapped in parks ( = 15) as well as in the city's sewage system ( = 85) in the winter of 2016/17. The protozoans were molecularly identified by means of a multiplex PCR (Allplex Gastrointestinal Panel-Parasite Assay). We also investigated the presence of co-infections among the species found. Four ZIP were identified, presenting significant prevalences in sewers, specifically (83.5%) (37.7%), spp. (34.1%), and (14.1%). Several co-infections among the detected ZIP were also detected. The reservoir role of ZIP that Norway rats play in cities as well as the role rats may play as sentinels of zoonotic parasites affecting humans in urban areas are strongly backed up by our findings. The increasing worldwide urbanization, climate change, and the COVID-19 pandemic are factors that are producing an increase in human-rat interactions. Our results should be considered a warning to the authorities to intensify rat control and surveillance in public health interventions.
PubMed: 33799948
DOI: 10.3390/pathogens10030311 -
Acta Tropica Jul 2020A test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a... (Comparative Study)
Comparative Study
INTRODUCTION
A test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a gold standard. Three different commercial kits were included in the comparison, with a range of 3-15 different PCR targets, while 14 targets were covered by in-house testing, so not all 16 target pathogens were covered by all assays.
METHODS
Residual materials from nucleic acid extractions of stool samples with very high likelihood of being colonized or infected by at least one enteric parasite species or microsporidia were tested. In all, 500 DNA samples were analyzed, but due to limited sample volume, only 250 of the 500 samples were tested per assay. Each sample was assessed with the qPCR platforms being compared and cycle threshold (Ct) values were included in a descriptive comparison.
RESULTS
Depending on the assay applied, qPCR detected per 250 tested samples Giardia duodenalis (184-205), Blastocystis spp. (174-183), Trichuris trichiura (118-120), Ascaris lumbricoides (79-96), Necator americanus (78-106), Hymenolepis nana (40-42), Cryptosporidium spp. (27-36), Dientamoeba fragilis (26-28), Schistosoma spp. (13-23), Enterobius vermicularis (8-14), Entamoeba histolytica (7-16), Strongyloides stercoralis (6-38), Cyclospora spp. (6-13), Taenia spp. (1-4), microsporidia (1-5), and Ancylostoma spp. (1-2). Inter-assay agreement kappa was almost perfect (0.81-1) for Dientamoeba fragilis, Hymenolepis nana, Cryptosporidium spp., and Ascaris lumbricoides, substantial (0.61-0.8) for Necator americanus, Blastocystis spp., Ancylostoma spp., Giardia duodenalis, Schistosoma spp., Trichuris trichiura, and Enterobius vermicularis, moderate (0.41-0.6) for Entamoeba histolytica, fair (0.21-0.4) for microsporidia, slight (0-0.2) for Cyclospora spp. and Strongyloides stercoralis, and poor (<0) for Taenia spp.
CONCLUSIONS
Varying inter-assay agreement makes interpretation of microsporidia and parasite PCR in stool samples challenging. Intra-assay agreement had been controlled during the developing of the assays. Future studies, e.g., with optimized nucleic acid procedures and including microscopically characterized samples, are advisable.
Topics: Animals; Feces; Humans; Intestinal Diseases, Parasitic; Microsporidia; Parasites; Real-Time Polymerase Chain Reaction
PubMed: 32371221
DOI: 10.1016/j.actatropica.2020.105516 -
Infectious Diseases (London, England) Feb 2020Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin...
Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of , spp., and from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results. SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region Jönköping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl-Neelsen staining on patients with certain symptoms and real-time PCR. We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR. In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.
