Multiple High-Risk HPV Types Contribute to Cervical Dysplasia in Ugandan Women Living With HIV on Antiretroviral Therapy.

BACKGROUND: Cervical cancer mortality remains high in sub-Saharan Africa, especially among women living with HIV (WLWH). Characterization of prevalent high-risk human papillomavirus (hrHPV) types and immune function in WLWH with cervical abnormalities despite antiretroviral therapy (ART) can inform prevention strategies. SETTING: Kampala, Uganda. METHODS: From 2017 to 2020, we enrolled Ugandan women with cervical dysplasia detected with visual inspection with acetic acid (VIA). WLWH were required to be on ART >3 months with plasma HIV RNA <1000 copies/mL. Biopsies from VIA-positive lesions underwent histopathologic grading and cervical swab specimens were tested for hrHPV. Clinical correlations were evaluated with Poisson regression to estimate adjusted prevalence ratios (aPR). RESULTS: One hundred eighty-eight WLWH and 116 HIV-seronegative women participated. Among WLWH, median ART duration was 6 years and median CD4 667 cells/µL. Cervical intraepithelial neoplasia (CIN) grade 2/3 was found in 29% of WLWH versus 9% of HIV-seronegative women. In women with CIN1 or without histopathology-confirmed dysplasia, hrHPV (aPR [95% confidence interval]: 2.17 [1.43 to 3.29]) and multiple hrHPV (aPR 3.73 [1.07 to 13.1]) were more common in WLWH, as were vaccine-targeted and vaccine-untargeted hrHPVtypes. Differences in hrHPV prevalence by HIV serostatus were not observed in women with CIN2/3 (interaction P < 0.01). Among WLWH, low CD4/8 ratio was associated with hrHPV while detectable plasma HIV RNA (20-1000 copies/mL) was associated with CIN2/3 or invasive cancer. CONCLUSION: Despite ART, WLWH with cervical VIA abnormalities remain at elevated risk for multiple hrHPV and high-grade dysplasia. Cervical cancer prevention and research tailored for WLWH are warranted in the ART era.

Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells.

Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (<1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine.IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We studied CMV acquisition in infants and found that infections are usually brief and self-limited and are successfully established relatively rarely. Thus, although most people eventually acquire CMV infection, it usually requires numerous exposures. Our analyses indicate that this is because the virus is surprisingly inefficient, barely replicating well enough to spread to neighboring cells in the mouth. Greater knowledge of why CMV infection usually fails may provide insight into how to prevent it from succeeding.

Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses.

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1-49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children.

Conventional fluorescent-antibody (FA) methods were compared to real-time PCR assays for detection of respiratory syncytial virus (RSV), influenza virus type A (FluA), parainfluenza virus types 1, 2, and 3 (PIV1, PIV2, and PIV3), human metapneumovirus (MPV), and adenovirus (AdV) in 1,138 specimens from children with respiratory illnesses collected over a 1-year period. At least one virus was detected in 436 (38.3%) specimens by FA and in 608 (53.4%) specimens by PCR (P<0.001). Specimen quality was inadequate for FA in 52 (4.6%) specimens; 13 of these (25%) were positive by PCR. In contrast, 18 (1.6%) specimens could not be analyzed by PCR; 1 of these was positive by FA. The number of specimens positive only by PCR among specimens positive by PCR and/or FA was 18 (7.0%) of 257 for RSV, 18 (13.4%) of 134 for FluA, 25 (64.1%) of 39 for PIV1, 8 (88.9%) of 9 for PIV2, 17 (30.1%) of 55 for PIV3, and 101 (76.5%) of 132 for AdV. MPV was detected in 6.6% of all specimens and in 9.5% of the 702 specimens negative by FA. The mean number of virus copies per milliliter in specimens positive by both PCR and FA was significantly higher, at 6.7x10(7), than that in specimens positive only by PCR, at 4.1x10(4) (P<0.001). The PCR assays were significantly more sensitive than FA assays for detecting respiratory viruses, especially parainfluenza virus and adenovirus. Use of real-time PCR to identify viral respiratory pathogens in children will lead to improved diagnosis of respiratory illness.

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR.

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.

High incidence of ganciclovir-resistant cytomegalovirus infection among lung transplant recipients receiving preemptive therapy.

Preemptive antiviral therapy in transplant patients is thought to be less likely to lead to antiviral resistance than is routine prophylaxis. Cytomegalovirus (CMV)-seropositive lung transplant patients (R+) were assigned to receive pp65 antigen-guided ganciclovir therapy, and seronegative recipients of organs from seropositive donors (D+/R-) were assigned to receive initially preemptive and then routine ganciclovir prophylaxis. The incidence of infection with ganciclovir-resistant (ganR) CMV was assessed retrospectively. GanR CMV infection developed in 4 (9%) of 45 patients, at a median of 4.4 months (range, 3.1-6.6 months) after transplantation, and was more common among D+/R- patients than among R+ patients (3 of 11 vs. 1 of 34; P =.04). The incidence among patients who received preemptive therapy was similar to that among patients who received routine prophylaxis. All ganR isolates contained a UL97 mutation. GanR CMV infection occurs in nearly 10% of lung transplant recipients, despite preemptive antiviral therapy, and is more common among D+/R- patients.