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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., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats., Results: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk., Conclusion: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.

Background: Pulmonary granulomas are sometimes resected because they resemble lung cancer and false-positive findings come through from positron emission tomography (PET) using 18fluorine-fluorodeoxyglucose (18F-FDG). Mycobacterial infection is a common cause of granulomas., Objective: The purpose of this study was to evaluate the radiopathological features and the methods for identifying mycobacterial infections in granulomatous nodules resected from the lung., Methods: Thirty-five solitary lesions resected because of suspected lung cancer were enrolled, including 22 nonfungal granulomatous lesions and 13 benign lesions as controls. The radiological, microbiological, and histological findings were reviewed. To identify mycobacterial infection, immunohistochemical (IHC) staining, IS6110 polymerase chain reaction (PCR), and real-time PCR for the detection of Mycobacterium tuberculosis (TB) were performed using formalin-fixed and paraffin-embedded tissue specimens. The correlations between the radiopathological features and the median maximum standardized uptake value (SUVmax) of 18F-FDG PET were also evaluated., Results: Mycobacteria were isolated from the cultures of 10 of the granulomatous lesions, including TB from 2 and Mycobacterium avium complex from 8. The mean size of the nodules in the culture-positive group was significantly larger than that of those in the culture-negative group (30.5 ± 13.1 vs. 15.1 ± 6.3 mm, p = 0.003). IHC stainings were positive in 15 granulomas. Eight granulomas were positive in IS6110 PCR, and 7 of them were also positive in real-time PCR. SUVmax was ≥2.5 in all of the PCR-positive granulomas., Conclusion: The most frequent cause of granulomatous lesions was mycobacterial infection. It seemed that the culture result was associated with nodule size and that the results of IS6110 were associated with 18F-FDG-uptake., (© 2017 S. Karger AG, Basel.)

Background: The differential diagnosis in a child with a swelling in the neck is broad. Chronic lymphadenopathy in children quite often leads to extensive blood tests. Cervicofacial non-tuberculous mycobacterial (NTM) lymphadenitis is, however, not revealed by blood tests. It is a rare condition, which is mostly seen in young children., Case Description: A 4-year-old girl had a 9-week history of a swelling in her neck; the skin covering the swelling had been red and flaking for the past week. Extensive blood tests did not provide a firm diagnosis and ultrasound revealed 3 heterogeneous abscessing lymph glands with fistulas to the subcutaneous layer, leading to a probable diagnosis of cervicofacial NTM lymphadenitis. The infected lymph node package was excised, and microbiological culture was positive for Mycobacterium haemophilum., Conclusion: NTM infections in young children are persistent infections that should be treated surgically at an early stage for the best cosmetic and functional result.

Introduction: Despite news reports, Food and Drug Administration disclaimers, and warnings from US plastic surgeons against the perils of cosmetic tourism, patients continue to seek care abroad and often present with infectious complications. Recent reports of Mycobacterium abscessus surgical site infection (SSI) is of particularly concern and its management, particularly surgical intervention, has been poorly documented., Methods: A retrospective review of 2 sisters who presented with M. abscessus SSI after cosmetic surgery in the Dominican Republic was performed. A comprehensive review of the literature was conducted to unveil similar cases after cosmetic tourism., Results: Both patients presented four months after index operation after definitive diagnoses have been reached. They were counselled to undergo immediate, aggressive debridement and antibiotic therapy. Although 1 patient agreed, the other patient opted for local wound care and oral antibiotics in hopes to avoid reoperation. When unsuccessful, she agreed to the initial plan which led to rapid convalescence of her infection. However, aesthetic result was far inferior to the first patient. Review of literature revealed 14 women with an average age of 40 years (range, 19-60 years). Most frequent cosmetic operations that resulted in M. abscessus SSI were abdominoplasty (41%), liposuction (27%), breast augmentation (14%), breast reduction (9%), and rejuvenation surgery (9%). Surgical interventions were performed in all cases except one. Antibiotic therapies focused on macrolides, particularly clarithromycin or azithromycin, with average time to complete recovery of 8 months (range, 2-22 months)., Conclusions: The 2 cases highlighted the importance of multidisciplinary approach of early aggressive surgical intervention and long-term intravenous antibiotics in treating M. abscessus SSI that is highly prevalent among those returning from medical tourism in cosmetic surgery.

