Detection of Cryptosporidium in stool samples by Taq-Man qPCR v1

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

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Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

Journal of Clinical Microbiology ◽

10.1128/jcm.01798-16 ◽

2016 ◽

Vol 55 (3) ◽

pp. 844-858 ◽

Cited By ~ 16

Author(s):

Per Sikora ◽

Sofia Andersson ◽

Jadwiga Winiecka-Krusnell ◽

Björn Hallström ◽

Cecilia Alsmark ◽

...

Keyword(s):

Parasite Burden ◽

Genomic Variation ◽

Loss Of Function ◽

Oocyst Wall ◽

Gene Encoding ◽

Cryptosporidium Hominis ◽

Content Type ◽

Successful Isolation ◽

Target Dna ◽

Stool Samples

ABSTRACTIn order to improve genotyping and epidemiological analysis ofCryptosporidiumspp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 differentCryptosporidium hominispatient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encodingCryptosporidiumoocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to theCryptosporidium parvumreference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in theC. hoministree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencingCryptosporidiumdirectly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes ofCryptosporidiumfrom human fecal samples, while alluding to the potential for a higher degree of genotyping withinCryptosporidiumepidemiology.

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Unusual cryptosporidiosis cases in Swedish patients: extended molecular characterization ofCryptosporidium viatorumandCryptosporidiumchipmunk genotype I

Parasitology ◽

10.1017/s003118201300084x ◽

2013 ◽

Vol 140 (14) ◽

pp. 1735-1740 ◽

Cited By ~ 21

Author(s):

MARIANNE LEBBAD ◽

JESSICA BESER ◽

MONA INSULANDER ◽

LILLEMOR KARLSSON ◽

JENS G. MATTSSON ◽

...

Keyword(s):

Molecular Characterization ◽

Cryptosporidium Parvum ◽

Geographical Variation ◽

Indian Subcontinent ◽

Common Species ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Cryptosporidium Hominis ◽

Genotype I ◽

Protein Locus

SUMMARYMost human cases of cryptosporidiosis are caused byCryptosporidium parvumorCryptosporidium hominis, but the use of molecular diagnostic methods has revealed that several other less common species or genotypes can also be involved. Here, we describe two unusual causes of cryptosporidiosis, one being the recently described speciesCryptosporidium viatorumand the otherCryptosporidiumchipmunk genotype I. Two Swedish patients who were infected withC. viatorumhad travelled to Kenya and Guatemala, respectively, and two others had been infected withCryptosporidiumchipmunk genotype I in Sweden. None of these four patients were immunocompromised, and all four showed classical symptoms of cryptosporidiosis. We performed extensive molecular characterization, including analysis of four loci. The twoC. viatorumisolates were found to differ slightly at the 70-kDa heat shock protein locus, which may indicate a local geographical variation in this species that has previously been described exclusively on the Indian subcontinent.

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Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in Fecal Samples by Using Multiplex Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.42.3.1220-1223.2004 ◽

2004 ◽

Vol 42 (3) ◽

pp. 1220-1223 ◽

Cited By ~ 255

Author(s):

J. J. Verweij ◽

R. A. Blange ◽

K. Templeton ◽

J. Schinkel ◽

E. A. T. Brienen ◽

...

Keyword(s):

Real Time ◽

Entamoeba Histolytica ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Giardia Lamblia ◽

Simultaneous Detection ◽

Fecal Samples

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Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652008005000003 ◽

2008 ◽

Vol 50 (3) ◽

pp. 139-143 ◽

Cited By ~ 18

Author(s):

Ana Julia Urias dos Santos Araújo ◽

Herminia Yohko Kanamura ◽

Marcos Eduardo de Almeida ◽

Aparecida Helena de Souza Gomes ◽

Thais Helena Lemos Pinto ◽

...

Keyword(s):

Fragment Length Polymorphism ◽

Genetic Characterization ◽

Cryptosporidium Oocyst ◽

Sao Paulo ◽

São Paulo ◽

Oocyst Wall ◽

Cryptosporidium Hominis ◽

Stool Samples ◽

Dna Fragment ◽

Genotypic Identification

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

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Cryptosporidium infection in non-human hosts in Malawi

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v76i4.19 ◽

2009 ◽

Vol 76 (4) ◽

Cited By ~ 7

Author(s):

Z. Banda ◽

Rosely A.B. Nichols ◽

A.M. Grimason ◽

H.V. Smith

Keyword(s):

Cryptosporidium Parvum ◽

Rainy Season ◽

18S Rrna ◽

Cryptosporidium Oocyst ◽

Dry Season ◽

Cool Season ◽

Cryptosporidium Hominis ◽

Adult Cattle ◽

Faecal Samples ◽

Cryptosporidium Oocysts

Of 1 346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3 % were from cattle (29.8 % of these were from calves < 6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6 % of adult cattle and 11.7 % of calves were infected, compared to 28.9 % of adult cattle and 36.7 % of calves in Thyolo. Dependent on season, between 7.8 % and 37.7 % (Chikwawa) and 16.7 % and 39.3 % (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n = 225], pigs [n = 92], sheep [n = 6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6 % of goat samples contained oocysts in Chikwawa, compared to between 16.7 % and 39.3 % in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7 %), whereas in Thyolo, infections occurred in all three seasons (17.9 % in the rainy season, 25 % in the cool season and 60 % in the dry season). Often diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and / or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.

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