Topics: Acetic Acid; DNA, Protozoan; Dientamoeba; Entamoeba histolytica; Feces; Formaldehyde; Giardia lamblia; Humans; Microscopy; Molecular Typing; Parasitology; Protozoan Infections; Sodium Acetate; Sweden
PubMed: 31656115
DOI: 10.1080/23744235.2019.1682188 -
Medicina (Kaunas, Lithuania) Apr 2024The guidelines for chronic urticaria in children contain recommendations that are often based on adult studies. The diagnostic pathway has not been standardized and the... (Observational Study)
Observational Study
The guidelines for chronic urticaria in children contain recommendations that are often based on adult studies. The diagnostic pathway has not been standardized and the effectiveness of anti-H1, omalizumab, montelukast, and systemic glucocorticoids is rarely reported in the pediatric population. There is a wide variation in the rate of remission of chronic urticaria between studies. The aim of this study is to enhance our understanding of pediatric chronic urticaria. This study enrolled 37 children with chronic urticaria aged from 0 to 18 years. Demographic parameters, medical history, clinical features, laboratory data and treatment information were collected. Children were treated with the recommended dosage of second-generation H1-antihistamines, which was increased by up to twofold. Omalizumab was added for refractory anti-H1 patients. A three-day course with systemic glucocorticoids was administered for severe exacerbations. Montelukast was administered to some children. : Wheals without angioedema were common. Chronic urticaria was spontaneous in 32 children (86.48%), inducible in 2 (5.41%), induced by a parasite in 1 and vasculitic in 2. Treatment of the potential causes of chronic urticaria was of no benefit, except for eradication of Dientamoeba fragilis. Chronic urticaria was resolved within three years in 45.9% of cases. Allergic diseases were present in nine children (24.32%) and autoimmune diseases were present in three (8.11%). All children were treated with anti-H1 at the licensed dose or at a higher dose. A partial or complete response to anti-H1 was observed in 29 (78.38%) patients. Montelukast showed no benefit. All children treated with omalizumab responded. Systemic glucocorticoids were successfully used to treat exacerbations. Our findings indicate that laboratory tests should not be routinely performed in children with chronic urticaria without clinical suspicion. However, comorbidities such as thyroid autoimmune disease and coeliac disease are suggested to be monitored over the chronic urticaria course. These clinical conditions could be diagnosed from the diagnostic framework of chronic urticaria. Increasing the dosage of anti-H1 and omalizumab was effective in children resistant to standard treatment but we still need further studies to generate a standard patient-centered treatment.
Topics: Humans; Child; Female; Male; Child, Preschool; Adolescent; Chronic Urticaria; Infant; Sulfides; Cyclopropanes; Quinolines; Acetates; Omalizumab; Histamine H1 Antagonists; Glucocorticoids; Anti-Allergic Agents; Infant, Newborn; Chronic Disease; Urticaria
PubMed: 38792886
DOI: 10.3390/medicina60050704 -
Parasite (Paris, France) 2022Commercial multiplex PCR assay panels were developed to overcome the limitations of microscopic examination for parasitological diagnosis on stool samples. However,...
Selecting a multiplex PCR panel for accurate molecular diagnosis of intestinal protists: a comparative study of Allplex (Seegene), G-DiaParaTrio (Diagenode), and RIDAGENE (R-Biopharm) assays and microscopic examination.
Commercial multiplex PCR assay panels were developed to overcome the limitations of microscopic examination for parasitological diagnosis on stool samples. However, given the increased supply of this diagnostic approach, these assays must be evaluated to position them in a diagnostic algorithm. Analytical performances of the multiplex PCR assay G-DiaParaTrio, Allplex GI parasite and RIDAGENE parasitic stool panel for detecting Blastocystis sp., Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., Dientamoeba fragilis, and Cyclospora cayetanensis, were assessed through a retrospective comparative study on 184 stool samples initially sent for parasitological investigation. The composite reference method for parasitological diagnosis was microscopic observation and Entamoeba histolytica-specific adhesion detection when necessary. Multiplex PCR assays were performed on extracted DNA from each stool, following the manufacturer's recommendations. Discrepant results with the composite reference method were investigated with species-specific PCR to approach a final parasitological diagnosis. Overall sensitivity/specificity for the multiplex PCR assays was 93.2%/100% for G-DiaParaTrio, 96.5%/98.3% for Allplex GI parasite and 89.6%/98.3% for RIDAGENE, whereas the composite reference method presented an overall sensitivity/specificity of 59.6%/99.8%. These results confirmed the added diagnostic value of the multiplex PCR approach for gastrointestinal protists. Nevertheless, the PCR procedure and the analytical performance for each protist of interest, variable depending on the multiplex PCR assay, must be considered when implementing a PCR-based diagnostic approach.
