Listeria monocytogenes MerR-Like Regulator NmlRlm: Its Transcriptome and Role in Stress Response.

NmlR, a negative transcription regulator in the MerR family, is involved in oxidative and nitrosative stress response in Neisseria gonorrhoeae and Haemophilus influenzae. In this study, the objective was to characterize the role and the regulon of NmlR in the foodborne Listeria monocytogenes. An L. monocytogenes nmlR null mutant strain was constructed. Transcriptomes of strain 10403S wild type (WT) and ΔnmlRlm strains grown to the stationary phase were determined by mRNA sequencing. Differential expression analyses revealed 74 genes with altered expression levels (>9-fold difference), comprising 46 negatively and 28 positively regulated genes. Twenty-four NmlRlm-dependent genes overlap with the members of previously identified regulons of HrcA, a negative regulator of heat response in L. monocytogenes, and of alternative sigma factor σ(H). Phenotypic characterization revealed that the ΔnmlRlm strain survived significantly less than the WT under acid stress (pH 2.5 for 1 h) and oxidative stress (3% hydrogen peroxide for 1 h). In addition, nmlRlm deletion also resulted in a significant decrease (p < 0.0005) of cell length and enhanced intracellular growth in a differentiated macrophage-like U937 cell line during entry into stationary phase. These findings indicate that NmlRlm is not only involved in oxidative stress response but also contributes to other characteristics such as acid tolerance and intracellular growth, either through direct regulation or co-regulation with other regulators such as HrcA and σ(H).

Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-ß-glucanase.

Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-ß-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a real-time (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1 × 10(-4) ng, respectively. In conclusion, the RT-PCR assay retained 100% sensitivity and 100% specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.

Pharmacogenetic markers of CYP2B6 associated with efavirenz plasma concentrations in HIV-1 infected Thai adults.

AIMS: To investigate the frequency of CYP2B6 polymorphisms and the influence of haplotype structure on plasma efavirenz concentrations in Thai adults with HIV-1 infection. METHODS: Genotyping of nine single nucleotide polymorphisms (SNPs, c.64C>T, c.499C>G, c.516G>T, c.785A>G, c.1375A>G, c.1459C>T, g.3003T>C, g.18492C>T and g.21563C>T) of CYP2B6 were performed using real-time PCR-based allelic discrimination on blood samples from 52 HIV-infected adults who had received an efavirenz-based regimen. Plasma efavirenz concentrations were measured by high performance liquid chromatography. RESULTS: The minor allele frequencies for c.64C>T, c.516G>T, c.785A>G, g.3003C>T, g.18492T>C and g.21563C>T were 0.087, 0.365, 0.413, 0.308 and 0.356, respectively. However, no variant alleles were identified for three SNPs (c.499 C>G, c.1375 A>G and c.1459 C>T). Efavirenz plasma concentrations were significantly associated with c.516G>T (P= 0.0095), c.785A>G (P= 0.0017), g.21563C>T (P= 0.0036) and g.18492C>T (P= 0.0011). The composite CYP2B6 of three SNPs (c.516G ≥ T, c.785A ≥ G and g.21563C ≥ T) genotypes were significantly associated with higher efavirenz concentrations. CONCLUSIONS: Our data indicate that the GAC-CYP2B6 haplotype is associated with higher plasma efavirenz concentrations in HIV-infected Thai adults.

An integrated bioinformatics approach to the characterization of influenza A/H5N1 viral sequences by microarray data: Implication for monitoring H5N1 emerging strains and designing appropriate influenza vaccines.

In order to characterize A/H5N1 viral sequences, a bioinformatics approach accurately identified viral sequences from discovery of a sequence signature, which provided enough distinctive information for sequence identification. Eight highly pathogenic H5N1 viral isolations were collected from different areas of Thailand between 2003 and 2006, and were used for analysis of H5N1 genotypic testing with a semiconductor-based oligonucleotide microarray. All H5N1 samples and H1N1, H4N8 negative controls were correctly subtyped. Sensitivity of the eight oligonucleotide probes, with optimized cut-offs, ranged from 70% (95% CI 65-75) to 87% (95% CI 84-91), and the corresponding Kappa values ranged from 0.76 (95% CI 0.72-0.80) to 0.86 (95% CI 0.83-0.89). Semi-conductor-based oligonucleotide array and oligonucleotide probes corresponded well when detecting H5N1. After fully correcting the subtype from the result of microarray signal intensity, the microarray output method combined with bioinformatics tools, identified and monitored genetic variations of H5N1. Capability of distinguishing different strains of H5N1 from Thailand was the outstanding feature of this assay. Ninety percent of HA and NA (4/5) genes were sequenced correctly, in accordance with previous examinations performed by classical diagnostic methods. The low-medium-high bioinformatics resolutions were able to predict an epidemic strain of H5N1. This study also showed the advantage of using a large genotypic database to predict the epidemic strain of H5N1. However, the monitoring protocol of this new strain has been recommended for further study with a large-scale sample.

Qualitative detection of avian influenza A (H5N1) viruses: a comparative evaluation of four real-time nucleic acid amplification methods.

The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers.

HIV-1 viral load is a basic marker to evaluate the severity of HIV-1 related diseases and to monitor the effectiveness of treatment. A method based on real-time RT-PCR technology has been developed to quantify HIV-1 RNA using self-quenched fluorogenic primers known as LUX primers. They were used in this study to recognize a low variable gag region of subtype E and B consensus sequences. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with 10 fold serial dilutions of synthetic HIV-gag RNA. A broad range linear relationship (10 to 10(6) copies/ml) was observed between the number of PCR cycles needed to detect a fluorescent signal and the number of RNA copies. The intra- and inter-assay coefficients of variation were 0.72 to 2.54% and 3.14 to 8.83%, respectively, thus indicating good reproducibility. Thirty out of fifty HIV-infected individual plasma samples were quantified by this method and compared with the AMPLICOR HIV-1 Monitor assay, which is widely considered the reference technique for HIV-RNA viral load measurement. The results indicate that the AMPLICOR HIV-1 Monitor assay and real-time RT-PCR using LUX primers are in good agreement (mean difference in log10 copies/ml+/-2 standard deviations = 0.21+/-1.34).

Detection of JC virus infection in progressive multifocal leukoencephalopathy: the first documented case in Thailand.

Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating brain disease caused by human polyoma JC virus (JCV). This disease is an important cause of morbidity and mortality in AIDS patients. Definite diagnosis currently requires a brain biopsy. PCR for JCV of CSF, an emerging diagnostic tool, has a high specificity for the diagnosis of PML in patients with characteristics on clinical and neuroradiological findings. The authors report a 36-year-old woman who presented with prolonged fever, progressive weakness, and slow speech for 2 months. Clinical features and MRI findings were compatible with PML. Qualitative PCR for JCV of CSF showed a positive result. This report emphasizes the yield of PCR, the CSF for JCV in a diagnosis of PML, which may reduce the need for a brain biopsy in such cases.