Sensitivity of Fluorochrome Microscopy for Detection of Mycobacterium tuberculosis versus Nontuberculous Mycobacteria

The results for 6,532 consecutive mycobacterial respiratory specimens collected from 1,040 patients from 1993 to 1995 in a Texas hospital were studied to determine the sensitivity of fluorescence microscopy for detection of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Smears were positive for acid-fast bacilli (AFB) in 63% (677 of 1,082) of specimens growing M. tuberculosis and 56% (638 of 1,148) of specimens growing the four most common species of NTM. Smear positivity by species was 58% (446 of 776) for M. avium complex, 51% (154 of 300) for rapidly growing mycobacteria (98% were M. abscessus), 78% (29 of 37) for M. kansasii, and 26% (9 of 35) for M. gordonae. Definite or probable disease by clinical criteria was present in 79% of patients with M. avium complex, 93% of patients with rapidly growing mycobacteria, 100% of patients with M. kansasii, and 0% of patients with M. gordonae. Patients with M. avium complex had a low incidence of AIDS (7%), and approximately 50% of non-AIDS patients had upper-lobe cavitary disease and 50% had nodular bronchiectasis. Only 23 of 6,532 (0.35%) of AFB smears were positive with a negative culture excluding patients on therapy for established mycobacterial disease. These studies suggest that NTM are as likely as M. tuberculosis to be detected by fluorescent microscopy in specimens from patients from areas endemic for NTM lung disease and at low risk for AIDS.     

Clinical significance of nontuberculous mycobacteria isolates in elderly Taiwanese patients

The aim of this study was to investigate the clinical significance of nontuberculous mycobacteria (NTM) isolates in elderly Taiwanese patients. From 2004 through 2008, patients >65 years old with NTM isolation were identified. The definitions of NTM disease followed the American Thoracic Society and Infectious Disease Society of America (ATS/IDSA) criteria. Among the 3,175 NTM isolates, Mycobacterium avium complex (MAC; n = 1,118, 35.2%) was the most prevalent species, followed by M. abscessus (n = 545, 17.2%). Among the 1,633 elderly patients with NTM isolates, the most prevalent NTM species were MAC (n = 592, 36.3%) and M. fortuitum complex (n = 311, 19.0%). NTM colonization was found in 1,339 (80.4%) patients and only 326 (19.6%) patients had NTM diseases. During the study period, the annual incidence rates (per 100,000 inpatients and outpatients) of NTM colonization and disease both increased significantly (p < 0.0001) from 10.5 to 15.8 and from 2.1 to 4.3, respectively. Isolated pulmonary NTM infections compromised 294 (90.2%) of the 326 elderly cases of NTM disease. In conclusion, this study found an increasing trend in the incidence of both NTM isolates and NTM diseases among elderly Taiwanese patients. MAC and M. abscessus were the most frequent species causing various types of NTM disease.     

High virulent clinical isolates of Mycobacterium abscessus from patients with the upper lobe fibrocavitary form of pulmonary disease

Pulmonary disease caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, can be classified into two distinct types of clinical disease; the upper lobe fibrocavitary (UC) form and nodular bronchiectatic (NB) form. However, the relationship between mycobacterial strain virulence and disease type in the pulmonary M. abscessus diseases has not been reported. To determine the differential virulence between strains causing two forms of disease, we obtained clinical isolates from patients with the UC and NB form of pulmonary disease caused by M. abscessus. In present study, we investigated the intracellular growth of clinical isolates in macrophages and their pathogenicity in C57BL/6 mice. For the isolates from the UC form, intracellular macrophage growth was faster and higher levels of cytokines were induced in macrophages than for those from NB form. Moreover, severe lung inflammation was only observed in mice intranasally infected with the isolate from the UC form with the increase of bacterial load. These findings suggest that M. abscessus isolates from the UC form of pulmonary disease are more virulent than those from NB form. This differential virulence of clinical strains may be one of the important factors involved in the determination of the disease form of pulmonary M. abscessus disease.     

