Locally Confined Clonal Complexes of Mycobacterium ulcerans in Two Buruli Ulcer Endemic Regions of Cameroon

Background Mycobacterium ulcerans is the causative agent of the necrotizing skin disease Buruli ulcer (BU), which has been reported from over 30 countries worldwide. The majority of notified patients come from West African countries, such as Côte d’Ivoire, Ghana, Benin and Cameroon. All clinical isolates of M. ulcerans from these countries are closely related and their genomes differ only in a limited number of single nucleotide polymorphisms (SNPs).ConclusionsOur comparative genomic analysis revealed that M. ulcerans clones diversify locally by the accumulation of SNPs. Case isolates coming from more recently emerging BU endemic areas, such as the Mapé river basin, may be less diverse than populations from longer standing disease foci, such as the Nyong river basin. Exchange of strains between distinct endemic areas seems to be rare and local clonal complexes can be easily distinguished by whole genome sequencing.     

Whole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana

Efforts to control the spread of Buruli ulcer – an emerging ulcerative skin infection caused by Mycobacterium ulcerans - have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30km² region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.     

Phage Therapy Is Effective against Infection by Mycobacterium ulcerans in a Murine Footpad Model

Background

Buruli Ulcer (BU) is a neglected, necrotizing skin disease caused by Mycobacterium ulcerans. Currently, there is no vaccine against M. ulcerans infection. Although the World Health Organization recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical management of advanced stages is still based on the surgical resection of infected skin. The use of bacteriophages for the control of bacterial infections has been considered as an alternative or to be used in association with antibiotherapy. Additionally, the mycobacteriophage D29 has previously been shown to display lytic activity against M. ulcerans isolates.

Methodology/Principal findings

We used the mouse footpad model of M. ulcerans infection to evaluate the therapeutic efficacy of treatment with mycobacteriophage D29. Analyses of macroscopic lesions, bacterial burdens, histology and cytokine production were performed in both M. ulcerans-infected footpads and draining lymph nodes (DLN). We have demonstrated that a single subcutaneous injection of the mycobacteriophage D29, administered 33 days after bacterial challenge, was sufficient to decrease pathology and to prevent ulceration. This protection resulted in a significant reduction of M. ulcerans numbers accompanied by an increase of cytokine levels (including IFN-γ), both in footpads and DLN. Additionally, mycobacteriophage D29 treatment induced a cellular infiltrate of a lymphocytic/macrophagic profile.

Conclusions/Significance

Our observations demonstrate the potential of phage therapy against M. ulcerans infection, paving the way for future studies aiming at the development of novel phage-related therapeutic approaches against BU.     

The Incubation Period of Buruli Ulcer (Mycobacterium ulcerans Infection)

Introduction

Buruli Ulcer (BU) is caused by the environmental microbe Mycobacterium ulcerans. Despite unclear transmission, contact with a BU endemic region is the key known risk factor. In Victoria, Australia, where endemic areas have been carefully mapped, we aimed to estimate the Incubation Period (IP) of BU by interviewing patients who reported defined periods of contact with an endemic area prior to BU diagnosis.

Method

A retrospective review was undertaken of 408 notifications of BU in Victoria from 2002 to 2012. Telephone interviews using a structured questionnaire and review of notification records were performed. Patients with a single visit exposure to a defined endemic area were included and the period from exposure to disease onset determined (IP).

Results

We identified 111 of 408 notified patients (27%) who had a residential address outside a known endemic area, of whom 23 (6%) reported a single visit exposure within the previous 24 months. The median age of included patients was 30 years (range: 6 to 73) and 65% were male. 61% had visited the Bellarine Peninsula, currently the most active endemic area. The median time from symptom onset to diagnosis was 71 days (range: 34–204 days). The midpoint of the reported IP range was utilized to calculate a point estimate of the IP for each case. Subsequently, the mean IP for the cohort was calculated at 135 days (IQR: 109–160; CI 95%: 113.9–156), corresponding to 4.5 months or 19.2 weeks. The shortest IP recorded was 32 days and longest 264 days (Figure 1 & 2). IP did not vary for variables investigated.Open in a separate windowFigure 1Geographic representation of Bellarine Peninsula, considered endemic for BU as of 2012.Bellarine Peninsula – east of line from Geelong to Torquay. Mornington and Westernport – southwest of line from Hampton to Tooradin (including Phillip Island).Open in a separate windowFigure 2Geographic representation of East Gippsland, considered endemic for BU as of 2012.East Gippsland: East of Sale and south of the great divide.

Conclusions

The estimated mean IP of BU in Victoria is 135 days (IQR: 109–160 days), 4.5 months. The shortest recorded was IP 34 days and longest 264 days. A greater understanding of BU IP will aid clinical risk assessment and future research.     

Single Nucleotide Polymorphism Typing of Mycobacterium ulcerans Reveals Focal Transmission of Buruli Ulcer in a Highly Endemic Region of Ghana

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted.     

