Early Outpatient Treatment for Covid-19 with Convalescent Plasma.

BACKGROUND: Polyclonal convalescent plasma may be obtained from donors who have recovered from coronavirus disease 2019 (Covid-19). The efficacy of this plasma in preventing serious complications in outpatients with recent-onset Covid-19 is uncertain. METHODS: In this multicenter, double-blind, randomized, controlled trial, we evaluated the efficacy and safety of Covid-19 convalescent plasma, as compared with control plasma, in symptomatic adults (≥18 years of age) who had tested positive for severe acute respiratory syndrome coronavirus 2, regardless of their risk factors for disease progression or vaccination status. Participants were enrolled within 8 days after symptom onset and received a transfusion within 1 day after randomization. The primary outcome was Covid-19-related hospitalization within 28 days after transfusion. RESULTS: Participants were enrolled from June 3, 2020, through October 1, 2021. A total of 1225 participants underwent randomization, and 1181 received a transfusion. In the prespecified modified intention-to-treat analysis that included only participants who received a transfusion, the primary outcome occurred in 17 of 592 participants (2.9%) who received convalescent plasma and 37 of 589 participants (6.3%) who received control plasma (absolute risk reduction, 3.4 percentage points; 95% confidence interval, 1.0 to 5.8; P = 0.005), which corresponded to a relative risk reduction of 54%. Evidence of efficacy in vaccinated participants cannot be inferred from these data because 53 of the 54 participants with Covid-19 who were hospitalized were unvaccinated and 1 participant was partially vaccinated. A total of 16 grade 3 or 4 adverse events (7 in the convalescent-plasma group and 9 in the control-plasma group) occurred in participants who were not hospitalized. CONCLUSIONS: In participants with Covid-19, most of whom were unvaccinated, the administration of convalescent plasma within 9 days after the onset of symptoms reduced the risk of disease progression leading to hospitalization. (Funded by the Department of Defense and others; CSSC-004 ClinicalTrials.gov number, NCT04373460.).

Hepatitis C Treatment in People With HIV: Potential to Eliminate Disease and Disparity.

Access to direct acting antivirals (DAAs) may be associated with reductions in hepatitis C virus (HCV) viremia prevalence among people with human immunodeficiency virus (PWH). Among 3755 PWH, estimated HCV viremia prevalence decreased by 94.0% from 36% (95% confidence interval [CI], 27%-46%) in 2009 (pre-DAA era) to 2% (95% CI, 0%-4%) in 2021 (DAA era). Male sex, black race, and older age were associated with HCV viremia in 2009 but not in 2021. Injection drug use remained associated with HCV viremia in 2009 and 2021. Targeted interventions are needed to meet the HCV care needs of PWH who use drugs.

Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo.

The Hunchback (Hb) transcription factor is crucial for anterior-posterior patterning of the Drosophila embryo. The maternal hb mRNA acts as a paradigm for translational regulation due to its repression in the posterior of the embryo. However, little is known about the translatability of zygotically transcribed hb mRNAs. Here, we adapt the SunTag system, developed for imaging translation at single-mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. Whereas the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

Symptom Duration and Resolution With Early Outpatient Treatment of Convalescent Plasma for Coronavirus Disease 2019: A Randomized Trial.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) reduces hospitalizations among outpatients treated early after symptom onset. It is unknown whether CCP reduces time to symptom resolution among outpatients. METHODS: We evaluated symptom resolution at day 14 by trial arm using an adjusted subdistribution hazard model, with hospitalization as a competing risk. We also assessed the prevalence of symptom clusters at day 14 between treatments. Clusters were defined based on biologic clustering, impact on ability to work, and an algorithm. RESULTS: Among 1070 outpatients followed up after transfusion, 381 of 538 (70.8%) receiving CCP and 381 of 532 (71.6%) receiving control plasma were still symptomatic (P = .78) at day 14. Associations between CCP and symptom resolution by day 14 did not differ significantly from those in controls after adjustment for baseline characteristics (adjusted subdistribution hazard ratio, 0.99; P = .62). The most common cluster consisted of cough, fatigue, shortness of breath, and headache and was found in 308 (57.2%) and 325 (61.1%) of CCP and control plasma recipients, respectively (P = .16). CONCLUSIONS: In this trial of outpatients with early COVID-19, CCP was not associated with faster resolution of symptoms compared with control. Overall, there were no differences by treatment in the prevalence of each symptom or symptom clusters at day 14. CLINICAL TRIALS REGISTRATION: NCT04373460.

Transfusing Convalescent Plasma as Post-Exposure Prophylaxis Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Double-Blinded, Phase 2 Randomized, Controlled Trial.

BACKGROUND: The efficacy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent plasma (CCP) for preventing infection in exposed, uninfected individuals is unknown. CCP might prevent infection when administered before symptoms or laboratory evidence of infection. METHODS: This double-blinded, phase 2 randomized, controlled trial (RCT) compared the efficacy and safety of prophylactic high titer (≥1:320 by Euroimmun ELISA) CCP with standard plasma. Asymptomatic participants aged ≥18 years with close contact exposure to a person with confirmed coronavirus disease 2019 (COVID-19) in the previous 120 hours and negative SARS-CoV-2 test within 24 hours before transfusion were eligible. The primary outcome was new SARS-CoV-2 infection. RESULTS: In total, 180 participants were enrolled; 87 were assigned to CCP and 93 to control plasma, and 170 transfused at 19 sites across the United States from June 2020 to March 2021. Two were excluded for screening SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) positivity. Of the remaining 168 participants, 12/81 (14.8%) CCP and 13/87 (14.9%) control recipients developed SARS-CoV-2 infection; 6 (7.4%) CCP and 7 (8%) control recipients developed COVID-19 (infection with symptoms). There were no COVID-19-related hospitalizations in CCP and 2 in control recipients. Efficacy by restricted mean infection free time (RMIFT) by 28 days for all SARS-CoV-2 infections (25.3 vs 25.2 days; P = .49) and COVID-19 (26.3 vs 25.9 days; P = .35) was similar for both groups. CONCLUSIONS: Administration of high-titer CCP as post-exposure prophylaxis, although appearing safe, did not prevent SARS-CoV-2 infection. CLINICAL TRIALS REGISTRATION: NCT04323800.