Continued Upward Trend in Non-Tuberculous Mycobacteria Isolation over 13 Years in a Tertiary Care Hospital in Korea

PurposeDespite decreased prevalence of tuberculosis, the incidence of the diseases associated with nontuberculous mycobacteria (NTM) has been increasing in South Korea and around the world. The present retrospective study was conducted to determine longitudinal changes in the epidemiology and distribution of NTM over 13 years at a tertiary care hospital in Korea.Materials and MethodsWe retrospectively analyzed data on Mycobacterium species over 13 years (January 2007 to December 2019) by utilizing the laboratory information system. Mycobacterium species were identified using biochemical tests and PCR-restriction fragment length polymorphism and Mycobacteria GenoBlot assays.ResultsAfter excluding duplicates from the initial pool of 17996 mycobacterial isolates, 7674 strains were analyzed and 2984 (38.9%) NTM were isolated. The proportion of NTM continuously increased over the 13-year period, from 17.0% in 2007 to 57.5% in 2019. Among the NTM isolates, the most common species were Mycobacterium intracellulare (50.6%), M. avium (18.3%), M. fortuitumcomplex (4.9%), M. abscessus (4.5%), M. gordonae (3.3%), M. kansasii (1.1%), M. chelonae (1.0%), and M. massiliense (0.9%). In patients over the age of 70 years, the proportion of NTM among the isolates increased from 26.6% in 2007 to 62.0% in 2019, and that of M. intracellulare isolates among the NTM increased from 13.9% (11/79) in 2007 to 37.4% (175/468) in 2019.ConclusionThe number of NTM isolates continuously increased over the study period, and the increase in the proportion of M.intracellulare in patients aged over 70 years was notable.     

Drug susceptibility of rapid and slow growing non-tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis,Central India

Background: Non-tuberculous mycobacteria (NTM) are emerging as important pathogens. Their treatment also differs from that of Mycobacterium tuberculosis. In India, any datum on them is scarce as species identification and drug susceptibility are not performed in most laboratories. Susceptibility also differs from one geographic area to another, and in our country, there are no data even to guide the clinicians to start treatment empirically. Methodology: The present study endeavours to generate drug susceptibility data on NTM isolated from sputum samples collected and stored from 6445 symptomatics for pulmonary tuberculosis during a prevalence survey and from specimens received from the hospital. Isolates were not necessarily associated with the disease. Species were identified and antibiotic susceptibility was performed using micro-broth dilution technique as per the standard Clinical and Laboratory Standards Institute guidelines. Results: A total of 65 NTM with 11 species were identified, of which 27 belonged to Mycobacterium fortuitum complex, 14 Mycobacterium gordonae, 9 Mycobacterium avium, 7 Mycobacterium flavescens, 4 Mycobacterium scrofulaceum and one each of others. Sensitivity to amikacin for M. fortuitum was 95.22% (20 out of 21), followed by ciprofloxacin (76.19%) and clarithromycin (71.42%). All the 9 M. avium isolates, 11 of M. gordonae (78.57%), 5 of M. flavescens and 2 of M. scrofulaceum were sensitive to clarithromycin. All NTM were resistant to first-line antitubercular drugs except 8, which were sensitive to streptomycin. Conclusions: Drug sensitivity of NTM varies from species to species. While amikacin was the best for rapidly growing mycobacteria, clarithromycin was the most active drug against M. avium and other slow growers.     

