Viable Bacilli Research Articles

Eradication of Drug-Tolerant Mycobacterium tuberculosis 2022: Where We Stand.

The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.

Feasibility of novel approaches to detect viable Mycobacterium tuberculosis within the spectrum of the tuberculosis disease.

Tuberculosis (TB) is a global disease caused by Mycobacterium tuberculosis (Mtb) and is manifested as a continuum spectrum of infectious states. Both, the most common and clinically asymptomatic latent tuberculosis infection (LTBI), and the symptomatic disease, active tuberculosis (TB), are at opposite ends of the spectrum. Such binary classification is insufficient to describe the existing clinical heterogeneity, which includes incipient and subclinical TB. The absence of clinically TB-related symptoms and the extremely low bacterial burden are features shared by LTBI, incipient and subclinical TB states. In addition, diagnosis relies on cytokine release after antigenic T cell stimulation, yet several studies have shown that a high proportion of individuals with immunoreactivity never developed disease, suggesting that they were no longer infected. LTBI is estimated to affect to approximately one fourth of the human population and, according to WHO data, reactivation of LTBI is the main responsible of TB cases in developed countries. Assuming the drawbacks associated to the current diagnostic tests at this part of the disease spectrum, properly assessing individuals at real risk of developing TB is a major need. Further, it would help to efficiently design preventive treatment. This quest would be achievable if information about bacterial viability during human silent Mtb infection could be determined. Here, we have evaluated the feasibility of new approaches to detect viable bacilli across the full spectrum of TB disease. We focused on methods that specifically can measure host-independent parameters relying on the viability of Mtb either by its direct or indirect detection.

Gene Expression Profile of Mycobacterium leprae Contribution in the Pathology of Leprosy Neuropathy.

Peripheral neuropathy is the main cause of physical disability in leprosy patients. Importantly, the extension and pattern of peripheral damage has been linked to how the host cell will respond against Mycobacterium leprae (M. leprae) infection, in particular, how the pathogen will establish infection in Schwann cells. Interestingly, viable and dead M. leprae have been linked to neuropathology of leprosy by distinct mechanisms. While viable M. leprae promotes transcriptional modifications that allow the bacteria to survive through the use of the host cell's internal machinery and the subvert of host metabolites, components of the dead bacteria are associated with the generation of a harmful nerve microenvironment. Therefore, understanding the pathognomonic characteristics mediated by viable and dead M. leprae are essential for elucidating leprosy disease and its associated reactional episodes. Moreover, the impact of the viable and dead bacteria in Schwann cells is largely unknown and their gene signature profiling has, as yet, been poorly explored. In this study, we analyzed the early differences in the expression profile of genes involved in peripheral neuropathy, dedifferentiation and plasticity, neural regeneration, and inflammation in human Schwann cells challenged with viable and dead M. leprae. We substantiated our findings by analyzing this genetic profiling in human nerve biopsies of leprosy and non-leprosy patients, with accompanied histopathological analysis. We observed that viable and dead bacteria distinctly modulate Schwann cell genes, with emphasis to viable bacilli upregulating transcripts related to glial cell plasticity, dedifferentiation and anti-inflammatory profile, while dead bacteria affected genes involved in neuropathy and pro-inflammatory response. In addition, dead bacteria also upregulated genes associated with nerve support, which expression profile was similar to those obtained from leprosy nerve biopsies. These findings suggest that early exposure to viable and dead bacteria may provoke Schwann cells to behave differentially, with far-reaching implications for the ongoing neuropathy seen in leprosy patients, where a mixture of active and non-active bacteria are found in the nerve microenvironment.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BackgroundThe current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed.MethodsTo evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results.ResultsThe susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST.ConclusionIn conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Detection of viable Mycobacterium tuberculosis in ocular fluids using mRNA-based multiplex polymerase chain reaction

