Short Signature rpoB Gene Sequence to Differentiate Species in Mycobacterium abscessus Group.

Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that consist of three closely related species: M. abscessus (Ma), M. bolletii (Mb), and M. massiliense (Mm). Differentiation of these species can be difficult but is increasingly requested owing to recent infectious outbreaks and their differential drug resistance. We developed a novel and rapid pyrosequencing method using short signature sequences (35 to 45 bp) at a hypervariable site in the rpoB gene to differentiate the three MAG species, along with M. chelonae (Mc), and M. immunogenum (Mi). This method was evaluated using 111 M. chelonae-abscessus complex (MCAC) isolates, including six reference strains. All isolates were successfully differentiated to the species level (69 Ma, four Mb, six Mm, 23 Mc, and nine Mi). The species identifications by this method had 100% agreement with Sanger sequencing as well as an in-silico rpoB typing method. This short signature sequencing (SSS) method is rapid (6 to 7 h), accurately differentiates MAG species, and is useful for informing antimicrobial therapy decision. IMPORTANCE Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that include three species: M. abscessus, M. massiliense, and M. bolletii. These species are among the leading causes of nontuberculosis mycobacteria infections in humans but difficult to differentiate using commonly used methods. The differences of drug resistance among the species shape the treatment regimens and make it significant for them to be differentiated accurately and quickly. We developed and evaluated a novel short signature sequencing (SSS) method utilizing a gene called rpoB to differentiate the three MAG species, as well as other two species (M. chelonae and M. immunogenum). The identification results had 100% agreement with both the reference method of Sanger sequencing and rpoB typing method via a computer-simulated analysis. This SSS method was accurate and quick (6 to 7 h) for species differentiation, which will benefit patient care. The technology used for this method is affordable and easy to operate.

Recovery and Its Dynamics of Filamentous Fungi from Clinical Specimen Cultures: An Extensive Study.

The culture method remains vital in diagnosing fungal infections, but extensive data-based evaluation of the method, especially for filamentous fungi (molds), is minimal. The purpose of this study was to characterize mold recoveries from fungal cultures and the impact of media and incubation duration. Clinical specimens for fungal cultures were submitted primarily from the eastern and central United States, and mold isolation data were prospectively collected and analyzed. A total of 1,821 molds in 59 genera were isolated from 1,687 positive specimens, accounting for approximately 5.6% of our cohort of 30,000 fungal cultures. Within 2 weeks, nearly 90% of molds and 97.3% of Aspergillus fumigatus complex were recovered (>95% confidence interval [CI]). All Mucorales fungi were recovered within 11 days of incubation. The recovery peak time was day 3 for Mucorales fungi, day 4 for hyaline molds, day 5 for dematiaceous molds, and day 7 for Onygenales fungi. The recovery of Histoplasma capsulatum and Trichophyton species in the fourth week of incubation reveals that a 3-week incubation time is insufficient. Inhibitory mold agar was the best medium for recovering all mold types among all tested specimen types, yielding nearly 78% of mold growth overall, indicating the necessity of selective medium for fungal cultures. IMPORTANCE Fungal culture is the gold standard method of diagnosing fungal infections, but important information, such as the impact of media and incubation times on fungal recovery, is not well documented. This study addressed these gaps using extensive data-based evaluation focused on molds. We identified the best medium types and incubation times for better fungal culture practice. We analyzed 1,821 molds from 1,687 positive specimens in our cohort of approximately 30,000 fungal cultures. Mold recovery peaked between 3 and 7 days of incubation, dependent upon the type of mold. Some well-defined fungal pathogens, such as Histoplasma capsulatum and Trichophyton species, were isolated in the fourth week of incubation. Inhibitory mold agar was identified as the best medium for recovering all mold types among all tested specimen sources. As we are aware, this is the largest study of fungal culture methods and supports 4 weeks of incubation for optimal mold recovery.

Current significance of the Mycobacterium chelonae-abscessus group.

Organisms of the Mycobacterium chelonae-abscessus group can be significant pathogens in humans. They produce a number of diseases including acute, invasive and chronic infections, which may be difficult to diagnose correctly. Identification among members of this group is complicated by differentiating at least eleven (11) known species and subspecies and complexity of identification methodologies. Treatment of their infections may be problematic due to their correct species identification, antibiotic resistance, their differential susceptibility to the limited number of drugs available, and scarcity of susceptibility testing.

Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method.

AIMS: Acid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated. METHODS: The method was validated using spiked cell-clotted paraffin blocks before use with patients' specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB. RESULTS: Both sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq. CONCLUSIONS: The PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.

Direct detection and identification of acid-fast bacteria from smear-positive broth cultures using a pyrosequencing method.

Broth culture is a standard method for detection of acid-fast bacteria (AFB) (e.g., Mycobacterium and Nocardia) from patient specimens. Direct nucleic acid-based identification from smear-positive broths expedites the infectious disease diagnosis. We developed and evaluated the performance of a pyrogram-based technique (direct-broth-pyrosequencing [DBP]) to identify AFB directly from smear-positive broths. One hundred thirteen AFB-positive broths from patient specimens were tested. Bacterial DNA was amplified by polymerase chain reaction and sequenced using the PyroMark ID system. The DBP method correctly identified the AFB species/group in 109 (97%) of the 113 broths, including 15 Mycobacterium species and 4 Nocardia species. Three broths that yielded indeterminate results were found to be AFB-AFB mixed broths and required purified colonies on solid media for definite identification. The 4th broth was repeatedly identified by sequencing to be Mycobacterium intracellulare, even though the organism was not isolated and the AccuProbe was negative. This method did not identify the AFB organisms from broths containing 2 AFB organisms, but did not produce false identification. No cross-reaction was observed when AFB-positive broths were spiked with non-AFB microorganisms, indicating that the DBP method was specific to AFB. The DBP method gives rapid (within 8 h), accurate AFB identification directly from broth cultures and provides another useful AFB identification tool in a clinical laboratory.

Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.

Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.

Identification of acid-fast bacilli using pyrosequencing analysis.

Pyrosequence identification of 117 isolates of acid-fast bacilli (AFB) was compared to both routine phenotypic methods and Sanger sequencing. Two (2) vendor-provided pyrosequencing primers specific for AFB were used for the study. Pyrosequence analysis correctly identified 114 (98%) of the tested 117 AFB isolates. Among the test Mycobacterium spp., 18 of 20 Mycobacterium spp. were identified correctly to the species level. All rapidly growing mycobacteria were correctly identified to species by pyrosequencing. Other slowly growing mycobacteria such as Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium-intracellulare, and others were easily identified by pyrosequencing. Only Mycobacterium simiae and Mycobacterium scrofulaceum were not identifiable by the pyrosequence method. Among the 25 Nocardia isolates, all were correctly identified to the genus level. Identification of AFB by pyrosequence analysis provides both a rapid and accurate method for this group of organisms.