The influence of the expanding HIV genetic diversity on molecular diagnosis in the Philippines.

Since the discovery in the Philippines of the first AIDS case in 1984, several subtypes of HIV-1 have been discovered. From the persons diagnosed in the early 1980s only subtype B was found and thereafter other subtypes, C, D, E, and F were also identified although HIV was not particularly prevalent at that time. In this paper, we determine whether the rapid expansion of genetic diversity will influence molecular diagnosis by polymerase chain reaction (PCR). First, we determine HIV-1 subtype on env (V3) and gag (p24) gene as a means of rapid genetic diversity. Secondly, we tried to analyse and identify homologous regions of gag (p24) gene of HIV genome for diagnostic purposes of designing primers. Out of 46 samples analysed, six subtypes were classified based on gag and env gene subtyping namely: 33 subtype B/B (71.2%), nine subtype A/E and one each subtype C/C, A/B and G/A (2.2% each). As a result, occurrence of non-subtype B and inter-subtype recombinant contributed to expanding genetic diversity. Based on inter- and intra-subtype gag alignment, oligonucleotides (>10 bases in length) could be easily selected as a universal primer to produce the PCR product composed of more than 100bp. This indicates that the PCR technology can be safely used with limited length of primers for the diagnosis of HIV infection in this country.

Survival of Shiga toxin-producing Escherichia coli O157 in marine water and frequent detection of the Shiga toxin gene in marine water samples from an estuary port.

Shiga toxin-producing Escherichia coli (STEC) O157 was investigated with respect to its halotolerance and whether it can survive in marine water. STEC O157 could multiply in a medium containing 5% NaCl and in sterilized marine water, and could survive in unsterilized marine water for at least 15 days. On the basis of these results, we postulated that STEC O157 may survive in natural marine water, and attempted to isolate the bacterium and Shiga toxin gene (stx) from marine water in Japan. The stx, comprising stx1 and stx2, was detected from marine water samples by PCR. STEC and other stx-positive bacteria, however, could not be isolated from these samples in this study. These results indicate that stx-positive bacteria may survive in marine water and suggest the necessity of a survey.

Targets of a protease inhibitor, KNI-272, in HIV-1-infected cells.

The targets of a protease inhibitor, KNI-272, in the HIV-1 life cycle were investigated in this study. Neither expression of HIV-1 Gag proteins nor production of virus particles was detected in cells infected acutely with HIV-1 cultured in the presence of KNI-272. Although HIV-1 proviral DNA was detected in the cells by PCR, the inhibitor depressed the amount of the proviral DNA in a concentration dependent manner. These results indicate that one of the targets of KNI-272 occurs in the stage before the expression of viral structural proteins. No direct inhibition of reverse transcription was found with the inhibitor. To confirm the inhibition of viral protease, persistently HIV-1-infected cells were cultured in the presence of the inhibitor and examined by electron microscopy for the morphology of HIV-1 particles. Doughnut-shaped immature particles were observed in the extracellular space of the cells, and disrupted semicircular shaped particles were also seen at the higher concentration of KNI-272. A bioassay for infectivity showed that the virus particles were not infectious, and immunofluorescent assay using anti-p17 antibody, that does not react with the precursor of Gag protein, revealed that Gag precursor p55 protein in the cells was not processed. Thus, KNI-272 blocked the maturation of viral particles. Consequently, KNI-272 has at least two inhibition targets in the stages of the HIV-1 life cycle.

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori.

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Disinfection potential of electrolyzed solutions containing sodium chloride at low concentrations.

Electrolyzed products of sodium chloride solution were examined for their disinfection potential against hepatitis B virus (HBV) and human immunodeficiency virus (HIV) in vitro. Electrolysis of 0.05% NaCl in tap water was carried out for 45 min at room temperature using a 3 A electric current in separate wells installed with positive and negative electrodes. The electrolyzed products were obtained from the positive well. The oxidation reduction potential (ORP), pH and free chlorine content of the product were 1053 mV, pH 2.34 and 4.20 ppm, respectively. The products modified the antigenicity of the surface protein of HBV as well as the infectivity of HIV in time- and concentration-dependent manner. Although the inactivating potential was decreased by the addition of contaminating protein, recycling of the product or continuous addition of fresh product may restore the complete disinfection against bloodborne pathogens.

