HBV lamivudine resistance among hepatitis B and HIV coinfected patients starting lamivudine, stavudine and nevirapine in Kenya.

Widespread use of lamivudine in antiretroviral therapy may lead to hepatitis B virus resistance in HIV-HBV coinfected patients from endemic settings where tenofovir is not readily available. We evaluated 389 Kenyan HIV-infected adults before and for 18 months after starting highly active antiretroviral therapy with stavudine, lamivudine and nevirapine. Twenty-seven (6.9%) were HBsAg positive and anti-HBs negative, 24 were HBeAg negative, and 18 had HBV DNA levels ≤ 10,000 IU/mL. Sustained HBV suppression to <100 IU/mL occurred in 89% of 19 evaluable patients. Resistance occurred in only two subjects, both with high baseline HBV DNA levels. Lamivudine resistance can emerge in the setting of incomplete HBV suppression but was infrequently observed among HIV-HBV coinfected patients with low baseline HBV DNA levels.

Comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients.

BACKGROUND: Sensitive detection of respiratory viruses is important for early diagnosis of infection in patients following hematopoietic cell transplantation (HCT). To evaluate the relative sensitivity of respiratory virus detection in specimens from HCT recipients, we compared the results of conventional and quantitative molecular methods. METHODS: We tested 688 nasal wash samples collected prospectively from 131 patients during the first 100 days after HCT by viral culture, fluorescent antibody staining (FA), and real-time quantitative reverse transcription-polymerase chain reaction (PCR) assay for detection of respiratory syncytial virus (RSV), influenza virus types A (FluA) and B (FluB), and parainfluenza virus types 1 (PIV1) and 3 (PIV3). Testing for human metapneumovirus (MPV) was performed only by PCR. Data regarding 10 respiratory symptoms were collected with each sample. RESULTS: By any method 37 specimens were positive for a respiratory virus; 34 were positive by PCR, 15 by culture, and 6 by FA. Four specimens were positive by all 3 methods (3 RSV, 1 FluA). One specimen was positive for PIV1, and 2 were positive for rhinovirus by culture alone. Specimens positive by PCR alone included 2 RSV, 2 PIV1, 8 PIV3, and 8 MPV. In 10 specimens positive for RSV, PIV, or influenza virus collected from patients reporting no respiratory symptoms, 9, 4, and 1 specimen were positive by PCR, culture, and FA, respectively. Overall, specimens positive only by PCR had significantly fewer viral copies/mL (mean log(10)=4.32) than specimens positive by both PCR and culture (mean log(10)=5.75; P=0.002) or PCR and FA (mean log(10)=6.83; P<0.001). CONCLUSIONS: FA testing alone did not detect a significant proportion of respiratory virus-positive samples in HCT recipients, especially in patients with no respiratory symptoms and patients with PIV detection. PCR increased the yield of positive specimens 2 times relative to culture and more than 4 times relative to FA. Detection of respiratory viruses by PCR alone was associated with lower virus quantities and with fewer reported respiratory symptoms compared with concomitant detection by both PCR and conventional methods, indicating that PCR may be important to detect asymptomatic or mildly symptomatic stages of respiratory viral infections.

Use of a glycoprotein G-based type-specific assay to detect antibodies to herpes simplex virus type 2 among persons attending sexually transmitted disease clinics.

BACKGROUND: Most genital herpes simplex virus type 2 (HSV-2) infections are unrecognized, thus, strategies to reduce the sexual transmission of HSV-2 are partly dependent on serologic screening. GOAL: To define performance characteristics of the Gull/ Meridian glycoprotein G-based HSV-2 enzyme-linked immunosorbent assay among sexually transmitted disease clinic attendees and correlates of test acceptance. STUDY DESIGN: The cross-sectional study was conducted during two periods. Serologic testing was offered at a US $15 charge during the first period and at no charge during the second period. Sera were tested by a type-specific glycoprotein G enzyme-linked immunosorbent assay and Western blot analysis, with the latter test used as the reference standard. RESULTS: Acceptance of HSV-2 testing was associated with free testing (odds ratio, 7.5; 95% CI, 6.0-9.9), older age, and white race. Sensitivity of the HSV-2 assay was 80.5% and specificity was 98.5%. The HSV-2 positive and negative predictive values were 95.8% (95% CI, 91.6-98.0%) and 92.2% (95 % CI, 89.6 -94.2%), respectively. Antibodies to HSV-2 were detected in 25.9% of 606 persons with no history of genital herpes. CONCLUSION: Acceptance of HSV-2 serologic testing was cost sensitive. In this high-prevalence population, the positive predictive value of the enzyme-linked immunosorbent assay was sufficient to warrant its use without a confirmatory test. This assay could be useful in the screening of sexually active adults to detect unrecognized HSV-2 infection.

High-throughput quantitative analysis of hepatitis B virus DNA in serum using the TaqMan fluorogenic detection system.