The use of bronchoscopes has increased in tuberculosis (TB) diagnostics to circumvent the diagnostic challenges that are associated with low sputum volume and smear-negative TB. In healthcare facilities situated in low income countries that have a high burden of TB, adequate decontamination of bronchoscopes is a challenge and often overlooked to save on time and costs. This amplifies the risk of outbreaks and pseudo-outbreaks due to Mycobacterium tuberculosis and nontuberculosis mycobacteria. In this minireview, we review published literature of contaminated bronchoscopes causing pseudo-outbreaks of M. tuberculosis and nontuberculosis mycobacteria in an effort to determine common sources, and possible mitigation strategies in low-resource settings., (Copyright © 2016. Published by Elsevier Ltd.)

Introduction: Mycobacterium abscessus is an emerging mycobacteria that is responsible for lung diseases and healthcare-associated extrapulmonary infections. Recent findings support its taxonomic status as a single species comprising 3 subspecies designated abscessus, bolletii and massiliense. We performed a review of English-language publications investigating all three of these subspecies. Areas covered: Worldwide, human infections are often attributable to environmental contamination, although the isolation of M. abscessus in this reservoir is very rare. Basic research has demonstrated an association between virulence and cell wall components and cording, and genome analysis has identified gene transfer from other bacteria. The bacteriological diagnosis of M. abscessus is based on innovative tools combining molecular biology and mass spectrometry. Genotypic and phenotypic susceptibility testing are required to predict the success of macrolide (clarithromycin or azithromycin)-based therapeutic regimens. Genotyping methods are helpful to assess relapse and cross-transmission and to search for a common source. Treatment is not standardised, and outcomes are often unsatisfactory. Expert commentary: M. abscessus is still an open field in terms of clinical and bacteriological research. Further knowledge of its ecology and transmission routes, as well as host-pathogen interactions, is required. Because the number of human cases is increasing, it is also necessary to identify more active treatments and perform clinical trials to assess standard effective regimens.

Mycobacterium tuberculous peritonitis is a less common cause of peritoneal dialysis related infection in developed countries. As both CAPD and APD are being used as renal replacement therapy in developing countries of South Asia, Mycobacterium tuberculous peritonitis are being reported. Any culture negative peritonitis should be investigated for this entity. In this manuscript, we report an index case and our experience with literature review of Mycobacterium tuberculous peritonitis. The diagnostic techniques, management and outcome are described., (Copyright © 2016 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.)

Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI-based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182 mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2min. Using a log confidence cut-off of ≥2.0, overall agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M. mucogenicum. Addition of spectra from a single clinical strain of each species to the existing database increased overall agreement to 93.1%., (Copyright © 2016. Published by Elsevier Inc.)

Mycobacteriosis is infrequently reported in free-ranging sea turtles. Nontuberculous Mycobacterium haemophilum was identified as the causative agent of disseminated mycobacteriosis in a juvenile leatherback turtle (Dermochelys coriacea) that was found stranded on the Atlantic coast of Florida. Disseminated granulomatous inflammation was identified histologically, most notably affecting the nervous system. Identification of mycobacterial infection was based on cytologic, molecular, histologic, and microbiologic methods. Among stranded sea turtles received for diagnostic evaluation from the Atlantic and Gulf of Mexico coasts of the United States between 2004 and 2015, the diagnosis of mycobacteriosis was overrepresented in stranded oceanic-phase juveniles compared with larger size classes, which suggests potential differences in susceptibility or exposure among different life phases in this region. We describe M. haemophilum in a sea turtle, which contributes to the knowledge of diseases of small juvenile sea turtles, an especially cryptic life phase of the leatherback turtle., (© 2016 The Author(s).)

