Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.

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“…PCRs were performed on the Rotor-Gene Q (Qiagen) real-time thermocycler using melting curve analysis. 24,25…”

Section: Discrepant Results Samplesmentioning

confidence: 99%

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System

Rocchetti

Silbert

Gostnell

et al. 2017

The Journal of Molecular Diagnostics

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“…agar and Lowenstein-Jensen, which provide culture in about one week), essential for the therapeutic decision because mycobacteria are resistant to many common drugs, and finally RNA probes. 1 , 6 , 7 …”

Section: Discussionmentioning

confidence: 99%

Mycobacterium chelonae cutaneous infection in a patient with mixed connective tissue disease

Lage

Biccigo

Santos

et al. 2015

An. Bras. Dermatol.

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Around 50 mycobacteria species cause human disease. Immunosuppressive states predispose to non-tuberculous mycobaterium infection, such as Mycobacterium chelonae: AFB, non-tuberculous, fast growth of low virulence and uncommon as a human pathogen. It may compromise the skin and soft tissues, lungs, lymph nodes and there is also a disseminated presentation. The diagnosis involves AFB identification and culture on Agar and Lowenstein-Jensen medium base. A 41-year-old female with MCTD (LES predominance) is reported, presenting painless nodules in the right forearm. She denied local trauma. Immunosuppressed with prednisone and cyclophosphamide for 24 months. Lesion biopsy has demonstrated positive bacilloscopy (Ziehl-Neelsen stain) and M.chelonae in culture (Lowenstein-Jensen medium base), therefore clarithromycin treatment has been started (best therapy choice in the literature).

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“…Based on methods previously published by Richardson et al (2009), we used a real-time PCR assay in which primers target the internal transcribed spacer of the M. abscessus-M. chelonae group and used a melting temperature analysis to distinguish M. chelonae from M. abscessus. This is more cost effective than sequence analysis, which is limited to reference laboratories and subject to amplicon contamination (Guarin et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Rapid diagnosis of Mycobacterium abscessus endophthalmitis

Rolfe

Garcia

Widen

et al. 2013

Journal of Medical Microbiology

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Nontuberculous mycobacteria are widely distributed in the environment and have the potential to cause a wide spectrum of infections including pulmonary, bone, soft tissue or ocular infections. They are a rare cause of endophthalmitis, a potentially devastating condition, which may be acquired through contamination of water or antiseptic solutions. Diagnosis is often delayed due to low clinical suspicion, resulting in poor clinical outcomes. Newer laboratory techniques such as real-time PCR can be used for rapid detection, identification and speciation of mycobacteria and allow for initiation of focused antibiotic therapy. We describe a case of Mycobacterium abscessus endophthalmitis that developed 30 years after traumatic loss of cornea in a patient with diabetes mellitus. Case ReportA 56-year-old woman with a history of non-insulin dependent diabetes mellitus presented to the emergency room with a 1 week history of worsening right eye pain with purulent drainage, headache, nausea and fever. Thirty years ago, she had suffered a traumatic injury to the right eye while living in India, which resulted in a blind eye. After a failed corneal transplant, she was fitted with a cosmetic lens (prosthesis) that fitted over her natural residual eye. She reported that she removed the prosthesis each night and stored it in a glass of tap water. The patient denied any prior upper respiratory infections or recent travel.On physical examination, the patient was afebrile. Examination of the eye revealed proptosis, uveal prolapse, conjunctival infection and yellowish discharge (Fig. 1). White blood cell count was 16.1610 3 ml 21, sedimentation rate was 37 mm h 21 and haemoglobin A1c was 6.8 %. Initial Gram stain of the drainage showed few polymorphonuclear cells and no organisms. Computed tomography (CT) scan of the orbits showed a 1.760.861.3 cm fluid collection with gas in the preseptal soft tissues raising concern about an abscess; multiple metallic foreign bodies were also present (Fig. 2). The right globe appeared proptotic. The patient was hospitalized and treated empirically with vancomycin and piperacillin-tazobactam, as well as trimethoprim-polymyxin ophthalmic solution.On hospital day 2, culture of the eye drainage grew few colonies of Staphylococcus aureus; piperacillin-tazobactam was discontinued. Despite treatment with vancomycin the patient continued to have persistent pain and drainage.On hospital day 5, Lowenstein-Jensen agar revealed one colony of a beaded, non-branching, Gram-positive bacillus which was Kinyoun stain positive. Initial PCR was positive for mycobacterial species. Given the speed of culture growth and PCR findings, it was felt that the organism was probably in the Mycobacterium fortuitum or M. abscessus-M. chelonae group of bacteria. Single-tube multiplex, real-time PCR testing (Richardson et al., 2009) for Mycobacterium tuberculosis complex, M. avium complex, the M. fortuitum group and the M. chelonae-M. abscessus group were performed. Results of this assay suggested M. abscessus.Antibiotics were ...

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Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

Cited by 6 publications

References 18 publications

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System

Mycobacterium chelonae cutaneous infection in a patient with mixed connective tissue disease

Rapid diagnosis of Mycobacterium abscessus endophthalmitis

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