PurposeIntra ocular tuberculosis (IOTB) can affect nearly any part of the eye and delayed diagnosis can result in permanent loss of vision. mRNA, with the ability to detect viable bacilli just like culture, could serve as an important tool to differentiate whether ocular manifestations of IOTB are due to active infection or hypersensitivity response to dead mycobacteria. MethodsA total of 19 patients with suspected IOTB and 20 controls (10 patients with active inflammation but no evidence of IOTB and 10 patients with no intraocular inflammation) were included. Vitreous fluid samples were collected from all patients and subjected to DNA and mRNA extraction followed by multiplex polymerase chain reaction (MPCR) using three specific targets for M. tuberculosis, i.e. IS6110, MPB64 and protein b. All samples were also subjected to Gene Xpert MTB/RIF assay. ResultsAmong the 19 patients in group 1, the highest test positivity was noted for DNA-MPCR (73.7%) followed by mRNA-MPCR (42.1%) and GeneXpert MTB/RIF assay (10.5%). All the three tests were negative in the 20 control samples demonstrating a 100% specificity. The negative predictive value was highest for DNA-MPCR (80%), followed by mRNA-PCR (64.5%) and Xpert MTB/RIF assay (48.8%). All patients with any one test positive were treated with anti-tubercular therapy (ATT) and there was good clinical response noted in all. ConclusionmRNA-based MPCR can be used as a marker for detecting viable M. tuberculosis in IOTB cases.

Determining Viability of M. ulcerans by 16S rRNA RT Reverse Transcriptase Real-Time PCR.

To overcome drawbacks of M. ulcerans culture in terms of incubation time and low sensitivity for the detection of viable bacilli from clinical specimens, a highly sensitive and M. ulcerans-specific RNA-based viability assay was developed. The assay combines a 16S rRNA reverse transcriptase real-time PCR (16S rRNA RT qPCR) to determine bacterial viability with an IS2404 quantitative real-time PCR (IS2404 qPCR) to ensure specificity as well as simultaneous quantification of bacilli. This proved to be highly efficient in detecting viable bacilli in clinical samples when implemented in the field.

Fluorescein Diacetate Vital staining for detecting viability of acid-fast bacilli in sputum of pulmonary tuberculosis patients

ObjectiveTo compare the performance of the Fluorescein diacetate (FDA) vital staining method with Ziehl-Neelsen staining method in detecting the viability of acid-fast bacilli using MGIT culture as “reference standard”. MethodsThis was a single centre prospective observational study conducted from October 2015 to November 2016. Microbiologically confirmed ZN-Smear positive (3+) sputum specimens were obtained from 30 pulmonary tuberculosis patients taking anti-tuberculosis treatment at DOTS centre of NITRD, New Delhi. Patients were made available to collect the first baseline sputum sample before commencing treatment, and an early morning sputum sample was collected as per RNTCP guidelines. After starting treatment, sputum specimens were collected weekly in the first month and thereafter twice-weekly until 18th week. All sputum specimens from patients receiving anti-tuberculosis treatment were examined using Ziehl-Neelsen (ZN) smear microscopy, FDA vital staining, and MGIT culture. ResultOut of 360 follow up sputum specimens collected from 30 adult microbiologically confirmed ZN- Smear (3+) pulmonary tuberculosis patients, 146 were ZN-positive and 130 FDA-positive. Of 130 FDA-positive sputum samples, mycobacteria tuberculosis (MTB) growth was found in 116 sputum samples, of which 116 sputum specimens were positive for FDA. Additionally, 14 culture-negative specimens were FDA positive. No FDA-negative sputum samples were positive for MGIT culture. Among ZN positive specimens, FDA had 100% sensitivity and 85.3% specificity with an accuracy of 96.58% for the detection of viable mycobacteria. Among ZN negative sputum specimens, FDA had comparatively high specificity (95.7%). Using positive MGIT culture as a reference for viability, negative predictive value (NPV) and positive predictive value (PPV) from FDA vital staining method were found to be 100 and 89% respectively. ConclusionFDA staining is a simple and rapid tool for identifying viable MTB bacilli. Because of its excellent NPV and encouraging specificity, FDA staining is useful to identify patients with non-viable bacilli (FDA negative) among retreatment cases at diagnosis and patients on anti-tuberculosis treatment for both drug-sensitive and drug-resistant tuberculosis for follow up for the response of treatment.

