Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

Genome Sequences of Two Klebsiella pneumoniae Isolates from Different Geographical Regions, Argentina (Strain JHCK1) and the United States (Strain VA360)

We report the sequences of two Klebsiella pneumoniae clinical isolates, strains JHCK1 and VA360, from a newborn with meningitis in Buenos Aires, Argentina, and from a tertiary care medical center in Cleveland, OH, respectively. Both isolates contain one chromosome and at least five plasmids; isolate VA360 contains the Klebsiella pneumoniae carbapenemase (KPC) gene

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system.

We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Cloning of a full-length cDNA encoding bovine interleukin 4 by the polymerase chain reaction

A human interleukin 4 (hIL-4)-encoding cDNA (hIL4) probe was used to screen a bovine genomic library, and three clones containing sequences with homology to the human and mouse IL4 cDNAs were isolated. Sequence information obtained from one of these genomic clones was used to design an oligodeoxyribonucleotide primer corresponding to the transcription start point region for use in the polymerase chain reaction (PCR). The PCR-RACE protocol, designed for the rapid amplification of cDNA ends, was successfully used to generate a full-length bovine IL4 (bIL4) cDNA clone from polyadenylated RNA isolated from concanavalin A-stimulated bovine lymph node cells. The bIL4 cDNA is 570 bp in length and contains an open reading frame of 405 nucleotides (nt), coding for a 15.1-kDa precursor of 135 amino acids (aa), which should be reduced to 12.6 kDa for unglycosylated bIL4 after cleavage of a putative hydrophobic leader sequence of 24 aa. The aa sequence contains one possible Asn-linked glycosylation site. Bovine IL4 is shorter than mouse (mIL4) and hIL4, because of a 51-nt deletion in the coding region. Comparison of the overall nt and deduced aa sequences shows a greater homology of bIL4 with hIL4 than with mIL4. This homology is not evenly distributed, however, with the nt sequences 5' and 3' of the coding region showing a much greater homology between all three species than the coding sequence.

Sexual Activity Without Condoms and Risk of HIV Transmission in Serodifferent Couples When the HIV-Positive Partner Is Using Suppressive Antiretroviral Therapy

Importance A key factor in assessing the effectiveness and cost-effectiveness of antiretroviral therapy (ART) as a prevention strategy is the absolute risk of HIV transmission through condomless sex with suppressed HIV-1 RNA viral load for both anal and vaginal sex. Objective To evaluate the rate of within-couple HIV transmission (heterosexual and men who have sex with men [MSM]) during periods of sex without condoms and when the HIV-positive partner had HIV-1 RNA load less than 200 copies/mL. Design, Setting, and Participants The prospective, observational PARTNER (Partners of People on ART-A New Evaluation of the Risks) study was conducted at 75 clinical sites in 14 European countries and enrolled 1166 HIV serodifferent couples (HIV-positive partner taking suppressive ART) who reported condomless sex (September 2010 to May 2014). Eligibility criteria for inclusion of couple-years of follow-up were condomless sex and HIV-1 RNA load less than 200 copies/mL. Anonymized phylogenetic analysis compared couples' HIV-1 polymerase and envelope sequences if an HIV-negative partner became infected to determine phylogenetically linked transmissions. Exposures Condomless sexual activity with an HIV-positive partner taking virally suppressive ART. Main Outcomes and Measures Risk of within-couple HIV transmission to the HIV-negative partner. Results Among 1166 enrolled couples, 888 (mean age, 42 years [IQR, 35-48]; 548 heterosexual [61.7%] and 340 MSM [38.3%]) provided 1238 eligible couple-years of follow-up (median follow-up, 1.3 years [IQR, 0.8-2.0]). At baseline, couples reported condomless sex for a median of 2 years (IQR, 0.5-6.3). Condomless sex with other partners was reported by 108 HIV-negative MSM (33%) and 21 heterosexuals (4%). During follow-up, couples reported condomless sex a median of 37 times per year (IQR, 15-71), with MSM couples reporting approximately 22 000 condomless sex acts and heterosexuals approximately 36 000. Although 11 HIV-negative partners became HIV-positive (10 MSM; 1 heterosexual; 8 reported condomless sex with other partners), no phylogenetically linked transmissions occurred over eligible couple-years of follow-up, giving a rate of within-couple HIV transmission of zero, with an upper 95% confidence limit of 0.30/100 couple-years of follow-up. The upper 95% confidence limit for condomless anal sex was 0.71 per 100 couple-years of follow-up. Conclusions and Relevance Among serodifferent heterosexual and MSM couples in which the HIV-positive partner was using suppressive ART and who reported condomless sex, during median follow-up of 1.3 years per couple, there were no documented cases of within-couple HIV transmission (upper 95% confidence limit, 0.30/100 couple-years of follow-up). Additional longer-term follow-up is necessary to provide more precise estimates of risk.