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Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5؊3 h for the QIAGEN extraction.Tuberculosis is a public health problem worldwide, and for optimal control, early diagnosis is necessary (4, 6, 7). Several researchers have developed real-time PCR assays that provide rapid detection of various target sequences of Mycobacterium tuberculosis complex (MTBC) and drug resistance genes in patient specimens (1,3,5,(16)(17)(18)(19). The ability of these assays to detect MTBC in clinical samples is dependent on both the target sequence selected and the efficiency of the DNA extraction procedure. Several methods of mycobacterial cell wall lysis and DNA extraction have been evaluated, including detergents, proteolytic enzymes, mechanical disruption, and temperature changes alone and in various combinations (1, 2, 5, 8-15, 17, 20). The objective of this study was to compare six methods of extracting M. tuberculosis DNA from respiratory specimens: Tris-EDTA (TE) boil extraction (10), PrepMan ultra extraction (Applied Biosystems, Inc., Foster City, CA), Infectio Diagnostics, Inc. (IDI) lysis extraction (Infectio Diagnostics, Inc. Quebec, Canada), QIAGEN QIAmp DNA mini kit (QIAGEN, Inc., Valencia, CA), sodium dodecyl sulfate (SDS)-Triton X extraction (9), and SDS-Triton X plus sonication. The effectiveness of each extraction method was assessed using two quantitative real-time PCR assays.Sample preparation. Digested and decontaminated (Nacetyl-cysteine-2% NaOH) sputum specimens that were culture negative for mycobacteria were pooled for use as the standard respiratory specimen. A suspension of M. tuberculosis ATCC 27294 was prepared in sterile saline and adjusted to the density of a 1.0 McFarland standard. The suspension was diluted 1:10 in saline and used to spike the pooled respiratory sp...

Mycobacterium massiliense is a rapidly growing mycobacterium that is indistinguishable from Mycobacterium chelonae/M. abscessus by partial 16S rRNA gene sequencing. We sequenced rpoB, sodA, and hsp65 genes from isolates previously identified as being M. chelonae/M. abscessus and identified M. massiliense from isolates from two patients with invasive disease representing the first reported cases in the United States.Rapidly growing mycobacterium infections are increasing in the United States (7) and are difficult to speciate by conventional methods. Partial 16S rRNA gene sequencing is the most widely used method for the identification of nontuberculous mycobacteria (6,8,12), but this gene target is often limited by the lack of sequence divergence among closely related Mycobacterium species (15). Mycobacterium chelonae and M. abscessus are two species that share the same 16S rRNA gene sequence, and since distinguishing these two species is clinically relevant, assays targeting base pair differences within the 16S-23S rRNA internal transcribed spacer (ITS) region have been developed (5

Several Mycobacterium-like organisms related to the Mycobacterium terrae complex have been isolated from clinical samples. In the clinical microbiology laboratory, partial 16S rRNA gene sequencing (approximately the first 500 bp) rather than full 16S rRNA gene sequencing is often used to identify Mycobacterium species. Partial 16S rRNA gene sequence analysis revealed 100 % similarity between 65 clinical isolates and Mycobacterium sp. MCRO 6 (GenBank accession no. X93032). Even after sequencing the nearly full-length 16S rRNA gene, closest similarity was only 99?6 % to Mycobacterium nonchromogenicum ATCC 19530 T . Sequencing of the nearly full-length 16S rRNA gene, the 16S-23S internal transcribed spacer region and the hsp65 gene did not reveal genotypic identity with the type strains of M. nonchromogenicum, M. terrae or Mycobacterium triviale. Although sequence analysis suggested that these clinical isolates represented a novel species, mycolic acid analysis by HPLC failed to distinguish them from M. nonchromogenicum. Therefore, phenotypic analysis including growth characterization, antibiotic susceptibility testing and biochemical testing was performed. These strains from clinical samples should be recognized as representing a novel species of the genus Mycobacterium, for which the name Mycobacterium arupense sp. nov. is proposed. The type strain is AR30097 T (=ATCC BAA-1242 T =DSM 44942 T ).At the time of writing, the genus Mycobacterium comprises 119 species with validly published names, at least 30 of which have been described within the last 5 years (http:// www.bacterio.cict.fr/m/mycobacterium.html). Despite this rapid increase in the number of newly recognized Mycobacterium species, many additional Mycobacterium species remain to be formally described (Pauls et al., 2003;Tortoli, 2003;Turenne et al., 2004). Many of these unnamed species have been isolated from clinical specimens and need to be correctly characterized for appropriate patient management.In the clinical microbiology laboratory, phenotypic and biochemical testing may not identify Mycobacterium species accurately, as the results of these tests may be identical between different species or may vary depending on the growth conditions employed. Sequencing the 16S rRNA gene of Mycobacterium species has improved the speed and accuracy of identification (Cloud et al., 2002;Turenne et al., 2001;Patel et al., 2000). Sequencing additional targets such as the hsp65 gene and the 16S-23S internal transcribed spacer region 1 (ITS1) has increased our ability to describe novel Mycobacterium species (Turenne et al., 2004;Tortoli, 2003;Ringuet et al., 1999; Mohamed et al., 2005).The purpose of this study was to describe a Mycobacteriumlike organism that appears to be a genotypic match to Abbreviations: FL-HPLC, fluorescence detection HPLC; ITS1, internal transcribed spacer region 1; NTM, non-tuberculous mycobacteria.

