Updating the PGMY primers and probes for improved detection of HPV68a: validation of version 2 of the PGMY-CHUV assay.

HPV68a is not efficiently detected by PCR with the PGMY primers. Version 2 of the PGMY-CHUV assay (PGv2) was developed from version 1 (PGv1) to evaluate HPV68-discordant results with the Anyplex II HPV28 assay. We now report that PGv2 is significantly more sensitive than PGv1 for HPV68a and as sensitive and specific for the other HPV genotypes during a 1-year prospective validation (n = 714 samples).

Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV assay for human papillomavirus genotyping.

The Anyplex II HPV28 (H28; Seegene) is a new semiquantitative real-time multiplex PCR assay for screening and genotyping 28 human papillomaviruses (HPV) in only 2 reaction wells. H28 was compared to the PGMY-CHUV assay (PG) with 309 archival DNA samples from cervical smears collected over 8 years in our laboratory. H28 and PG were fully concordant at the genotypic level on 228 (73.8%) out of 309 samples: 27 HPV negative and 201 HPV positive. The 201 fully concordant positive samples corresponded to single infections (n = 145) and to multiple infections (2 genotypes, n = 38; 3 to 5 genotypes, n = 18). The remaining 81 samples (26.2%) were either partially concordant (n = 64, 20.7%) or fully discordant (n = 17, 5.5%). While genotype-specific agreement was nearly perfect (κ = 0.877), HPV51 was significantly less well detected by H28 and the converse was observed for HPV40, -42, -54, and -68. Sequencing of PG amplicons confirmed HPV51 discordants and suggested the involvement of a possibly local HPV51 subtype. Mismatches in the PGMY09 primers to HPV68a explained most of the HPV68 discordants, confirming the specificity of H28 toward HPV68. With PG as a reference, the sensitivity and specificity of H28 were 93.4% and 99.0%, respectively. Considering H28 as a reference, the sensitivity and specificity of PG were 83.8% and 99.6%, respectively. H28 is a very sensitive and specific HPV genotyping assay suitable for research and clinical use as an adjunct to a clinically validated test. H28 semiquantitative readout ought to be evaluated for primary cervical cancer screening.

Validation of a low-cost human papillomavirus genotyping assay based on PGMY PCR and reverse blotting hybridization with reusable membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

Physiological and biological factors associated with a 24 h treadmill ultra-marathon performance.

The purpose of this study was to examine the physiological and biological factors associated with ultra-endurance performance. Fourteen male runners volunteered to run on a treadmill as many kilometers as possible over a 24-h period (24TR). Maximal oxygen uptake (VO(2max)), velocity associated with VO(2max)(VO(2max)) and running economy (RE) at 8 km/h were measured. A muscle biopsy was also performed in the vastus lateralis muscle. The subjects ran 149.2 ± 15.7 km in 18 h 39 ± 41 min of effective attendance on the treadmill, corresponding to 39.4 ± 4.2% of . Standard multiple-regression analysis showed that performance was significantly (R(2) = 0.82; P = 0.005) related to VO(2max) and specific endurance, i.e. the average speed sustained over the 24TR expressed in . VO(2max) was associated with a high capillary tortuosity (R(2) = 0.66; P = 0.01). Specific endurance was significantly related to RE and citrate synthase activity. It is concluded that a high VO(2max) and an associated developed capillary network are essential for ultra-endurance running performance. The ability to maintain a high %VO(2max) over a 24TR is another factor associated with performance and is mainly related to RE and high mitochondrial oxidative capacity in the vastus lateralis.

A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

Heterogeneous distribution of a fatty acid analogue uptake in the myocardium of aged rats.

The aim of this study was to determine the extent and location of damaged myocardial areas in senescent rats. The viability of myocardial cells was evaluated in virgin young (4 months old) and aged (29 months old) female Wistar rats by analysing the uptake of a slowly metabolisable radiolabelled fatty acid analogue, 15-p-iodophenyl-beta-methylpentadecanoic acid (IMPPA). The biodistribution of IMPPA was measured in various organs, and regional myocardial uptake was specifically assessed using quantitative autoradiography. Myocardial enzymatic activity and DNA content were also evaluated with nitro blue tetrazolium (NBT) and propidium iodide (PI) staining, respectively. In senescent rats, cardiac and renal IMPPA uptake showed a significant (50%) reduction compared with young adult rats and the uptake was not significantly changed in the liver, spleen, lungs, and skeletal muscle. Total ventricular NBT staining and IMPPA uptake were almost homogeneous in young adult rats, whereas they were very heterogeneous in aged rats. In the latter, approximately 11% of the total ventricular volume showed a significantly decreased (by 60% or more) IMPPA uptake compared with normal values, and this reduction was greater in ventricle base than in apex. The myocardial areas unlabelled or poorly labelled by IMPPA represented 4, 5, 6, and 21% of the right ventricular, left ventricular epicardial, septal, and left ventricular endocardial volume, respectively, and were poorly stained with NBT. In some of these areas, PI staining indicated the presence of living cells unable to pick up NBT staining. In conclusion, in young adult rats, no myocardial lesions were observed using three different labelling techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Granules of cytolytic T-lymphocytes contain two serine esterases.