Capsular hyaluronic acid of group A streptococci hampers their invasion into human pharyngeal epithelial cells.

Group A streptococci (GAS) cause various diseases, from uncomplicated noninvasive, to severe invasive infections. Capsular hyaluronic acid (HA) is known to resist phagocytosis, however, interaction between HA and epithelial cells have not been clearly understood. In this study, both HA-producing wild strains and HA-nonproducing mutants were employed to examine their invasiveness into confluent cultures of HEp-2, a nonphagocytic human epithelial cell line. Invasion of HEp-2 cells by GAS strains increased over time. The hasA gene encoding hyaluronate synthase of GAS strains was inactivated by allelic replacement. It was found that hasA-inactivated mutants were internalized into HEp-2 cells more efficiently than their parent strains under various conditions in terms of incubation time and inoculum size. Taken together, these findings indicate that GAS can be internalized into HEp-2 cells with considerably high frequencies and that the presence of HA of GAS decreased the invasion efficiency.

Inhibition of salivary fluid secretion by occlusion of the intercellular space.

The effects of HCO3- on fluid secretory rate, cell volume and tissue structure were studied in perfused submandibular salivary glands under hyposmotic conditions (240 mosM). Fluid secretion was elicited by acetylcholine (ACh) in a hyposmotic HCO3(-)-free solution. Upon switching the perfusate from the HCO3(-)-free to the HCO3(-)-containing solution during ACh stimulation, the fluid secretory rate exhibited a small transient increase followed by a sharp decrease. ACh stimulation evoked rapid cell shrinkage in the absence of HCO3-, but upon switching from the HCO3(-)-free to the HCO3(-)-containing solution during ACh stimulation, the acinar cells exhibited increases in volume. In laser confocal microscopic examination of isolated acini, fluorescence of lucifer yellow was detected in the lateral intercellular spaces during ACh stimulation in the absence of HCO3-, but not in the presence of HCO3-. Electron microscopic examination revealed similar findings. We propose that the occlusion of the lateral intercellular space induced by cell swelling may result in an increase in the resistance to fluid flow in the space and consequently a decrease in the fluid secretion rate. Maintenance of an open lateral intercellular space may be an important requirement for fluid secretion. These observations suggest that a paracellular pathway may play a significant role in salivary fluid secretion.

Contrast-enhancement for the image of human immunodeficiency virus from ultrathin section by immuno electron microscopy.

A simple contrast-enhancement method is described for electron microscopic imaging of the human immunodeficiency virus (HIV) from a sample embedded in Lowicryl K4M resin, by immuneelectron microscopy. Ultrathin sections were treated with a mixture of ruthenium red dye (RR) and osmium tetroxide (OSO4). This treatment provided good contrast enhancement of the entire ultrastructural image of virus particles without the loss of immunolabelling. RR/OsO4 solution is simple to prepare and provides a better contrast than that which is achieved during conventional post-embedding immunoelectron microscopy. Treatment of ultrathin sections from low temperature-embedded samples with RR/OsO4 solution is recommended.

Staphylococcus species on the skin surface of infant atopic dermatitis patients.

The Staphylococcus species present in microbiological samples from the facial skin of 10 infants with atopic dermatitis and eight healthy infants were studied. S. aureus colonization was detected on the cheek and nose skin of most of the infants with atopic dermatitis (9/10 and 7/10, respectively). Among the coagulase-negative staphylococci found, S. epidermidis was detected commonly on the skin of healthy infants; several other species of coagulase-negative staphylococci were found on the skin of infants with atopic dermatitis and of healthy infants.