Corticosteroid-Induced Immunosuppression Ultimately Does Not Compromise the Efficacy of Antibiotherapy in Murine Mycobacterium ulcerans Infection

Background

Buruli ulcer (BU) is a necrotizing disease of the skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans. It has been suggested that the immune response developed during the recommended rifampicin/streptomycin (RS) antibiotherapy is protective, contributing to bacterial clearance. On the other hand, paradoxical reactions have been described during or after antibiotherapy, characterized by pathological inflammatory responses. This exacerbated inflammation could be circumvented by immunosuppressive drugs. Therefore, it is important to clarify if the immune system contributes to bacterial clearance during RS antibiotherapy and if immunosuppression hampers the efficacy of the antibiotic regimen.

Methodology/Principal Findings

We used the M. ulcerans infection footpad mouse model. Corticosteroid-induced immunosuppression was achieved before experimental infection and maintained during combined RS antibiotherapy by the administration of dexamethasone (DEX). Time-lapsed analyses of macroscopic lesions, bacterial burdens, histology and immunohistochemistry were performed in M. ulcerans-infected footpads. We show here that corticosteroid-immunosuppressed mice are more susceptible to M. ulcerans, with higher bacterial burdens and earlier ulceration. Despite this, macroscopic lesions remised during combined antibiotic/DEX treatment and no viable bacteria were detected in the footpads after RS administration. This was observed despite a delayed kinetics in bacterial clearance, associated with a local reduction of T cell and neutrophil numbers, when compared with immunocompetent RS-treated mice. In addition, no relapse was observed following an additional 3 month period of DEX administration.

Conclusions/Significance

These findings reveal a major role of the RS bactericidal activity for the resolution of M. ulcerans experimental infections even during immunosuppression, and support clinical investigation on the potential use of corticosteroids or other immunosuppressive/anti-inflammatory drugs for the management of BU patients undergoing paradoxical reactions.     

Identification of Mycobacterium ulcerans in the Environment from Regions in Southeast Australia in Which It Is Endemic with Sequence Capture-PCR

We recently described the use of PCR to identify the environmental source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the M. ulcerans-specific insertion sequence, IS2404. In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two M. ulcerans sequences (IS2404 and IS2606) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of M. ulcerans infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for M. ulcerans. Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of M. ulcerans cells in positive samples from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.     

Human African Trypanosomiasis Diagnosis in First-Line Health Services of Endemic Countries,a Systematic Review

While the incidence of Human African Trypanosomiasis (HAT) is decreasing, the control approach is shifting from active population screening by mobile teams to passive case detection in primary care centers. We conducted a systematic review of the literature between 1970 and 2011 to assess which diagnostic tools are most suitable for use in first-line health facilities in endemic countries. Our search retrieved 16 different screening and confirmation tests for HAT. The thermostable format of the Card Agglutination Test for Trypanosomiasis (CATT test) was the most appropriate screening test. Lateral flow antibody detection tests could become alternative screening tests in the near future. Confirmation of HAT diagnosis still depends on visualizing the parasite in direct microscopy. All other currently available confirmation tests are either technically too demanding and/or lack sensitivity and thus rather inappropriate for use at health center level. Novel applications of molecular tests may have potential for use at district hospital level.     

Sero-epidemiology as a tool to screen populations for exposure to Mycobacterium ulcerans

Background

Previous analyses of sera from a limited number of Ghanaian Buruli ulcer (BU) patients, their household contacts, individuals living in BU non-endemic regions as well as European controls have indicated that antibody responses to the M. ulcerans 18 kDa small heat shock protein (shsp) reflect exposure to this pathogen. Here, we have investigated to what extent inhabitants of regions in Ghana regarded as non-endemic for BU develop anti-18 kDa shsp antibody titers.

Methodology/Principal Findings

For this purpose we determined anti-18 kDa shsp IgG titers in sera collected from healthy inhabitants of the BU endemic Densu River Valley and the Volta Region, which was so far regarded as BU non-endemic. Significantly more sera from the Densu River Valley contained anti-18 kDa shsp IgG (32% versus 12%, respectively). However, some sera from the Volta Region also showed high titers. When interviewing these sero-responders, it was revealed that the person with the highest titer had a chronic wound, which was clinically diagnosed and laboratory reconfirmed as active BU. After identification of this BU index case, further BU cases were clinically diagnosed by the Volta Region local health authorities and laboratory reconfirmed. Interestingly, there was neither a difference in sero-prevalence nor in IS2404 PCR positivity of environmental samples between BU endemic and non-endemic communities located in the Densu River Valley.

Conclusions

These data indicate that the intensity of exposure to M. ulcerans in endemic and non-endemic communities along the Densu River is comparable and that currently unknown host and/or pathogen factors may determine how frequently exposure is leading to clinical disease. While even high serum titers of anti-18 kDa shsp IgG do not indicate active disease, sero-epidemiological studies can be used to identify new BU endemic areas.     

