Mireille M. Kattar

Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts

ABSTRACT Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.

Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Human Brucella Isolates in a Region of Brucellosis Endemicity

ABSTRACT Brucellosis remains an important anthropozoonosis worldwide. Brucella species are genetically homogeneous, and thus, the typing of Brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive. Although many methods could segregate isolates into the phylogenetically recognized taxa, limited within-species genetic diversity has been identified. Recently, multilocus variable-number tandem-repeat analysis (MLVA) was found to have a high degree of resolution when it was applied to collections of Brucella isolates from geographically widespread locations, and an assay comprising 16 such loci (MLVA-16) was proposed. This scheme includes eight minisatellite loci (panel 1) and eight microsatellites (panel 2, which is subdivided into panels 2A and 2B). The utility of MLVA-16 for the subtyping of human Brucella isolates from geographically restricted regions needs to be further evaluated, and genotyping databases with worldwide coverage must be progressively established. In the present study, MLVA-16 was applied to the typing of 42 human Brucella isolates obtained from 41 patients recovered from 2002 to 2006 at a tertiary-care center in Lebanon. All isolates were identified as Brucella melitensis by MLVA-16 and were found to be closely related to B. melitensis isolates from neighboring countries in the Middle East when their genotypes were queried against those in the web-based Brucella2007 MLVA database (http://mlva.u-psud.fr/ ). Panel 2B, which comprised the most variable loci, displayed a very high discriminatory power, while panels 1 and 2A showed limited diversity. The most frequent genotype comprised seven isolates obtained over 7 weeks in 2002, demonstrating an outbreak from a common source. Two isolates obtained from one patient 5 months apart comprised another genotype, indicating relapsing disease. These findings confirm that MLVA-16 has a good discriminatory power for species determination, typing of B. melitensis isolates, and inferring their geographical origin. Abbreviated panel 2B could be used as a short-term epidemiological tool in a small region of endemicity.

Central venous catheter-related bacteremia due to Tsukamurella species in the immunocompromised host: a case series and review of the literature.

We report 6 cases of bacteremia due to Tsukamurella species, all of which were in immunosuppressed patients with indwelling central venous catheters (CVCs). Fewer than 20 cases of serious illness due to these gram-positive bacilli have been reported in the medical literature; these cases have mostly been ascribed to the species Tsukamurella paurometabola. Tsukamurella species are frequently misidentified as Rhodococcus or Corynebacterium species. We used high-performance liquid chromatography to identify these organisms to the genus level and 16S ribosomal RNA gene sequencing and DNA-DNA dot blots for species identification. Three of our isolates were identified as Tsukamurella pulmonis, 1 was identified as Tsukamurella tyrosinosolvans, and 1 was identified as a unique species. One isolate was not maintained long enough for species identification. All patients were successfully treated with antimicrobial therapy and CVC removal. Infection with this organism should be considered in the immunosuppressed patient with an indwelling CVC and gram-positive bacilli in the blood.

Tsukamurella strandjordae sp. nov., a Proposed New Species Causing Sepsis

ABSTRACT We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of Tsukamurellaspecies by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genusTsukamurella for which we propose the nameTsukamurella strandjordae sp. nov. Our study also revealed that Tsukamurella paurometabola ATCC 25938 represents a misnamed Tsukamurella inchonensis isolate and confirms thatTsukamurella wratislaviensis belongs to the genusRhodococcus.

Oxacillinase-mediated resistance to carbapenems in Klebsiella pneumoniae from Lebanon

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Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

ABSTRACT PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis.

Spontaneous complete regression of hepatic epithelioid haemangioendothelioma

A 75-year-old woman presented with a 3-month history of upper abdominal pain associated with distention and change in bowel habits. She also reported fatigue and weight loss of 5 kg in the preceding 4 months. She had no history of hepatitis, blood transfusions, or alcohol intake. Her family history was negative for malignant disease, and she was not taking any prescribed drugs. On examination, the patient was pale, but not icteric; vital signs were within normal limits. She had mild tender ness in the right upper quadrant of the abdomen without hepatosplenomegaly. Laboratory results were as follows (reference values in parentheses): alanine amino transferase 13 U/L (<50 U/L); aspartate aminotransferase 9 U/L (<50 U/L); γ glutamyl transferase 10 U/L (<50 U/L); alkaline phosphatase 105 U/L (<120 U/L); α feto protein 1·7 μg/L (1·0–9·0 U/mL); cancer antigen (CA) 19-9 6·6 U/mL (0–37 U/mL); carcino embryonic antigen 1·8 μg/L (0–5 μg/L); and CA 125 16·5 U/mL (0–40 U/mL). Further investigation included helical CT of the chest, abdomen, and pelvis, which showed multiple suspicious lesions in the liver (fi gure 1A) and a few lesions in the lung bases. Fine-needle aspiration and core biopsy of the liver lesions were done, and the biopsy sample showed epithelioid tumour cells arranged in small nests and short cords in desmoplastic stroma. The tumour cells had abundant eosinophilic cytoplasm and moderate nuclear pleiomorphism; multinucleated cells and occasional mitoses were noted (fi gure 2A). Some cells had intracellular lumina that were thought at fi rst to be vacuoles or glandular lumina. On the basis of these fi ndings, the tumour was initially diagnosed as a poorly diff erentiated carcinoma. Work-up—including upper and lower gastrointestinal endoscopy, ultrasonography, and mammography—were within normal limits. The patient refused any type of treatment, and was discharged home. Nevertheless, the patient was followed up regularly by her primary physician. 20 months later, after an improvement in her clinical condition and a weight gain of more than 10 kg, a repeat CT showed resolution of all hepatic lesions (fi gure 1B). The patient was followed up for more than 3·5 years and continues to do well. In view of the clinical behaviour of this neoplasm, path ological material was reassessed and imm u nostaining was done. Tumour cells were positive for vascular markers CD34 (fi gure 2B) and CD31, and were negative for epithelial markers (including cytokeratin 7, cytokeratin 20, cyto keratins 8/18 (Cam 5·2), and cytokeratins AE1/AE3); α feto protein; oestrogen receptors and progesterone receptors; and thyroid transcription factor 1 (TTF1, which is usually expressed in lung carcinoma). The morphological and immunophenotypic features are diagnostic of epithelioid haemangioendothelioma. Epithelioid haemangioendothelioma is an indolent mesenchymal neoplasm that arises from endothelial cells. This rare vascular neoplasm occurs frequently in soft tissues, liver, lung, bone, and spleen. It is a solid tumour of low-grade malignancy, consisting of epithelioid-like endothelial cells, and can be misdiagnosed as carcinoma—primary or metastatic. The clinical features of epithelioid haemangioendothelioma of the liver have been reviewed by several researchers. Makhlouf and colleagues analysed 137 patients with epithelioid haemangioendothelioma Lancet Oncol 2006; 7: 439–41

Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

BackgroundThe detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA.MethodsFive kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN), peqGold™ Tissue DNA Mini Kit (PeqLab), UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin™ Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control.ResultsKits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared.ConclusionsWe observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

Fatal disseminated Mycobacterium simiae infection in a non-HIV patient

We report a fatal case of disseminated Mycobacteriumsimiae infection in an immunocompetent elderly man with fever of unknown origin. The diagnosis was based on culture isolation of non-tuberculous mycobacteria from cerebrospinal fluid and bronchoalveolar lavage. Both culture isolates were identified as M. simiae by 16S rRNA gene sequencing.

Preliminary validation of real-time PCR assays for the identification of Yersinia pestis

Abstract Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples. Clin Chem Lab Med 2008;46:1239–44.