Development of a cost-effective line probe assay for rapid detection and differentiation of Mycobacterium species

Abstract

Background Line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different Mycobacterium species. There are several commercial kits based on line probe assay for detection of Mycobacterium species. Because of their high cost, especially for underdeveloped and developing countries, and the discrepancy of NTM prevalence across geographic regions, it would be reasonable to consider the development of an in-house LPA. The aim of this study was to develop a LPA to detect and differentiate mycobacterial species, and also to evaluate the usefulness of PCR-LPA for direct application on clinical samples. Method One pair of biotinylated primers and fifteen designed DNA oligonucleotide probes were used based on multiple aligned ITS sequences. Specific binding of the PCR amplified products to the probes immobilized on a strip of nitrocellulose membrane was evaluated by hybridization method. Experiments were performed three times on separate days to evaluate the repeatability of the assay. Further, evaluation of the PCR-LPA was carried out directly on 9 clinical samples and cultivated isolates. Results All the fifteen probes used in this study were hybridized specifically to ITS sequences of the corresponding standard species. Results were reproducible for all of the strains in different days. Mycobacterium species of nine clinical specimens and their relevant cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing. Conclusions In this study, we described a PCR-LPA which is readily applicable in clinical laboratory. This assay is a fast, cost-effective and highly specific method which requires no radioactive materials.

Mycobacterium simiae pulmonary infection: a case series and literature review

“…Also, a mycobacterial culture of the patients' samples on Lowenstein-Jensen medium showed positive results for AFB (Table 1). Cases were identified using the native reverse line probe assay (LPA) [5]. The results were also confirmed by the sequencing of the ITS (16S-23S) rRNA spacer region with an accession number (Table 1).…”

“…Symptoms of this infection include cough, sputum production, haemoptysis, fever, night sweats, and weight loss. Several previous studies reported production of sputum, severe cough, and weight loss as the most common clinical symptoms of M. simiae pulmonary infection [5]. Moreover, the infection is more common in the elderly people (age range: 57-73 years), especially elderly women (Table 3).…”

“…There is limited information on the relationship between the in vitro sensitivity of this organism and the in vivo response to drugs. Mycobacterium simiae infections have usually shown a poor therapeutic response in vivo [4], and most clinical isolates are resistant to firstline TB drugs such as isoniazid and rifampicin [5]. The treatment regimen for M. simiae infections is quite different from the TB treatment regimen, and drugs such as moxifloxacin, clarithromycin, and cotrimoxazole are effective against this pathogen [4].…”

“…The treatment regimen for M. simiae infections is quite different from the TB treatment regimen, and drugs such as moxifloxacin, clarithromycin, and cotrimoxazole are effective against this pathogen [4]. Mycobacterium simiae has been previously reported from Iran [5]. In the current study, we surveyed the clinical features, risk factors, diagnosis, and treatment of M. simiae pulmonary infection in Mashhad, Iran.…”

“…Mycobacterium simiae infection often occurs in immunocompromised patients with underlying diseases. In addition, factors such as a previous history of TB, being infected with HIV, having malignancies, older ages, cardiovascular disease, diabetes mellitus (DM), smoking, and structural abnormalities of the respiratory system increase the risk of infection [5]. COPD, bronchiectasis, and a history of TB were the most common risk factors for M. simiae pulmonary infection [6].…”