Evaluation of Granada agar plate for detection of Streptococcus agalactiae in urine specimens from pregnant women

The Granada agar plate (GAP; Biomedics SL, Madrid, Spain) was evaluated for the detection of group B streptococci (GBS) in urine specimens from pregnant women submitted for testing for asymptomatic bacteriuria and was compared with blood agar (BA [Columbia agar with 5% sheep blood]; bioMérieux, Marcy l'Etoile, France). The GAP detected 103 out of 105 GBS, whereas BA detected only 50. Use of the GAP could be a good method for the detection of GBS in urine specimens from pregnant women.     

Prolonged hepatitis A infection in an HIV-1 seropositive patient

Hepatitis A virus (HAV) is a worldwide disease; in most cases, it causes an acute self-limited illness that does not lead to a chronic state. The course of HAV viremia in a homosexual male with human immunodeficiency virus type 1 (HIV-1) and the correlation between HIV and HAV viral load, alanine aminotranferase (ALT) level, and CD4(+) lymphocyte count were investigated during the course of the infection. HAV RNA was detected quantitatively up to 256 days after clinical onset. To our knowledge, this specific case is the first report of a prolonged infection with hepatitis A in a male with HIV-1. The ALT levels decreased gradually; however, 286 days after clinical onset of hepatitis, ALT levels were three times higher than normal values. HIV viral load was not affected by the infection with HAV and CD4(+) cell count was stable during the course of the co-infection. The duration and the high-titer viremia of hepatitis A virus in an immunodeficient patient constitute a serious risk of the spread of hepatitis A within this population. As inactivated HAV vaccine is safe in HIV-positive subjects, it would be wise to establish a strategy of preventive vaccination in this high-risk group.     

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci

In a context of worldwide emergence of resistance among Streptococcus pneumoniae strains, early detection of strains with decreased susceptibility to beta-lactam antibiotics is important for clinicians. If the 1-microgram oxacillin disk diffusion test is used as described by the National Committee for Clinical Laboratory Standards, no interpretation is available for strains showing zone sizes of /=2.0 microgram/ml) to penicillin. For ceftriaxone, among 98 strains with no zone of inhibition in response to oxacillin, 68 had intermediate resistance (MIC, 1.0 microgram/ml), and 22 were resistant (MIC, >/=2.0 microgram/ml). To optimize the use of the disk diffusion method, we propose that the absence of a zone of inhibition around the 1-microgram oxacillin disk be regarded as an indicator of nonsusceptibility to penicillin and ceftriaxone and recommend that such strains be reported as nonsusceptible to these antimicrobial agents, pending the results of a MIC quantitation method.     

Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells in patients with chronic hepatitis C treated with interferon alpha

PCR was used to detect hepatitis C virus (HCV) RNA in serum and peripheral blood mononuclear cells (PBMCs) for evaluation of a six-month course of Interferon therapy in 18 patients with histologically confirmed chronic hepatitis C. At follow-up six months after the end of therapy positive-stranded (genomic) and negative-stranded (anti-genomic, presumptive replicative intermediate) HCV RNA could be detected in PBMCs of all ten patients who either did not respond to therapy or suffered a relapse; genomic strand RNA was detected in five patients who responded but then relapsed. The study confirms that interferon therapy leads to inhibition of HCV replication but not eradication of the virus. Persistence of the virus at extrahepatic sites may explain its reactivation after cessation of interferon therapy.     

Effect of transport medium and transportation time on culture of Helicobacter pylori from gastric biopsy specimens.

To determine whether transportation time and use of a low budget transport medium (NaCl 0.9%) would influence culture of Helicobacter pylori from gastric biopsy specimens, upper gastrointestinal endoscopy was performed on 42 patients. The specimens were cultured and examined histologically, and H pylori antibodies were determined using an ELISA technique. Patients were regarded as H pylori positive when culture was positive or when histology or IgG anti-H pylori antibodies indicated H pylori infection. Rapid transportation of gastric biopsy specimens in NaCl 0.9%, at room temperature resulted in a high diagnostic yield (23 H pylori positive cultures in 26 patients with H pylori infection). A 24 hour delay in plating gastric biopsy specimens after transportation in NaCl 0.9%, at room temperature, did not seriously affect results (22 instead of 23 H pylori positive cultures). The culture results after transportation in Cairy-Blair medium were comparable with those after transportation in NaCl 0.9%, but because of availability, low cost, and ease of handling in the endoscopy department, NaCl 0.9% was preferred as transport medium. This study shows that for culture of H pylori from gastric biopsy specimens sterile saline is an adequate medium, and that transportation can be delayed for 24 hours without a significant loss of diagnostic yield.