Extrapulmonary infections caused by a dominant strain of Mycobacterium massiliense (Mycobacterium abscessus subspecies bolletii)

A single strain of Mycobacterium massiliense (BRA 100), a subspecies of the Mycobacterium abscessus complex, has been responsible for an epidemic of post-surgical infections in Brazil. Outside Brazil, this is the first report to describe a single emerging strain of M. massiliense (TPE 101) associated with extrapulmonary infections. This phenomenon may be underestimated because sophisticated molecular typing of M. abscessus is not routinely performed. Our molecular epidemiology study was triggered by an outbreak investigation. Nine case isolates were grown from the surgical sites of nine mostly paediatric patients receiving operations from 2010 to 2011. All available non-duplicated isolates of M. abscessus during this period were obtained for comparison. Mycobacteria were characterized by multilocus sequence analysis (MLSA), repetitive sequence PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). Of 58 isolates of M. abscessus overall, 56 were clinical isolates. MLSA identified 36 of the isolates as M. massiliense. All case isolates were indistinguishable by PFGE and named the TPE 101 pulsotype. Of the stored strains of M. abscessus, TPE 101 strains were over-represented among the control surgical wound (7/7, 100%) and subcutaneous tissue isolates (4/5, 80%) but rare among the respiratory isolates (1/16, 6%) and absent from external skin, ocular and environmental samples. In conclusion, a unique strain of M. massiliense has emerged as a distinctive pathogen causing soft tissue infections in Taiwan. Further study to identify whether this is due to an occult common source or to specific virulence factors dictating tissue tropism is warranted.     

Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.     

ISOLATION AND IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA FROM WATER AND SOIL IN CENTRAL INDIA

Nontuberculous mycobacteria (NTM), important organisms in the Genus Mycobacterium and commonly present in the environment, are known to cause disseminated disease in AIDS patients. In this study, NTM were isolated from environment (soil and water) of the AIDS patients with disseminated NTM disease to know the prevalence of environmental NTM species and their correlation with clinical isolates from patients of the same area. Paraffin baiting technique was used to isolate NTM from environmental samples. Once isolated, subcultures were made on Lowenstein Jensen and Middlebrook 7H10 media and the species were identified using phenotypic and genotypic techniques. A total of 26 NTM isolates belonging to seven different species could be identified. Mycobacterium avium was the only species isolated from both clinical and environmental samples of the same patient; but the isolates did not match using PCR for IS 1311 and IS 1245 spacer sequences.     

Clinical and demographic study of non-tuberculous mycobacterial ocular infections in South India

PurposeTo describe demographics, risk factors, antibiotic susceptibility, management and outcomes of ocular infections caused by non-tuberculous mycobacteria (NTM).MethodsA retrospective review of medical case records and microbiology records of patients with ocular infections that were culture positive for non-tuberculous Mycobacteria from January 2014 to December 2018 was done. Antibiotic susceptibility profile was done based on the CLSI guidelines. Laboratory diagnosis for the NTM Species was done by conventional microbiological methods. The species identification was done for stored isolated utilizing polymerase chain reaction targeting 16S rDNA and rpoB gene, followed by DNA sequencing and phylogenetic analysis.ResultsTwenty patients with NTM ocular infections were identified during the study period. A majority of cases presented as 12 infectious keratitis (60%) and three suture-related corneal infiltrates (15%). Common risk factors were history of trauma in 9 (45%) patients and history of ocular surgery in 5 (25%) patients. Patients were treated with combination of amikacin and flouroquinolones/chloramphenicol (70%) and surgical interventions were performed in 25% cases. Only twelve isolates were stored and ten isolates were identified as the M. abscessus subsp. abscessus and two isolates as M. abscessus subsp. massiliense by sequencing and phylogenetic analysis. Majority of the NTM were sensitive to amikacin (75%) followed by moxifloxacin, ciprofloxacin, cephotaxime and tobramycin (35%).ConclusionHigh degree of clinical suspicion, multidrug antibiotic therapy and timely surgical intervention in patients with NTM infections, are advised for better clinical outcomes. Prior ocular trauma, prior ocular surgery and presence of biomaterials were the major predisposing factors. Earlier surgical intervention in cases where abscesses or biomaterials are involved, is necessary for rapid recovery.     

Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study

Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy.     

Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates

Background/purposeThe World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes.MethodsRabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293 T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods.ResultsWe obtained five rabbit mAbs against LAM, four of which had high sensitivities (100 pg/ml) and affinities (1.16–1.73 × 10^?9 M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified.ConclusionThe novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application.     

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.     

Classification Algorithm for Subspecies Identification within the Mycobacterium abscessus Species,Based on Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.     

Nontuberculous mycobacterial infection after lung transplantation: A single-center experience in South Korea

PurposeNontuberculous mycobacteria (NTM) infection is an important issue after lung transplantation. However, a large-scale epidemiological study on this issue in Korea is lacking. We aimed to evaluate the epidemiology of NTM infection after lung transplant surgery in Korea.MethodsBetween October 2012 and December 2018, we retrospectively evaluated lung transplant recipients in a referral hospital in South Korea. A total of 215 recipients were enrolled. The median age at transplantation was 56 years (range, 17–75), and 62% were men. Bronchoscopy was performed according to the surveillance protocol and clinical indications. A diagnosis of NTM infection was defined as a positive NTM culture from a bronchial washing, bronchoalveolar lavage sample, or two separate sputum samples. We determined NTM pulmonary disease (NTM-PD) according to the American Thoracic Society/Infectious Disease Society of America 2007 guidelines. The Kaplan–Meier method and log-rank test were used for conditional survival analysis in patients with follow-up of ≥12 months.ResultsFourteen patients (6.5%) were diagnosed with NTM infection at a median of 11.8 months (range, 0.3–51.4) after transplantation. Nine patients (4.2%) were diagnosed with NTM-PD, and the incidence rate was 1980/100,000 person-years. Mycobacterium abscessus was the most common species causing NTM-PD (66%), followed by M. avium complex (33%). The presence of NTM infection did not influence all-cause mortality among those who underwent follow-up for ≥12 months (N = 133, log-rank P = 0.816).ConclusionThe incidence of NTM-PD was considerably high among lung-transplant recipients. M. abscessus was the most common causative species of NTM-PD after lung transplantation.     

Incidence of tuberculous and non-tuberculous mycobacteria,differentiated by multiplex PCR,in clinical specimens of a large general hospital

OBJECTIVE:

To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment.

METHODS:

Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117).

RESULTS:

In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria.

CONCLUSIONS:

Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.     

Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977^T. Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.     

Epidemiology and Risk Factors Associated with NTM Pulmonary and Extrapulmonary Infections in a High Tuberculosis Endemic Region

Introduction: Non-tuberculous mycobacteria, although identified as pathogenic to humans long time ago, are emerging as the new threat in the past two decades. Even in tuberculosis endemic country such as India, they are being isolated from the clinical specimens more often than previously. This change in trend is of concern, because they are often misdiagnosed as Mycobacterium tuberculosis or even as drug-resistant tuberculosis. Objectives: A prospective, observational study was planned to identify the frequency and risk factors associated with pulmonary and extrapulmonary non-tuberculous mycobacterial (NTM) infections. Agreement between two commercially available molecular systems, namely GenoType Mycobacteria CM assay and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) used in the identification of mycobacterial species is also analysed. Materials and Methods: NTM isolated from pulmonary and extrapulmonary clinical specimens over a period of 1½ year was included in the study. Patient demographics were collected, and the risk factors associated with NTM infections were analyzed. NTM grown on culture was speciated using GenoType Mycobacteria CM assay (HAIN Life Sciences, Germany) and MALDI TOF MS (bioMerieux, France). Drug-susceptibility tests were done for rapid-growing NTM using E-test (bioMerieux, France). Results: Eight hundred and fifty-four mycobacteria were isolated from 5009 specimens processed during the study period. Out of the mycobacteria grown, 74 (8.7%) were NTM and 780 (91.3%) were Mycobacterium tuberculosis complex. The NTM isolated from pulmonary specimens were 46 (62.16%) and from extrapulmonary sources were 28 (37.84%). The most common species isolated from pulmonary specimens was Mycobacterium intracellulare and from extrapulmonary specimens was Mycobacterium abscessus. Concordance between the two commercial assays used for the identification was 96.49%. The most common risk factor associated with pulmonary NTM was previous lung pathology, while with extrapulmonary NTM infection was previous surgical intervention. Drug-susceptibility tests for rapid growers showed amikacin and clarithromycin as the most active drugs in vitro. Conclusions: NTM plays a significant role in causing pulmonary and extrapulmonary infections even in our part of the country with high endemicity of tuberculosis. NTM has emerged as important pathogens even in the immunocompetent patients. There is a need for rapid diagnosis and susceptibility testing of NTM to aid physicians administer timely and appropriate treatment to the patients.     

