Loop-mediated isothermal amplification (LAMP) assay targeting RLEP for detection of Mycobacterium leprae in leprosy patients.

OBJECTIVE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and it remains a significant health problem in several parts of the world. Early and accurate diagnosis of this disease is therefore essential. Previously published loop-mediated isothermal amplification (LAMP) protocols for detecting mycobacterial species used conventional primers targeting the 16S rRNA, gyrB and insertion sequence genes. METHODS: In this study, we conducted a LAMP assay for leprosy and compared it with quantitative polymerase chain reaction (q-PCR) and conventional PCR assays to determine the efficiency, sensitivity and specificity of each technique. We chose conserved sequence RLEP as a suitable molecular target for assays. RESULTS: The LAMP assay provided rapid and accurate results, confirming leprosy in 91/110 clinical skin tissue samples from leprosy patients and amplifying the target pathogen in <60 min at 65 °C. The assay was more sensitive than conventional PCR and more straightforward and faster than the q-PCR assay. CONCLUSIONS: The LAMP assay has the potential for developing quicker, more accessible visual methods for the detection of M. leprae, which will enable early diagnosis and treatment and prevent further infection in endemic areas.

The high resolution melting analysis (HRM) as a molecular tool for monitoring parasites of the wildlife.

In an interconnected world, the international pet trade on wild animals is becoming increasingly important. As a consequence, non-native parasite species are introduced, which affect the health of wildlife and contribute to the loss of biodiversity. Because the investigation of parasite diversity within vulnerable host species implies the molecular identification of large samples of parasite eggs, the sequencing of DNA barcodes is time-consuming and costly. Thereby, the objectives of our study were to apply the high resolution melting (HRM) approach for species determination from pools of parasite eggs. Molecular assays were validated on flatworm parasites (polystomes) infecting the Mediterranean pond turtle Mauremys leprosa and the invasive red-eared slider Trachemys scripta elegans in French natural environments. HRM analysis results indicated that double or multiple parasitic infections could be detected from wild animal populations. They also showed that the cycle of parasite eggs production was not regular over time and may depend on several factors, among which the ecological niche and the target species. Thereby, monitoring parasites from wild endangered animals implies periodic parasitological surveys to avoid false negative diagnostics, based solely on eggs production.

Mycobacterium leprae specific genomic target in the promoter region of probable 4-alpha-glucanotransferase (ML1545) gene with potential sensitivity for polymerase chain reaction based diagnosis of leprosy.

With the absence of an effective diagnostic tool for leprosy, cases with negative bacteriological index and limited clinical manifestations often pose diagnostic challenges. In this study, we investigated the utility of a novel Mycobacterium leprae specific 112-bp DNA sequence in the promoter region of probable 4-alpha-glucanotransferase (pseudogene, ML1545) for polymerase chain reaction (PCR) based diagnosis of leprosy in comparison to that of the RLEP gene. DNA was extracted from slit skin scrapings of 180 newly diagnosed untreated leprosy cases that were classified as per Ridley Jopling classifications and bacteriological index (BI). Primers were designed using Primer Blast 3.0 and PCR was performed with annealing temperatures of 61°C for ML1545 and 58°C for the RLEP gene using conventional gradient PCR. The results indicated a significant increase in PCR positivity of ML1545 when compared to RLEP across the study groups (164/180 [91.11%] were positive for ML1545 whereas 114/180 (63.33%) were positive for RLEP [p<.0001, z=6.3]). Among 58 leprosy cases with negative BI, 28 (48.28%) were positive for RLEP and 48 (82.76%) were positive for ML1545 (p=.0001, z=3.8). Of the 42 borderline tuberculoid leprosy cases, 23 (54.76%) were positive for RLEP whereas 37 (88.09%) were positive for ML1545 (p<.0001, z=3.9). Increase in PCR positivity for ML1545 was also noted in lepromatous leprosy and BI-positive groups. ML1545 can be a potential gene target for PCR-based diagnosis of leprosy especially in cases where clinical manifestations were minimal.

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

Development of species-specific primers for rapid identification of Debaryomyces hansenii.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

[Rapid detection of mutations related to Mycobacterium leprae drug resistance by using Hp-rPCR (hairpin primer- real time PCR) method].

Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.

Application of ISSR-PCR for rapid strain typing of Debaryomyces hansenii isolated from dry-cured Iberian ham.