A 19-year old woman with acute myeloid leukemia presented with newly observed liver lesions during ongoing consolidation therapy. Due to unexplained cholestasis during induction, biliary duct drainage was performed. Microbiologic and histologic examinations revealed the presence of atypical mycobacteria, namely Mycobacterium abscessus. With an appropriate antiinfective regime which was continuously administered using a portable pump in the outpatient setting, further mycobacterial spread during simultaneous chemotherapy-associated neutropenia was prevented. Despite multiple bacterial resistance mechanisms, proper treatment of leukemia with curative intention could be ensured.

Opportunistic infections are a major concern in renal and transplant medicine. We present the case of a renal transplant recipient with a generalised Mycobacterium haemophilum infection after an increase in immunosuppressive therapy and treatment with a tumour necrosis factor-α (TNF-α) inhibitor. Infection involved skin and soft tissue, joints and bones, as well as the renal transplant with an interstitial nephritis. Rapid diagnosis using PCR and DNA sequencing allowed early appropriate treatment. Triple antibiotic therapy and reduction in immunosuppression resulted in a slow but sustained recovery. Immunosuppression causes severe opportunistic infections. TNF-α inhibitors are very effective and well tolerated but have an increased susceptibility to infections with mycobacteria. Mycobacterial infections represent a significant clinical risk to transplant recipients because of their aggressive clinical course and the need for complex toxic antibiotic treatments. In these patients, M. haemophilum is a cause of skin infections., Competing Interests: Conflicts of Interest: None declared., (2016 BMJ Publishing Group Ltd.)

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[(18)F]fluoro-d-trehalose ((18)F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[(19)F]fluoro-d-trehalose ((19)F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several (19)F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the (19)F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that (19)F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived (18)F-FDTre PET radiotracers, we carried out (19)F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.

Purpose: To describe a case of postoperative Mycobacterium manitobense endophthalmitis with good visual outcome that is the first report of endophthalmitis by this organism., Methods: Clinical and microbiological description of a patient with postoperative endophthalmitis., Results: A 50-year-old chronic alcoholic man was referred to us with decreased vision in his right eye for 4 days. He had undergone cataract surgery with intraocular lens implantation in his right eye a month ago. The presenting vision in his right eye was counting fingers close to face. He was diagnosed as a case of postoperative endophthalmitis. Subsequently, the patient underwent pars plana vitrectomy and vitreous biopsy. The microbiologic investigation of the vitreous biopsy showed poorly stained gram-positive beaded bacilli that were acid fast, and growth in culture was identified as Corynebacterium pseudodiphtheriticum by Vitek 2 bacterial identification system. However, DNA analysis confirmed the organism to be M. manitobense. The patient responded well to repeat interventions with intravitreal antibiotics with a final reported visual acuity of 20/30 at 4 months after first intervention., Conclusion: Although known to be an organism causing soft-tissue infections, M. manitobense can also cause postsurgical endophthalmitis. The diagnosis can be confused with Corynebacterium sp. on smear and culture. Subsequent DNA sequencing of the culture provides definite identification of the organism.

The immunocompromised host is at increased risk of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infection. Although Mycobacterium tuberculosis complex is a significant mycobacterial pathogen, nontuberculous mycobacteria causes substantial disease in those with suppressed immune responses. Mycobacterial infections can cause significant morbidity and mortality in this patient population, and rapid identification and susceptibility testing of the mycobacterial species is paramount to patient management and outcomes. Mycobacterial diagnostics has undergone some significant advances in the last two decades with immunodiagnostics (interferon gamma release assay), microscopy (light-emitting diode), culture (automated broth-based systems), identification (direct PCR, sequencing and matrix-assisted laser-desorption ionization-time of flight mass spectrometry) and susceptibility testing (molecular detection of drug resistance from direct specimens or positive cultures). Employing the most rapid and sensitive methods in the mycobacterial laboratory will have a tremendous impact on patient care and, in the case of Mycobacterium tuberculosis complex, in the control of tuberculosis.

