National HIV prevalence surveillance among TB patients through periodic surveys: experience in Cambodia.

SETTING: The National Tuberculosis Programme (NTP) in Cambodia, one of the countries most affected by tuberculosis (TB) and human immunodeficiency virus (HIV) infection in Asia. OBJECTIVE: To conduct national HIV prevalence surveillance among TB patients, to estimate HIV prevalence among TB patients and to determine the potential of the NTP as a source for antiretroviral treatment (ART) scale-up. DESIGN: Anonymous unlinked cross-sectional seroprevalence surveys including all TB patients registered by the NTP in January 2003 and January 2005. RESULTS: HIV prevalence among all TB patients fell from 11.8% in 2003 to 9.9% in 2005 (P < 0.05). In 2003 and 2005, respectively 265 and 261 TB patients were identified as HIV-positive in a given month. Among new smear-positive pulmonary TB patients, the prevalence dropped from 8.2% to 5.2% (P < 0.01). CONCLUSION: The two periodic surveys demonstrated a high prevalence of HIV among TB patients in Cambodia. However, the declining incidence of HIV from the late 1990s might now be reflected in the HIV prevalence among new smear-positive TB patients. The NTP is a potential source of ART if HIV counselling and testing are made more widely available to TB patients.

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

SETTING: The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. OBJECTIVE: To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. DESIGN: A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. RESULTS: We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. CONCLUSION: The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.

Band heterotopia or double cortex in a male: bridging structures suggest abnormality of the radial glial guide system.

A moderately retarded Japanese boy, with a normal male karyotype (46,XY), was diagnosed to have a subcortical band heterotopia or double cortex syndrome. The band heterotopia was relatively thick compared with that of other patients reported. On T2-weighted coronal MR sections, there were numerous radial linear structures between the cortex and the band, probably representing the trace of radial fibers. He had no family members with seizures or mental retardation. Over 50 described patients with this malformation have been female except two patients briefly mentioned by several investigators. Band heterotopia or the double cortex syndrome is inherited as a sex-linked dominant condition. Affected mothers may have affected daughters or sons with lissencephaly, suggesting a link between these disorders. This is the first detailed description of a male with band heterotopia.

Inactivation of human viruses by povidone-iodine in comparison with other antiseptics.

Inactivation of a range of viruses, such as adeno-, mumps, rota-, polio- (types 1 and 3), coxsackie-, rhino-, herpes simplex, rubella, measles, influenza and human immunodeficiency viruses, by povidone-iodine (PVP-I) and other commercially available antiseptics in Japan was studied in accordance with the standardized protocol in vitro. In these experiments, antiseptics such as PVP-I solution, PVP-I gargle, PVP-I cream, chlorhexidine gluconate, alkyldiaminoethyl-glycine hydrochloride, benzalkonium chloride (BAC) and benzethonium chloride (BEC) were used. PVP-I was effective against all the virus species tested. PVP-I drug products, which were examined in these experiments, inactivated all the viruses within a short period of time. Rubella, measles, mumps viruses and HIV were sensitive to all of the antiseptics, and rotavirus was inactivated by BAC and BEC, while adeno-, polio- and rhinoviruses did not respond to the other antiseptics. PVP-I had a wider virucidal spectrum, covering both enveloped and nonenveloped viruses, than the other commercially available antiseptics.

Early diagnosis of vertical HIV infection in infants by rapid detection of immune complex-dissociated HIV p24 antigen.

Conventional HIV antibody detection was problematic for diagnosis of HIV infection in young infants < 18 months of age who were born to HIV-infected mothers. The HIV p24 antigen (Ag) is mainly bound to the antibody as an immune complex which causes underdetection by conventional methods. Attempts were made to dissociate these immune complexes to release free p24 Ag for detection. The current study's objective was to evaluate the rapid assays for detection of immune complex-dissociated p24 Ag (ICD p24 Ag) for early identification of HIV-infected infants as compared to the detection of HIV RNA by polymerase chain reaction (PCR) assay. The ICD was performed by acid dissociation and heat-denatured dissociation, and then the released ICD p24 Ag were detected. Tested were 41 HIV-infected children who acquired the infection perinatally and who had positive PCR and 30 HIV noninfected children with negative PCR. The overall sensitivity of the ICD p24 Ag detection after acid- and heat-denatured dissociation in the infected children was 85.4% and 87.8%, respectively, compared to 34.2% of p24 Ag without pretreatment for dissociation of the serum samples. The specificity of nonimmune complex dissociation and both methods of immune complex dissociation test were 100%. The sensitivity of ICD-p24 Ag test using these two methods showed excellent agreement (K = 0.893). Besides the relatively high sensitivity and specificity of the ICD p24 Ag test, its advantages include simplicity, rapidity, and relatively low cost--indicating ICD p24 Ag detection as a promising method for early diagnosis of vertical HIV infection in infants.