Reproducible quantitative assays to detect viral nucleic acids have proven useful in defining disease progression and following response to therapy in a wide variety of viral infections. We describe the development of a quantitative assay to detect hepatitis B virus (HBV) DNA using real-time fluorescent-probe polymerase chain reaction (PCR) (TaqMan). The assay is highly reproducible, highly automated, and much more sensitive than the currently used branched-chain DNA (bDNA) assay for HBV. The quantitative PCR assay accurately detected samples ranging from 10 to 10(9) copies of HBV DNA per milliliter. Of 157 serum samples submitted for HBV quantitation, 119 were positive by TaqMan PCR versus only 55 by bDNA (P <.001). All 55 bDNA-positives were positive by TaqMan. Of the 77 samples with detectable HBV-DNA titers below 3.75 x 10(5) copies by TaqMan, only 13 were detected by bDNA. We tested 119 patients negative for all HBV serologic markers, and all tested negative in the TaqMan assay. HBV DNA was detected by TaqMan in 164 of 195 (84%) of hepatitis B surface antigen (HBsAg)-positive samples. Among hepatitis B e antigen (HBeAg)-positive samples, median titers were 4. 3 x 10(6) copies/mL versus 322 copies/mL in HBeAg-negative samples (P =.012). The TaqMan assay for HBV DNA is highly sensitive and reproducible and thus appears useful in accurately defining levels of viral replication among persons with HBV infection.

Risk factors for HIV-1 shedding in semen.

Semen is the body fluid most commonly associated with sexual transmission of human immunodeficiency virus type-1 (HIV-1). Because the male genitourinary tract is distinct immunologically from blood, compartment-dependent factors may determine HIV-1 shedding in semen. To identify these factors, the authors obtained 411 semen and blood specimens from 149 men seen up to three times. Seminal plasma was assayed for HIV-1 RNA and semen was cocultured for HIV-1 and cytomegalovirus (CMV), which may up-regulate HIV-1 replication. The best multivariate model for predicting a positive semen HIV-1 coculture included two local urogenital factors, increased seminal polymorphonuclear cell count (odds ratio (OR) = 12.6 for each log10 increase/mL, 95% confidence interval (CI) 12.2, 134.5) and a positive CMV coculture (OR = 3.0, 95% CI 1.2, 7.7). The best multivariate model for predicting semen HIV-1 RNA included two systemic host factors, CD4+ cell counts <200/microliter (OR = 3.0, 95 percent CI 1.3, 6.9) and nucleoside antiretroviral therapy (monotherapy: OR = 0.5, 95% CI 0.3, 1.0; combination therapy: OR = 0.4, 95% CI 0.2, 0.9), and a positive CMV coculture (OR = 1.7, 95% CI 1.0, 3.0). Thus, both systemic and local genitourinary tract factors influence the risk of semen HIV-1 shedding. These findings suggest that measures of systemic virus burden alone may not predict semen infectivity reliably.

Inability of enzyme immunoassays to discriminate between infections with herpes simplex virus types 1 and 2.

OBJECTIVE: To determine the accuracy of three commercial enzyme immunoassays in detecting and subtyping antibodies to herpes simplex virus type 1 or 2. DESIGN: Cross-sectional. SETTING: Referral medical center. PATIENTS: Ninety patients with culture-positive lesions caused by infection with herpes simplex virus type 1 or 2. The results of Western blot and glycoprotein G immunodot enzyme assays showed that an additional 53 patients had subclinical herpes simplex virus 2 infection, that another 20 patients had subclinical herpes simplex virus type 1 infection, and that 23 patients were seronegative. MEASUREMENTS: Three commercial enzyme immunoassays were used to determine herpes simplex virus antibody subtypes. MAIN RESULTS: All three commercial assays performed poorly in all patient groups (except in patients who were seronegative for herpes simplex virus). Among the 40 patients with a first episode of genital herpes, seroconversion to the appropriate viral type was shown by the three assays in only 33%, 55%, and 75% of cases. Among patients with recurrent genital herpes, the three commercial assays identified more than 90% of patients with only herpes simplex virus type 2 antibodies but failed to identify herpes simplex virus type 2 infections in 58% to 76% of patients with antibodies to both virus subtypes. The three assays correctly identified only 55%, 75%, and 85% of the 53 "silent carriers" of herpes simplex virus type 2. Overall, the three enzyme immunoassays detected herpes simplex virus type 2 antibodies in 60%, 62%, and 93% of patients with subtype 2 infections and falsely detected type 2 antibodies in 8%, 27%, and 49% of patients with type 1 infections. CONCLUSION: Currently licensed enzyme immunoassays give inaccurate or misleading results about the correct herpes simplex virus infecting subtype.

Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a high-prevalence population.

One hundred thirty specimens from 108 consecutive patients with a history of recurrent genital ulcerations were used in a study comparing herpes simplex virus (HSV) isolation with a direct fluorescent-antibody (DFA) technique using mouse monoclonal antibodies. HSV was isolated from 70% of vesicular lesions, 67% of pustular lesions, 32% of ulcerative lesions, and 17% of crusted lesions, whereas the DFA technique detected HSV antigen in 87, 67, 30, and 10% of lesions in similar stages, respectively. When both methods were used, HSV was identified in 97, 79, 45, and 17% of vesicles, pustules, ulcers, and crusted lesions, respectively. The overall sensitivity and specificity of the DFA technique in comparison with virus isolation (VI) were 74 and 85%, respectively. Of the 17 patients from whom DFA-positive, VI-negative samples were obtained, HSV was subsequently isolated from a genital lesion in 14, suggesting that they were not DFA false-positives. Similarly, of the 46 patients whose initial lesion samples were DFA and VI negative, 37 (80%) had HSV identified from subsequent genital lesions during follow-up. Thus, a single sample for VI or DFA testing from a recurrent genital lesion had a sensitivity of only 53 and 51%, respectively. Combining the DFA technique and VI increased the sensitivity of laboratory diagnosis of a single recurrent episode of genital HSV; however, repetitive laboratory testing was often required to confirm the diagnosis of recurrent genital HSV infection.