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology—including the tendency to clump, and “differential” culturability—and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.

Probing the in-vivo reservoir of latent tuberculosis infection

For decades the unanswered question of where viable bacilli reside in latently infected individuals has been a central conundrum in tuberculosis. The hitherto insurmountable challenge of identifying dormant Mycobacterium tuberculosis bacilli in asymptomatic latently infected individuals is why diagnosis of latent tuberculosis infection relies on measurement of the cell-mediated immune response to M tuberculosis, by tuberculin skin test or interferon-γ release assay (IGRA).1 The first clues about M tuberculosis's anatomical niche in latent infection stretch back a century when it was shown that post-mortem cadaveric tissue homogenates, from healthy lung as well as fibrotic and granulomatous lesions, could propagate tuberculosis in challenged guinea pigs, a finding replicated in several subsequent studies.

Role of routine nephrectomy for non-functioning kidneys due to genitourinary tuberculosis: Data from an Indian subcontinent

Objective: To evaluate the role of routine nephrectomy for tuberculous non-functioning kidney (TNFK) after receiving anti-tubercular therapy (ATT) by demonstrating whether live tubercle bacilli persist in nephrectomy specimens after treatment or not. Materials and methods: Patients with TNFK who underwent nephrectomy after completion of at least 6 months of ATT were included in this prospective cohort study. We sent tissue/pus from a nephrectomy specimen for acid-fast bacilli (AFB) staining, polymerase chain reaction (PCR) and culture to look for live bacilli. Bacilli were considered alive only if AFB culture was positive. Results: Twenty-four patients underwent nephrectomy for TNFK between April 2015 and October 2017 (18 laparoscopic and 6 open nephrectomy). Laparoscopic nephrectomy was associated with lower blood loss (225 ml versus 408 ml, p = 0.0003) and shorter hospital stay (3 versus 3.8 days, p = 0.06) compared with open nephrectomy; however, mean operative time and overall complications were similar. Eight specimens were AFB smear and/or tuberculosis PCR positive, out of which three showed viable bacilli upon culture. Drug sensitivity testing showed multi-drug resistant strain in all three patients who were treated with second-line ATT. Conclusion: It is preferable to do routine nephrectomy for TNFKs as they are more likely to harbour live bacilli and lead to disease recurrence. Viability testing for AFB must be performed on all operated specimens to identify drug resistant bacilli so that patients may be treated with second-line therapy if required. Level of evidence: 4.

A man in his seventies with dyspnoea and ataxia after BCG instillation for bladder cancer.

BACKGROUND This case report introduces one of the first documented incidents of infectious complications of intravesical BCG immunotherapy in Norway. It took time to feel satisfied with the final diagnosis due to the diagnostic challenges related to obtaining positive results on microbiological analyses. CASE PRESENTATION A man in his seventies presented with malaise, fever and elevated liver enzymes after intravesical administration of BCG. He was given glucocorticoids against a potential hypersensitive reaction on the liver, but the patient soon returned with worsening general condition, persistent fever, cough and ataxia. CT thorax showed a miliary pattern in the lungs and brain MRI showed dural/meningeal enhancement. We were unable to detect viable bacilli in microbiological samples from the lungs, cerebral fluid or blood. Histologic finding of granulomas on lung biopsy, together with no other reasonable alternative diagnosis, was crucial for the decision to start antitubercular drugs. INTERPRETATION Although a rare complication, BCG infections should be considered early in patients developing illness after intravesical BCG instillation. The procedure is relatively common in Norwegian hospitals and adverse results do occur.

