Routine expectorated sputa were collected and cultured as necessary for acid-fast bacilli (AFB) [20] [21] . For patients unable to produce sputum by spontaneous expectoration, sputum induction was performed with nebulized hypertonic saline. Samples were processed using standard decontamination procedures, fluorochrome microscopy and cultured on solid media on a plate of Middlebrook 7H10 agar with and without antibiotics and a broth culture (ESP, Thermofisher, formerly Trek Diagnostic Systems, and Cleveland, OH) as described [20] [21] [22] [23] . MAC isolates were identified using AccuProbe (Hologic Gen-Probe Inc.) [21] . For decontamination, N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method was used [24] . Alternatively, a combination of N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and Oxalic acid was also used as necessary for decontamination [24] . All methods were performed in accordance with the relevant guidelines and regulations. As the environmental control, we utilized a 15 ml falcon tube containing PBS and kept it open for the time period when the sputa were processed. We were unable to isolate bacteria from this culture and as a reason, the sample was not processed further.
Accuprobe
Manufactured by Hologic
Sourced in United States
The AccuProbe is a diagnostic lab equipment used for the detection and identification of specific microbial organisms. It utilizes nucleic acid hybridization technology to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
8 protocols using accuprobe
1
Sputum Collection and Culture for AFB
2
Mycobacterial Culture and Identification
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Routine expectorated sputa were collected and cultured as necessary for detection of AFB67 (link)–69 (link). Samples were processed using standard decontamination procedures, fluorochrome microscopy and cultured on solid and liquid media as recommended by the Clinical and Laboratory Standards Insititute (CLSI) guidelines for mycobacteria detection and culture67 (link),69 (link). MAC isolates were identified using AccuProbe (Hologic Gen-Probe Inc)68 (link). For decontamination, the N-acetyl-L-cysteine-sodium hydroxide method alone or in combination with oxalic acid was used70 (link). All methods were performed in accordance with relevant guidelines and regulations.
3
Evaluation of MTBRP PCR Assay for Mycobacteria Identification
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Twenty-six Mtbc culture isolates and 92 nontuberculous mycobacterial (NTM) culture isolates were used to evaluate the ability of the MTBRP PCR assay to correctly differentiate Mtbc from NTM and to evaluate the detection of INH resistance mediated by the katG(S315T) mutation. The isolates were cultured from clinical specimens using standard techniques and were identified using the nucleic acid hybridization probe specific for Mtbc (Hologic AccuProbe). NTM isolates were identified using either a hybridization probe specific for M. avium complex or M. gordonae, or sequencing of a 500 bp region of the 16S rDNA gene as previously described [6] (link). Following identification, phenotypic broth susceptibility testing of Mtbc isolates was performed using the VersaTREK® platform (Thermo Fisher, Waltham, MA).
4
Fungal Isolate Characterization Protocol
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A total of 75 unique fungal isolates obtained from clinical specimens or reference collections were used, including 22 yeasts, 52 molds, and 1 mushroom-forming fungus. Clinical isolates (46/75) collected from 2016–2020 and stored at −70 °C were analyzed retrospectively. Reference isolates (29/75) were obtained from the ATCC (Manassas, VA, USA) or CDC Antimicrobial Resistance (AR) isolate bank. All isolates (100%, 75/75) had genus-level IDs and most (79%, 59/75) had species-level IDs. A variety of identification methods were used, including microscopic morphology, VITEK® MS (bioMérieux, Hazelwood, MO, USA), API® (bioMérieux), and AccuProbe (Hologic, San Diego, CA, USA). Additionally, 6 isolates were sent to the UT Health San Antonio Fungus Testing Laboratory for identification by DNA sequence analysis. Additional information regarding source, identification method(s) used, and both the original and WGS taxonomic ID are found in Supplemental Table S1 .
5
Mycobacterial Culture and Identification from Respiratory Specimens
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The respiratory specimens were first decontaminated with N-acetyl-L-cysteine-sodium hydroxide (NACL), and were concentrated with centrifugation (3000 g for 15 minutes), according to standard procedures [13] . After centrifugation, the supernatant was decanted, and phosphate buffer was added to the pellet. Part of the sediment was used to prepare an AFB fluorochrome smear. Approximately 0.5 ml was used to inoculate into MB/BacT bottle and incubated in the BacT/ALERT 3D system (bioMérieux, Durham), and about 0.25 ml onto a Middlebrook 7H11 plate. The cultures were incubated at 37°C for 5–6 weeks. Isolates of mycobacteria were identified by DNA probes (AccuProbe, Hologic Gen-Probe, San Diego, CA) or by conventional biochemical tests, according to standard protocol [13] . The remaining sediments were stored at 2–8°C for up to 3 days until they were tested for MTD, or at −70°C if they needed to be stored for more than 3 days.
6
Bacterial Identification Protocols
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Bacterial organisms were obtained from routine specimen submissions at the Nevada State Public Health Laboratory. The organisms were identified through validated and FDA cleared methods. Most bacterial identification was performed using BD Phoenix M50 and MALDI-TOF (Siruis, Bruker). Mycobacterium were identified using AccuProbe (Hologic). Additional species identification was performed on enteric bacteria using ANI (Average Nucleotide Identity) in BioNumerics 7.6 (Applied Maths).
7
M. tuberculosis Complex Identification
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All isolates were identified as members of the M. tuberculosis complex, using gene probes (ACCUProbe; Hologic, San Diego, CA, USA) or the GenoType MTBC assay (Hain Lifescience, Nehren, Germany). The differentiation between the species of the M. tuberculosis complex was based on genotyping data of each individual isolate [7 (link)–9 (link)]. Drug susceptibility testing was determined by means of the proportioning method on Löwenstein–Jensen medium and/or by means of the modified proportioning method with the BACTEC MGIT 960 system (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA).
8
Respiratory Pathogen Culture and Identification
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Respiratory samples were processed at the Lankenau Medical Center Microbiology Laboratory by use of standard methods, including a commercial hybridization assay (AccuProbe; Hologic, Inc., http://www.hologic.ca ). For each patient for whom respiratory culture was positive, 1 MAC isolate was subcultured and sent for further analysis to the University of Texas Health Science Center (Tyler, TX, USA) and Virginia Polytechnic Institute and State University (Blacksburg, VA, USA) (Table 2 ). Species identification was performed by the same methods used for biofilm isolates.
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