Comparison of target enrichment strategies for ancient pathogen DNA.

In ancient DNA research, the degraded nature of the samples generally results in poor yields of highly fragmented DNA; targeted DNA enrichment is thus required to maximize research outcomes. The three commonly used methods - array-based hybridization capture and in-solution capture using either RNA or DNA baits - have different characteristics that may influence the capture efficiency, specificity and reproducibility. Here we compare their performance in enriching pathogen DNA of Mycobacterium leprae and Treponema pallidum from 11 ancient and 19 modern samples. We find that in-solution approaches are the most effective method in ancient and modern samples of both pathogens and that RNA baits usually perform better than DNA baits.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Detection of antibiotic resistance in leprosy using GenoType LepraeDR, a novel ready-to-use molecular test.

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy. METHODOLOGY/PRINCIPAL FINDINGS: A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method. CONCLUSIONS/SIGNIFICANCE: The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.

Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species.

The aim of this study was to develop a PCR and reverse line blot hybridization (PCR-RLB) macroarray assay based on 16S-23S rRNA gene internal transcribed spacer sequences for the identification and differentiation of 34 mycobacterial species or subspecies. The performance of the PCR-RLB assay was assessed and validated by using 78 reference strains belonging to 55 Mycobacterium species, 219 clinical isolates which had been identified as mycobacteria by high-performance liquid chromatography or gas chromatography, three skin biopsy specimens from patients with suspected leprosy which had been shown to contain acid-fast bacilli, and isolates of 14 nonmycobacterial species. All mycobacteria were amplified in the PCR and hybridized with a genus-specific probe (probe MYC). The 34 species-specific probes designed in this study hybridized only with the corresponding Mycobacterium species. The mycobacterial PCR-RLB assay is an efficient tool for the identification of clinical isolates of mycobacteria; it can reliably identify mixed mycobacterial cultures and M. leprae in skin biopsy specimens.

Análisis de sondas y técnicas de amplificación de genes para el diagnóstico y control del tratamiento en la lepra infantil / No disponible

Se detectaron secuencias de ácidos nucleídos de Mycobacterium leprae utilizando sonda de genes que hibridan con secuencias diana RNA ribosómicas (16SrRNA), DNA ribosómico (16S rDNA) y técnicas de amplificación de genes (PCR) en lesiones cutáneas de pacientes de lepra infantil y el efecto del tratamiento farmacológico sobre estas técnicas. Se incluyeron en este trabajo 80 pacientes de lepra infantil. La mayoría de caso (79%) tenía entre 9-16 años. Se dividieron los caso en 3 grupos de acuerdo con el tratamiento, sin tratar (30) en tratamiento (39) y al finalizar el tratamiento (20). Se efectuaron exámenes clínicos y baciloscopia para la detección de bacilos ácido-alcohol resistente BAAR y de las biopsias se extrajeron y fraccionaron los ácidos nucleídos. Se detectó rRNA y rDNA 16S específico de M.leprae mediante hibridación con sondas, mientras que la secuencia del gen 36 kDa se detectó mediante técnicas de amplificación de genes (PCR). Los casos se clasificaron en lepra paucibacilar (PB) y multibacilar (MB) de acuerdo a los criterios de la OMS (1988). La positividad del rRNA 16S en los casos PB disminuyó desde 60% en los casos no tratados al 10,5% después de 4-8 meses de tratamietno, mientras el rDNA 16S disminuyó del 50% al 21% y con PCR desde 70% al 36.8% para la misma muestra y todos se negativizaron al año. La misma tendencia se observó en el grupo MB, donde la positividad en los casos baciloscopia positivos decreció desde el 100% al 56,2% con rRNA 16S y al 42,8% con rDNA 16S y PCR respectivamente, después de los 9-12 meses de tratamiento, siendo a los 2 años todos negativos, menos un caso que permaneció positivo con PCR. Los casos MB con baciloscopia negativa siguieron la misma tendencia, 100% de positividad detectado por rRNA 16S y PCR, 75% detectado por rDNA 16S y decreció hasta la negatividad a los 9-12 meses de tratamiento. Estos resultados apuntan hacia un posible potencial de estas técnicas como apoyo molecular para el diagnóstico de casos MB baciloscopia negativos y el control de la respuesta al tratamiento. Sin embargo, la prueba definitiva exige ser valorada mediante estudios prospectivos de seguimiento

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 to treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated form skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36kKa 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 rDNA, it decreased from 50% to 21% for PCR form 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 observation however, needs to be validated in prospective follow up studies

Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans.