Cough-independent production of viable Mycobacterium tuberculosis in bioaerosol

BackgroundSymptoms of infectious respiratory illnesses are often assumed to drive transmission. However, production and release of Mycobacterium tuberculosis (Mtb) bioaerosols is poorly understood. We report quantitation of Mtb exhaled during specific respiratory manoeuvres. MethodsDirect capture of nascent bioaerosol particles and indirect collection of aged particles was performed in 10 healthy subjects. Indirect and direct capture of exhaled viable Mtb bacilli was compared in 38 PTB patients and directly captured viable Mtb during cough and bronchiole-burst manoeuvres in 27 of the PTB patients. ResultsDirect sampling of healthy subjects captured larger bioaerosol volumes with higher proportions of 2–5 μm particles than indirect sampling. Indirect sampling identified viable Mtb in 92.1% (35 of 38) of PTB patients during 60-min relaxed breathing, median bacillary count 7.5 (IQR: 3.25–19). Direct sampling for 10-min identified Mtb in 97.4% (37 of 38) of PTB patients with higher bacilli counts (p < 0.001), median 24.5 (IQR:11.25–37.5). A short 5-min sampling regimen of 10 coughs or 10 bronchiole-burst manoeuvres yielded a median of 11 (IQR: 4–17) and 11 (IQR: 7–17.5) Mtb bacilli, respectively (p = 0.53). ConclusionsPeripheral lung bioaerosol released through deep exhalations alone contained viable Mtb suggesting non-cough transmission is possible in PTB.

Pb(II) Bio-Removal, Viability, and Population Distribution of an Industrial Microbial Consortium: The Effect of Pb(II) and Nutrient Concentrations

This study presents the effect of aqueous Pb(II) and nutrient concentrations on the Pb(II)-removal, biomass viability, active species identities, and population distribution of an industrial Pb(II) resistant microbial consortium. The studied consortium has previously shown to be highly effective at precipitating Pb(II) from solution. At all conditions tested (80 and 500 ppm Pb(II), and varying nutrients conditions) it was found that circa 50% of Pb(II) was removed within the first 3 h, with the absence of any visual changes, followed by a slower rate of Pb(II) removal accompanied by the formation of a dark precipitate. The Pb(II) removal was found to be independent of microbial growth, while growth was observed dependent on the concentration of Pb(II), nutrients, and nitrates in the system. SEM analysis indicated viable bacilli embedded in precipitate. These findings indicate that precipitation occurs on the surface of the biomass as opposed to an internal excretion mechanism. BLAST (Basic Local Alignment Search Tool) results indicated Klebsiella pneumoniae as the active species responsible for Pb(II) bioprecipitation for both the 80 and 500 ppm isolated colonies, while a diverse population distribution of organisms was observed for the streak plate analyses. A quicker microbial generation rate was observed than what was expected for Klebsiella pneumoniae, indicating that the overall consortial population contributed to the growth rates observed. This study provided insights into the factors affecting Pb(II) bio-removal and bioprecipitation by the investigated industrially obtained consortium, thereby providing invaluable knowledge required for industrial application.

BCG shortage: Reassessing the clinical viability of Bacillus Calmette-Guerin (BCG) after reconstitution.

534 Background: To address the current worldwide shortage of BCG, the AUA and SUO recommend reducing the dose to 1/3rd vial and enabling 3 patients to be treated in one setting. The manufacturer states that BCG must be used immediately after reconstitution, despite literature in the bacteriological world suggesting that M. Bovis might be viable for longer than a few hours. Herein we sought to study the viability of BCG after re-constitution at time points relevant to clinical practice. Methods: TICE BCG from separate lots was reconstituted per the manufacturer’s guidance and stored at 4 °C without light exposure. At predetermined time points, BCG was inoculated on Selective Middlebrook 7H11 agar in triplicate and incubated at 37°C with 5% CO2. M. smegmatis served as a positive control and un-inoculated media was incubated for 2 weeks as a negative control. CFUs were assessed between 3 and 4 weeks from plating. Acid-fast staining confirmed the presence of BCG. Data was analyzed as the mean of replicated experiments and compared to the reference (Time 0) using Student’s T-tests. Results: No significant difference in CFUs was observed for BCG between 0 and 8 hours after reconstitution (Table). Colony forming units significantly declined starting 24 hours after reconstitution, though the magnitude of this difference was less than 10 fold (which falls within the range of CFUs listed for the vial by manufacturer). Viability remained constant for both lots analyzed. Conclusions: Given the recurrent shortages of BCG, split dosing may become a clinical necessity. This often presents logistic quandaries since the manufacturer recommends each vial must be used within 2 hours. We have shown that the viability of TICE BCG is unaltered at least 8 hours after reconstitution and only begins to decline at 24 hours after reconstitution. This should allow pharmacists and physicians administering BCG more leeway in scheduling patients for split dose therapy. [Table: see text]