Electronmicroscopic study of human herpesvirus 6-infected human T cell lines superinfected with human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as one of the co-factors responsible for the development of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV) carriers. We analyzed the interaction between HHV-6 and HIV-1 in superinfected cells. Cell-free HIV-1 could superinfect human T cell lines, MT-4 and Molt-4, which had been previously infected with HHV-6. Both HHV-1 and HHV-6 replicated in the same cells. We observed two types of morphologically distinguished cells as early as 4 days after superinfection. One type (D) was degenerate cells with intracellular and extracellular HHV-6 and with less HIV-1 virions. The other type (I) was relatively intact cells with both HIV-1 and HHV-6 virions. Replication of HIV-1 was more active in the type I as compared with type D cells. The level of HIV-1 reverse transcriptase (RT) activity in the culture supernatants of cells superinfected on day 0 declined after day 7, while that in the supernatants of cell cultures infected with HIV-1 alone remained high between days 12 and 40. These results suggest that the superinfection of the HHV-6-infected cells with HIV-1 may induce a degenerative process in these cells.

Susceptibility of Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis to 10 Kampo formulations.

We examined the in vitro sensitivities of three bacteria: Propionibacterium acnes, and Staphylococcus epidermidis, commonly detected in acne lesions, and Staphylococcus aureus, a common cause of skin infections, to 10 Kampo formulations (Chinese herbal medicines; combinations of powdered extracts of crude drugs). Both Staphylococcus species showed similar sensitivities to all 10 formulations, with minimum inhibitory concentrations (MICs) ranging from 25 to 400 mg/ml. P. acnes, however, was particularly sensitive to one formulation, keigai-rengyo-to (MIC, 0.78-25 mg/ml), prompting speculation that it might contain components with strong antibacterial activity to P. acnes. P. acnes showed similar sensitivities to all the other formulations (MIC 6.25-200 mg/ml). The ranges of MICs and the MIC50S (concentrations that inhibit 50% of isolates) were very similar to those previously recorded in 1990 for the two Staphylococcus species.

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

[An effective concentration method for human immunodeficiency virus type 1 (HIV-1)].

We designed an effective virus concentration method to prevent the infection with human immunodeficiency virus type 1 (HIV-1) in laboratories. The absorbent of Minicon concentrators (Amicon Division, M.R. Grace & Co.-Conn.) was changed to chitin, a mucopolysaccharide extracted from the shells of Japanese pink crab. HIV-1 in the supernatant of HIV-1 infected Molt-4 cells was concentrated by Minicon and the new concentrators. The new concentrator showed good concentration rate and equality of concentration speed.

[Detection of HTLV-I/ATLV by using the culture of lymph node lymphocytes with adult T-cell leukemia/lymphoma].

We cultured the cervical lymph node lymphocytes of a patient suffering from cutaneous T-cell lymphoma. His anti-ATLV antibody was positive by indirect immunofluorescent method (IF). ATLV was detected on these cultured cells by IF. Type C particles were observed in the cultured cells by electron microscopy. These particles were measured to be 60 to 120 nm in diameter with electron dense core, and were considered as ATLV. This case showed a possibility of detecting ATLV by culture of lymph node lymphocytes from such a patient.

[Comparative evaluation of markers with reverse transcriptase inhibiting antibody in human immunodeficiency virus type 1 infection].

To investigate a non-RI test which is equivalent to the reverse transcriptase inhibiting antibody test, a reverse transcriptase inhibiting antibody was compared to the absolute number of CD-4 or CD-8 cells or CD-4/-8 ratio and also to photodensitometric analysis for western blotting. There is no correlation of the reverse transcriptase with any test for cell numbers and their ratio. In photodensitometry, relative units of anti-p65 and anti-p51 were compared with reverse transcriptase inhibiting antibody. The reverse transcriptase inhibiting antibody showed a higher correlation to the relative unit of p65 antibodies than that of p51 antibodies. The photodensitometric analysis of western blotting for a serum test may be a possible method to find a prognostic marker in HIV-1 infection.