The severe acute respiratory syndrome coronavirus in tears.

BACKGROUND: Severe acute respiratory syndrome (SARS) is a new infectious disease that caused a global outbreak in 2003. Research has shown that it is caused by a novel coronavirus. A series of cases is reported where polymerase chain reaction (PCR) testing on tears had demonstrated the presence of the virus. Detection of ocular infection from tears using the PCR technique has been widely used by ophthalmologists to diagnose infections for other viruses. METHODS: This is a case series report from cases classified as probable or suspect SARS cases. Tear samples were collected from 36 consecutive patients who were suspected of having SARS in Singapore over a period of 12 days (7-18 April 2003), and analysed by PCR using protocols developed by the WHO network of laboratories. RESULTS: Three patients with probable SARS (one female and two male patients) had positive results from their tear samples. Tear samples were used to confirm SARS in the female patient, who was positive only from her tears. The positive specimens were found in cases sampled early in their course of infection. CONCLUSIONS: This is the first case series reported with the detection of the SARS coronavirus from tears, and has important implications for the practice of ophthalmology and medicine. The ability to detect and isolate the virus in the early phase of the disease may be an important diagnostic tool for future patients and tear sampling is both simple and easily repeatable. Many healthcare workers are in close proximity to the eyes of patients and this may be a source of spread among healthcare workers and inoculating patients. Ophthalmic practices may need to change as more stringent barrier methods, appropriate quarantine, and isolation measures are vital when managing patients with SARS.

Prevention of human immunodeficiency virus (HIV) transmission from mother to child--a cohort study in Singapore.

INTRODUCTION: The landmark Paediatric AIDS Clinical Trials Group (PACTG) trial 076 showed in 1994 that antiretroviral therapy (ART) was effective in reducing maternal-child transmission of human immunodeficiency virus (HIV). This trial included antenatal oral zidovudine (ZDV), intrapartum intravenous ZDV, 6 weeks of oral ZDV to the babies and no breastfeeding. MATERIALS AND METHODS: This study is an on-going, prospective, open-label trial conducted from 1995 in which we enrolled HIV-infected pregnant women using the above strategy. Since 1997, the antenatal component of the regimen was modified to include lamivudine with ZDV. All babies had serial HIV polymerase chain reaction (PCR) and antibody tests including enzyme-linked immunosorbent assay (EIA), particle agglutination (PA) and Western blot (WB) at day 1, 1 week, 1, 2, 3, 6, 12 and 18 months. RESULTS: A total of 16 out of 19 eligible women were recruited from 1995 to 1999. The median age was 26 years (range 22 to 38 years), 38% were Singaporeans, median CD4 was 421 cells/mL (range 18 to 713 cells/mL) and median baseline gestational age was 23.5 weeks (range 8 to 32 weeks). None of the 16 children was infected as evidenced by 2 negative HIV PCRs including 1 done > 4 months old with a follow-up of 6 months to 2 years. There was a statistically significant difference between the 3 HIV antibody tests at 12 months of age (P = 0.003), there being more negative results with WB as compared to PA (P = 0.02). However, the difference between the 3 tests at 18 months was not statistically significant. No long-term side effects in these children were seen. CONCLUSION: Although the number of patients in this study is small, the absolute prevention of transmission (95% confidence intervals 0%-17%) in this cohort supports the recommendation of antenatal HIV screening and treatment of those infected.

Routine screening for Chlamydia trachomatis in subfertile women--is it time to start?

UNLABELLED: Genital Chlamydia trachomatis infection has long been recognised as the major cause of pelvic disease and subsequently infertility. The diagnosis of this infection has traditionally relied on tissue culture. The availability of DNA amplification methods like ligase chain reaction promises faster and more sensitive results. This study was conducted to evaluate the prevalence of chlamydial infection in a subfertile population subgroup. AIM: A case control longitudinal study of 100 subfertile women in a tertiary teaching hospital were analyzed for the prevalence of genital Chlamydia trachomatis infection using ligase chain reaction test kit. RESULTS: A prevalence rate of 8% was detected, the majority being 25 years old or less (33.3%), p = 0.007. All patients gave no prior history of abnormal PAP smears, hospitalisation for pelvic inflammatory disease or abnormal vaginal discharge at the time of investigation. CONCLUSION: Our infertile group of patients has a relatively high incidence of silent genital Chlamydia trachomatis infection. This being highest in the below 25 years old age group. This finding indicates that screening for chlamydia may be necessary for the subfertile couple presenting to clinic. This is especially so if the patient is of the younger age group.

