Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

Annals of Laboratory Medicine ◽

10.3343/alm.2015.35.3.306 ◽

2015 ◽

Vol 35 (3) ◽

pp. 306-313 ◽

Cited By ~ 10

Author(s):

Abdullah Kilic ◽

Mohammad J. Alam ◽

Naradah L. Tisdel ◽

Dhara N. Shah ◽

Mehmet Yapar ◽

...

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Real Time Pcr ◽

Stool Samples ◽

Pcr Method ◽

Simultaneous Identification

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Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor

Annals of Saudi Medicine ◽

10.5144/0256-4947.2021.293 ◽

2021 ◽

Vol 41 (5) ◽

pp. 293-298

Author(s):

Mehmet Karabey ◽

Hüseyin Can ◽

Tülay Öncü Öner ◽

Mert Döşkaya ◽

Sedef Erkunt Alak ◽

...

Keyword(s):

Sample Size ◽

Real Time ◽

Solid Tumors ◽

Real Time Pcr ◽

Tertiary Care ◽

Diagnostic Methods ◽

Cross Sectional ◽

Cryptosporidium Spp ◽

Stool Samples ◽

Pcr Targeting

BACKGROUND: Cryptosporidium spp . is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp . can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp . in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp . prevalence was determined using Ziehl–Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp . in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl–Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl–Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.

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Genotype-specific detection of Giardia lamblia in stool samples of diarrhoeal and non-diarrhoeal patients in Dhaka, Bangladesh

Dhaka University Journal of Biological Sciences ◽

10.3329/dujbs.v20i2.8979 ◽

1970 ◽

Vol 20 (2) ◽

pp. 183-189 ◽

Cited By ~ 1

Author(s):

Md Masud Alam ◽

Mohammad Ilias ◽

Md Abdullah Siddique ◽

Md Mamun Kabir ◽

Farida Nazib ◽

...

Keyword(s):

Positive Result ◽

Real Time ◽

Real Time Pcr ◽

Giardia Lamblia ◽

Specific Antigen ◽

Diarrhoeal Disease ◽

Giardia Cyst ◽

Positive Stool ◽

Stool Samples ◽

Assemblage B

Two major genotypic assemblages (A and B) of Giardia lamblia infect humans. A single-vessel multiplex real-time PCR assay was used that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. In this study, 157 diarrhoeal (symptomatic) and non-diarrhoeal (asymptomatic) stool samples collected from the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and Bangabandhu Sheikh Mujib Medical University (BSMMU) hospital, respectively were analyzed to determine whether an association exists between infections with G. lamblia assemblages A or B and diarrhea in Bangladesh. Of the 157 stool samples, Giardia cysts were observed in 35 by microscopy and 127 showed positive result for Giardia cyst specific antigen. The 127 ELISA positive samples were assayed for genotyping by real?time polymerase chain reaction. Of the 117 real-time PCR positive stool samples, 15 were positive for G. lamblia assemblage A, 96 were positive for assemblage B and 6 samples showed positive result for both G. lamblia assemblage A and B infections. Higher ratios for diarrhea were observed for assemblage A infections, whereas higher parasite DNA loads and a higher overall rate were observed for assemblage B infections in both diarrhoeal and non-diarrhoeal patients. Real-time PCR is, therefore, useful as an additional test supplementary to microscopy or enzyme immunoassay to detect genotypes of Giardia. Key words: Giardia lamblia; Genotypes; Multiplex real-time PCR; Immunoassay DOI: http://dx.doi.org/10.3329/dujbs.v20i2.8979 DUJBS 2011; 20(2): 183-189

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Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽

10.3390/pathogens8030152 ◽

2019 ◽

Vol 8 (3) ◽

pp. 152 ◽

Cited By ~ 2

Author(s):

Vivornpun Sanprasert ◽

Ruthairat Kerdkaew ◽

Siriporn Srirungruang ◽

Sarit Charuchaibovorn ◽

Kobpat Phadungsaksawasdi ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Simultaneous Detection ◽

Diagnostic Tools ◽

Multiple Infections ◽

Parasitic Infections ◽

Soil Transmitted Helminths ◽

Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

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Molecular Characterization of Prader–Willi Syndrome by Real-Time PCR

Genetic Testing ◽

10.1089/gte.2007.0105 ◽

2008 ◽

Vol 12 (2) ◽

pp. 319-324 ◽

Cited By ~ 6

Author(s):

Teresa Munce ◽

Robert Simpson ◽

Francis Bowling

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Prader Willi Syndrome

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Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods

Journal of Medical Microbiology ◽

10.1099/jmm.0.007732-0 ◽

2009 ◽

Vol 58 (7) ◽

pp. 905-911 ◽

Cited By ~ 27

Author(s):

Matthew W. Gilmour ◽

Linda Chui ◽

Theodore Chiu ◽

Dobryan M. Tracz ◽

Kathryn Hagedorn ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Molecular Methods ◽

Genetic Profile ◽

Pcr Assay ◽

Suspension Array ◽

Stool Samples ◽

Pcr Targeting

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

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Advances in molecular surveillance of Clostridium difficile in Bulgaria

Journal of Medical Microbiology ◽

10.1099/jmm.0.058149-0 ◽

2013 ◽

Vol 62 (9) ◽

pp. 1428-1434 ◽

Cited By ~ 5

Author(s):

Elina G. Dobreva ◽

Ivan N. Ivanov ◽

Rossitza S. Vathcheva-Dobrevska ◽

Katucha I. Ivanova ◽

Galina D. Asseva ◽

...