NSC 18725, a Pyrazole Derivative Inhibits Growth of Intracellular Mycobacterium tuberculosis by Induction of Autophagy.

The increasing incident rates of drug-resistant tuberculosis (DR-TB) is a global health concern and has been further complicated by the emergence of extensive and total drug-resistant strains. Identification of new chemical entities which are compatible with first-line TB drugs, possess activity against DR-, and metabolically less active bacteria is required to tackle this epidemic. Here, we have performed phenotypic screening of a small molecule library against Mycobacterium bovis BCG and identified 24 scaffolds that exhibited MIC99 values of at least 2.5 μM. The most potent small molecule identified in our study was a nitroso containing pyrazole derivative, NSC 18725. We observed a significant reduction in viable bacilli load of starved Mycobacterium tuberculosis upon exposure to NSC 18725. The action of NSC 18725 was “synergistic” with isoniazid (INH) and “additive” with other drugs in our checkerboard assays. Structure-activity relationship (SAR) studies of the parent compound revealed that pyrazole derivatives without a functional group at fourth position lacked anti-mycobacterial activity in vitro. The derivative with para-chlorophenyl substitution at the first position of the pyrazole ring was the most active scaffold. We also demonstrate that NSC 18725 is able to induce autophagy in differentiated THP-1 macrophages. The induction of autophagy by NSC 18725 is the major mechanism for the killing of intracellular slow and fast-growing mycobacteria. Taken together, these observations support the identification of NSC 18725 as an antimycobacterial compound, which synergizes with INH, is active against non-replicative mycobacteria and induces autophagy in macrophages.

Minimum Bactericidal Concentration Techniques in Mycobacterium tuberculosis: A Systematic Review.

Minimum bactericidal concentration (MBC) assay is an accepted parameter for evaluating new antimicrobial agents, and it is frequently used as a research tool to provide a prediction of bacterial eradication. To the best of our knowledge, there is no standardization among researchers regarding the technique used to detect a drug's MBC in Mycobacterium tuberculosis. Thus, the aim of this systematic review is to discuss the available literature in determining a drug's MBC in M. tuberculosis, to find the most commonly used technique and standardize the process. A broad and rigorous literature search of three electronic databases (PubMed, Web of Knowledge, and LILACS) was performed according to the PRISMA statement. We considered studies that were published from January 1, 1990 to February 19, 2019. Google Scholar was also searched to increase the number of publications. We searched for articles using the MeSH terms "microbiological techniques," "Mycobacterium," "antibacterial agents." In addition, free terms were used in the search. The search yielded 6,674 publications. After filter application, 5,348 publications remained. Of these, we evaluated the full text of 187 publications. By applying the inclusion criteria, 69 studies were included in the present systematic review. In the literature analyzed, a great variety in the techniques used to determine a drug's MBC in M. tuberculosis was observed. The most common variability is related to the culture media used, culture incubation time, and the percentage of bacterial death for the drug to be considered as bactericidal. The most commonly used technique for drug's MBC determination was carried out using the drug's minimum inhibitory concentration (MIC) assay. Aliquots from prior MIC values were subcultured in Middlebrook agar and incubated for 4 weeks at 35°C for determining the colony forming unit (CFU) with relevance to detect 99.9% bacilli killed or reduction in 3 log10 viable bacilli.