Background: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by inborn errors of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex. From the first year of life onward, most affected patients display multiple, severe, and recurrent infections caused by bacteria and fungi. Mycobacterial infections have also been reported in some patients., Objective: Our objective was to assess the effect of mycobacterial disease in patients with CGD., Methods: We analyzed retrospectively the clinical features of mycobacterial disease in 71 patients with CGD. Tuberculosis and BCG disease were diagnosed on the basis of microbiological, pathological, and/or clinical criteria., Results: Thirty-one (44%) patients had tuberculosis, and 53 (75%) presented with adverse effects of BCG vaccination; 13 (18%) had both tuberculosis and BCG infections. None of these patients displayed clinical disease caused by environmental mycobacteria, Mycobacterium leprae, or Mycobacterium ulcerans. Most patients (76%) also had other pyogenic and fungal infections, but 24% presented solely with mycobacterial disease. Most patients presented a single localized episode of mycobacterial disease (37%), but recurrence (18%), disseminated disease (27%), and even death (18%) were also observed. One common feature in these patients was an early age at presentation for BCG disease. Mycobacterial disease was the first clinical manifestation of CGD in 60% of these patients., Conclusion: Mycobacterial disease is relatively common in patients with CGD living in countries in which tuberculosis is endemic, BCG vaccine is mandatory, or both. Adverse reactions to BCG and severe forms of tuberculosis should lead to a suspicion of CGD. BCG vaccine is contraindicated in patients with CGD., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

To date, more than 170 species of mycobacteria have been described, of which more than one third may be pathogenic to humans, representing a significant workload for microbiology laboratories. These species must be identified in clinical practice, which has long been a major problem due to the shortcomings of conventional (phenotypic) methods and the limitations and complexity of modern methods largely based on molecular biology techniques. The aim of this review was to briefly describe different aspects related to the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) for the identification of mycobacteria. Several difficulties are encountered with the use of this methodology in these microorganisms mainly due to the high pathogenicity of some mycobacteria and the peculiar structure of their cell wall, requiring inactivation and special protein extraction protocols. We also analysed other relevant aspects such as culture media, the reference methods employed (gold standard) in the final identification of the different species, the cut-off used to accept data as valid, and the databases of the different mass spectrometry systems available. MS has revolutionized diagnosis in modern microbiology; however, specific improvements are needed to consolidate the use of this technology in mycobacteriology., (Copyright © 2016 Elsevier España, S.L.U. All rights reserved.)

We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To establish a multiplex polymerase chain reaction (mPCR) method with high sensitivity and specificity for rapid detection of common nontuberculous mycobacterium(NTM) infection in the hand., Methods: Application of primer design software to the mycobacterium marinum, mycobacterium avium, mycobacterium kansasii and mycobacterium fortuitum, the specific gene sequences were used to design construction of multiplex PCR and detection of DNA from the non tuberculous mycobacterial standard strains of each bacterium of single PCR and multiplex DNA accuracy and sequence contrast evaluation to verify the specificity of multiple PCR primers.26 clinical specimens were identified by this method., Results: Detection of 26 cases of clinical samples, positive detection of more than 7/8, the identification time is shorter than the traditional method., Conclusion: The research method can be rapid, specific, sensitive and effective to detect the common hand of mycobacterium tuberculosis infection, can be used for clinical identification of mycobacterium tuberculosis infection.

Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: The establishment of therapeutic regimens for mycobacteriosis depends on the accurate identification of Mycobacterium species, and misdiagnosis can result in inappropriate treatment and increased mortality of patients. Differential diagnosis among Mycobacterium species has been made by conventional phenotypic and biochemical tests after a long culture period. Specialized molecular diagnostics of mycobacteria allows rapid detection and species identification; however, such tests are not available in public health programs. Our aim was to demonstrate the clinical implications of erroneous diagnosis by performing molecular genotyping of mycobacterial infections in patients that were diagnosed based on symptoms, culture and bacilloscopy., Methods: Culture samples of mycobacterial infections from 55 patients clinically diagnosed as tuberculosis in 2013 and 2014, based on conventional methods, were identified by PCR -RFLP and results are discussed., Results: We have confirmed 35 (63.6%) positive samples as M. tuberculosis, but 18 (32.7%) were identified as non-tuberculous mycobacteria (M. avium type 1, M. avium type 2, M. kansasii type 1 type 1, M. mucogenicum, M. chelonae, M. terrae type 3, and 1 unknown RFLP pattern) and two were negative. Regarding clinical diagnosis, 61.8% (34/55) was classified as pulmonary tuberculosis. It is important to emphasize that 36.4% (20/55) of samples were misdiagnosed by conventional methods, and 11 (61.1%) of the HIV positive patients (18/55) were NTM-coinfected., Conclusion: The identification of species in mycobacterial infections is essential for correct diagnosis and choice of treatment regimen, and misdiagnosis by conventional tools can lead to chronic disease, increased resistance and death.