A Sero-epidemiological Approach to Explore Transmission of Mycobacterium ulcerans

The debilitating skin disease Buruli ulcer (BU) is caused by infection with Mycobacterium ulcerans. While various hypotheses on potential reservoirs and vectors of M. ulcerans exist, the mode of transmission has remained unclear. Epidemiological studies have indicated that children below the age of four are less exposed to the pathogen and at lower risk of developing BU than older children. In the present study we compared the age at which children begin to develop antibody responses against M. ulcerans with the age pattern of responses to other pathogens transmitted by various mechanisms. A total of 1,352 sera from individuals living in the BU endemic Offin river valley of Ghana were included in the study. While first serological responses to the mosquito transmitted malaria parasite Plasmodium falciparum and to soil transmitted Strongyloides helminths emerged around the age of one and two years, sero-conversion for M. ulcerans and for the water transmitted trematode Schistosoma mansoni occurred at around four and five years, respectively. Our data suggest that exposure to M. ulcerans intensifies strongly at the age when children start to have more intense contact with the environment, outside the small movement range of young children. Further results from our serological investigations in the Offin river valley also indicate ongoing transmission of Treponema pallidum, the causative agent of yaws.     

Clinical,Microbiological and Pathological Findings of Mycobacterium ulcerans Infection in Three Australian Possum Species

Background

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors'' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia.

Methodology/Principal Findings

Twenty-seven clinical cases of M. ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998–2011. Common ringtail possums (Pseudocheirus peregrinus), a common brushtail possum (Trichosurus vulpecula) and a mountain brushtail possum (Trichosurus cunninghami) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both Trichosurus spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS2404, animals typically had significant levels of M. ulcerans DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums v others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums.

Conclusions/Significance

There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for M. ulcerans disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of M. ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas.     

Mycobacterium ulcerans Fails to Infect through Skin Abrasions in a Guinea Pig Infection Model: Implications for Transmission

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.     

Rapid,Serial, Non-invasive Assessment of Drug Efficacy in Mice with Autoluminescent Mycobacterium ulcerans Infection

Background

Buruli ulcer (BU) caused by Mycobacterium ulcerans is the world''s third most common mycobacterial infection. There is no vaccine against BU and surgery is needed for patients with large ulcers. Although recent experience indicates combination chemotherapy with streptomycin and rifampin improves cure rates, the utility of this regimen is limited by the 2-month duration of therapy, potential toxicity and required parenteral administration of streptomycin, and drug-drug interactions caused by rifampin. Discovery and development of drugs for BU is greatly hampered by the slow growth rate of M. ulcerans, requiring up to 3 months of incubation on solid media to produce colonies. Surrogate markers for evaluating antimicrobial activity in real-time which can be measured serially and non-invasively in infected footpads of live mice would accelerate pre-clinical evaluation of new drugs to treat BU. Previously, we developed bioluminescent M. ulcerans strains, demonstrating proof of concept for measuring luminescence as a surrogate marker for viable M. ulcerans in vitro and in vivo. However, the requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments.

Methodology/Principal Finding

For this study, we engineered M. ulcerans strains that express the entire luxCDABE operon and therefore are autoluminescent due to endogenous substrate production. The selected reporter strain displayed a growth rate and virulence similar to the wild-type parent strain and enabled rapid, real-time monitoring of in vitro and in vivo drug activity, including serial, non-invasive assessments in live mice, producing results which correlated closely with colony-forming unit (CFU) counts for a panel of drugs with various mechanisms of action.

Conclusions/Significance

Our results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity.     

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse

Background

Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain.

Methods

We evaluated BCG vaccine efficacy against challenge with ∼3×10⁵ M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly.

Principal Findings

BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice.

Conclusions

The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.     

Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.     

Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water

A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).Water exposure (15) is one source of human infection caused by nontuberculous mycobacteria (NTM). Nevertheless, the isolation and enumeration of NTM from water is difficult because other microorganisms overgrow NTM colonies (22). Consequently, the development of an alternative detection and enumeration method is essential for monitoring NTM sources in the environment.Two real-time quantitative PCR (qPCR) methods for NTM measurement have been described (7, 29). The primer pair used in the first real-time qPCR method (7) targets 16S rRNA and was previously used to track mycobacterial growth in industrial water samples by conventional PCR (31). It was presented as a sensitive test for members of the Mycobacterium genus because it detected 34 species of mycobacteria (19, 25). However, the primer specificity was only measured by conventional PCR against DNA of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus (31) or by in silico analysis (7). The second real-time qPCR method, targeting the hsp65 gene (29), was also sensitive (detection of 34 out of 37 Mycobacterium spp. tested). Although the primers showed high specificity (no detection of 16 different nonmycobacterial species) by conventional PCR (21), their specificity combined with the qPCR probe was only tested against Candida albicans DNA (29).We sought to develop a reliable real-time qPCR method to detect Mycobacterium spp. in water samples. The development involved in silico primer screening followed by a specificity study by conventional PCR. Furthermore, the efficiency (Ef), correlation coefficient (r²), limit of quantification (LOQ), specificity (Sp), and sensitivity (Ss) of this new method targeting 16S rRNA were compared with those of the two previously described methods (7, 29).