Optimization and Local Cost-Effectiveness of Nasopharyngeal Carcinoma Screening Strategies in Southern China: Secondary Analysis of the Guangdong Randomized Trial.

BACKGROUND: Screening with anti-Epstein-Barr Virus (EBV) serology and endoscopy decreased nasopharyngeal carcinoma (NPC) mortality in Guangdong in a randomized trial. We conducted a secondary analysis of this trial using local incidence and cost data to optimize screening programs, hypothesizing that screening could be cost-effective in southern China. METHODS: Screening costs and life-years after NPC diagnosis were obtained from the Guangdong trial's intent-to-screen population (men and women age 30-69). Seropositive subjects were rescreened annually for five years. Thereafter, we evaluated 12 screening strategies in Guangdong and Guangxi using a validated model. Strategies used combinations of serology, nasopharyngeal swab PCR (NP PCR), endoscopy, and MRI from trial sub-cohorts. Incidence data and costs were obtained from local cancer registries and the provincial healthcare system. RESULTS: In the intent-to-screen population, screening with serology and endoscopy was cost-effective (¥42,366/life-year, 0.52 GDP per-capita). Screening for 5-15 years between ages 35-59 met a willingness-to-pay threshold of 1.5 GDP/QALY in all modeled populations. Despite doubling costs, adding MRI could be cost-effective via improved sensitivity. NP PCR triage reduced endoscopy/MRI referrals by 37%. One lifetime screen could reduce NPC mortality by pproximately 20%. CONCLUSIONS: EBV-based serologic screening for NPC is likely to be cost-effective in southern China. Among seropositive subjects, the preferred strategies use endoscopy alone or selective endoscopy triaged by MRI with or without NP PCR. These data may aid the design of screening programs in this region. IMPACT: These findings support population-based screening in southern China by defining the target population, cost effectiveness, and optimized screening approach.

BA.5 bivalent booster vaccination enhances neutralization of XBB.1.5, XBB.1.16 and XBB.1.9 variants in patients with lung cancer.

This study reports that most patients with NSCLC had a significant increase in the nAb response to the currently circulating Omicron variants after bivalent booster vaccination and had Ab titers comparable to healthy participants. Interestingly, though the durability of the nAb response persisted in most of the healthy participants, patients with NSCLC had significantly reduced nAb titers after 4-6 months of vaccination. Our data highlight the importance of COVID-19 bivalent booster vaccination as the standard of care for patients with NSCLC given the evolution of new variants of concern.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Identification and confirmation via in situ hybridization of Merkel cell polyomavirus in rare cases of posttransplant cutaneous T-cell lymphoma.

BACKGROUND: Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens. METHODS: Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip. RESULTS: Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes. CONCLUSIONS: Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.

Comparison of Real-Time PCR and Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma.

Plasma Epstein-Barr virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma. However, existing real-time quantitative PCR (qPCR) assays are limited by poor interlaboratory reproducibility. This is a barrier to biomarker integration into staging systems and management. It was hypothesized that EBV digital PCR (dPCR) would have similar sensitivity but improved precision relative to qPCR. Using the World Health Organization EBV standard and patient specimens, the NRG-HN001 BamHI-W qPCR, two commercial EBNA-1 qPCR assays, and two laboratory-developed dPCR assays amplifying the BamHI-W, EBNA-1, and EBER targets were compared. Testing was conducted in the North American reference laboratory for the NRG-HN001 randomized trial. The EBV dPCR assays achieved similar performance compared with qPCR. Although dPCR does not require quantitation standards, different dPCR thresholding algorithms yielded significant qualitative and quantitative variation. This was most evident with low levels of EBV DNA. No-template control-informed thresholding (ddpcRquant) mitigated false-positive/false-negative findings. The NRG-HN001 BamHI-W qPCR and laboratory-developed BamHI-W droplet dPCR offered higher sensitivity, lower limit of blank, higher precision at low plasma EBV DNA levels (≤1500 IU/mL), and higher overall agreement with clinical specimens versus single-copy qPCR/dPCR targets (EBNA-1/EBER). These data confirm the rationale for using the BamHI-W target to define prognostic thresholds and indicate that both qPCR and dPCR methods harmonized to the World Health Organization standard can provide the necessary analytical performance.