Background: Rapidly growing mycobacteria (RGM) bloodstream infections (BSI) are an emerging problem often associated with therapeutic challenges. We review the epidemiology, treatment and outcomes over a 5-year period of a heterogeneous group presenting to our institution with RGM BSI., Materials and Methods: A retrospective cohort study of patients with primary RGM BSI from January 2006-December 2011 was conducted. Patient characteristics (age, race, sex and comorbidities), infection characteristics (catheter associated, hospital acquired, microbiology and antimicrobial susceptibilities), therapy and outcomes were recorded and compared by species., Results: Among 32 patients, 33 RGM BSI occurred. Patients had an average of 3-4 comorbidities, most commonly malignancy (45.5%). Most isolates (30.3%) were Mycobacterium fortuitum or Mycobacterium mucogenicum (27.2%), followed by Mycobacterium abscessus/chelonae (18.2%) and Mycobacterium immunogenum (12.2%). In all, 85% were catheter associated and 27.3% were hospital acquired. Empiric therapy was started in 19 (57.6%) patients and among these, it was adequate (at least 2 active agents based on susceptibilities) in 12 (63.2%). Among 21 patients with outcome data, cure was assumed for 14 (66.7%). One death was attributable to RGM BSI. Cure rates were higher among those who received adequate empiric therapy compared to those who did not (83.3% versus 42.9%). In general, antibiotic susceptibility was favorable across species for clarithromycin, amikacin and imipenem., Conclusions: RGM BSI occurred in a population with multiple comorbidities, most commonly malignancy, and most were catheter associated. Higher cures were seen among those who received adequate empiric therapy and based on susceptibility data, a broad empiric regimen of clarithromycin, amikacin and imipenem would be expected to be adequate., (Copyright © 2016 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Mycobacteria cause a range of diseases in both immunocompetent and immunosuppressed individuals. An increase in non-tuberculous mycobacterial (NTM) infections targeting skin has been described. Many hypotheses have been developed in order to explain it: the increasing burden of immunocompromised individuals, immigration from endemic countries, improved laboratory identification techniques, and changes inhuman behavior that expose individuals to this NTM. Mycobacterium mucogenicum group comprises M. mucogenicum, Mycobacterium aubagnense, and Mycobacterium phocaicum. This group of organisms was first named Mycobacterium chelonae-like organism in 1982. Most clinically significant cases of those organisms involved catheter-related infections. Nevertheless, we report an interesting patient with a cutaneous infection produced by M. mucogenicum mimicking a squamous cell carcinoma; an excellent response to combined therapy with rifampicin and clarythromicin was observed.

Background: When administered to an immune-compromised patient, BCG (Bacille Calmette-Guérin) can cause disseminated and life-threatening infections., Objective: To describe the imaging findings in children with primary immunodeficiency and BCG-related infections., Materials and Methods: We reviewed the imaging findings of children with primary immunodeficiency treated at a children's hospital during 2012-2014 with localized or disseminated BCG infection. Imaging modalities included US, CT and radiography., Results: Nine children with primary immunodeficiency had clinical signs of post-vaccination BCGitis; seven of these children showed disseminated disease and two showed only regional lesions with characteristic ipsilateral lymphadenopathy. Overall, lymphadenopathy was the most prevalent feature (n = 8) and characteristically appeared as a ring-enhancing hypodense (CT) or hypoechoic (US) lesion. Visceral involvement with multiple abscesses appeared in the spleen (n = 2), liver (n = 1) and bones (n = 1). All lesions regressed following appropriate anti-tuberculosis treatment., Conclusion: BCG infection needs to be considered in children with typical findings and with suspected primary immunodeficiency.