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.

Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

Simplified PCR detection method for nasal Mycobacterium leprae.

We report here a simplified method for the detection of nasal carriage of Mycobacterium leprae. DNA extracted from nasal swabs was analyzed by PCR, and M. leprae specific amplicons detected by means of a novel peptide-nucleic-acid-ELISA (PNA-ELISA) method. Parameters for the method were established using swabs taken from untreated lepromatous leprosy patients. We have developed this method to study nasal carriage in endemic populations. However, due to the sensitivity of PCR based techniques, we wished to assess the possibility of false positive samples arising in our method. We therefore examined samples taken from individuals in Norway, a country non-endemic for leprosy, using our technique. A total of 219 nasal swabs were collected and tested in our laboratory in London. All of these were found to be negative by our criteria. In order to corroborate our results, and also to assess the specificity of the method, a small number of these samples were randomly selected, and a known amount of M. leprae DNA added to them. All 219 samples were then retested using the same techniques under "double blind" conditions in our laboratory in India. All of the samples to which M. leprae DNA had been added were successfully identified by this method whereas all other swabs were negative. Taken together, these results suggest that the technique described here is simple, sensitive, and specific for use in large-scale epidemiological studies. This study, part of the larger MILEP 2 study, represents the first use of a PNA-PCR method for an epidemiological study of infection. The method using PNA-ELISA is significantly simpler and more rapid than gel based detection methods. The supply of laboratory consumables and overall detection procedure were simplified and standardized by use of PCR Ready-to-Go beads.

Linfocitos Th1, Th2 y enfermedad / Lymphocytes Th1, Th2 and disease

Al activarse, los linfocitos T CD4+ muestran una producción de citocinas que usualmente sigue tres patrones distintos y estereotipados; estos patrones son producidos por subpoblaciones discretas que se denominan Th1, Th2 y Th0. Las células Th1 secretan principalmente interferón- (IFN-), interleucina-2 (IL-2) y factor de necrosis tumoral (FNT), citocinas que tienen un papel clave en la respuesta inmune mediada por células y el fenómeno de hipersensibilidad de tipo tardío. Las células Th2 producen IL-4, IL-5, IL-6, IL-9, IL-10 e IL-13, las cuales juegan un papel clave en la generación de la respuesta inmune humoral. Existe una regulación negativa recíproca entre las células Th1 y Th2 ya que ciertas citocinas de las primeras (principalmente IFN-) y de las segundas (IL-4, IL-10) tienen un efecto inhibitorio sobre células Th2 y Th1 respectivamente. Las células Th0 producen citocinas tipo Th1 y Th2. Es posible determinar el patrón de citocinas que están siendo producidas en un tejido, lo que permite inferir si ocurre una activación preferencial de células Th1, Th2 o Th0 en ese sitio. La activación preferencial de subpoblaciones de linfocitos CD4+ tiene un papel importante en la patogenia de enfermedades infecciosas y autoinmunes. Los pacientes con lepra lepromatosa tienen una activación preferencial de células Th2, lo cual conduce a una enfermedad diseminada con ausencia de respuesta inmune celular. La activación preferencial de células Th2 puede ser de importancia en la patogenia de enfermedades autoinmunes generalizadas, en tanto que las células Th1 tienen importancia en padecimientos autoinmunes órgano-específicos. Resulta factible el manipular in vivo la activación preferencial de linfocitos Th1 y Th2, lo cual puede tener implicaciones terapéuticas importantes

Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.