Yeast populations of dry-cured Iberian ham isolated from seven industries in the province of Badajoz were characterized by ISSR-PCR using the (CAG)4 primer and PCR-RFLP of the ITS1-5.8S rRNA-ITS2 fragment, and identified by DNA sequencing. A total of 242 isolates were analyzed, indicating the primary species present was Debaryomyces hansenii at 80.9% of the isolates followed by Candida zeylanoides at 10.3% of the isolates. The remainders of isolates were identified as Yamadazyma triangularis, Sporobolomyces roseus, Meyerozyma guilliermondii, Rhodotorula slooffiae, and Cryptococcus victoriae. The ISSR-PCR method was a fast and reliable method which was able to discriminate species at a level comparable to restriction analyses of the ITS1-5.8S rRNA-ITS2 region. This method allowed for strain typing of D. hansenii, yielding 29 different PCR patterns within 196 isolates. Moreover, ISSR-PCR using the (CAG)4 primer indicated that this technique could be a promising tool for rapid discrimination of yeast starter cultures and spoilage species in dry-cured Iberian ham.

Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes.

The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 106 cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

BACKGROUND: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. METHODOLOGY/PRINCIPAL FINDINGS: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L · h to 0.75 g/L · h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L · h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies.

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.

Characterization of the yeast ecosystem in grape must and wine using real-time PCR.

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.

Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears.

Abstract Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases.

Molecular determination of Mycobacterium leprae viability by use of real-time PCR.

Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (MPhi), or within immune-activated MPhi. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.

Are variable-number tandem repeats appropriate for genotyping Mycobacterium leprae?

Comparative genomics analysis of the Tamil Nadu strain of Mycobacterium leprae has uncovered several polymorphic sites with potential as epidemiological tools. In this study we compared the stability of two different markers of genomic biodiversity of M. leprae in several biopsy samples isolated from the same leprosy patient. The first type comprises five different variable-number tandem repeats (VNTR), while the second is composed of three single nucleotide polymorphisms (SNP). Contrasting results were obtained, since no variation was seen in the SNP profiles of M. leprae from 42 patients from 7 different locations in Mali whereas the VNTR profiles varied considerably. Furthermore, since variation in the VNTR pattern was seen not only between different isolates of M. leprae but also between biopsy samples from the same patient, these VNTR may be too dynamic for use as epidemiological markers for leprosy.

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR.

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.

Variation in MICA and MICB genes and enhanced susceptibility to paucibacillary leprosy in South India.

In a study of mainly paucibacillary leprosy-affected sib-pair families from South India, in addition to the expected associations with the HLA-DRB1 locus, we have identified significant association with a functional variant of the MICA gene as well as a microsatellite in the flanking region of the MICB gene. The associations with MICA and MICB cannot be accounted for by linkage disequilibrium with the HLA class II locus indicating a role in genetic susceptibility to leprosy that is independent of HLA-DRB1. Previous studies have shown that MICA and MICB are expressed on the surface of cells in response to infection, where they are recognized by the NKG2D receptor on gammadelta T cells, CD8+ alphabeta T cells and natural killer cells, all of which contribute to defense against mycobacteria. The MICA*5A5.1 allele, associated here with leprosy susceptibility, encodes a protein lacking a cytoplasmic tail providing a possible mechanism for defective immune surveillance against mycobacteria.

Protective responses against experimental Mycobacterium leprae infection in mice induced by recombinant Bacillus Calmette-Guérin over-producing three putative protective antigen candidates.

The components of Ag85 (Ag85A, Ag85B, and Ag85C) are putative protective antigen candidates against mycobacterial infection. A recombinant Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) over-producing Ag85A, Ag85B, and MPB51 (rBCG/BA51) was constructed. rBCG/BA51 could secrete these antigens at levels more than five times higher than parental BCG. Immunization of C57BL/6 and BALB/c mice with this rBCG reduced the multiplication of Mycobacterium leprae in the foot pads of both strains of mice. The inhibition by rBCG/BA51 was more evident than that by parental BCG.

DNA encoding a single mycobacterial antigen protects against leprosy infection.

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

Repetitive sequences in Mycobacterium leprae and their impact on genome plasticity.

About 2% of the genome of Mycobacterium leprae is composed of repetitive DNA. There are more than 26 extinct IS elements together with four families of dispersed repeats, present in five copies or more, RLEP (37 copies), REPLEP (15 copies), LEPREP (eight copies), and LEPRPT (five copies). Although there is no sequence similarity to known transposable elements, RLEP occurs predominantly at the 3'-end of genes and, in several cases, within pseudogenes, suggesting that it was capable of dissemination. Strikingly, on comparison of the genome sequences of M. leprae and the closely related tubercle bacillus, Mycobacterium tuberculosis H37Rv, many of these repetitive sequences were found at sites of discontinuity in gene order. Evidence is presented that loss of synteny, inversion and genome downsizing may have resulted from recombination between dispersed copies of these repetitive elements.