Molecular Analysis of the 18S rRNA Gene of Cryptosporidium Parasites from Patients with or

An 840-bp fragment of the 18S rRNA gene was used to identify Cryptosporidium spp. recovered from human

J OURNAL OF C LINICAL M ICROBIOLOGY,Apr.2003,p.1458–1462Vol.41,No.4 0095-1137/03/$08.00ϩ0DOI:10.1128/JCM.41.4.1458–1462.2003

Copyright©2003,American Society for Microbiology.All Rights Reserved.

Molecular Analysis of the18S rRNA Gene of Cryptosporidium Parasites from Patients with or without Human Immunodeficiency Virus Infections Living in Kenya,Malawi,Brazil,the

United Kingdom,and Vietnam

Wangeci Gatei,1,2Julie Greensill,2Richard W.Ashford,1Luis E.Cuevas,1

Christopher M.Parry,2Nigel A.Cunliffe,2Nicholas J.Beeching,1

and C.Anthony Hart2*

Liverpool School of Tropical Medicine1and Department of Medical Microbiology and Genitourinary

Medicine,University of Liverpool,2Liverpool,United Kingdom

Received31July2002/Returned for modification11November2002/Accepted23January2003

An840-bp fragment of the18S rRNA gene was used to identify Cryptosporidium spp.recovered from human

immunodeficiency virus(HIV)-infected and-uninfected patients from Kenya,Malawi,Brazil,the United King-

dom,and Vietnam.Initial identification was by Ziehl-Neelsen acid-fast staining.Confirmation was by nested

PCR,targeting the most polymorphic region of the18S rRNA gene.Genotyping was by restriction endonuclease

digestion of the PCR product followed by nucleotide sequencing.Among63isolates analyzed,four genotypes

of Cryptosporidium were identified;75%of the isolates were of the C.parvum human genotype,while the

potentially zoonotic species were of the C.parvum bovine genotype(21.7%),the C.meleagridis genotype(1.6%

[one isolate]),and the C.muris genotype(1.6%[one case]).HIV-infected individuals were more likely to have

zoonotic genotypes than the HIV-uninfected individuals.Among the C.parvum group,strains clustered dis-

tinctly into either human or bovine genotypes regardless of the geographical origin,age,or HIV status of the

patients.The intragenotypic variation observed in the C.parvum human genotype was extensive compared to

that within the C.parvum bovine genotype group.The variation within genotypes was conserved in all geo-

graphical regions regardless of the patients’HIV status.The extensive diversity within genotypes at the18S

rRNA gene locus may limit its application to phylogenetic analyses.

The use of molecular methods in the taxonomy of Crypto-sporidium spp.has led to increased recognition of the diversity of the species infecting humans.The main causative agents of human cryptosporidiosis are strains of human and bovine ge-notypes of the species C.parvum(also called genotype1and genotype2,respectively)(3).Recent reports document human infections with zoonotic species,including C.parvum dog and cat types(now renamed C.canis and C.felis,respectively)and C.meleagridis and C.muris(mainly avian and murine parasites, respectively)(4,5,11,13,24).A recently recognized Crypto-sporidium cervine genotype has been identified in both immu-nocompromised and immunocompetent people(9).However, the prevalence and significance of the different species and genotypes in both immunocompetent and immunodeficient people are not yet clear.Experimental studies of both humans and animals suggest that diverse species and genotypes show different levels of infectivity and virulence(8).Others have pointed out that zoonotic strains of C.parvum produce more severe infections in humans than the strains found only in humans(7).Moreover,the potential reservoir hosts and trans-mission pathways for the novel species infecting humans are unclear(3).These issues underscore the importance of the precise identification of species recovered from humans.

So far,a limited number of isolates have been typed from developing countries and especially from human immunodefi-ciency virus(HIV)-infected people(5,12,16,17,19,20,24), yet cryptosporidiosis is endemic in most tropical regions(2). Extensive genotyping of isolates from different parts of the world is therefore crucial for a more precise mapping of the epidemiology of Cryptosporidium.Our study has targeted the hypervariable region of the18S rRNA gene in Cryptosporidium for precise parasite identification.Samples analyzed were re-covered from children and adult human patients,with or with-out HIV infection,living in diverse geographical regions.Our study also assessed the extent of variation in sequences from the isolates within each genotype and their effect on the ap-plication of this gene target to a phylogenetic analysis of Cryp-tosporidium.

MATERIALS AND METHODS

Sample collection and DNA extraction.Cryptosporidium oocysts were recov-ered from human fecal samples collected from Kenya(32samples)and stored in 2.5%potassium dichromate at4°C.Other isolates were from Malawi(11sam-ples),Vietnam(3samples),Brazil(7samples),and the United Kingdom(10 samples;9samples from humans and1sample from a captive monkey)and were recovered from fecal samples stored atϪ80°C without preservatives.Specimens stored in potassium dichromate were washedfive times in cold distilled water to remove traces of the preservative prior to DNA extraction.A pea size sample of the frozen fecal sample or approximately a200-␮lfinal suspension of each sample in potassium dichromate was suspended in200␮l of lysis buffer supplied in a QIAamp kit(QIAGEN Ltd.,Crawley,West Sussex,United Kingdom). Oocysts were ruptured by subjecting them to a freeze-thaw cycle ofϪ80°C for30 min andϩ80°C for15min.DNA was then extracted from the suspension with a

*Corresponding author.Mailing address:Department of Medical Microbiology and Genitourinary Medicine,The University of Liver-pool,Duncan Building,Daulby St.,Liverpool L693GA,United King-dom.Phone:01517064381.Fax:01517065805.E-mail:cahmm@liv

.ac.uk.

1458 at Penn State Univ on February 8, 2008 http://doc.xuehai.net Downloaded from

第1页下一页