Alteration of virus entry mode: a neutralisation mechanism for Dengue-2 virus.

Polyclonal antibodies derived from dengue virus immune sera and 3H5 monoclonal antibody showed potent neutralisation effect on dengue-2 virus in the plaque reduction neutralisation assay. This study demonstrated that antibodies present in immune human sera and 3H5 monoclonal antibody neutralised dengue-2 virus by altering the virus entry pathway into cells. In the presence of neutralising antibodies, dengue-2 virus was endocytosed by LLC-MK2 cells. The endocytosis process involved ruffling of antibody-coated virions by cellular pseudopodia and invagination of cell membrane. This mode of entry is atypical as compared to direct fusion of dengue-2 virus with cell membrane in the absence of antibody. The virions were internalised in the form of virion-antibody complexes consisting of single or clumps of virions. After 3 minutes of incubation, neutralised virions were detected in cellular vesicles, and signs of intra-endosomal penetration into cytoplasm were not evident even after a prolonged incubation of 10 minutes, suggesting that viral uncoating was compromised. Vesicle-bound virions were no longer detected after 20 minutes of incubation. In addition, no sign of viral replication was detected in cells infected with "neutralised" virions by immunofluorescence assay. This indicated that internalised virions had been degraded leading to abortive infection. In conclusion, antibodies present in 3H5 monoclonal antibody and human immune sera rendered dengue-2 virus non-infective by neutralising the viral fusion site and causing alteration of viral entry mode. Antibodies in immune sera but not 3H5 monoclonal antibody also exerted minimal inhibitory effect on virus binding and internalisation.

Failure of routine HIV-1 tests in a case involving transmission with preseroconversion blood components during the infectious window period.

CONTEXT: Current screening practices for blood donations have been successful in reducing human immunodeficiency virus (HIV) transmission through receipt of contaminated blood products. However, HIV-infected blood donations made prior to seroconversion and before high levels of viral replication occur could test negative using both serologic antigen and antibody tests. Testing based on nucleic acid amplification (NAT) is being implemented to screen for HIV-infected blood donated during this period, yet the issue of single vs minipool donation screening remains unresolved. OBJECTIVES: To determine HIV-1 genetic linkage between virus in 2 HIV-1-infected recipients of blood components and virus in the donor, who was HIV antigen and antibody negative at the time of donation; to screen the blood donor's plasma with HIV NAT assays, including those currently proposed for use in US blood donation screening. DESIGN AND SETTING: Case study conducted in October 1997 involving the Communicable Disease Centre, Singapore General Hospital, and the Singapore Blood Transfusion Service, Singapore. SUBJECTS: The blood donor and the 2 recipients of donor platelets and red blood cells. MAIN OUTCOME MEASURES: Genetic analysis of the HIV-1 p17 coding region of gag and the C2V5 region of env to determine the genetic relatedness of virus from the donor and recipients; reactivity in quantitative and qualitative assays, and reactivity in donor screening HIV NAT assays in single donation and minipool screening contexts. RESULTS: Direct DNA sequencing demonstrated identical HIV-1 subtype E viral sequences in the donor and recipients. Based on comparisons of a qualitative and quantitative assay for HIV-1 RNA levels, a low level of viremia (range, 5-39 copies/mL in plasma) was estimated to be in the donor's undiluted blood at the time of donation. Additional testing using donor-screening NAT assays showed consistent detection of HIV RNA in the undiluted donor plasma whereas detection was inconsistent at the 1:16 and 1:24 dilution levels currently used in minipool screening of blood donations in the United States. CONCLUSIONS: Transmission of HIV from a blood donor to a platelet recipient and a red blood cell recipient occurred in the preseroconversion infectious window period. The viral load in the implicated donation was estimated to be less than 40 copies/mL of plasma. Current US minipool HIV NAT screening protocols may not be sufficiently sensitive to detect all infectious window-period donations. JAMA. 2000;284:210-214

