Nucleic Acid Amplification Tests for Diagnosis of Smear-Negative TB in a High HIV-Prevalence Setting: A Prospective Cohort Study

Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28-51) and that of secA1 was 24% (95% CI 15-35). Both tests had specificities of 95% (95% CI 90-98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations

Multiple Copies of the 16S rRNA Gene in Nocardia nova Isolates and Implications for Sequence-Based Identification Procedures

Molecular investigation of two Nocardia patient isolates showed unusual restriction fragment length polymorphism patterns with restriction endonuclease assays (REA) using an amplified portion of the 16S rRNA gene. Patterns typical of Nocardia nova were obtained with REA of an amplified portion of the 65-kDa heat shock protein gene. Subsequent sequence analysis of the 16S rRNA gene regions of these isolates showed the presence of ambiguous bases within an expected restriction endonuclease recognition site which were not able to be resolved on repeat testing. Cloning of amplified regions of the 16S rRNA genes and subsequent sequencing of the resulting clones from the two patient isolates showed three different 16S rRNA gene sequences which corresponded to sequences found in N. nova, a molecular variant of N. nova, and a previously undescribed sequence. Hybridization studies using a DNA probe corresponding to an 89-bp conserved region of the 16S rRNA gene confirmed the presence of at least two copies of the 16S rRNA gene in the N. nova type strain, in a patient isolate identical to the molecular variant of N. nova, and in the two other patient isolates. All isolates were found to belong to the species N. nova as determined by DNA-DNA hybridization. Because minimal variation has been found in the 16S rRNA gene sequences of different species of Nocardia, those laboratories employing molecular methods for identification of these species must be aware of the potential identification complications that may be caused by the presence of differing 16S rRNA genes in the same isolate

Analysis of secA1 Gene Sequences for Identification of Nocardia Species

Molecular methodologies, especially 16S rRNA gene sequence analysis, have allowed the recognition of many new species of Nocardia and to date have been the most precise methods for identifying isolates reliably to the species level. We describe here a novel method for identifying Nocardia isolates by using sequence analysis of a portion of the secA1 gene. A region of the secA1 gene of 30 type or reference strains of Nocardia species was amplified using secA1-specific primers. Sequence analysis of 468 bp allowed clear differentiation of all species, with a range of interspecies similarity of 85.0% to 98.7%. Corresponding 16S rRNA gene sequences of a 1,285-bp region for the same isolates showed a range of interspecies similarity of 94.4 to 99.8%. In addition to the type and reference strains, a 468-bp fragment of the secA1 gene was sequenced from 40 clinical isolates of 12 Nocardia species previously identified by 16S rRNA gene sequence analysis. The secA1 gene sequences of most isolates showed >99.0% similarity to the secA1 sequences of the type or reference strain to which their identification corresponded, with a range of 95.3 to 100%. Comparison of the deduced 156 amino acid sequences of the SecA1 proteins of the clinical isolates showed between zero and two amino acid residue differences compared to that of the corresponding type or reference strain. Sequencing of the secA1 gene, and using deduced amino acid sequences of the SecA1 protein, may provide a more discriminative and precise method for the identification of Nocardia isolates than 16S rRNA gene sequencing

Evaluation of the Integrated Database Network System (IDNS) SmartGene Software for Analysis of 16S rRNA Gene Sequences for Identification of Nocardia Species▿

16S rRNA gene sequences of 102 Nocardia isolates were analyzed using the Integrated Database Network System (IDNS) SmartGene centroid database. A total of 76% of the isolates were correctly identified. Discordant identifications were due to inadequate centroid length (3 species), inaccurate or insufficient entries in the public databases (5 species), and heterogeneous sequences among members of a species (1 species)

Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy

The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification

Evaluation of Partial 16S Ribosomal DNA Sequencing for Identification of Nocardia Species by Using the MicroSeq 500 System with an Expanded Database

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i) conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii) molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii) when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana