High frequency of M. leprae DNA detection in asymptomatic household contacts.

BACKGROUND: Characterization of the Mycobacterium leprae genome has made possible the development of Polymerase Chain Reaction (PCR) systems that can amplify different genomic regions. Increased reliability and technical efficiency of quantitative PCR (qPCR) makes it a promising tool for early diagnosis of leprosy. Index cases that are multibacillary spread the bacillus silently, even before they are clinically diagnosed. Early detection and treatment could prevent transmission in endemic areas. METHODS: In this study, the qPCR technique is used to detect DNA of M. leprae in samples of slit skin smears (SSS) of the ear lobe and blood of leprosy patients and their asymptomatic household contacts residing in Governador Valadares, MG, Brazil, a hyperendemic area for leprosy. A total of 164 subjects participated in the study: 43 index cases, 113 household contacts, and, as negative controls, 8 individuals who reported no contact with patients nor history of leprosy in the family. The qPCR was performed to amplify 16S rRNA fragments and was specifically designed for M. leprae. RESULTS: Of asymptomatic household contacts, 23.89% showed bacillary DNA by qPCR in samples of SSS and blood. Also, 48.84% of patients diagnosed with leprosy were positive for qPCR while the bacillary load was positive in only 30.23% of patients. It is important to note that most patients were already receiving treatment when the collection of biological material for qPCR was performed. The level of bacillary DNA from household contacts was similar to the DNA levels detected in the group of paucibacillary patients. CONCLUSION: Considering that household contacts comprise a recognizable group of individuals with a high risk of disease, as they live in close proximity to a source of infection, qPCR can be used to estimate the risk of progress towards leprosy among household contacts and as a routine screening method for a chemoprophylactic protocol.

Relative Abundance in Bacterial and Fungal Gut Microbes in Obese Children: A Case Control Study.

BACKGROUND: Differences in relative proportions of gut microbial communities in adults have been correlated with intestinal diseases and obesity. In this study we evaluated the gut microbiota biodiversity, both bacterial and fungal, in obese and normal-weight school-aged children. METHODS: We studied 28 obese (mean age 10.03 ± 0.68) and 33 age- and sex-matched normal-weight children. BMI z-scores were calculated, and the obesity condition was defined according to the WHO criteria. Fecal samples were analyzed by 16S rRNA amplification followed by denaturing gradient gel electrophoresis (DGGE) analysis and sequencing. Real-time polymerase chain reaction (PCR) was performed to quantify the most representative microbial species and genera. RESULTS: DGGE profiles showed high bacterial biodiversity without significant correlations with BMI z-score groups. Compared to bacterial profiles, we observed lower richness in yeast species. Sequence of the most representative bands gave back Eubacterium rectale, Saccharomyces cerevisiae, Candida albicans, and C. glabrata as present in all samples. Debaryomyces hansenii was present only in two obese children. Obese children revealed a significantly lower abundance in Akkermansia muciniphyla, Faecalibacterium prausnitzii, Bacteroides/Prevotella group, Candida spp., and Saccharomyces spp. (P = 0.031, P = 0.044, P = 0.003, P = 0.047, and P = 0.034, respectively). CONCLUSION: Taking into account the complexity of obesity, our data suggest that differences in relative abundance of some core microbial species, preexisting or diet driven, could actively be part of its etiology. This study improved our knowledge about the fungal population in the pediatric school-age population and highlighted the need to consider the influence of cross-kingdom relationships.

Viability of Mycobacterium leprae in the environment and its role in leprosy dissemination.

BACKGROUND: Leprosy, a chronic disease caused by Mycobacterium leprae, is a public health concern in certain countries, including India. Although the prevalence of the disease has fallen drastically over time, new cases continue to occur at nearly the same rate in many regions. Several endemic pockets have been observed in India and elsewhere. The precise dynamics of leprosy transmission are still not clearly understood. Both live bacilli as well as M. leprae DNA have been detected in the soil and water of endemic areas; they possibly play an important role in disease transmission. AIMS: To study the occurrence of viable M. leprae in environmental samples collected from areas of residence of patients with active leprosy. METHODS: The study was conducted on 169 newly diagnosed leprosy patients in Ghatampur, Uttar Pradesh, India. Soil and water samples were collected from their areas of residence using a standardized protocol. An equal number of soil and water samples were also collected from non-patient areas of the same or adjoining villages. The environmental samples collected from the patients surroundings were subjected to 16S ribosomal RNA gene analysis after obtaining informed consent. RESULTS: About a quarter of the environmental samples collected from patient areas, (25.4% of soil samples and 24.2% of water samples) were found to be positive for specific 16S ribosomal RNA genes of M. leprae. Environmental samples collected from non-patient areas were all found negative for M. leprae 16S ribosomal RNA genes. LIMITATIONS: The major limitation of the study was that the sample size was small. CONCLUSION: The study demonstrated the presence of viable strains of M. leprae in skin smear samples of paucibacillary patients and multibacillary patients, as well as in the environmental samples obtained from around their houses. This could play an important role in the continued transmission of leprosy.

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

PURPOSE: PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. METHOD: Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RESULTS: RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. CONCLUSION: Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes.

What is the evidence that the putative Mycobacterium lepromatosis species causes diffuse lepromatous leprosy?

Han et al. have made a retrospective isolation of DNA from two patients with fatal Lucio's phenomenon. This DNA does have some molecular differences to M. leprae and may constitute a variant of M. leprae. However the experiments and data needed to confirm that this is a new leprosy-causing species have not yet been done. We have outlined the work that does need to be done. For the moment the assertion that 'M. lepromatosis' is a new leprosy-causing species is not proven.

A new Mycobacterium species causing diffuse lepromatous leprosy.

Mycobacterium leprae causes leprosy. M leprae strains collected worldwide have been genetically clonal, which poorly explains the varying severity and clinical features of the disease. We discovered a new Mycobacterium species from 2 patients who died of diffuse lepromatous leprosy (DLL). The Mycobacterium was purified from heavily infected, freshly frozen autopsy liver tissue followed by DNA extraction in 1 case. Paraffin-embedded skin tissue was used for DNA extraction in another case. Six genes of the organism were amplified by polymerase chain reaction, sequenced on cloning or from amplicons, and analyzed. Significant genetic differences with M leprae were found, including a 2.1% divergence of the 16S ribosomal RNA (rRNA) gene, a highly conserved marker of bacterial evolution, and 6% to 14% mismatches among 5 less conserved genes. Phylogenetic analyses of the genes of 16S rRNA, rpoB, and hsp65 indicated that the 2 most related organisms evolved from a common ancestor that had branched from other mycobacteria. These results and the unique clinicopathologic features of DLL led us to propose Mycobacterium lepromatosis sp nov. This species may account for some of the clinical and geographic variability of leprosy. This finding may have implications for the research and diagnosis of leprosy.

Reclassification of Gluconacetobacter hansenii strains and proposals of Gluconacetobacter saccharivorans sp. nov. and Gluconacetobacter nataicola sp. nov.

Ten strains previously assigned to Acetobacter hansenii (=Gluconacetobacter hansenii), Acetobacter pasteurianus LMG 1584 and eight reference strains of the genus Gluconacetobacter were reclassified by 16S rRNA gene sequencing, DNA-DNA similarity, DNA base composition and phenotypic characteristics. The A. hansenii strains and A. pasteurianus LMG 1584 were included in the cluster of acetic acid bacteria (family Acetobacteraceae) by 16S rRNA gene sequences. Further, they were separated into seven distinct groups by DNA-DNA similarity. DNA-DNA similarity group I was identified as G. hansenii. DNA-DNA similarity group II was retained as Gluconacetobacter sp., because DNA-DNA similarity between the strain and Gluconacetobacter entanii LTH 4560(T) could not be determined. This was due to a lack of availability of the type strain from any source. DNA-DNA similarity group III was regarded as a novel species, for which the name Gluconacetobacter saccharivorans sp. nov. (type strain, LMG 1582(T)=NRIC 0614(T)) is proposed. DNA-DNA similarity group IV included the type strains of Gluconacetobacter oboediens and Gluconacetobacter intermedius, and three A. hansenii strains. This group was identified as G. oboediens because high values of DNA-DNA similarity were obtained between the type strains and G. oboediens has priority over G. intermedius. DNA-DNA similarity group V was identified as Gluconacetobacter europaeus. DNA-DNA similarity group VI was regarded as a novel species, for which the name Gluconacetobacter nataicola sp. nov. (type strain, LMG 1536(T)=NRIC 0616(T)) is proposed. DNA-DNA similarity group VII was reclassified as Gluconacetobacter xylinus. The description of G. hansenii is emended.

Analysis of gene probes and gene amplification techniques for diagnosis and monitoring of treatment in childhood leprosy.

Nucleic acid sequences of Mycobacterium leprae were detected using gene probes hybridizing with targeting ribosomal RNA (16S rRNA), ribosomal DNA (16S rDNA) and gene amplification techniques (PCR) in skin lesion of paediatric leprosy patients and the effect of treatment on the by these methods. Eighty paediatric leprosy patients were included in the study. Most cases (79%) were between 9 and 16 years of age. Cases were divided into three groups according to treatment status, viz. untreated (30), undergoing treatment (30), and at the end of treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated from skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36 kDa gene sequence was detected by a gene amplification assay (PCR). The cases were classified as paucibacillary (PB) and multibacillary (MB) by the standard criteria of WHO (1988). Positivity of 16S rRNA in PB cases decreased from 60% in untreated to 10.5% after 4-8 months of treatment whereas for 16S r DNA, it decreased from 50% to 21%, for PCR from 70% to 36.8% for the same specimen, and all became negative at 1 year. Similar trends were seen in MB cases where positivity in smear positive untreated cases decreased from 100% to 56.2% with 16S rRNA and 42.8% with 16S rDNA and PCR, respectively, after 9-12 months of treatment and all became negative at 2 years, except one case which remained positive with PCR. Similar results were observed in smear negative MB cases, 100% positivity detected by 16S r RNA and PCR, 75% detected by 16S rDNA decreased to zero after 9-12 months of therapy. This study suggests the potential usefulness of gene probes targeting 16S rRNA and 16S rDNA and PCR as supportive molecular tools for diagnosis of smear negative evolving MB disease and also monitoring the response to treatment, these observations however, needs to be validated in prospective follow up studies.

Colorimetric microtitre plate hybridization assay for the detection of Mycobacterium leprae 16S rRNA in clinical specimens.

We have developed a colorimetric microtitre plate hybridization assay in order to simplify detection of Mycobacterium leprae in clinical specimens. This system detects the products amplified by a sensitive RT-PCR assay targeting a species-specific sequence of the bacterial 16S rRNA. The assay detected as few as 10 bacilli isolated from infected nude mouse lymph nodes or human skin biopsies. Sensitivity for diagnosis of clinical specimens was assessed for 58 tissue biopsies from untreated leprosy patients. The assay detected M. leprae RT-PCR products in 100% of biopsies from patients with multibacillary disease and 80% of biopsies from patients with paucibacillary disease, for an overall sensitivity of 91.3%. The test was highly specific as no RT-PCR products were amplified from skin biopsies of normal individuals or patients with skin diseases other than leprosy. The colorimetric assay is faster, more sensitive, and simplifies detection of RT-PCR products compared to Southern blot analysis. It may be useful for diagnosis of difficult cases of leprosy, and, since RNA is rapidly degraded after cell death, it may be appropriate for assessing response to therapy and for distinguishing relapse from reaction.

Staphylococcus equorum subsp. linens, subsp. nov., a starter culture component for surface ripened semi-hard cheeses.