Topics: Animals; Cryptosporidiosis; Cryptosporidium; Entamoeba histolytica; Feces; Giardia lamblia; Multiplex Polymerase Chain Reaction; Retrospective Studies; Scrapie; Sensitivity and Specificity; Sheep
PubMed: 35138245
DOI: 10.1051/parasite/2022003 -
Microorganisms Apr 2020This study aims at evaluating the performances of the multiplex PCR Allplex Gastrointestinal Panel-Parasite Assay (GIPPA), which detects , spp., , , , and , by...
This study aims at evaluating the performances of the multiplex PCR Allplex Gastrointestinal Panel-Parasite Assay (GIPPA), which detects , spp., , , , and , by comparison to microscopy. A retrospective evaluation was conducted on a series of positive clinical samples ( = 99) stored at -80 °C or at +4 °C. A five-month prospective study was then conducted on all samples sent to our lab for parasite detection ( = 586). In the retrospective cohort, sensitivity was 81% for both (26/32) and (21/26) and 100% for spp. (26/26, including 6 different species), (26/26), and (4/4). During the prospective study, 95 samples were positive by microscopy and 207 by multiplex PCR assay. The molecular assay showed a significantly higher sensitivity of PCR, especially for (100% vs. 60.7%, < 0.01), (97.2% vs. 14.1%, < 0.001), and (99.4% vs. 44.2%, < 0.001) but also for (100% vs. 50.0%). The sensitivity of the Allplex GIPPA on the first stool sample was equivalent to the sensitivity of microscopy on multiple stool samples but inferior to multiplex PCR on multiple stool samples. Taken together, the Allplex GIPPA is suitable for the routine detection of protozoa in fecal samples.
PubMed: 32326453
DOI: 10.3390/microorganisms8040569 -
Mikrobiyoloji Bulteni Oct 2019Aim of the present study was to identify protozoones which are difficult to define through wet slide in fresh fecal samples by using different fixatives with modified...
Aim of the present study was to identify protozoones which are difficult to define through wet slide in fresh fecal samples by using different fixatives with modified Trichrome stain within five minutes. Two different fixatives prepared for the alternative approach. The slides were fixed by two different fixatives, one of them (fixative-1) was based ethylalcohol, formalin, acetic acid, distilled water and the other one (fixative-2) based ethylalcohol, formalin, citric acid, distilled water included a mordant [divalent or polyvalent metals which make coordination complex with some dyes] consisted copper sulphate pentahydrate (CuSO4 .5H2 O). Slides prepared by the two different fixatives were stained by a different modification of Gomori's trichrome stain that we made. Samples fixed by Schaudinn fixative including mercury chloride were stained by Wheatley modification of Gomori's trichrome stain as a gold standard for control and comparison. We worked with 50 fecal samples which we thought included human intestinal protozoones after the wet slide examination. Comparing the methods, slides prepared with the method including citric acid gave almost similar results with the classical method excluded Entamoeba coli cystes. Slides prepared with the methode including acetic acid gave low performance compared with the classical method especially E.coli cystes and Blastocystis spp., Endolimax nana, Iodamoeba bütschlii, E.hartmanni. Both new fixatives gave superior performance at the slides included Dientamoeba fragilis and approximately shorten the procedure process ten times than the classical method. When the both alternative methods compared in each other, the slides prepared with fixative-2 exposed better performance for the protozoones Blastocystis spp., E.nana, I.bütschlii and E.hartmanni while the fixative-1 displayed minimal superiority for D.fragilis including criterias that we based. The fixative-2 and modified stain methode that we used in our study, makes available the diagnostic phase ten times faster than the classical method in human stool parasitological tests excluding the E.coli cystes at parasitology and microbiolgy laboratories. It seems to be a good option to the classical method for routine usage.
Topics: Eukaryota; Feces; Fixatives; Formaldehyde; Humans; Microscopy; Parasitic Diseases; Parasitology
PubMed: 31709939
DOI: 10.5578/mb.68633 -
New Microbes and New Infections Apr 2024Gastrointestinal pathogens (GPs) contribute significantly to the burden of illness worldwide with diarrhoea being the most common among gastrointestinal symptoms (GSs)....