Evaluation of a Semiautomated System for the Quantitation of Human Adenovirus DNA from Clinical Samples.

Human adenoviruses (HAdVs) cause severe disease in immunocompromised patients. Quantitation of HAdV DNA in peripheral blood is used to assess the risk of disseminated disease and to monitor response to therapy. The lower limit of detection, precision, and linearity of the semiautomated AltoStar adenovirus quantitative PCR (qPCR) was evaluated using reference HAdV-E4 in EDTA plasma and respiratory virus matrix. Qualitative and quantitative agreement was determined using 122 clinical EDTA plasma specimens previously tested using a laboratory-developed HAdV qPCR. The 95% lower limit of detection (LLOD) was 33 IU/mL (95% confidence interval [CI], 10 to 56) for EDTA plasma and 188 IU/mL (95% CI, 145 to 304) for respiratory swab matrix. In both matrices, the AltoStar HAdV qPCR was linear from 7.0 to 2.0 log10 IU/mL. For the clinical specimens, overall agreement was 96.7% (95% CI, 91.8 to 99.1), positive percent agreement was 95.5% (95% CI, 87.6 to 98.5), and negative percent agreement was 98.2% (95% CI, 88.5 to 99.7). Passing-Bablok analysis of specimens quantifiable by both methods revealed a regression line of Y = 1.11 · X + 0.00; there was positive proportional bias (95% CI of the slope, 1.05 to 1.22) but no systematic bias (95% CI of the Y-intercept, -0.43 to 0.23) compared to the reference. The AltoStar platform provides accurate quantitation of HAdV DNA and provides a semiautomated option for the clinical monitoring of HAdV following transplantation. IMPORTANCE Accurate quantification of human adenovirus DNA in the peripheral blood plays a critical role in the management of adenovirus infections in transplant recipients. Many laboratories utilize in-house laboratory-based PCR assays for the quantification of human adenovirus, as there are few commercial options available. Here, we describe the analytical and clinical performance of the semiautomated AltoStar adenovirus quantitative PCR (Altona Diagnostics). This platform provides sensitive, precise, and accurate quantification of adenovirus DNA that is well suited for virological testing following transplantation. Prior to implementing a new quantitative test in the clinical laboratory, a rigorous evaluation is required to determine assay performance characteristics and to correlate results to current in-house methods of quantitation.

Recommendations for Epstein-Barr virus-based screening for nasopharyngeal cancer in high- and intermediate-risk regions.

A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Outpatient human coronavirus associated conjunctivitis in India.

BACKGROUND: Viral conjunctivitis (pink eye) can be highly contagious and is of public health importance. There remains significant debate whether SARS-CoV-2 can present as a primary conjunctivitis. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge. METHODS: This prospective study was conducted in the spring and summer months of 2021. 106 patients with acute conjunctivitis who presented to the Aravind Eye Center in Madurai, India were included. One anterior nasal swab and one conjunctival swab of each eye were obtained for each enrolled patient. Samples were subsequently processed for unbiased metagenomic RNA deep sequencing (RNA-seq). Outcomes included clinical findings and codetection of other pathogens with SARS-CoV-2 in patients with conjunctivitis. RESULTS: Among the 13 patients identified with human coronavirus RNA fragments in their swabs, 6 patients had SARS-CoV-2 infection, 5 patients had coinfections of SARS-CoV-2 and human adenovirus (HAdV), 1 patient had a coinfection with human coronavirus OC43 and HAdV, and 1 patient had a coinfection of Vittaforma corneae and SARS-CoV-2. 30% had bilateral disease and symptoms on presentation. Petechial hemorrhage was noted in 33% of patients with SARS-CoV-2 infection. No patients with SARS-CoV-2 or SARS-CoV-2 and HAdV infections had subepithelial infiltrates on presentation. All patients denied systemic symptoms. CONCLUSIONS: Among the patients presented with conjunctivitis associated with human coronavirus infection, over 50% of the patients had co-infections with other circulating pathogens, suggesting the public-health importance of broad pathogen testing and surveillance in the outpatient conjunctivitis population.