The DiversiLab System, which includes microfluidics-based detection, reagent kits, and software for data processing and analysis, is an automated method using repetitive sequence-based PCR (rep-PCR) for microbial strain typing. To assess the reliability of the DiversiLab System for strain characterization of Staphylococcus aureus, we tested clinical isolates sent to ARUP Laboratories for typing and compared results to those of pulsed field electrophoresis (PFGE) aided by the cluster analysis provided by BioNumerics software. spa typing was performed when the results of these two methods for an outbreak were not concordant. The study included 89 S. aureus isolates (65 mecA positive, 24 mecA negative) from 19 outbreaks (2 to 11 isolates/ outbreak). The DiversiLab and PFGE-BioNumerics results were concordant for 15 of the 19 outbreaks. For the remaining four outbreaks, there was partial concordance between the two methods. spa typing results were the same as or more similar to rep-PCR results for three of those outbreaks and were more similar to PFGE results for one. With regard to performance, the DiversiLab system was considerably less labor intensive than PFGE and provided results in less than 24 h, compared with 2 to 3 days for PFGE. Additionally, the Web-based DiversiLab software provides standardized comparisons among isolates almost instantaneously and generates user-friendly, customized reports.

Fungal infections are increasing, particularly among immunocompromised hosts, and a rapid diagnosis is essential to initiate antifungal therapy. Often fungi cannot be identified by conventional methods and are classified as nonsporulating molds (NSM).We sequenced internal transcribed spacer regions from 50 cultures of NSM and found 16 potential pathogens that can be associated with clinical disease. In selected clinical settings, identification of NSM could prove valuable and have an immediate impact on patient management.Fungal infections are increasing, particularly among immunocompromised hosts, and a rapid, accurate diagnosis is essential for the initiation of targeted antifungal therapy. Diagnosis of fungal infections usually depends on recovery of fungi from culture of clinical specimens, and their identification requires the presence of reproductive structures. Often fungi cannot be characterized fully because the mold does not sporulate, making identification by microscopic morphology not possible and potentially increasing the time to report an inconclusive result to 21 days. While many laboratorians and clinicians assume that these fungal isolates are environmental organisms and not clinically significant, to our knowledge no study has systematically attempted to classify these previously unidentifiable fungi in a clinical microbiology laboratory.Amplification and sequencing of target regions within the ribosomal DNA gene complex has emerged as a useful, adjunctive tool for the identification of fungi and does not depend on mold sporulation for identification (3,5,9,11). The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (2,3,5,9,11,15). The goals of this study were to determine if sequencing the ITS 1 and 2 regions of nonsporulating molds (NSM) could identify fungi that were not identifiable by conventional methods and could serve as an approach to detect clinically relevant pathogens.Sample selection. Identification of molds directly from a specimen or submitted as an isolated culture to Associated Regional and University Pathologists, Inc., Laboratories (ARUP Laboratories) was attempted by growth on inhibitory mold agar, modified Sabouraud agars, or potato dextrose agar. Microscopic structures were observed on tease or tape preparations and slide cultures for up to 21 days. NSM were defined as molds without reproductive structures and that could not be further characterized.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of dermatophytes commonly isolated in a clinical laboratory was assessed by comparing results to those of conventional tests (colony morphology, microscopic examination of slide cultures, and, for suspected Trichophyton species, use of additional media). Sixty-one cultures were tested in phase 1, the feasibility portion of the study; 64 additional cultures were tested in phase 2, the validation portion conducted to assess reproducibility and confirm accuracy. Discrepancies were resolved by repeating rep-PCR and conventional tests and, in phase 2, sequencing the internal transcribed spacers. After initial testing of the cultures in phase 1 (excluding one contaminated culture), agreement between conventional tests and rep-PCR was 90% (54 of 60). Agreement was 98.3% after resolution of discrepancies, and in all but one case the initial rep-PCR result was correct. After initial testing of cultures in phase 2 (excluding one discarded and one contaminated culture), agreement between rep-PCR and conventional testing was 88.7% (55 of 62). After discrepancies were resolved, agreement was 100%. Initial rep-PCR results were correct, except for one Microsporum canis culture containing two colony variants, which could not be initially identified by rep-PCR. The performance of the DiversiLab system for identification of the dermatophytes commonly encountered in a clinical mycology laboratory-Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, and M. canis-was excellent. Moreover, the DiversiLab system is technically simple and provides results in <24 h once a pure culture is available for testing, which is considerably more rapid than conventional identification tests.Dermatophytes are keratinophilic fungi that cause infections of skin, hair, and nails. The three genera of dermatophytes generally are identified in the laboratory based on colony morphology and the microscopic appearance of conidia (8, 10). Trichophyton species have numerous microconidia and rare thin-walled smooth macroconidia; Microsporum species have many rough, thick-walled macroconidia, and microconidia usually are also present; Epidermophyton floccosum has numerous thin-and thick-walled smooth macroconidia but no microconidia. Identification of Trichophyton to the species level also may require inoculation of urea and Trichophyton agars. Slide cultures and use of special media is time-consuming; results often are not available for several weeks. Additionally, relying on phenotypic features for identification occasionally is problematic because the distinguishing characteristics of these fungi are not stable (1).The DiversiLab system (Spectral Genomics, Houston, TX) is a rapid, technically simple method that uses repetitive-sequence-based PCR (rep-PCR) to determine relatedness of many organisms and to identify Aspergillus and Candida to the species level (2, 7). This system has three components: rep-PCR reagent kits; the Agilent 2...

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