A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of <i>Cryptosporidium parvum</i> genotypes 1 and 2

Gile, M; Warhurst, David C.; Webster, K. A.; West, Donna; Marshall, J. A.

doi:10.1017/s0031182002001786

Cited by 28 publications

(24 citation statements)

References 20 publications

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“…A TaqMan PCR assay targeting the 18S ribosomal DNA (rDNA) has allowed sensitive detection of Cryptosporidium species (155). Various other PCR assays for the detection of Cryptosporidium species in stool specimens have also been developed (18,107,136,293). While being more expensive and time-consuming, PCR and ELISA have shown superior sensitivity for the detection of Cryptosporidium species compared to conventional staining and microscopy (158,199).…”

Section: Cryptosporidiummentioning

confidence: 99%

Clinical Significance of Enteric Protozoa in the Immunosuppressed Human Population

et al. 2009

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SUMMARY Globally, the number of immunosuppressed people increases each year, with the human immunodeficiency virus (HIV) pandemic continuing to spread unabated in many parts of the world. Immunosuppression may also occur in malnourished persons, patients undergoing chemotherapy for malignancy, and those receiving immunosuppressive therapy. Components of the immune system can be functionally or genetically abnormal as a result of acquired (e.g., caused by HIV infection, lymphoma, or high-dose steroids or other immunosuppressive medications) or congenital illnesses, with more than 120 congenital immunodeficiencies described to date that either affect humoral immunity or compromise T-cell function. All individuals affected by immunosuppression are at risk of infection by opportunistic parasites (such as the microsporidia) as well as those more commonly associated with gastrointestinal disease (such as Giardia). The outcome of infection by enteric protozoan parasites is dependent on absolute CD4+ cell counts, with lower counts being associated with more severe disease, more atypical disease, and a greater risk of disseminated disease. This review summarizes our current state of knowledge on the significance of enteric parasitic protozoa as a cause of disease in immunosuppressed persons and also provides guidance on recent advances in diagnosis and therapy for the control of these important parasites.

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Section: Cryptosporidiummentioning

confidence: 99%

Clinical Significance of Enteric Protozoa in the Immunosuppressed Human Population

et al. 2009

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“…In immunosuppressed patients, the disease is often more severe, is usually associated with chronic diarrhea and wasting, and can be life threatening (1). Laboratory diagnosis of cryptosporidiosis traditionally relies on special staining techniques, while alternative diagnostic techniques have also been employed, including ELISA and various PCR assays (2,9,12,32). A number of studies have demonstrated that PCR has superior sensitivity for the detection of Cryptosporidium species compared to conventional staining, microscopy, and ELISA (14,16).…”

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confidence: 99%

Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis , Entamoeba histolytica , and Giardia intestinalis in Clinical Stool Samples

et al. 2011

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The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.Enteric protozoa continue to be the most commonly encountered parasitic diseases and to cause significant morbidity and mortality throughout both developed and developing regions of the world, affecting millions of people each year (22). While Blastocystis hominis is the most common protozoan parasite detected in stool samples, the major etiological agents of parasitic diarrhea are considered to be Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis (22).E. histolytica is a pathogenic amoeboid protozoan parasite for which humans are the primary reservoir (27). It is a potentially invasive pathogen and the causative agent of amebiasis, with approximately 50 million cases and 100,000 deaths annually (28). The clinical presentation can range from asymptomatic carriage to gastrointestinal disease and invasive disease. E. histolytica is morphologically identical to the nonpathogenic species Entamoeba dispar and Entamoeba moshkovskii, though genetic differences have confirmed their separation into independent species (4, 20). Due to this conserved morphology, stained smears of stool specimens are insufficient for differentiation of the species. Currently, the only way to differentiate the pathogenic E. histolytica from the nonpathogenic strains via microscopy is to visualize ingested red blood cells within the E. histolytica trophozoite; however, this is rarely seen i...

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“…One portion in a 15ml Falcon tube was stained with Lugol's iodine. Smear preparation of the other portion on clean dry 76mm x 26mm microscopy slide was stained with the modified Ziehl-Neelsen stain (23) .…”

Section: Sample Processingmentioning

confidence: 99%

“…The assay was performed on species-specific DNA controls and a range of well-defined stools samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic infections and could improve patient management and infection control (23) .…”

Section: Introductionmentioning

confidence: 99%

“…A Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) with specific primers and probes represents an alternative to conventional PCR for increasing the speed of sample analysis while decreasing the potential risks for contamination of the laboratory environment with amplicons (23) . The causative agent of amebic colitis and liver abscess is E. histolytica (54) .…”

Section: Introductionmentioning

confidence: 99%

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DETECTION OF HUMAN INTESTINAL PROTOZOA BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City

Mohammad

2013

Zagazig University Medical Journal

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Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. During Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays, 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stools samples containing various bacterial, viral, and other protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites.

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A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2

Cited by 28 publications

References 20 publications

Clinical Significance of Enteric Protozoa in the Immunosuppressed Human Population

Clinical Significance of Enteric Protozoa in the Immunosuppressed Human Population

Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis , Entamoeba histolytica , and Giardia intestinalis in Clinical Stool Samples

DETECTION OF HUMAN INTESTINAL PROTOZOA BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City

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