Shoulder position influences the location of the musculocutaneous nerve in the axillary fossa.

In the axillary fossa, the musculocutaneous nerve (MC) is generally distant from the axillary artery and from the other brachial plexus nerves. In that way, MC requires a specific block. We observed that the location of MC is influenced by the position of the patient's arm and shoulder. Abduction of the shoulder significantly reduced the distance between the MC and the axillary artery. This change in the location of the MC is probably due to the moving of the nerve because of muscle rearrangements and the ability to achieve better proximity of the probe in the axillary fossae.

Evidence for population-specific positive selection on immune genes of Anopheles gambiae.

Host-pathogen interactions can be powerful drivers of adaptive evolution, shaping the patterns of molecular variation at the genes involved. In this study, we sequenced alleles from 28 immune-related loci in wild samples of multiple genetic subpopulations of the African malaria mosquito Anopheles gambiae, obtaining unprecedented sample sizes and providing the first opportunity to contrast patterns of molecular evolution at immune-related loci in the recently discovered GOUNDRY population to those of the indoor-resting M and S molecular forms. In contrast to previous studies that focused on immune genes identified in laboratory studies, we centered our analysis on genes that fall within a quantitative trait locus associated with resistance to Plasmodium falciparum in natural populations of A. gambiae. Analyses of haplotypic and genetic diversity at these 28 loci revealed striking differences among populations in levels of genetic diversity and allele frequencies in coding sequence. Putative signals of positive selection were identified at 11 loci, but only one was shared by two subgroups of A. gambiae. Striking patterns of linkage disequilibrium were observed at several loci. We discuss these results with respect to ecological differences among these strata as well as potential implications for disease transmission.

Diverged alleles of the Anopheles gambiae leucine-rich repeat gene APL1A display distinct protective profiles against Plasmodium falciparum.

Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A(2)) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A(1)) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum.

Evaluation of a new disinfection method for ultrasound probes used for regional anesthesia: ultraviolet C light.

OBJECTIVES: Ultrasound-guided regional anesthesia is commonly used for block placement. At present, the risk of cross contamination from probes is not well documented. To avoid transmission of infectious agents, several methods have been used for probe disinfection and protection. The aim of this study was to evaluate the antibacterial efficacy of a new high-level disinfection method based on ultraviolet C (UV-C) light under routine conditions after block placement with an unprotected probe. METHODS: The study was after approval by the local Ethics Committee. In the first part of the study, 15 ultrasound probes were exposed to a large inoculum of 3 bacteria. Ultraviolet C disinfection consisted of cleaning the probe with dry and disinfectant-impregnated paper followed by a 90-second UV-C disinfection cycle in a decontamination chamber. A protocol was established to retrieve the probe with sterile gloves after opening the door of the chamber. In the second part, 50 blocks were placed with ultrasound-guided regional anesthesia. The skin was first prepared with an antiseptic solution, and sterile gel was applied; no covers were used to protect the probes. The blocks were then disinfected with UV-C light. Bacteriologic samples were collected before and after the UV-C method and inoculated on chocolate agar plates. RESULTS: During the first part of the study, all probes were infected after inoculation (>150 colony-forming units) but were considered sterile (<10 colony-forming units) after disinfection. During the second part of the study, all probes were considered sterile before and after disinfection. CONCLUSIONS: Ultraviolet C disinfection seems relevant for ultrasound-guided regional anesthesia just before block placement. It offers simple, fast, and effective high-level disinfection. Moreover, this method should obviate the use of sterile probe covers, which can improve echogenicity.

A cryptic subgroup of Anopheles gambiae is highly susceptible to human malaria parasites.

Population subgroups of the African malaria vector Anopheles gambiae have not been comprehensively characterized owing to the lack of unbiased sampling methods. In the arid savanna zone of West Africa, where potential oviposition sites are scarce, widespread collection from larval pools in the peridomestic human habitat yielded a comprehensive genetic survey of local A. gambiae population subgroups, independent of adult resting behavior and ecological preference. A previously unknown subgroup of exophilic A. gambiae is sympatric with the known endophilic A. gambiae in this region. The exophilic subgroup is abundant, lacks differentiation into M and S molecular forms, and is highly susceptible to infection with wild Plasmodium falciparum. These findings might have implications for the epidemiology of malaria transmission and control.

Comfort of the patient during axillary blocks placement: a randomized comparison of the neurostimulation and the ultrasound guidance techniques.

BACKGROUND: Axillary brachial plexus block under neurostimulation is commonly used for upper limb surgery, but it is sometimes recognized as an uncomfortable technique, with most patients identifying electrical stimulation as an unpleasant moment. Ultrasound-guided regional anaesthesia, which becomes an increasingly popular technique, does not require electrical stimulation and then should theoretically improve axillary block placement comfort. The aim of this study was to compare the comfort of the patients during axillary block placement with neurostimulation and ultrasound guidance using either the out-of-plane or the in-plane approach. METHODS: Consecutive patients were prospectively enrolled in three equal groups: neurostimulation, ultrasound out-of-plane and ultrasound in-plane approaches. A score was used to measure the comfort of the patients during axillary blocks placement. This score included three criteria: maximum pain intensity perceived during block placement measured using a visual analogue scale (0, no pain and 100, maximal or worse imaginable pain), the number of unpleasant events declared by the patients and the satisfaction of the patient (unsatisfied, acceptable, satisfied, very satisfied). The comfort score was calculated as the sum of each criterion, which was attributed a value of 0 or 1: visual analogue scale (30/100, 0), number of unpleasant events (0, 1; >or=1, 0) and satisfaction (satisfied or very satisfied, 1; acceptable or unsatisfied, 0). Procedures of axillary blocks placement resulting in a comfort score of 3 and 2 were arbitrary considered as very comfortable and comfortable, respectively. Success rate of axillary blocks, time to perform block and complications related to procedures were noted. RESULTS: One hundred and twenty patients were included. In the ultrasound out-of-plane group, 55% (22/40) and 25% (10/40) of the procedures were very comfortable and comfortable as compared with 32% (13/40, P < 0.05) and 20% (8/40, P < 0.01) in the ultrasound in-plane group and 25% (10/40, P < 0.01) and 8% (3/40, P < 0.01) in the neurostimulation group, respectively. Duration of axillary placement was significantly smaller in the ultrasound out-of-plane group as compared with that of in-plane approaches (P < 0.05) and neurostimulation (P < 0.01). CONCLUSION: The present study showed that the ultrasound approaches were less painful and more comfortable than neurostimulation to place axillary blocks. We also showed that, although pain intensity resulting from blocks placement was similar with the ultrasound approaches, very comfortable procedures were more frequent with the out-of-plane than with the in-plane approach.

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

A comprehensive survey of single nucleotide polymorphisms (SNPs) across Mycobacterium bovis strains and M. bovis BCG vaccine strains refines the genealogy and defines a minimal set of SNPs that separate virulent M. bovis strains and M. bovis BCG strains.

To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovis BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovis and M. bovis BCG. SNPs were assayed in M. bovis isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovis strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovis strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovis strains and the attenuated BCG strain that will underpin future functional analyses.

Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP.

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.

Spread of injectate associated with radial or median nerve-type motor response during infraclavicular brachial-plexus block: an ultrasound evaluation.

