Stockman, L

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

Four commercially available acridinium ester-labeled DNA probes directed against rRNA were evaluated for their ability to identify Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans in culture. rRNA was extracted by sonication of 1- to 2-mm2 portions of cultures of fungi in two chaotropic reagents with glass beads. Following a heat inactivation step, the extracts were hybridized in solution with probes specific for each pathogen. The acridinium ester reporter moiety of nonhybridized probe was selectively hydrolyzed, and chemiluminescence of specific DNA:RNA hybrids was quantitated in relative light units with a luminometer. A positive identification required a relative light unit value of > or = 50,000. Sensitivity and specificity of the probes were determined by probing cultures of the respective pathogenic fungi (target) and nontarget fungi. Both mycelial and yeast forms of the dimorphic fungi (B. dermatitidis and H. capsulatum) were tested. For B. dermatitidis, sensitivity and specificity were 87.8 and 100%, respectively (74 target and 219 nontarget fungi tested). For C. immitis, sensitivity and specificity were 99.2 and 100%, respectively (122 target and 164 nontarget fungi tested). For H. capsulatum, sensitivity and specificity were 100 and 100%, respectively (86 target and 154 nontarget fungi tested). For C. neoformans, sensitivity and specificity were 97 and 100%, respectively (100 target and 230 nontarget fungi tested). For B. dermatitidis, C. immitis, and C. neoformans, repeat testing increased the respective sensitivities to 97.3, 100, and 100%. The high sensitivities and specificities of the probes, the relatively short time (less than 1 h) required to perform the assay, and the availability of standardized reagent kits make the acridinium ester-labeled DNA probes well suited to laboratories in need of a rapid method to identify these fungal pathogens. Further, use of the probes to identify pathogenic fungi as soon as colonies appear on primary recovery media significantly shortens the time to reporting.

Commercially available kits (Mycobacterium avium Complex Rapid Diagnostic System and Mycobacterium tuberculosis Complex Rapid Diagnostic System; Gen-Probe, Inc., San Diego, Calif.) utilizing nucleic acid hybridization for the rapid identification of members of the M. avium-M. intracellulare complex and M. tuberculosis complex were evaluated by using 339 clinical and American Type Culture Collection (Rockville, Md.) isolates. The tests, which can be performed in approximately 2 h, use specific [125I]DNA probes complementary to the rRNAs of M. avium, M. intracellulare, and M. tuberculosis complex, the latter of which includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti. The M. avium-M. intracellulare probes correctly identified 99 of 114 M. avium-M. intracellulare isolates, with 7 false-negatives and 8 false-positives, for a sensitivity of 93.4% and a specificity of 96.6%. After repeat testing, 110 of 114 were correctly identified, with 4 false-negatives and no false-positives, for a sensitivity of 96.5% and a specificity of 100%. The M. tuberculosis complex probe correctly identified 99 of 102 M. tuberculosis isolates, with 1 false-negative and 2 false-positives, for a sensitivity of 99% and a specificity of 99.2%. After repeat testing, 100 of 102 isolates were correctly identified, with no false-negatives and 2 false-positives, for a sensitivity of 100% and a specificity of 99.2%. Overall, there were 15 discrepant M. avium-M. intracellulare results, with 4 such results after repeat testing, and 3 discrepant M. tuberculosis complex results, with 2 such results after repeat testing. The Gen-Probe kits are highly sensitive and specific for use in identifying M. avium-M. intracellulare complex and M. tuberculosis complex isolates and will be useful in the clinical laboratory which can use the present radionuclide-containing kits cost effectively.

An enzymatic method involving a protease (pronase) for the elimination of interference factors in the latex test for cryptococcal antigen was developed and compared with dithiothreitol treatment. The two were equivalent in their ability to remove interference factors; however, the enzymatic method generally yielded higher titers. The method is simple, requires only 20 min, and makes the latex test for cryptococcal antigen specific.

ABSTRACT Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples ( n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (≥11,000 versus ≥30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.

ABSTRACT The ability to identify yeast isolates by the new enzymatic RapID Yeast Plus System was compared to the ability to identify yeast isolates by the API 20C system. A total of 447 yeast isolates representing Blastoschizomyces capitatus , 17 Candida spp., 5 Cryptococcus spp., Geotrichum spp., 2 Hanseniaspora spp., Hansenula anomala , Hansenula wingei , 3 Rhodotorula spp., Saccharomyces cerevisiae , Sporobolomyces salmonicolor , Trichosporon beigelii , and 2 Prototheca spp. were evaluated. Also, five quality control strains ( Candida spp. and Cryptococcus laurentii ) with well-documented reactivities by the RapID Yeast Plus System were used. Each isolate was evaluated by both methods with a 48-h culture grown at 30°C on Sabouraud dextrose agar (Emmons modification) by following the recommendations of the manufacturers. The RapID Yeast Plus System enzymatic reactions were read after 4 h of incubation, and the API 20C carbohydrate assimilation identification profiles were obtained after 72 h of incubation. There was good (95.7%) agreement between the identifications obtained by the two methods with the eight common Candida spp. and with Cryptococcus neoformans . The agreement was lower when the emerging Candida spp. and other yeast-like pathogens were tested (79.1 and 75.2%, respectively). These preliminary data suggest the potential utility of the RapID Yeast Plus System for use in the clinical laboratory for the rapid identification of common yeast pathogens as well as certain new and emerging species.

ABSTRACT Mycobacterium triplex was first named in 1996 as an acid-fast bacillus with features that most resemble Mycobacterium simiae and Mycobacterium avium-intracellulare complex but which possesses a distinct mycolic acid pattern as well as a distinctive 16S rRNA hypervariable region. It has been isolated from lymph node, sputum, and cerebrospinal fluid specimens, but to date only rare clinical cases of this organism have been reported in the literature. The following is a case report of M. triplex that was isolated from the pericardial and peritoneal fluid of a 13-year-old female liver transplant patient.