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Variable Number ◽

Innovative Methods ◽

Pcr Ribotyping ◽

Typing Methods ◽

Stool Samples

The increasing incidence of Clostridium difficile infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 C. difficile isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the gluD and tcdB genes was developed for rapid C. difficile identification. Four toxigenic profiles were distinguished by PCR: A+B+CDT− (53.1 %, 17/32), A−B+CDT− (28.1 %, 9/32), A+B+CDT+ (9.4 %, 3/32) and A−B−CDT− (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical C. difficile isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most C. difficile cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of gluD and tcdB proved to be very effective and improved C. difficile identification and confirmation of clinical C. difficile isolates.

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Molecular characterization of toxigenic clostridium difficile by multiplex and real-time PCR

10.5353/th_b4663139 ◽

2011 ◽

Author(s):

Chi-chiu, Elvin Chan

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Molecular Characterization ◽

Real Time Pcr

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Human Bocavirus Prevalence In Children With Acute Gastroenteritis From Rural Communities In The Northen Region Of South Africa

10.1101/830281 ◽

2019 ◽

Author(s):

Mpumelelo Casper Rikhotso ◽

Ronewa Khumela ◽

Jean Pierre Kabue ◽

Afsatou Ndama Traoré ◽

Natasha Potgieter

Keyword(s):

South Africa ◽

Rural Communities ◽

Real Time ◽

Real Time Pcr ◽

Acute Gastroenteritis ◽

Enteric Viruses ◽

Health Care Facilities ◽

Human Bocavirus ◽

Stool Samples ◽

Pcr Targeting

AbstractBACKGROUNDAcute gastroenteritis (AGE) is a leading cause of morbidity and mortality in young children worldwide. Human Bocavirus (HBoV) is an emerging virus globally associated with diarrhea. The aim of this study was to demonstrate the prevalence of HBoV genotypes in children (≤5 years) from rural communities in South Africa (SA) suffering from AGE.MATERIAL AND METHODA total of 141 fecal samples of children ≤5 years with acute gastroenteritis (AGE) were collected from rural Primary Health Care facilities in the Vhembe district of SA between June 2017 and July 2018. Clinical symptoms and demographic data were also recorded. A total of 102 (72%) were outpatients and 39 (28%) were hospitalized patients. Human Bocavirus (HBoV) genotypes were determined using Real-Time Multiplex PCR. DNA extracts of positive samples were confirmed by conventional PCR targeting the NS1 gene. Co-infection with other enteric viruses were determined in HBoV positive samples using Real-Time PCR.RESULTSHBoV was detected in 8 (5.7%) children with AGE. Children were in the age group between 1-24 months. HBoV1 and HBoV3 genotypes were each detected in 3 (37.5%) stool samples and HBoV2 in 2 (25%) stool samples. Co-infection with other enteric viruses included Rotavirus (37.5%); Adenovirus (37.5%); Norovirus (25%) and Astrovirus (12.5%).CONCLUSIONHBoV infections could be seen as a potential emerging diarrheal pathogen in South Africa. Further studies are required to understand the role of HBoV infections in children and adults with acute gastroenteritis.Author summaryAcute gastroenteritis (AGE) is recognized as a major cause for mortality in children ≤5 years of age in Africa and other developing countries. Viruses known to be involved in AGE includes Rotavirus, Norovirus, Astrovirus and Adenovirus and have been reported globally. Recently the Human Bocavirus (HBoV) have been reported in numerous studies globally as a potential cause of diarrhea. In this study, the prevalence and genetic diversity of human Bocavirus in children with AGE from rural communities in Limpopo, South Africa were investigated. In total, 141 stool samples from children ≤ 5 years with AGE were assessed for the presence of HBoV using Real-Time PCR. HBoV were detected in 8 (5.7%) patients and included 3 positive samples for HBoV1 and HBoV3 respectively and 2 positive for HBoV2. No HBoV4 were detected. Among the 8 positive HBoV samples, co-infection with other enteric viruses were found in 7 (87.5%) samples, while mono infection with HBoV alone was detected in 1 (12.5%) patient. HBoV mixed infection with Rotavirus (3/8; 37.5%); Adenovirus (3/8; 37.5%); Norovirus (2/8; 25%) and Astrovirus (1/8; 12.5%) were observed in this study. This study reported for the first time on the prevalence of human Bocavirus in children with AGE from rural communities in South Africa.

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