The diagnosis of cutaneous tuberculosis poses a serious challenge due to many skin diseases of different etiology resembling the lesions caused by the TB (tuberculosis) bacillus, and difficulties in confirming the disease. The presented case concerns skin lesions in a hobby aquarist stung in the finger of the left hand by a fish. The resulting inflammatory infiltration was to be cutaneous tuberculosis or mycobacteriosis caused by MOTT (Mycobacterium other than tuberculosis). Laboratory, pathomorphologic, genetic and microbiologic tests of samples obtained from the patient, fish and water in the aquarium gave ambiguous results. A multidisciplinary discussion is presented on the difficulties in the differential diagnosis, problems with a clear interpretation of the results of various conducted tests, and possible ways of transmission of the infection, relevant to the described example.

Mycobacterial infection is a rare complication associated with breast surgery using implants. Over the course of 5 months, one center experienced 12 such cases, 10 of which were linked to a single surgeon. Most presented 3 to 6 weeks postoperatively with clear serous drainage from the incision, minimal local redness, no fever or other systemic signs of infection, and negative standard bacterial cultures. Patients were given empiric broad-spectrum oral antibiotic therapy. In eight cases, implants were removed and exchanged for new devices after irrigation of the pocket with antibiotics; these patients nonetheless experienced recurrent infection, which led to explantation (without immediate exchange for new implants). The last two patients proceeded straight to explantation. Because mycobacteria grow in water, the water supply and air-conditioning system were initially suspected as the source, and both were disinfected. However, this did not stop the outbreak. Eventually, the source was traced to a new species of mycobacteria isolated from a garden hot tub. These bacteria had then been unwittingly transferred to patients during surgery. A Triclosan-containing shampoo effectively ended the outbreak. This series is unique in several respects: the novelty of the pathogen, the heavy colonization of the surgeon, and the mechanism of transmission (the first occurrence of human-to-human mycobacterial transfer published in the plastic surgery literature). Surgeons who perform breast surgery with implants should be aware of the possibility of mycobacterial infection. Proactive culturing of the organism, use of antibiotics, and reoperation are essential to good outcomes.

We report a case of disseminated Mycobacterium haemophilum osteomyelitis in a patient with advanced HIV infection, who later developed recurrent immune reconstitution inflammatory syndrome after commencement of antiretroviral therapy. We review previous reports of M. haemophilum bone and joint infection associated with HIV infection and describe the management of M. haemophilum-associated immune reconstitution inflammatory syndrome, including the role of surgery as an adjunctive treatment modality and the potential drug interactions between antiretroviral and antimycobacterial agents., (© The Author(s) 2015.)

Background: Mycobacterium yongonense, as a novel member of the M. avium complex (MAC), was recently reported to be isolated from human specimens in South Korea and Italy. Due to its close relatedness to other MAC members, particularly M. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes., Methods: Using the Mycobacterium yongonense genome information, we have identified a novel IS-element, ISMyo2. Targeting the ISMyo2 sequence, we developed a real-time PCR method and applied the technique to Mycobacterial genomic DNA., Results: To identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence (IS) elements of 3 M. intracellulare and 3 M. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the M. yongonense-specific IS element, ISMyo2 (2,387 bp), belonging to the IS21 family. Next, we developed a real-time PCR method using SYBR green I for M. yongonense-specific detection targeting ISMyo2, producing a 338-bp amplicon. When this assay was applied to 28 Mycobacterium reference strains and 63 MAC clinical isolates, it produced amplicons in only the 6 M. yongonense strains, showing a sensitivity of 100 fg of genomic DNA, suggesting its feasibility as a diagnostic method for M. yongonense strains., Conclusions: We identified a novel ISMyo2 IS element belonging to the IS21 family specific to M. yongonense strains via genome analysis, and a real-time PCR method based on its sequences was developed.

