Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Raman spectroscopic analysis of the clonal and horizontal spread of CTX-M-15-producing Klebsiella pneumoniae in a neonatal intensive care unit.

Nosocomial outbreaks of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5 months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 ß-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.

Cryptosporidium parvum (Eucoccidiorida: Cryptosporiidae) in calves: results of a longitudinal study in a dairy farm in Sfax, Tunisia.

A longitudinal study was undertaken to determine the prevalence of Cryptosporidium in a dairy farm in Sfax, Tunisia. 480 faecal samples were obtained from 30 calves under one month of age. All faecal samples were analysed for Cryptosporidium oocysts by microscopic examination of smears stained by modified Ziehl Neelsen technique. The parasite was detected in 26 calves (86.7%). Infection was significantly associated with diarrhoea. A molecular characterization, performed in seven calves, confirmed that isolates were C. parvum. This work is the first report on Cryptosporidium in calves in Tunisia.

Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia.

1,001 faecal samples were obtained from 89 sheep (lambs and adult), 184 goats, 190 horses, 178 rabbits, 110 camels, 200 broiler chicken and 50 turkeys housed in farms from different localities in Tunisia. All samples were analysed for Cryptosporidium oocysts by microscopic examination of smears stained by modified Ziehl Neelsen technique. The parasite was detected in ten lambs and adult sheep (11.2 %) and nine broiler chicken (4.5 %). Molecular characterization, performed in four animals, identified C. bovis in three lambs and C. meleagridis in one broiler chicken. This work is the first report on Cryptosporidium in farm animals in Tunisia.

PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin.

Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

Molecular characterization of Cryptosporidium isolates obtained from humans in France.

Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.

[Cryptosporidium and wildlife: a risk for humans?]. / Cryptosporidium et faune sauvage: un risque pour l'homme?

The present review underlines the knowledge of Cryptosporidium, especially its biodiversity and transmission. The presence of the parasite in different mammal host species is discussed with real, potential risk of transmission to humans. The potential role of insects in mechanical transmission of the parasite is evaluated by experimental protocols. The cost of cryptosporidiosis at health and economic levels are mentioned, which emphasises the importance of detection and identification of the parasite in the environment and in wild mammal species, using specific molecular tools. Potential measures to be accomplished in order to fight off cryptosporidiosis are also noted.

Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii.

Genetic divergence at the SODA (manganese-dependent superoxide dismutase, MnSOD) locus were compared in six Pneumocystis carinii formae speciales isolated from mouse, rabbit, human, macaque and pig. A degenerate oligonucleotide primer strategy was designed to amplify 85-90% of the full-length SODA gene from P. carinii genomic DNA isolates. DNA sequence analysis revealed an A/T bias in the nucleotide composition (71-77.2%) and the presence of seven small introns (41-142 bp), interrupting each P. carinii open reading frame (ORF) at the same position. The MnSOD deduced amino acid sequences from all P. carinii isolates shared residues which were conserved within the MnSOD family and which are required for enzymatic activity and binding of the cofactor metal. Phylogenetic analysis including MnSOD sequences from representatives of the fungal phyla Basidiomycota and Ascomycota indicated that the P. carinii formae speciales form a monophyletic group that is related to the budding yeasts (subphylum Saccharomycotina, previously called class Hemiascomycetes) in the Ascomycota. In the whole Pneumocystis group, P. carinii f. sp. hominis, P. carinii f. sp. macacae and P. carinii f. sp. oryctolagi MnSOD sequences clustered together, as did the rat-derived P. carinii and P. carinii f. sp. muris sequences.

Molecular cloning and characterization of a superoxide dismutase (sod) gene in Pneumocystis carinii.

This work reports the isolation and characterization of a gene encoding a superoxide dismutase (SOD, EC.1.15.1.1.) from Pneumocystis carinii derived from rat. Sense and antisense oligonucleotides, deduced from SOD amino acid sequences from a wide variety of organisms, allowed amplification of a 669 bp genomic DNA fragment specific to this P. carinii. RACE-PCR was used to obtain the major part of the complementary DNA; the 5'- and 3'-genomic regions were obtained respectively from a Mbol subgenomic library and from an amplified fragment using oligonucleotides designed from the cDNA sequence. Comparison of genomic and cDNA sequences showed an open reading frame of 660 bp interrupted by seven small introns. The deduced amino acid sequence contained 220 residues. Protein sequence alignment demonstrated the highest homology (50.5% identity; 70.3% similarity) with Saccharomyces cerevisiae manganese-SOD (MnSOD) suggesting that P. carinii SOD belongs to the mitochondrial MnSOD group. A putative targeting peptide found at the 5'-end of the P. carinii SOD sequence also suggested its mitochondrial localization.

Isoenzyme diversity in Pneumocystis carinii from rats, mice, and rabbits.

Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised patients. To investigate the genetic diversity of P. carinii populations, multilocus enzyme electrophoresis was used to analyze five enzyme systems (malate dehydrogenase, glucose phosphate isomerase, leucine aminopeptidase, malic enzyme, and 6-phosphogluconate dehydrogenase). Only five different multilocus associations (zymodemes) were recorded for the 70 isolates studied. While only one multilocus combination was found in mice and rabbits, three different multilocus associations were recorded in rats. Population genetic tests and phylogenetic analysis strongly suggest that P. carinii genotypes are host-specific, in agreement with molecular study results, and that no genetic exchange occurs between genotypes from different host species. This hypothesis could be verified only by the evolutionary genetic approach, which relies here on multilocus analysis.

Biodiversity of Pneumocystis carinii hominis: typing with different DNA regions.

The purpose of this study was to identify the most useful gene for the detection of biodiversity of Pneumocystis carinii hominis isolates and to compare samples from French and Italian subjects. We studied 20 bronchoalveolar lavage fluid specimens from 20 human immunodeficiency virus-infected patients (10 French and 10 Italian patients) with Pneumocystis carinii pneumonia by DNA sequencing of the thymidylate synthase (TS), 5S rRNA, large-subunit mitochondrial rRNA (mt LSU rRNA), and internal transcribed spacer (ITS1 and ITS2) genes. Thirteen of the 20 sequenced samples had the prototype TS gene sequence. Fourteen of the 20 samples showed the prototype sequence of the 5S rRNA gene, and 6 had variant sequences of the 5S rRNA gene. The mt LSU rRNA gene was sequenced for 18 of the 20 samples; all sequences were different from the prototype sequence and were classified into four groups. Thirteen of the 20 ITS1 and ITS2 sequences were analyzed, and all the sequences were found to be different from the prototype sequence and were classified into 10 groups. The internal transcribed spacer regions thus appear to be the most discriminatory region of DNA for analysis of the biodiversity of P. carinii hominis isolates.