Correlation of baseline quantitative plasma human immunodeficiency (HIV) type 1 RNA viral load with clinical status and CD4+ T-cell counts in treatment-naïve HIV-positive patients in Singapore.

INTRODUCTION: Quantitative measurement of plasma HIV-1 RNA viral load has been available in Singapore since 30 November 1996. This study investigates the relationship, in antiretroviral-naïve, HIV-positive Singapore residents, between the baseline HIV-1 RNA viral load and clinical status at the time of quantification. The association of HIV-1 RNA viral load with CD4+ T-cell counts was also studied. MATERIALS AND METHODS: HIV-1 RNA viral load was determined using Amplicor HIV-1 Monitor Test. One hundred and eighty subjects had baseline plasma HIV-1 RNA levels quantified during the period 30 November 1996 to 27 July 1998. They were classified into three clinical groups: A for asymptomatic infection (n = 110), B for symptomatic infection (n = 29) and C for AIDS (n = 41). RESULTS: The differences between mean HIV-1 RNA levels were statistically significant (P < 0.001) for groups A and B (mean difference = -0.61 log10), and for groups A and C (mean difference = -0.75 log10). However, there was no statistically significant difference (P = 0.68) between groups B and C (mean difference = -0.13 log10). Of those subjects with CD4+ T-cell counts measured within 30 days of viral load quantification, there were statistically significant negative correlations between HIV-1 viral load and CD4+ T-cell counts for groups A (n = 91, r = -0.536, P < 0.01) and C (n = 34, r = -0.446, P < 0.01) but not group B (n = 26, r = -0.297, P > 0.05). CONCLUSION: This suggest that the more advanced the phase of HIV infection, the higher the baseline plasma viral load and the lower the CD4+ T-lymphocyte counts.

Retrospective study of Western blot profiles in immune sera of natural dengue virus infections.

The Western blot (WB) assay was used to determine dengue virus antibodies present in human immune sera arising from recent primary and secondary dengue virus infections in Singapore. Cell lysates of dengue-2 virus-infected C6/36 and Vero cells were used. Antibodies directed against structural proteins of dengue-2 virus including envelope (E, gp60/50), capsid-premembrane (C-PrM, gp35), and premembrane (PrM, gp20) were detected, with antibody against envelope protein being most dominant. Similar WB profiles were detected in both primary and secondary dengue virus infections. The reactivity rate of antibodies to dengue-2 virus proteins was higher in infected Vero cell lysate than in infected C6/36 cell lysate, with the exception of antibodies to nonstructural proteins of NS1 and NS3, which were detected predominantly in infected C6/36 cell lysate. More than 75% of "normal" individuals (with no complaint of recent dengue virus infection) examined had low levels of dengue virus antibodies, but all presented with similar WB profiles as patients with recent dengue virus infections. This finding reflects a high seroprevalence of dengue virus infections and the long lasting nature of E, C-PrM, and PrM antibodies. Results from this study indicate that in natural dengue virus infections, native E, C-PrM, and PrM antigens of dengue virus are immunogenic and elicit long-lasting antibodies.

Evaluation of Amplicor- and IS6110-PCR for direct detection of Mycobacterium tuberculosis complex in Singapore.

