The functional polymorphism Ala258Ser in the innate receptor gene ficolin-2 in the donor predicts improved renal transplant outcome.

BACKGROUND: Innate immunity plays a role in controlling adaptive immune responses. METHODS: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set. RESULTS: Polymorphisms in TLR-3 (rs3775296) in the recipients and in ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed ficolin-2. Donor grafts with the ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of ficolin-2 to N-acetylglucosamine. CONCLUSIONS: Presence of the ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses.

Array-based MLPA to detect recurrent copy number variations in patients with idiopathic mental retardation.

Microdeletions, either subtelomeric or interstitial, are responsible for the mental handicap in approximately 10-20% of all patients. Currently, Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used to detect these small aberrations in a routine fashion. Although cost-effective, the throughput is low and the degree of multiplexing is limited to maximally 40-50 probes. Therefore, we developed an array-based MLPA method, with probes identified by unique tag sequences, allowing the simultaneous analysis of 180 probes in a single experiment thereby covering all known mental retardation loci with at least two probes. We screened 120 patients with idiopathic mental retardation. In this group we detected 6 aberrations giving a detection rate of 5%, consistent with similar studies. In addition we tested 293 patients with mental retardation who were negative for fragile X syndrome and commercially available subtelomeric MLPA. We found seven causative rearrangements in this group (detection rate of 2.4%) thereby illustrating the value of including probes for interstitial microdeletion syndromes and additional probes in the telomeric regions in targeted screening sets for mental retardation. Array-based MLPA may thus be a good candidate to develop probe sets that rapidly detect copy number changes of disease associated loci in the human genome. This method may become a valuable tool in a routine diagnostic setting as it is a fast, user-friendly and relatively low-cost technique providing straightforward results requiring only 125 ng of genomic DNA.

Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico.

BACKGROUND: Little information is available on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites in humans. This study used molecular methods to determine the Mycobacterium species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease. METHODS: Bacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the Mycobacterium species present. DNA samples positive for M. tuberculosis complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the rpoB gene associated with rifampicin resistance. RESULTS: Of the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for Mycobacterium species by PCR or line probe assay, respectively. In tissues, M. tuberculosis complex and M. genus were found in lymph nodes, and M. genus was found in brain and vagina specimens. In body fluids, M. tuberculosis complex was found in synovial fluid. M. gordonae, M. smegmatis, M. kansasii, M. genus, M. fortuitum/M. peregrinum complex and M. tuberculosis complex were found in urine. M. chelonae/M. abscessus was found in pericardial fluid and M. kansasii was found in gastric aspirate. Two of M. tuberculosis complex isolates were also PCR and LiPA positive for the rpoB gene. These two isolates were from lymph nodes and were sensitive to rifampicin. CONCLUSION: 1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found.

Gene polymorphisms of Toll-like and related recognition receptors in relation to the vaginal carriage of Gardnerella vaginalis and Atopobium vaginae.

Host genetic factors have previously been found to act as determinants of differential susceptibility to major infectious diseases. It is less clear whether such polymorphisms may also impose on pathogen recognition in mucosal overgrowth conditions such as bacterial vaginosis, an anaerobic overgrowth condition characterised by the presence of a vaginal biofilm consisting of the Gram-positive anaerobes Gardnerella vaginalis and Atopobium vaginae. We selected 34 single nucleotide polymorphisms pertaining to 9 genes involved with Toll-like receptor-mediated pathogen recognition and/or regulation (LBP, CD14, TLR1, TLR2, TLR4, TLR6, MD2, CARD15 and SIGIRR) and assessed in a nested case-control study their putative association with bacterial vaginosis, as diagnosed by Gram staining, and with the vaginal carriage of A. vaginae and G. vaginalis, as determined by species-specific PCR, among 144 pregnant women. Carriage of G. vaginalis during early pregnancy was associated with the -1155A>G substitution in the promoter region of the MD2 gene (p=0.041). The presence of A. vaginae during the first half of the pregnancy was significantly associated with the CD14 intron 2 1342G>T (p=0.039), the TLR1 exon 4 743A>G (p=0.038), and the CARD15 exon 4 14772A>T (p=0.012) polymorphisms, and marginally significantly associated with the LBP exon13 26842C>T (p=0.056), the CD14 promoter -260C>T (p=0.052), and the TLR1 promoter -7202A>G (p=0.062) polymorphisms. However, no association between gene polymorphisms and bacterial vaginosis as such could be documented. Our data suggest that some degree of genetic susceptibility involving pathogen recognition may occur with the key bacterial vaginosis organism, A. vaginae.