BACKGROUND AND OBJECTIVES: We have compared ultrasound characteristics of spread during infraclavicular brachial-plexus blocks by use of electrically evoked radial-nerve- or median-nerve-type distal motor responses to guide the injection of 30 mL of 1.5% mepivacaine. METHODS: Consecutive patients who required surgery distal to the upper arm were prospectively included in this study. With radial- or median-evoked distal motor response at a stimulating current intensity of less than 0.5 mA, patients were distributed into 2 equal groups. An independent investigator blinded to the evoked response described ultrasound characteristics of the spread of local anesthetic and assessed block quality 30 minutes after placement. A quality diffusion score proportional to the extent and intensity of spread around the axillary artery was used, and dynamic movements during injection were noted. RESULTS: Thirty-two patients were included. With radial-nerve-type motor response, the success rate of infraclavicular plexus block was 100%, but 3 supplemental axillary blocks were requested with median-nerve-type motor response. Quality diffusion scores were significantly higher with radial-nerve-type as compared with median-nerve-type motor response (P = .03). Injection after radial-nerve-type motor response resulted in a typical and reproducible ultrasound feature of posterior local-anesthetic spread associated with medial and upper movement of the axillary artery. With median-nerve-type motor response, failed blocks were associated with a specific posterior displacement of the axillary artery that resulted from superficial spread. CONCLUSION: We have demonstrated that as compared with median-nerve-type motor response, injection performed after a radial-nerve-type motor response promoted reproducible and remarkable ultrasound spread characteristics associated with complete sensory block of the 3 cords at 30 minutes.

Genome plasticity of BCG and impact on vaccine efficacy.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

Single-stimulation, low-volume infraclavicular plexus block: influence of the evoked distal motor response on success rate.

BACKGROUND AND OBJECTIVES: We compared the success rate of single-injection infraclavicular plexus block by using electrically evoked radial, ulnar, or median nerve-type distal motor response to guide the injection of local anesthetic. METHODS: Consecutive patients requiring surgery distal to the upper arm were prospectively included in this study over a 6-month period. No search for predetermined distal motor responses was performed. The first qualifying distal motor response evoked for a stimulating current intensity of <0.5 mA distributed patients into 3 groups of patients. The study was continued until 3 groups of 60 patients were fulfilled. Twenty to 25 minutes after the injection of 30 mL of 1.5% mepivacaine, blinded evaluation of block quality was performed. A successful block was defined by the presence of a complete sensory block of the 5 major nerve distal distributions of the arm. RESULTS: Five hundred patients were included. The first evoked distal motor response was of radial, median, and ulnar nerve type in 46% (n = 230), 42% (n = 210), and 12% (n = 60) cases, respectively. The success rate of the infraclavicular plexus block was significantly higher when the injection was performed on a radial nerve-type response (90%) as compared with the median (74%) or ulnar (68%) nerve distal motor response. Intraoperative sedation and general anesthesia were not needed. None of the patients experienced specific complications. CONCLUSION: We showed that evoked distal motor response influenced the success rate of single-injection infraclavicular plexus block. The highest success rate was obtained when injection was performed after radial nerve-type motor response.

On the origin of leprosy.

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae. This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

Differential effects of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG strain expressing RD1 antigens.

In silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovis BCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccae on the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized with M. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection with M. tuberculosis, compared to BCG. In contrast, animals exposed to M. scrofulaceum or M. vaccae prior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized with M. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.

Common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans.

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

On the origin of leprosy

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae. This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.

The complete genome sequence of Mycobacterium bovis.

Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host-bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis.

Bacterial artificial chromosome-based comparative genomic analysis identifies Mycobacterium microti as a natural ESAT-6 deletion mutant.

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that causes tuberculosis in voles. Most strains of M. microti are harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent M. microti but present in human pathogen M. tuberculosis or Mycobacterium bovis were searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microti OV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovis AF2122/97 and M. tuberculosis H37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microti OV254 and M. tuberculosis. A 14-kb chromosomal region (RD1(mic)) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested M. microti strains. This region covers 13 genes, Rv3864 to Rv3876, in M. tuberculosis, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5(mic), a region that contains three phospholipase C genes (plcA to -C), was missing from only the vole isolates and was present in M. microti strains isolated from humans. Apart from RD1(mic) and RD5(mic) other M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microti and thus contributes to the characteristic spoligotype of M. microti strains.