Deep sequencing analysis of clinical samples from patients with acute infectious conjunctivitis during the COVID-19 delta surge in Madurai, India.

BACKGROUND: Seasonal outbreaks of infectious conjunctivitis remain a public health issue. Determination of outbreak etiologies in the context of a worldwide pandemic may provide useful information to guide public health strategies. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge. METHODS: This prospective study was conducted from April 2021 to September 2021. All outpatients presenting to the Aravind Eye Center (Madurai, India) with signs and symptoms consistent with acute infectious conjunctivitis were eligible. Three swabs were obtained from each participant: one from each conjunctiva and one from the anterior nares. Samples were processed for metagenomic RNA deep sequencing (RNA-seq). RESULTS: Samples from 106 study participants were sequenced. The most common presenting symptoms were tearing (86%) and itching (71%). Preauricular lymphadenopathy was present in 38% of participants. 20% of participants had close contacts with similar symptoms. Systemic symptoms such as coughing, runny nose, vomiting or diarrhea were uncommonly reported. 60% of all participants used some medicated eye drops upon enrollment. 75% of study participants demonstrated infection with human adenovirus D (HAdV-D). 11% of conjunctivitis was associated with SARS-CoV-2. 15% had no definitive pathogen detected. 8% of all participants had codetection of more than one pathogen on RNA-seq. CONCLUSIONS: During the COVID-19 Delta surge in India, HAdV-D was the most common pathogen associated with infectious conjunctivitis. SARS-CoV-2 was the second most common associated pathogen. Seasonal surveillance may be necessary for the determination of emerging and reemerging pathogens responsible for infectious conjunctivitis.

Multiplex Epstein-Barr virus BALF2 genotyping detects high-risk variants in plasma for population screening of nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) exhibits unusual geographic restriction despite ubiquitous lifelong infection. Screening programs can detect most NPC cases at an early stage, but existing EBV diagnostics are limited by false positives and low positive predictive value (PPV), leading to excess screening endoscopies, MRIs, and repeated testing. Recent EBV genome-wide association studies (GWAS) suggest that EBV BALF2 variants account for more than 80% of attributable NPC risk. We therefore hypothesized that high-risk BALF2 variants could be readily detected in plasma for once-lifetime screening triage. METHODS: We designed and validated a multiplex genotyping assay to detect EBV BALF2 polymorphisms in human plasma. Targeted next-generation sequencing was used to validate this assay, conduct association studies with clinical phenotype, and longitudinally genotype plasma to assess within-host haplotype stability. We examined the association between NPC and BALF2 haplotypes in a large non-endemic population and three prior EBV GWAS. Finally, we estimated NPC mortality reduction, resource utilization, and cost-effectiveness of BALF2 variant-informed screening using a previously-validated cohort model. RESULTS: Following analytical validation, the BALF2 genotyping assay had 99.3% concordance with sequencing in a cohort of 24 NPC cases and 155 non-NPC controls. BALF2 haplotype was highly associated with NPC in this non-endemic population (I613V: odds ratio [OR] 7.9; V317M: OR 178.8). No other candidate BALF2 polymorphisms were significantly associated with NPC or hematologic disorders. Longitudinal genotyping revealed 97.8% within-host haplotype concordance, indicative of lifelong latent infection. In a meta-analysis of 755 NPC cases and 981 non-NPC controls, BALF2 I613V and V317M were significantly associated with NPC in both endemic and non-endemic populations. Modeled variant-informed screening strategies achieved a 46% relative increase in PPV with 7% decrease in effective screening sensitivity, thereby averting nearly half of screening endoscopies/MRIs among endemic populations in east/southeast Asia. CONCLUSIONS: EBV BALF2 haplotypes are temporally stable within hosts and can be readily detected in plasma via an inexpensive multiplex genotyping assay that offers near-perfect sequencing concordance. In endemic and non-endemic populations, I613V and V317M were highly associated with NPC and could be leveraged to develop variant-informed screening programs that mitigate false positives with small reductions in screening sensitivity.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study.

BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response. METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naïve individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination. RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P < 0.001), sphingosine 1-phsophate (S1P) receptor modulators (P < 0.001), mycophenolate (P = 0.002), and B cell lymphoma (P = 0.004); those associated with low cellular response rates included S1P receptor modulators (P < 0.001) and mycophenolate (P < 0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination. CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection.

Background: COVID-19 manifests with respiratory, systemic, and gastrointestinal (GI) symptoms.1, SARS-CoV-2 RNA is detected in respiratory and fecal samples, and recent reports demonstrate viral replication in both the lung and intestinal tissue.2, 3, 4 Although much is known about early fecal RNA shedding, little is known about long-term shedding, especially in those with mild COVID-19. Furthermore, most reports of fecal RNA shedding do not correlate these findings with GI symptoms.5. Methods: We analyzed the dynamics of fecal RNA shedding up to 10 months after COVID-19 diagnosis in 113 individuals with mild to moderate disease. We also correlated shedding with disease symptoms. Findings: Fecal SARS-CoV-2 RNA is detected in 49.2% [95% confidence interval, 38.2%-60.3%] of participants within the first week after diagnosis. Whereas there was no ongoing oropharyngeal SARS-CoV-2 RNA shedding in subjects at 4 months, 12.7% [8.5%-18.4%] of participants continued to shed SARS-CoV-2 RNA in the feces at 4 months after diagnosis and 3.8% [2.0%-7.3%] shed at 7 months. Finally, we found that GI symptoms (abdominal pain, nausea, vomiting) are associated with fecal shedding of SARS-CoV-2 RNA. Conclusions: The extended presence of viral RNA in feces, but not in respiratory samples, along with the association of fecal viral RNA shedding with GI symptoms suggest that SARS-CoV-2 infects the GI tract and that this infection can be prolonged in a subset of individuals with COVID-19. Funding: This research was supported by a Stanford ChemH-IMA grant; fellowships from the AACR and NSF; and NIH R01-AI148623, R01-AI143757, and UL1TR003142.

Novel utilization of strand-specific reverse transcription polymerase chain reaction in perioperative clinical decision making for SARS-CoV-2 polymerase chain reaction positive patients.

In order to prevent in-hospital transmission and potential complications related to SARS-CoV-2 in the perioperative patient, most healthcare institutions require preoperative testing for SARS-CoV-2 prior to proceeding with elective surgery. The Centers for Disease Control and Prevention (CDC) recommends a time and symptom-based duration of isolation for the presumed infectious period. The guidance to avoid retesting of asymptomatic patients in the 90 days following a positive reverse transcription polymerase chain reaction (RT-PCR) test is because of the possibility of detection of non-infectious viral shedding. When to reschedule asymptomatic patients who test RT-PCR positive for SARS-CoV-2 preoperatively is of considerable debate, both from the perspective of ensuring a patient's full preoperative fitness, as well as reducing the risk of viral transmission within the hospital. We describe the novel perioperative use of a strand-specific assay to detect minus strand ribonucleic acid (RNA) in a clinical decision-making algorithm to determine optimal timing of elective surgery after a patient tests RT-PCR positive for SARS-CoV-2. This is the first description in the literature of an attempt to further stratify patients who repeatedly test positive for SARS-CoV-2 into infectious versus non-infectious for perioperative planning.

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Comprehensive investigation of sources of misclassification errors in routine HIV testing in Zimbabwe.

