Comparison of three non-nested RT-PCR for the detection of influenza A viruses.

BACKGROUND: The viral isolation technique (VIT) is largely used as a gold standard for the detection of influenza A and B viruses in respiratory samples. Some recent studies have pointed out that the polymerase chain reaction (PCR) assays allow sensitive and rapid detection of influenza viruses, also providing excellent correlation with traditional methods. OBJECTIVES AND DESIGN STUDY: The aim of this study was to evaluate the efficiency of three non-nested PCR, two PCR-hybridization assays using primers defined in M and NS genes, and one PCR which uses primers defined in NP, NS and HA genes and combines the detection of H3N2 and H1N1 hemagglutinin genes using defined primers in NP, NS and HA genes (PCR3), in comparison with an IF assay (IFA) and viral isolation technique (VIT). The study was carried out on 244 nasal samples collected mainly by practitioners of the GROG surveillance network during winter 1998-1999 for the detection of influenza A virus. RESULTS: Overall influenza viruses were detected more frequently by PCR techniques in 157 (64.3%), 147 (60.2%), 110 (45%) cases for PCR1, PCR2, PCR3, respectively, than by VIT or IFA, in 100 (40.9%) and 74 (30.3%) cases, respectively. Taking the positive culture samples as a reference, 100 (41.8%) samples were found to be positive for influenza A, and the sensitivity of IFA, PCR 1, PCR 2 and PCR3 techniques were 70, 100, 99, and 90%, respectively as compared with viral isolation cultures. On the other hand, as 86.5% of positive samples were positive with at least two different techniques, the sensitivity, specificity, VPP and VPN of each technique were recalculated taking into account a further criterion defining a positive sample: positivity with two techniques. We observe that techniques PCR 2 and particularly PCR 1 have very good sensitivity, respectively 98.6 and 100%, far better than the traditional techniques, IFA and culture, whilst maintaining acceptable specificity: 94.1 and 86.1%, respectively. In both cases they enable 141 (57.7%) A-positive influenza samples to be detected instead of the 100 (40.9%) obtained when culture is the reference test. IFA, culture and PCR 3 are highly specific (VPP=100%), but in comparison with PCR 1 and 2 their sensitivity, respectively 51.7, 69. 9, 77.6%, and negative predictive value are unsatisfactory. PCR 1 and 2 are superior to the other techniques to a statistically highly significant degree in terms of sensitivity, but the difference between the two is not significant.

Sperm survival during short-term storage and after cryopreservation of semen from striped trumpeter (Latris lineata).

Methods of short-term storage and cryopreservation were examined for semen from striped trumpeter (Latris lineata). For fresh semen at 18 degrees C, the percentage of motile sperm declined rapidly from over 80% immediately after activation with sea water to less than 2% within 9 min after activation. The motility after activation of undiluted fresh sperm stored at 5 degrees C was maintained for two days and then declined markedly so that by the eighth day, sperm were mostly immotile after activation. The post-thawing motility was higher for sperm frozen with a non-activating diluent containing 2.84 M DMSO in saline (117 mM NaCl) than in an activating glycerol (2 M) medium in dilute sea water (300 mOsm). Post-thawing motility was higher for a dilution rate of 1:5 (semen:diluent) than 1:2 or 1:11 but was similar when frozen semen was thawed at 10 degrees, 20 degrees or 30 degrees C. For semen stored at a range of volumes as pellets frozen on dry ice (0.2 to 2.0 mL) or straws frozen in liquid nitrogen vapor (0.25 to 0.5 mL) and thawed in a waterbath at 20 degrees C, the post-thawing motilities were similar even though the patterns of cooling and thawing differed markedly between methods of freezing and sizes of pellets and straws.

[Epidemiology of respiratory virus infections]. / Epidémiologie des infections virales respiratoires.

Respiratory viral infections are very common in young children. They sometimes occur as primary infections (and sometimes re-infections) by influenza and parainfluenza virus, respiratory syncytial virus (VRS), adenovirus, rhinovirus and coronavirus. The clinical pictures are very varied and without strict clinico-virological correlation. In adults the role of the site (frail lung, aged persons) and the type of virus play an important part. Many viral infections develop in an epidemiological way (influenza, VRS bronchiolitis, rhinovirus infections...) and several epidemics by different viruses overlap from September-October to March-April making it very difficult to decide the precise cause. Epidemics are followed thanks to networks of medical practitioners (GROG, SENTINELLE...) and by data from hospitalised patients, but precise identification of epidemic viruses is only possible and validated by virological analysis of samples taken from patients.

Evaluation of virological procedures to detect fetal human cytomegalovirus infection: avidity of IgG antibodies, virus detection in amniotic fluid and maternal serum.

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection and fetal damage largely due to maternal primary infection. Virological procedures which are able to detect HCMV fetal infection were evaluated. HCMV IgG antibodies were detected in 62.5% of the pregnant women and 1.47% had a primary infection. From March, 1992 to August, 1995, 29 seroconversions were observed, and in 64 other cases. HCMV IgM antibodies were detected in the first serological test. The mean IgG antibody avidity test (AI) was 31% for the 11 seroconversions tested and 74% in 32 cases where IgG and IgM HCMV antibodies were detected in the first serum. In the 29 HCMV seroconversions, 19 amniocentesis were carried out and 12 fetuses (41.4%) were infected in utero. In four amniotic fluids positive in culture and PCR, the fetus or newborns were infected and in one out of the two cordocentesis undertaken, hepatitis, anemia, and thrombocytopenia were noted. In four other cases, investigations seeking HCMV in amniotic fluid were negative whereas infants were infected at birth. Among the 64 cases with positive HCMV IgM and IgG antibodies detected in the first serological test, three fetuses were infected in utero, but no amniotic fluid was available in these cases. Amniotic fluids were studied in 39 cases, and HCMV detection by culture and PCR-hybridization was negative. HCMV DNA was detected in the maternal sera of five out of 21 pairs of seroconversions and in two cases on the first negative serum. The assay was also carried out on 50 of the 64 HCMV IgM positive sera. Two had detectable HCMV DNA.