[Detection of HIV-1 in plasma samples using microplate hybridization after RT-PCR].

We have developed a microplate hybridization(MH) technique, which utilizes the non-isotopic method of enzyme-linked assay for detection of HIV in the amplified product after PCR. HIV RNA extracted from plasma was amplified by RT-Nested-PCR using biotinylated-inner primers of gag, pol and env regions, respectively. The PCR product was visualized by 5% polyacrylamide gel electrophoresis and ethidium bromide staining. The heat-denatured PCR product was hybridized with HIVcDNA of each region which was immobilized to a microplate. The hybridized microplate was reacted with streptavidin-conjugate peroxidase and then the optical density(O.D.) was read at 490nm. The cut off value was determined at O.D. 0.25. The results of the electrophoresis of gag, pol and env regions were all positive in 53 HIV-1 seropositive samples from Japan and the USA, and all negative in 55 HIV-1 seronegative samples. Using the MH technique, USA samples showed a higher O.D. than the Japan samples, particularly in the pol region. The results of MH technique in gag and pol regions coincided with that of electrophoresis. But, one of 27 Japanese HIV-1 seropositive samples showed O.D. of less than 0.2 in only env region. This particular sample was classified by V3 peptide-based enzyme immunoassay as subtype E, which differs from the typical subtype B of Japan and USA samples. This suggests the presence of several genotypes in HIV-1 seropositive individuals in Japan. Based on this data, the MH technique using gag, pol and env region is a simple, sensitive, safe and specific assay for detection of HIV-1 RNA in plasma, and would be useful in clinical testing.

[ELISA for diagnosis of infections by viruses].

The assay used most widely to detect or diagnose virus infection, especially infection of blood borne viruses e.g. HBV, HCV, HIV and HTLV, is the enzyme linked immunosorbent assay (ELISA), whose sensitivity and practicability have rendered it the most common primary screening assay. ELISA can be mass screening used automatic or semiautomatic machines. ELISAs can be indirect assay, competition assays or sandwich assays. In indirect and sandwich assays, the development of color indicates the presence of antigen or antibody, whereas in competition assays the absence of color development signifies a positive reaction. Alkaline phosphatase and horseradish peroxidase the most commonly used enzymes, are associated with their respective substrates, usually p-nitro-phenyl phosphate and hydrogen peroxide. The ELISA for antigen detection is used polyclonal antibody or monoclonal antibodies. The other hand, the ELISA for antibody screening is used whole virus, synthetic peptides or recombinant antigens.

[Clinical usefulness of urinary anti HIV antibody test--a large scale study from 11 institutes in Japan].

An enzyme immuno assay kit has been developed to detect anti-HIV antibody in urine. In order to examine the clinical utility of the kit, 1333 urine samples were assayed. These samples consisted of 233 urine samples from HIV infected patients, 472 samples from HIV uninfected patients including 203 samples from patients with urogenital diseases, and 628 samples from normal subjects. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV infection was 100% with no false negative cases. A variety of anti-HIV antibody titers were found in the urine samples from HIV infected patients. However, no significant differences were found in the distribution patterns of urinary anti-HIV antibody titers among AC, ARC and AIDS patients. False positives were determined in only five samples in 628 healthy subjects (0.8%), one in 19 patients with hepatitis (5.3%), one in 45 patients with hemophilia (2.2%) and two in 105 pregnant women (1.9%). The antibody titers of all the false positive samples in these groups were less than the cut-off index multiplied by two. However, relatively high positive rates were demonstrated in the samples from urogenital diseases (11.8%), diabetes mellitus (20.0%) and auto-immune diseases (7.3%). False positive results were found to be directly correlated to the protein concentration of urinary protein, especially the immunoglobulin concentration in urine. The assay system was also evaluated by various reproducibility tests performed by different operators at different laboratories. The test results were satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of HIV-1 proviral DNA in peripheral leukocytes by AMPLICOR HIV-1 test kit].