INTRODUCTION: Misclassification errors have been reported in rapid diagnostic HIV tests (RDTs) in sub-Saharan African countries. These errors can lead to missed opportunities for prevention-of-mother-to-child-transmission (PMTCT), early infant diagnosis and adult HIV-prevention, unnecessary lifelong antiretroviral treatment (ART) and wasted resources. Few national estimates or systematic quantifications of sources of errors have been produced. We conducted a comprehensive assessment of possible sources of misclassification errors in routine HIV testing in Zimbabwe. METHODS: RDT-based HIV test results were extracted from routine PMTCT programme records at 62 sites during national antenatal HIV surveillance in 2017. Positive- (PPA) and negative-percent agreement (NPA) for HIV RDT results and the false-HIV-positivity rate for people with previous HIV-positive results ("known-positives") were calculated using results from external quality assurance testing done for HIV surveillance purposes. Data on indicators of quality management systems, RDT kit performance under local climatic conditions and user/clerical errors were collected using HIV surveillance forms, data-loggers and a Smartphone camera application (7 sites). Proportions of cases with errors were compared for tests done in the presence/absence of potential sources of errors. RESULTS: NPA was 99.9% for both pregnant women (N = 17224) and male partners (N = 2173). PPA was 90.0% (N = 1187) and 93.4% (N = 136) for women and men respectively. 3.5% (N = 1921) of known-positive individuals on ART were HIV negative. Humidity and temperature exceeding manufacturers' recommendations, particularly in storerooms (88.6% and 97.3% respectively), and premature readings of RDT output (56.0%) were common. False-HIV-negative cases, including interpretation errors, occurred despite staff training and good algorithm compliance, and were not reduced by existing external or internal quality assurance procedures. PPA was lower when testing room humidity exceeded 60% (88.0% vs. 93.3%; p = 0.007). CONCLUSIONS: False-HIV-negative results were still common in Zimbabwe in 2017 and could be reduced with HIV testing algorithms that use RDTs with higher sensitivity under real-world conditions and greater practicality under busy clinic conditions, and by strengthening proficiency testing procedures in external quality assurance systems. New false-HIV-positive RDT results were infrequent but earlier errors in testing may have resulted in large numbers of uninfected individuals being on ART.

Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.

BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity.

Background: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. Methods: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. Findings: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. Interpretation: Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered. Funding: The work was partially funded by The Saban Research Institute at Children's Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

Cost-Effectiveness of Nasopharyngeal Carcinoma Screening With Epstein-Barr Virus Polymerase Chain Reaction or Serology in High-Incidence Populations Worldwide.

BACKGROUND: The incidence of endemic Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) varies considerably worldwide. In high-incidence regions, screening trials have been conducted. We estimated the mortality reduction and cost-effectiveness of EBV-based NPC screening in populations worldwide. METHODS: We identified 380 populations in 132 countries with incident NPC and developed a decision-analytic model to compare 10 unique onetime screening strategies with no screening for men and women aged 50 years. Screening performance and the stage distribution of undiagnosed NPC were derived from a systematic review of prospective screening trials. RESULTS: Screening was cost-effective in up to 14.5% of populations, depending on the screening strategy. These populations were limited to East Asia, Southeast Asia, and North Africa or were Asian, Pacific Islander, or Inuit populations in North America. A combination of serology and nasopharyngeal polymerase chain reaction was most cost-effective, but other combinations of serologic and/or plasma polymerase chain reaction screening were also cost-effective. The estimated reduction in NPC mortality was similar across screening strategies. For a hypothetical cohort of patients in China, the 10-year survival improved from 71.0% (95% confidence interval = 68.8% to 73.0%) without screening to a median of 86.3% (range = 83.5%-88.2%) with screening. This corresponded to a median 10-year reduction in NPC mortality of 52.9% (range = 43.1%-59.3%). Screening interval affected absolute mortality reduction and cost-effectiveness. CONCLUSIONS: We observed decreased NPC mortality with EBV-based screening. Screening was cost-effective in many high-incidence populations and could be extended to men and women as early as age 40 years in select regions. These findings may be useful when choosing among local public health initiatives.