One hundred and seventy-eight samples from 168 individuals were tested for Mycobacterum tuberculosis complex (Mtc) using Amplicor PCR, IS6110-PCR (in-house), acid fast (AF)-staining and culture. Thirty-one samples were positive by culture, but 37 samples were later resolved to be truly positive for Mtc. Of these, Amplicor detected 32 (86.5%), IS6110-PCR detected 31 (83.6%), and AF-staining 21 (56.8%). None of the 141 Mtc-negative samples was positive by these tests, thus giving 100% specificity. Although the IS6110-PCR was more sensitive than Amplicor in detecting spiked Mtc DNA, it was not more sensitive than the latter in detecting Mtc in clinical samples. Reasons likely to account for the PCR false negativity were (i) sample inoculum size, (ii) nonuniform samples due to clumping effect of Mtc and (iii) the absence of target gene sequences for IS6110-PCR. Culture negativity, on the other hand, was likely to be associated with nonviable Mtc. Amplicor PCR is promising for direct detection of Mtc. The IS6110-PCR, however, may not be as suitable because of possible existence of IS6110-deleted Mtc strain in Singapore.

Human immunodeficiency virus (HIV) antigen testing to detect HIV infection in female sex workers in Singapore.

Human immunodeficiency virus (HIV) infection is characterised by seroconversion after a ¿window¿ period of 2 to 3 months. After this period antibodies are usually detectable by screening tests (enzyme immunoassay or particle agglutination) confirmed by Western blot analysis. We studied 1000 newly enrolled female sex workers who had not been previously tested for HIV to assess the usefulness of HIV antigen testing to improve the efficacy of HIV infection detection. Blood was taken at enrollment for HIV antigen and HIV antibody testing. The Abbott HIVAG-1 test was used to detect antigen; antibody detection was by the Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay (EIA) test, the Fujirebio Serodia-HIV particle agglutination (PA) test for screening, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot (WB) test for antibody confirmation. Of the 1000 samples, 26 were positive for HIV antibody testing (26/26 for EIA, 25/25 for PA, 26/26 for WB), giving a prevalence rate of 2.6%, Of these 26 seropositive samples 1 was positive on HIV antigen testing. There were no samples which were antigen-positive and antibody-negative. HIV antigen testing does not add to increased efficacy of HIV detection among female sex workers in Singapore.

PIP: Since the HIV p24 antigen appears a few weeks before the HIV antibody, researchers conducted a study to determine whether the HIV antigen test Abbott HIV AG-1 would identify recently HIV-infected female sex workers in Singapore whose infection might be missed if only HIV antibody tests were used. During April 1993-April 1994, blood samples were taken from 1000 female sex workers newly enrolled in the Department of STD (sexually transmitted disease) Control of Singapore General Hospital to test for the HIV p24 antigen. Results of the Abbott HIV AG-1 test were compared with 3 HIV antibody tests (Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay [EIA] test, the Fujirebio Serodia-HIV particle agglutination [PA] test, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot [WB] test). All 3 HIV antibody tests found 25 female prostitutes to be HIV positive. The EIA and WB tests found 26 women to be HIV positive for a prevalence rate of 2.6%. Only 1 specimen tested positive for HIV antigen. This specimen also tested positive for HIV antibodies. There was no HIV antigen positive specimen that was HIV antibody negative or indeterminate. These findings show that the HIV antigen test did not improve the detection rate of HIV infection in these female sex workers, since there were no HIV antigen specimens that were HIV antibody negative or indeterminate.

Elevated secretory IgA antibodies to Epstein-Barr virus (EBV) and presence of EBV DNA and EBV receptors in patients with cervical carcinoma.

Epstein-Barr virus (EBV) receptors (EBV/C3d receptors) were detected, using the monoclonal antibody HB5, on 23 ectocervical and 5 endocervical biopsies of the uterine cervix. Elevated IgA titers against the viral capsid antigen and early antigen of EBV were also found in the cervical secretions from cervical carcinoma patients (83%), compared with samples from patients with cervical intraepithelial neoplasia (75%), herpes simplex virus-infected patients (0%), and gynecologic patients with nonmalignant conditions (0%). EBV DNA was present in 63% of cervical carcinoma biopsies detected by in situ hybridization. These observations suggest a positive association between EBV and carcinoma of the cervix.

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology.

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.