A low cost ingenious approach for ultraviolet decontamination of N95 filtering face-piece respirators to deal with dwindling supply during the COVID-19 pandemic

Introduction: COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 is rapidly evolving and hospitals are facing the issue of shortage of personal protective equipment (PPE) due to stringent requirements of PPE use. For reuse of N95 filtering face-piece respirators (N95FFR), we prepared an ingenious cabinet with ultraviolet light C (UV-C) using scarce material available during the lockdown period. Materials and Methods: Since like many other microbiology laboratories in medical colleges, we did not have access to viruses, we could not test our product with them. We thus tested the efficacy of the cabinet to decontaminate material using 0.5 McFarland standard broth of Escherichia coli 25922. The broth was exposed to UV-C inside the cabinet for 15 and 30 minutes in Petri dishes, with and without lids. The broth was subcultured on nutrient agar plates, both pre and post exposure. We also could not test integrity and static charge of the respirators; we relied on CDC data on the same. Results: It was observed that there was at least 4 log reduction (99.99%) in the number of viable E. coli on exposure to UV-C for 15 as well as 30 minutes. The plates with glass lids on did not show any reduction in number of viable bacilli. The reduction in number of E. coli was taken as surrogate marker for the reduction of ssRNA viruses. Conclusions: UV-C inside an ingeniously made cabinet can be used to decontaminate N95 FFR in exceptional circumstances of reduced supply under lockdown conditions of a pandemic.

Mycobacterium tuberculosis DNA in living donor transplanted livers and donor-related tuberculosis in recipients: A retrospective longitudinal cohort study.

Mycobacterium tuberculosis DNA has been detected in multiple organs in people without active tuberculosis or a history of tuberculosis. Molecular testing for metabolic activity has suggested that M tuberculosis DNA represents viable bacilli. Whether transplanted organs with M tuberculosis DNA can result in tuberculosis in recipients has not been assessed. Biopsies obtained at the time of living donor liver transplantation were tested for the presence of M tuberculosis DNA using in situ PCR. The cohort of recipients was longitudinally followed for the development of tuberculosis. Living donor liver transplantation was performed for 270 patients. Mean age was 33years (median: 41years, range: 1-80years). Recipients were followed for a mean of 68months (median: 72months, range: 1-138months) after transplantation. Mycobacterium tuberculosis DNA was detected in 25 of 155 donated livers (16%) with liver biopsies available for testing. None of the recipients of these livers received tuberculosis chemoprophylaxis and only one (4%) developed tuberculosis 15months after transplantation. Among the entire cohort of 270 patients, post-transplant tuberculosis was diagnosed in four patients (1.48%) at an incidence rate of 2.61 cases per 1000 transplant-years. No factors associated with developing tuberculosis were identified, including positive M tuberculosis DNA in transplanted livers. Mycobacterium tuberculosis DNA in living donor transplanted livers did not result in tuberculosis despite post-transplant immunosuppression.

Development of a combined RLEP/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples

BackgroundLeprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures.MethodsValidation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on “must not detect”/“must detect” samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo.ResultsBoth assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%).ConclusionThe combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection.

Report on an unusual case of leprosy from Germany: just an exception of the rule?

Case presentationWe report on a German leprosy patient originating from Pakistan who had a relapse more than 5 years after completion of multi-drug therapy (MDT) of his first episode of multibacillary (MB) leprosy. State-of-the-art laboratory techniques (histopathology, PGL-I serology, microscopy and DNA/RNA qPCR) were applied for laboratory confirmation and monitoring of treatment outcome. Serology indicated the relapse long before the presence of unambiguous clinical signs. At the time of diagnosis of the relapse the patient had a remarkably high bacterial load suggesting increased risk for a second relapse. Furthermore, unexpectedly prolonged excretion of viable bacilli through the upper respiratory tract for more than 3 months after onset of MDT was shown. Therefore, MDT was administered for 2 years.Discussion and conclusionsThe clinical course of the patient, as well as the prolonged excretion of viable bacilli, underlines the usefulness of laboratory assessment. Laboratory tools including up-to-date molecular assays facilitate rapid diagnosis, timely MDT, identification of individuals excreting viable bacilli and patients at risk for relapses, monitoring of treatment outcome and respective adaptation of treatment where appropriate.