Clinical and Microbiological Differences between Mycobacterium abscessus and Mycobacterium massiliense Lung Diseases

In recent years, many novel nontuberculous mycobacterial species have been discovered through genetic analysis. Mycobacterium massiliense and M. bolletii have recently been identified as species separate from M. abscessus. However, little is known regarding their clinical and microbiological differences in Japan. We performed a molecular identification of stored M. abscessus clinical isolates for further identification. We compared clinical characteristics, radiological findings, microbiological findings, and treatment outcomes among patients with M. abscessus and M. massiliense lung diseases. An analysis of 102 previous isolates of M. abscessus identified 72 (71%) M. abscessus, 27 (26%) M. massiliense, and 3 (3%) M. bolletii isolates. Clinical and radiological findings were indistinguishable between the M. abscessus and M. massiliense groups. Forty-two (58%) patients with M. abscessus and 20 (74%) patients with M. massiliense infections received antimicrobial treatment. Both the M. abscessus and M. massiliense groups showed a high level of resistance to all antimicrobials, except for clarithromycin, kanamycin, and amikacin. However, resistance to clarithromycin was more frequently observed in the M. abscessus than in the M. massiliense group (16% and 4%, respectively; P = 0.145). Moreover, the level of resistance to imipenem was significantly lower in M. abscessus isolates than in M. massiliense isolates (19% and 48%, respectively; P = 0.007). The proportions of radiological improvement, sputum smear conversion to negativity, and negative culture conversion during the follow-up period were higher in patients with M. massiliense infections than in those with M. abscessus infections. Patients with M. massiliense infections responded more favorably to antimicrobial therapy than those with M. abscessus infections.     

Genitourinary infections caused by nontuberculous mycobacteria at a university hospital in Taiwan, 1996–2008

Genitourinary infections caused by nontuberculous mycobacteria (NTM) are rarely reported. The medical records of all patients with genitourinary NTM infections treated at National Taiwan University Hospital from 1996–2008 were retrospectively reviewed. Fifteen patients were identified, of whom 10 (67%) were male. More than two-thirds of patients had underlying conditions, the most common of which was chronic renal disease. Only one patient had AIDS. Acid-fast smears of urine were negative in all patients. Eleven isolates were available for further confirmation by sequencing of the 16S rRNA gene. Mycobacterium avium complex was the most common (n = 5, 33%), followed by both Mycobacterium abscessus (n = 2; 13%) and Mycobacterium fortuitum (n = 2; 13%). Of the 12 patients receiving anti-NTM treatment, only four received adequate prescribed regimens and none died of NTM infections. Two patients died of refractory urosepsis before the urinary NTM infections were diagnosed. The clinical characteristics of the 15 patients were also compared with 43 previously reported patients with genitourinary tuberculosis. Patients with genitourinary NTM infections were more likely to report constitutional symptoms, seek medical help within 1 month after the onset of symptoms and develop leukocytosis. Patients with genitourinary tuberculosis were more likely to have ureteral strictures and abnormal chest radiographs associated with active or inactive tuberculosis. Although rare, genitourinary NTM infections pose a significant threat to life and should be considered in the differential diagnosis of genitourinary infections, especially when patients are unresponsive to conventional antibiotic treatment.     

Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in Clinical Isolates by Multiplex PCR

The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.