Digital PCR cluster predictor: a universal R-package and shiny app for the automated analysis of multiplex digital PCR data.

Digital polymerase chain reaction (dPCR) is an emerging technology that enables accurate and sensitive quantification of nucleic acids. Most available dPCR systems have two channel optics, with ad hoc software limited to the analysis of single and duplex assays. Although multiplexing strategies were developed, variable assay designs, dPCR systems, and the analysis of low DNA input data restricted the ability for a universal automated clustering approach. To overcome these issues, we developed dPCR Cluster Predictor (dPCP), an R package and a Shiny app for automated analysis of up to 4-plex dPCR data. dPCP can analyse and visualize data generated by multiple dPCR systems carrying out accurate and fast clustering not influenced by the amount and integrity of input of nucleic acids. With the companion Shiny app, the functionalities of dPCP can be accessed through a web browser.

Investigation of a staphylococcal food poisoning outbreak combining case-control, traditional typing and whole genome sequencing methods, Luxembourg, June 2014.

In June 2014, a staphylococcal food poisoning outbreak occurred at an international equine sports event in Luxembourg requiring the hospitalisation of 31 persons. We conducted a microbiological investigation of patients and buffet items, a case-control study and a carriage study of catering staff. Isolates of Staphylococcus aureus from patients, food and catering staff were characterised and compared using traditional typing methods and whole genome sequencing. Genotypically identical strains (sequence type ST8, spa-type t024, MLVA-type 4698, enterotoxin A FRI100) were isolated in 10 patients, shiitake mushrooms, cured ham, and in three members of staff. The case-control study strongly suggested pasta salad with pesto as the vehicle of infection (p<0.001), but this food item could not be tested, because there were no leftovers. Additional enterotoxigenic strains genetically unrelated to the outbreak strain were found in four members of staff. Non-enterotoxigenic strains with livestock-associated sequence type ST398 were isolated from three food items and two members of staff. The main cause of the outbreak is likely to have been not maintaining the cold chain after food preparation. Whole genome sequencing resulted in phylogenetic clustering which concurred with traditional typing while simultaneously characterising virulence and resistance traits.

The distribution of hepatitis B virus genotypes in Thailand.

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Swine influenza virus antibodies in humans, western Europe, 2009.

Serologic studies for swine influenza viruses (SIVs) in humans with occupational exposure to swine have been reported from the Americas but not from Europe. We compared levels of neutralizing antibodies against 3 influenza viruses--pandemic (H1N1) 2009, an avian-like enzootic subtype H1N1 SIV, and a 2007-08 seasonal subtype H1N1--in 211 persons with swine contact and 224 matched controls in Luxembourg. Persons whose profession involved contact with swine had more neutralizing antibodies against SIV and pandemic (H1N1) 2009 virus than did the controls. Controls also had antibodies against these viruses although exposure to them was unlikely. Antibodies against SIV and pandemic (H1N1) 2009 virus correlated with each other but not with seasonal subtype H1N1 virus. Sequential exposure to variants of seasonal influenza (H1N1) viruses may have increased chances for serologic cross-reactivity with antigenically distinct viruses. Further studies are needed to determine the extent to which serologic responses correlate with infection.

Possible new hepatitis B virus genotype, southeast Asia.

We conducted a phylogenetic analysis of 19 hepatitis B virus strains from Laos that belonged to 2 subgenotypes of a new genotype I. This emerging new genotype likely developed outside Southeast Asia and is now found in mixed infections and in recombinations with local strains in a geographically confined region.

Proteomic analysis of the cortisol-mediated stress response in THP-1 monocytes using DIGE technology.

The glucocorticoid (GC) cortisol, the main mediator of the hypothalamic-pituitary-adrenal axis has many implications in metabolism, stress response and the immune system. Its function is mediated via binding to the glucocorticoid receptor (GR), a member of the superfamily of ligand-activated nuclear hormone receptors. The activity of the ligated GR results from its binding as a transcription factor to glucocorticoid response elements (GREs). Two-dimensional gel electrophoresis with DIGE (fluorescence difference gel electrophoresis) technology was applied to study the effects of cortisol on the human THP-1 monocytic cell line. A total of 28 cortisol-modulated proteins were identified belonging to five functional groups: cytoskeleton (8), chaperones (9), immune response (4), metabolism (3) and transcription/translation (4). Their corresponding genes were screened for putative GREs in their + 10 kb/- 0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS). FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein, and contains the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggests GC inducibility of all transcripts. Additionally, proteomics (2D DIGE and 2D immunoblotting) revealed the existence of several FKBP51 isoforms, which were not previously described. To our knowledge this is the first proteomic study that addresses the effects of cortisol on immune cells. FKBP51 isoforms found on the gel map were linked to alternative promoter usage on the genetic level, successfully correlating both the specific proteomic and genomic findings.