Cytoplasmic granules from cytolytic T-lymphocytes (CTL) contain two proteins which react with the serine esterase-specific affinity label diisopropylfluorophosphate (DFP). One of these is a trypsin-like esterase which consists of two disulfide-linked 35-kd subunits. The other consists of a single 29-kd chain. Both molecules are induced concomitantly with cytolytic activity and perforin activity in a CTL-derived T-cell hybrid.

[Demonstration of human spermatozoa chromosomes in a heterospecific system: technical difficulties]. / Mise en évidence des chromosomes de spermatozoïdes humains dans un système hétérospécifique: difficultés techniques.

The incidence of chromosomal abnormalities at conception has been estimated to be very high about 40%; these estimations have been made on indirect evidence provided by karyotyping spontaneous abortions products. Now direct evidence is available, consisting in sperm chromosome analysis by using fertilization of zona-free eggs of the golden hamster. In our experience 175 assays have been performed with modifications of the technique described by Martin et al. (1982) and by Brandriff et al. (1984). Consistent results are obtained since the last 30 assays, using for the first time an R banding technique. Chromosomal analysis of 48 spermatozoa from 7 normal males is reported. The frequency of abnormal sperm complements (21%) is higher than reported by previous reports.

[Cytogenetic analysis of the pronucleus after an interspecies man-hamster model of fertilization]. / Analyse cytogénétique des pronucleus à partir du modèle de fécondation interspécifique homme-hamster.

A cytogenetic analysis of seventy human sperms originating from seven normal subjects is described. Results are compared to those of previously reported studies. The share of male and female contributions in the genesis of chromosomal abnormalities in human zygotes is discussed.

The effect of specific antibody on antibody-independent interactions between E. coli J5 and human complement.

We previously reported that Escherichia coli J5, the galactose epimerase-deficient mutant of E. coli O111:B4, can bind and activate purified human C1. The effects of hyperimmune rabbit anti-J5 IgG or IgM on E. coli J5 interactions with human C have been examined. Specific IgG or IgM increased the binding of 125I-C1 by J5. However, the rate of C1 activation, as determined by SDS-PAGE of eluted 125I-C1s, was decreased if bacteria were preincubated with immune IgG. Complexes formed between J5 preincubated with immune Ig and C1, under conditions in which all of the C1 was allowed to activate, consumed more C4 than J5 alone plus C1. However, the amount of C4 consumed per C1 molecule was identical for all bacteria preparations. Concentrations of specific IgG or IgM that significantly increased C1 binding did not appear to enhance C3b deposition upon incubation of E. coli J5 in NHS. Thus, although specific antibody may enhance C1 binding by E. coli J5, the ability of these additional C1 molecules to alter later events in the C cascade may depend on the control of C1 activation and its subsequent activity when bound to different membrane components.

Expression of "early" and "late" viral functions in a somatic cell hybrid between a mouse cell and a spontaneous yielder SV 40-transformed Chinese hamster cell.

A somatic cell hybrid (Cl. 6d) was originated from the fusion of mouse 3T3-4E) and spontaneous yielder SV 40-transformed Chinese hamster (CHK/SVLP AG) cells. During the early stages of its history, the C1. 6d hybrid underwent a rapid chromosome loss, preferentially loosing hamster chromosomes. This was not a constant tendency of the hybrid cells. As the parental CHK)SVLP AG cells, the hybrid cells were always found 100 per cent SV40 T-antigen positive. While CHK/SVLP AG cells infectious SV 40 DNA, V-antigen and virus were regularly detected, in the hybrid cells only infectious DNA was occasionally detected. This was not due either to the loww of an essential Chinese hamster gene(s) or to the presence of an inhibiting mouse cell component(s); it was apparently the consequence of inability of the cells to properly activate the resident SV 40 genome(s). After superinfection with SV 40 DNA, the hybrid cells-though capable of synthesizing SV 40 V-antigen--were unable to ensure virus assembly. Experimental evidence was obtained suggesting that SV 40 maturation is dependent of a cellular function(s).