Two staphylococcal strains, RP29T and RP33, were isolated from the main microflora of a surface ripened Swiss mountain cheese made from raw milk. These two strains were differentiated from the most closely related species Staphylococcus equorum on the basis of DNA-DNA hybridisation and phenotypic characteristics and are proposed as Staphylococcus equorum subsp. linens subsp. nov. They could be distinguished phenotypically from S. equorum by their sensitivity to all 14 tested antibiotics, especially to novobiocin, their incapability to ferment alpha-D-lactose, maltose, sucrose, D-trehalose, D-xylose, L-arabinose, salicin, D-ribose, D-raffinose, D-mannitol, and D-alanine. The GenBank accession numbers for the reference sequences of the 16S rDNA and the hsp60 gene used in this study are AF527483 and AF527484, respectively. 30 tons of a semi-hard Swiss cheese were produced with Staphylococcus equorum subsp. linens DSM 15097T as starter culture component in addition to Debaryomyces hansenii, Geotrichum candidum, Brevibacterium linens, Corynebacterium casei for surface ripened cheeses. The products were sensorically and hygienically perfect. Therefore, Staphylococcus equorum subsp. linens DSM 15097T can be proposed as starter culture component for surface ripened cheeses without any detected antibiotic resistances. The type strain of Staphylococcus equorum subsp. linens is DSM 15097T (CIP 107656T).

A genomic study on the cultivable and nerve invading Mycobacterium HI-75 after the recovery 3 months of the inoculation to nude mice.

The sequence of the polymerase chain reaction (PCR) product of 16S ribosomal RNA (16S rRNA) of the leproma-derived and cultivable Mycobacterium HI-75 (M. HI-75) which was obtained from the infected regions of inoculated mice, was examined and compared with that of the cultured bacteria by the direct sequencing techniques. The sequence was completely consistent with the cultured bacilli in the comparable 837 bases of 16S rRNA. The mycobacterium examined in this study was originally isolated as M. leprae (ML) by Skinsnes, et al. in 1975 from leproma of a lepromatous type Hansen's disease patient and therefore named as Mycobacterium leprae HI-75 by them, and was maintained from 1984 using either Ogawa's or Sauton's media in the beginning and Ogawa's medium enriched with glucronic acid and N-acetyl-D-glucosamine recently. Sasaki and Hamit reported the nerve invasion and the growth of the inoculated bacilli either to the nude mice or the I131 treated immunocompromised Swiss mice. We previously reported that cultured HI-75 was most similar to M. scrofulaceum by the direct sequencing of the gene of 16S rRNA. The 16S rRNA obtained from the mouse tissue in the present study indicated that M. HI-75 would be a variant of M. scrofulaceum possessing an ability to invade into peripheral nerve. The results suggest that the HI-75 strain claims a nature as a pathogen to develop a leproma-like lesion.

Use of NASBA RNA amplification for detection of Mycobacterium leprae in skin biopsies from untreated and treated leprosy patients.

This study was performed to assess the value of NASBA RNA amplification of a 16S rRNA target for the detection of presumably viable Mycobacterium leprae in sections of skin biopsies from leprosy patients. The NASBA positivity rate was 90.4% (84/93) for untreated multibacillary (MB) patients [bacterial index (BI) > or = 2] and 16.7% (8/48) for the untreated paucibacillary (PB) patients (BI < 2). NASBA positivity showed a good concordance with the presence of solidly stained M. leprae [morphological index (MI)] in skin biopsies from leprosy patients, but no relationship could be demonstrated between the strength of the NASBA signals and the BI. Furthermore, the usefulness of the detection of 16S rRNA by NASBA to monitor the efficacy of leprosy treatment was investigated using an additional 154 biopsy specimens analyzed from 80 MB patients during the course of treatment. The NASBA positivity rate declined during treatment. A significant decrease was observed after only 1-3 months. These results favor the view that detection of RNA by NASBA may reflect the viability of M. leprae.