BACKGROUND
Gastrointestinal pathogens (GPs) contribute significantly to the burden of illness worldwide with diarrhoea being the most common among gastrointestinal symptoms (GSs). In the COVID-19 disease, diarrhoea, could be one of the initial presenting symptoms. However, no data on the potential correlation between diarrhoea-causing pathogens and SARS-CoV-2 infection are available. Therefore, we carried out a 2-years retrospective study aimed to evaluate the prevalence of "classic" GPs among SARS-CoV-2 infected and non-infected patients with diarrhoea in Italy.
METHODS
Results of SARS-CoV-2 research from nasopharyngeal and detection of GPs from stool swab samples by Allplex™ SARS-CoV-2 and GI Virus, Bacteria and Parasite Assay were analysed for all patients with diarrhoea referring to Policlinico Ospedaliero Universitario, Foggia, (Italy) from February 2022 to October 2023.
RESULTS
Out of the 833 involved patients, 81 (3.9%) were COVID-19 positive, while 752 (90.3%) were COVID-19 negative. Among COVID-19-positive patients, 37% (n = 30/81) were found positive for one or more GPs with a higher prevalence of protozoan parasites (18.5%) ( ST1-ST4 subtypes, genotype I), followed by bacteria (7.4%) ( sp., sp.). Viral pathogens were more frequent among COVID-19 negative patients (Adenovirus, Norovirus). Among GPs, ST3 subtype was the most prevalent registered in the 16% of patients (p = 0.0001).
CONCLUSIONS
Based on obtained results, a likely interaction between the classic GPs and SARS-CoV-2 infection can be speculated, driven by protozoan parasites. Moreover, these results also provide baseline data to understand more deeply sp. role in this scenario of dysbiosis, particularly in those cases of SARS-CoV-2 co-infection.
PubMed: 38406386
DOI: 10.1016/j.nmni.2024.101228 -
Revista Espanola de Quimioterapia :... Feb 2023Microscopic examination of the intestinal parasites, from the patient's concentrated feces, has a lower sensitivity when compared to molecular diagnostic techniques....
OBJECTIVE
Microscopic examination of the intestinal parasites, from the patient's concentrated feces, has a lower sensitivity when compared to molecular diagnostic techniques. Therefore, the objective of this study has been to compare both techniques, as well as to evaluate whether there is a correlation between the microscopic examination and the threshold cycles (Ct) obtained for Blastocystis hominis.
METHODS
Retrospective study of the samples received in the Microbiology laboratory during September 2021. The MiniParasep SF® concentration test was performed for microscopic visualization and then PCR was performed with the Seegene AllplexTM Parasite Assay panel.
RESULTS
A 27% (n=74) of the samples were positive by molecular diagnosis, with a total of 87 parasites detected. 53% (n=39) were women with a mean age of 47 ± 24 years. In 76% (n=56) of the cases the service of origin was Primary Care. The most frequently found parasite was B. hominis, 85% (n=64), followed by Dientamoeba fragilis 20% (n=15) and Giardia lamblia 11% (n=8). Co-infection by two parasites was detected in 13 cases (B. hominis + D. fragilis in 6 cases, and B. hominis + G. lamblia in 7 cases). In the microscopic diagnosis, 9.5% (n=26) positivity was obtained. The most frequently found parasite was B. hominis, 84% (n=23), followed by G. lamblia, which was seen in three cases by microscopy. D. fragilis was not seen in any case. Coinfection of B. hominis + G. lamblia was observed in one sample.
CONCLUSIONS
Techniques for molecular diagnosis of intestinal parasites are fast, reliable and more sensitive than microscopic techniques, improving microbiological diagnosis and quality of care.
Topics: Humans; Female; Young Adult; Adult; Middle Aged; Aged; Male; Retrospective Studies; Microscopy; Intestinal Diseases, Parasitic; Giardia lamblia; Feces; Molecular Biology
PubMed: 36453171
DOI: 10.37201/req/088.2022