To report a case of a ruptured mycotic abdominal aortic aneurysm (MAA) after intravesical Bacille Calmette-Guerin (BCG) therapy because of bladder carcinoma. A 57-year-old male patient was admitted to our hospital for follow-up computed tomography 14 months after transurethral resection of a papillary carcinoma of the bladder and intravesical BCG therapy. The CT scan revealed a ruptured MAA aneurysm and the patient underwent an endovascular repair with an aorto-bi-iliac stent graft. A ruptured MAA is a rare but lethal complication after BCG instillation therapy. The standard therapy is the open reconstruction but according to the literature an endovascular therapy in combination with long-term antibiotics should be considered as a bridging or a definite solution., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mycobacterium haemophilum is a slow-growing non-tuberculous mycobacterium that is rarely known to cause human skin infection, particularly in immunocompromised patients. We recently experienced a 69-year-old Japanese woman with this infection who had been under immunosuppressive treatment for recalcitrant rheumatoid arthritis. The patient showed disseminated erythematous plaques and subcutaneous nodules on the face and extremities, and interestingly, the face manifested with a striking "facies leontina" appearance. Biopsy revealed abscess and granulomatous dermatitis with the involvement of peripheral nerve bundles and the presence of innumerable acid-fast bacilli, thus necessitating differentiation from lepromatous leprosy. M. haemophilum was identified by molecular characterization as well as by successful culture with iron supplements. Although drug susceptibility testing indicated responsiveness to multiple antibiotics administrated simultaneously for the treatment, it took over 6 months to achieve significant improvement, and we also employed concurrent oral potassium iodide administration and repeated surgical excision. This case highlights the importance of continuous combination therapy for successful outcome in this rare infection. Furthermore, application of potassium iodide for mycobacterial infection warrants further evaluation by accumulating more cases., (© 2015 Japanese Dermatological Association.)

Until recently, the study of mycobacterial diseases was trapped in culture-based technology that is more than a century old. The use of nucleic acid amplification is changing this, and powerful new technologies are on the horizon. Metabolomics, which is the study of sets of metabolites of both the bacteria and host, is being used to clarify mechanisms of disease, and can identify changes leading to better diagnosis, treatment, and prognostication of mycobacterial diseases. Metabolomic profiles are arrays of biochemical products of genes in their environment. These complex patterns are biomarkers that can allow a more complete understanding of cell function, dysfunction, and perturbation than genomics or proteomics. Metabolomics could herald sweeping advances in personalized medicine and clinical trial design, but the challenges in metabolomics are also great. Measured metabolite concentrations vary with the timing within a condition, the intrinsic biology, the instruments, and the sample preparation. Metabolism profoundly changes with age, sex, variations in gut microbial flora, and lifestyle. Validation of biomarkers is complicated by measurement accuracy, selectivity, linearity, reproducibility, robustness, and limits of detection. The statistical challenges include analysis, interpretation, and description of the vast amount of data generated. Despite these drawbacks, metabolomics provides great opportunity and the potential to understand and manage mycobacterial diseases.

We report the case of a 36-year-old woman who presented with a surgical site infection with atypical mycobacteria several months after liposuction in the Caribbean. Postoperative wound infections with Mycobacterium abscessus present as subcutaneous nodular abscesses with partly putrid secretion. Due to the necessity of specific diagnostic measures, diagnosis is often delayed. Treatment is difficult because Mycobacterium abscessus is resistant to conventional tuberculostatics and many other antibiotics. Clarithromycin combined with amikacin is the commonly used empirical treatment. Treatment duration is several months.

Background: Recently, we introduced a novel peptide nucleic acid (PNA) multi-probe real time PCR method targeting the hsp65 gene (hsp65 PNA RT-PCR) to distinguish Mycobacterium abscessus groups., Methods: Here, we evaluated the usefulness of the hsp65 PNA RT-PCR for the direct identification of the M. abscessus group at the subspecies and genotype levels from sputa samples. The method was applied to total sputa DNA from 60 different patients who were identified as having mycobacterial infections via rpoB PCR restriction analysis of the same cultures., Results: The hsp65 PNA RT-PCR method had higher sensitivity than the multi-probe real-time PCR assay targeting hsp65 (HMPRT-PCR) for the detection of M. abscessus from sputum [96.7 % (29/30 samples) vs. 70 % (21/30 samples); 100 % specificity]., Conclusions: These results suggest that the PNA-based method is feasible for the detection of M. abscessus members not only from cultures but also directly from sputa.