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay.

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.

Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and 'M. avium subsp. hominissuis' for the human/porcine type of M. avium.

In an attempt to clarify the taxonomy of the Mycobacterium avium complex, the relationship between IS1245 RFLP, growth temperature, 16S rDNA signature sequences and the 16S-23S rDNA internally transcribed spacer (ITS) of 160 M. avium-complex isolates from different sources was investigated. All 70 isolates identified as M. avium by INNO-LiPA MYCOBACTERIA (Innogenetics, Belgium), a DNA probe test that targets the ITS, and by 16S rDNA analysis carried multiple copies of IS1245. Three isolates with multiple copies of IS1245 were identified by 16S rDNA analysis as Mycobacterium intracellulare and by LiPA as M. intracellulare (n = 1) and M. avium-intracellulare complex (n = 2). A dichotomy among the M. avium isolates was found on the basis of a C and a G signature nucleotide at position 228 of the 16S-23S rDNA spacer sequence, and this grouping was largely confirmed on the basis of similarities in IS1245 RFLPs. Strains with the characteristic three-band IS1245 'bird-type', as well as M. avium subsp. silvaticum or 'wood-pigeon' strains, invariably contained the C signature. A third characteristic that separated the M. avium bird-type isolates from M. avium isolates from humans and other mammals was growth-temperature tolerance: in contrast to bird isolates, human/porcine isolates grew at 24 and 45 degrees C. Based on differences in IS1245 RFLP, 16S-23S rDNA ITS and growth temperature, M. avium isolates originating from birds should be considered as a separate, evolutionarily conserved taxon. Because all M. avium isolates from birds are invariably of this type, the designation M. avium subsp. avium should be reserved for these bird-type strains. For clarity in the epidemiology of M. avium-related disease, isolates from humans and pigs with multibanded IS1245 RFLPs merit a separate designation. The designation 'M. avium subsp. hominissuis' is suggested for this group of bacteria.

Characterization of rpoB gene mutations in rifampicin

Objetivo. Caracterizar las mutaciones del gen rpoB de las cepas de M. tuberculosis resistentes a rifampicina aisladas en pacientes con tuberculosis pulmonar de México. Material y métodos. Se analizaron 37 cepas de M. tuberculosis cultivadas en el medio de Löwenstein-Jensen y aisladas de pacientes tuberculosos consecutivos de 5 hospitales públicos. Las cepas fueron analizadas mediante PCR e INNO LiPA Rif TB para amplificación y detección de mutantes asociadas con resistencia a rifampicina, respectivamente. Resultados. Veintitrés de las 37 cepas (62.2 por ciento) resultaron ser de tipo salvaje (sensibles a rifampicina), y 14 cepas (37.8 por ciento) contenían mutaciones asociadas con resistencia a rifampicina. Siete de las 37 cepas (18.9 por ciento) tenían una mutación ?S1, en la posición del nucleótido número 511; una (2.7 por ciento) tenía una mutación R4b, en el nucleótido H526D; cinco contenían una mutación R5, en el nucleótido S531L; y una (2.7 por ciento) mostró una doble mutación ?S1/R4b. Conclusión. De acuerdo al marcador usado (resistencia a rifampicina), por lo menos cinco cepas diferentes de M. tuberculosis circulan entre los pacientes con tuberculosis pulmonar en México. Las mutaciones del gen rpoB asociadas con resistencia a rifampicina son comunes en México. Una mutación única en el nucleótido 511 fue la más frecuentemente observada, seguida por mutaciones únicas en los nucleótidos S531L y H526D.