Publications (2)8.12 Total impact
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Article: Bartonella koehlerae, a new cat-associated agent of culture-negative human endocarditis.
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ABSTRACT: Bartonella koehlerae is reported for the first time to be a human pathogen that causes culture-negative endocarditis. It is also shown that this species, isolated twice before from domestic cats, can be recovered as well from a stray cat population in Israel. This work follows a recent report of the same case in which the causative agent was misidentified as B. henselae, based on serology and PCR-restriction fragment length polymorphism (RFLP) analysis (A. Schattner, O. Zimhony, B. Avidor, and M. Gilad, Lancet 361:1786, 2003). B. koehlerae was identified in the valvular tissue of an endocarditis patient by DNA sequencing of the PCR products of two Bartonella genes: the genes for citrate synthase (gltA) and riboflavin synthase (ribC). The commonly used PCR-RFLP analysis of the TaqI-digested gltA PCR product did not distinguish between B. koehlerae and B. quintana or between B. elizabethae and B. clarridgeiae. PmlI digestion of the gltA amplification product failed to differentiate between B. quintana, B. clarridgeiae, and B. elizabethae. RFLP analysis of the heat shock protein (htrA) gene by TaqI digestion misidentified B. koehlerae as B. henselae. However, RFLP analysis of the ribC PCR product, digested with TaqI, was able to distinguish between the human endocarditis-associated Bartonella species tested, B. henselae, B. quintana, B. elizabethae, and B. koehlerae, as well as between the cat-associated Bartonella species, B. henselae and B. clarridgeiae. Given the expanding number of Bartonella species emerging as human pathogens, it is suggested that PCR-RFLP analysis for the diagnosis of Bartonella infections target several genes and be coupled with DNA sequencing to avoid species identification.Journal of Clinical Microbiology 09/2004; 42(8):3462-8. · 4.15 Impact Factor -
Article: Insight into the intrinsic sensitivity of the PCR assay used to detect CMV infection in amniotic fluid specimens.
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ABSTRACT: PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3 x 10(6) viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2 x 10(4) copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8 x 10(3) copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. Conclusions: AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.Journal of Clinical Virology 05/2004; 29(4):260-70. · 3.97 Impact Factor