Central African Republic is part of the West-African hepatitis B virus genotype E crescent.

BACKGROUND: Recent studies have shown that Hepatitis B virus (HBV) genotype E predominates in a vast crescent in West-Africa spanning from Senegal to Angola. OBJECTIVES: To determine whether HBV strains in the Central African Republic (CAR) belong predominately to the homogeneous West-African genotype E or whether they are more closely related to genotypes found in East Africa. STUDY DESIGN: Serum samples were randomly collected from 196 patients admitted with symptoms of acute or chronic hepatitis to the Central Hospital in Bangui. Thirty complete and 36 partial sequences of HBV strains were obtained. RESULTS: Ninety-four percent (62/66) of the strains belonged to genotype E, while genotype A1, most closely related to a strain from Tanzania and genotype D were detected in only one and three samples, respectively. One strain presented a recombination between the S and X gene of a genotype E precursor and a partial PreC/C gene of a genotype D precursor. CONCLUSIONS: Genotype E is predominant in CAR with little overlap with genotypes from Eastern Africa, extending the West-African HBV genotype E crescent further to the East.

Hepatitis B virus genotype E surface antigen detection with different immunoassays and diagnostic impact of mutations in the preS/S gene.

The major neutralizing epitope, the "a" determinant of the hepatitis B virus (HBV) genotype E surface antigen (HBsAg) is most divergent from that of genotype A, which is used for preparing monoclonal antibodies used in commercially available HBV reagents. To evaluate the performance of the latest generation of HBsAg detection assays with respect to genotype E HBsAg. Three commercial assays were evaluated using sera from 200 Nigerian patients compared to the preS/S sequence of DNA positive samples. Out of 200 samples, 61 and 103 gave concordant positive and negative results between the three HBsAg assays. Of 36 samples with discordant results, 35 were confirmed negative by neutralisation. One of the three assays showed significantly high rate of false positives (29 of 35). DNA positive samples with no detectable HBsAg or reduced HBsAg detection signals (<75% of mean signal obtained with HBsAg positive samples) revealed several mutations (V14A, F46S, N48T, L49R, I49T, D51G, A53V, P54L, Q82P, F83C, L127P, A184V, T189I, S204N, V224A), mostly outside the a-determinant. Several of these mutations are found as wild type nucleotides normally in genotype A and only exceptionally in genotype E. All three assays showed comparable sensitivities for genotype E HBsAg detection (98.4-100%) but differed considerably in specificity (84-99%). Failure to detect HBsAg antigen and differences in signal intensity were mainly associated with mutations in the preS/S gene outside the "a" determinant.

Phylogenetic analysis of the precore/core gene of hepatitis B virus genotypes E and A in West Africa: new subtypes, mixed infections and recombinations.

One hundred and twenty-two new hepatitis B virus (HBV) preC/C sequences and three complete genomes from three major countries in West Africa were analysed. The majority of sequences were of genotype E and the only other genotype found was genotype A. Although for genotype E sequences, the genetic diversity of the preC/C gene was about two to three times higher than that of the preS/S gene, it was still considerably lower than that for genotype A sequences. The HBV/E preC/C gene was related most closely to subgenotype D1 and D2 sequences. Evidence of recombination was found in two strains that were of genotype A in the preS/S gene and of genotype E in the preC/C gene. The genotype A strains from Cameroon, Mali and Nigeria could be divided phylogenetically into three subtypes, A3 and two new subtypes, tentatively designated A4 and A5. Each subtype presented a genetic diversity of 2.19-3.85 % and intersubtype distances of 4.47-5.97 %. Interestingly, one sample from Nigeria showed evidence of a triple recombination of genotypes E/D and A, separated by a genotype G-specific insert of 36 bp. Of 110 patients, 19 (17.3 %) showed a coinfection of genotypes A and E, mostly in human immunodeficiency virus-positive children from Cameroon. Thus, in Cameroon, where both genotypes coexist, 37 % of all individuals tested had mixed infections. The low genetic variability in the preC/C gene of genotype E supports our previous speculation about a relatively short evolutionary history of this genotype, in contrast to the subtype-rich African genotype A strains.

Measles virus genotyping by nucleotide-specific multiplex PCR.

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.