AMPLICOR HIV-1 test kits, which had been developed as an HIV-1 provirus detection test by PCR method, have been evaluated for its clinical diagnostic application. Sixty-six of HIV-1 antibody positive and 67 of HIV-1 antibody negative blood samples derived from hemophiliacs, who had received blood products, have been tested by AMPLICOR HIV-1. All of the results from AMPLICOR HIV-1 were consistent with those from antibody test and clinical aspects. Thirty-nine of HIV-1 antibody positive samples have been tested by AMPLICOR HIV-1 and virus isolation (culture method). Twelve of 39 (30.8%) were positive by virus isolation, and 39 of 39 (100%) were positive by AMPLICOR HIV-1. Two of new born infants from HIV-1 sero-positive mothers were tested by AMPLICOR HIV-1, and the result suggested that the kit would be useful for diagnosis of infants from sero-positive mothers. Based on these studies, AMPLICOR HIV-1 is considered as useful clinical diagnostic for HIV-1 proviral DNA detection.

Absence of nonpercutaneous transmission of hepatitis C virus in a colony of chimpanzees.

Transmission of hepatitis C virus (HCV) was studied in a colony of 85 chimpanzees using assays for anti-HCV and HCV-RNA. Thirteen of the 85 sera were positive for anti-HCV, and 12 of the 13 were also positive for HCV-RNA. All of the anti-HCV positive sera except one were obtained from chimpanzees which had been inoculated with non-A, non-B hepatitis virus. On the other hand, only one of 63 sera of chimpanzees without history of experimental infection of the virus was positive for anti-HCV. Transmission to this chimpanzee was thought to be a needle contaminated with HCV. All 39 samples of chimpanzees born in the center were negative for both anti-HCV and HCV-RNA. Sixteen of their mothers had undergone experimental infection, and 6 of them were positive for both anti-HCV and HCV-RNA. These results suggest that nonpercutaneous transmission, including sexual and mother-to-infant transmissions, is not an important mode of transmission. If these findings apply to humans, definition of inapparent sources of the infection is needed.

Sequence analysis of V3 loop region of HIV-1 strains prevalent in India.

The third variable (V3) domain of human immunodeficiency virus type 1 (HIV-1) env gene has been found to elicit type-specific neutralizing antibodies as well as a cytotoxic and helper T-cell response in both humans and animals. We analyzed the V3 domain of 8 HIV-1 isolates from India by using polymerase chain reactions. The V3 loops of 7 Indian isolates contained the apical tetra peptide GPGQ, while the V3 loop of one Indian isolate carried the apical tetra peptide GPGK. The amino acid sequences of the seven Indian isolates were closely related to each other, with an average of the nucleotide sequence homology of 96.0% (94.6 to 97.6%). The marked relatedness of the amino acid sequences among the seven Indian HIV-1 isolates indicated a recent and very rapid spread of this HIV-1 variant in Bombay. The amino acid sequence of the C2/V3 region of env gene of the 7 Indian isolates were homologous to the C subtype reported by Meyers et al. These findings could be useful in assessing the sources of infection and developing an AIDS vaccine.

[Risk of hepatitis C virus infection by needlestick among medical employees].

We have experienced 99 medical employees who had stuck themselves by the needles used by patients in the past 5 years. Sixteen of the 99 cases (16.2%) were of the patients who had hepatitis C Virus (HCV) antibodies. We followed up these 16 medical employees during 24.8 +/- 12.0 months. We could not find any case among them who had seroconverted HCV antibody positive in their serums. We concluded that the risk of HCV infection by the needlestick accident is not so high.

[HIV infection of CD4-CD8+ T-cell line derived from patients with HAM].

This studies have attempted human immunodeficiency virus (HIV) infection in four CD4+ or CD8+ T-cell lines derived from HTLV-I associated myelopathy virus (HAM) patients. Not only CD4+ cell line but also CD8+ cell line could be infected with HIV and CD4+ cell line showed a higher susceptibility than CD8+ cell line on HIV infection. HIV antigen in early stage after HIV inoculation was detected by using enzyme-linked immunosorbent assay (ELISA) rather than indirect immunofluorescence assay (IFA). HTLV-I producing CD4+ and CD8+ cell lines became to express two viral antigens (HTLV-I and HIV) after HIV inoculation. The results indicated that CD4-CD8+ T-cell line from patient with HAM can be infected with HIV. So that, we have found that other epitopes except for CD4 antigen may be associated with HIV infection.