When mycobacteria are recovered in clinical specimens, timely species-level identification is required to establish the clinical significance of the isolate and facilitate optimization of antimicrobial therapy. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been reported to be a reliable and expedited method for identification of mycobacteria, although various specimen preparation techniques and databases for analysis are reported across studies. Here we compared two MALDI-TOF MS instrumentation platforms and three databases: Bruker Biotyper Real Time Classification 3.1 (Biotyper), Vitek MS Plus Saramis Premium (Saramis), and Vitek MS v3.0. We evaluated two sample preparation techniques and demonstrate that extraction methods are not interchangeable across different platforms or databases. Once testing parameters were established, a panel of 157 mycobacterial isolates (including 16 Mycobacterium tuberculosis isolates) was evaluated, demonstrating that with the appropriate specimen preparation, all three methods provide reliable identification for most species. Using a score cutoff value of ≥1.8, the Biotyper correctly identified 133 (84.7%) isolates with no misidentifications. Using a confidence value of ≥90%, Saramis correctly identified 134 (85.4%) isolates with one misidentification and Vitek MS v3.0 correctly identified 140 (89.2%) isolates with one misidentification. The levels of accuracy were not significantly different across the three platforms (P = 0.14). In addition, we show that Vitek MS v3.0 requires modestly fewer repeat analyses than the Biotyper and Saramis methods (P = 0.04), which may have implications for laboratory workflow., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mycobacterium species are exposed to oxidative and nitrosylative stress from environments within and outside the host cells. After the host is infected with the bacilli, macrophages produce superoxide molecules via NADPH oxidase activity and nitric oxide (NO) via inducible NO synthase activity to kill the bacilli. The pathogenic bacilli can successfully survive in host cells via anti-oxidative and anti-nitrosylative mechanisms. In particular, Mycobacterium tuberculosis persisters pose a great problem for chemotherapy because most anti-mycobacterial drugs are ineffective against mycobacteria that are in the persistent state. In accordance with the changes in redox balance, the bacilli change their metabolic pathways from aerobic to anaerobic ones, thereby leading to a change from an actively growing state to a dormant state. Therefore, M. tuberculosis is expected to be equipped with sensors that detect redox stress in the environment such that it can switch to the dormant state and change its metabolic pathways accordingly. In this review, roles of the mycobacterial O2, NO, and CO gas sensors, DosS and DosT, consisting of the DosR regulon, and mycobacterial DNA binding proteins WhiBs, which contain iron-sulfur clusters, in latent infection are discussed.

Aims: To directly identify clinically relevant mycobacteria from clinical specimens, we have developed a multiplex real-time PCR assay with hydrolysis probes that can identify 20 mycobacterial species., Methods and Results: The assay was initially evaluated using 248 strains, including both reference strains and clinical isolates. Then, the assay was implemented according to a scheme in our laboratory. The scheme based on the clinical differences between the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) consisted of three stepwise PCRs. MTC and NTM were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and step 3 PCRs. During a 2·5-year period, 1877 isolates of MTC (1142 directly recovered from clinical specimens) and 596 isolates of NTM (143 directly recovered from clinical specimens) were detected, and the species of 590 (99·0%) of the 596 NTM isolates were identified., Conclusions: Our experience shows that this is a new paradigm for rapidly and accurately identifying clinically relevant mycobacteria, in which a multiplex real-time PCR assay is directly applied to clinical specimens in a stepwise fashion., Significance and Impact of the Study: This study is the first report of a multiplex real-time PCR assay for identifying clinically important mycobacterial species directly from clinical specimens and its application in a clinical microbiology laboratory., (© 2015 The Society for Applied Microbiology.)

Mycobacterium riyadhense is a newly described slowly growing, non-tuberculous mycobacterium species. We describe 2 new cases of Mycobacterium riyadhense infections presenting with extra-pulmonary involvement, and reviewed all previously reported cases in the literature. We also describe the spectrum of the disease and explore treatment options based on the experience with the current and previously reported cases.