Transfusion reactions associated with COVID-19 convalescent plasma in outpatient clinical trials.

BACKGROUND: COVID-19 convalescent plasma (CCP) is an important therapeutic option for outpatients at high risk of hospitalization from SARS-CoV-2 infection. We assessed the safety of outpatient CCP transfusions administered during clinical trials. STUDY DESIGN AND METHODS: We analyzed data pertaining to transfusion-related reactions from two randomized controlled trials in the U.S. that evaluated the efficacy of CCP versus control plasma in various ambulatory settings. Multivariable logistic regression was used to assess whether CCP was associated with transfusion reactions, after adjusting for potential confounders. RESULTS: The combined study reported 79/1351 (5.9%) adverse events during the transfusion visit, with the majority 62/1351 (4.6%) characterized by mild, allergic-type findings of urticaria, and/or pruritus consistent with minor allergic transfusion reactions; the other reported events were attributed to the patients' underlying disease, COVID-19, or vasovagal in nature. We found no difference in the likelihood of allergic transfusion reactions between those receiving CCP versus control plasma (adjusted odds ratio [AOR], 0.75; 95% CI, 0.43-1.31). Risk of urticaria and/or pruritus increased with a pre-existing diagnosis of asthma (AOR, 2.33; 95% CI, 1.16-4.67). We did not observe any CCP-attributed antibody disease enhancement in participants with COVID-19 or increased risk of infection. There were no life-threatening severe transfusion reactions and no patients required hospitalization related to transfusion-associated complications. DISCUSSION: Outpatient plasma administration was safely performed for nearly 1400 participants. CCP is a safe therapeutic option for outpatients at risk of hospitalization from COVID-19.

Nonadherence to Ledipasvir/Sofosbuvir Did Not Predict Sustained Virologic Response in a Randomized Controlled Trial of Human Immunodeficiency Virus/Hepatitis C Virus Coinfected Persons Who Use Drugs.

BACKGROUND: Eliminating hepatitis C virus (HCV) will require effective treatment delivery to persons with substance use disorders (SUDs). We evaluated the relationship between ledipasvir/sofosbuvir treatment persistence (receiving 84 tablets), adherence, and sustained virologic response (SVR) in persons with human immunodeficiency virus (HIV)/HCV coinfection. METHODS: Of the 144 participants with HIV/HCV and SUDs, 110 initiated a 12-week treatment course under 1 of 3 conditions (usual care, peer mentors, and cash incentives). We used self-report, pharmacy pill counts, and expected date of refill to examine adherence. Persistent participants were categorized as high adherence (taking ≥90% of doses) or low adherence (taking <90% of doses). RESULTS: Most participants persisted on treatment after initiation (n = 105), with 95% (n = 100) achieving SVR. One third (34%) of participants had moderate/heavy alcohol use by the biomarker phosphatidylethanol ([Peth] ≥50 ng/mL), and 44% had urine toxicology positive for cocaine or heroin at enrollment. The proportion of persons with high adherence was 72% (n = 76), and the proportion of persons with low adherence was 28%. Although low adherence was associated with moderate/heavy alcohol use by PEth (relative risk = 2.77; 95% confidence interval, 1.50-5.12), SVR did not vary according to adherence (P = .702), and most participants (97%) with low adherence achieved SVR. CONCLUSIONS: Treatment persistence led to high SVR rates among persons with HIV/HCV, despite imperfect adherence and SUDs.

Factors associated with antiretroviral therapy use among pregnant women in rural and urban settings in Southern Province, Zambia: 2016-2019.

OBJECTIVES: To assess antiretroviral therapy (ART) coverage among pregnant women living with HIV and compare the characteristics of women who received and did not receive ART during pregnancy in Zambia. METHODS: A cross-sectional study was conducted at urban and rural health facilities in Southern Province, Zambia, from 2016 to 2019. Pregnant women living with HIV delivering at study sites were enrolled and administered a questionnaire, and the results of infant diagnostic testing for HIV at birth was documented. RESULTS: About 1184 mother/infant pairs were enrolled. ART coverage was 93.7%. Most women who did not receive ART during pregnancy reported HIV diagnosis at delivery (18.0%) or during pregnancy (57.7%). The primary reported reason for not receiving ART was not wanting to take the drugs. Women who did not receive ART during pregnancy were significantly younger, less likely to have disclosed their HIV-infection status to others, and less likely to have received antenatal care than women who received ART. ART use correlated with higher levels of education in urban but not rural sites. Overall, 1.0% of infants were infected with HIV at birth, including 0.8% of infants born to women who received ART and 4.1% of infants born to women who did not. CONCLUSIONS: Most women received ART according to guidelines, resulting in low perinatal transmission rates of HIV to infants. Efforts to increase ART coverage and prevent vertical transmission should focus on identifying incident HIV infections during pregnancy and strengthening counselling for newly diagnosed pregnant women.

Respiratory viruses in rural Zambia before and during the COVID-19 pandemic.

OBJECTIVES: With the emergence of the COVID-19 pandemic, restrictions were implemented globally to control the virus. Data on respiratory pathogens in sub-Saharan Africa during the COVID-19 pandemic are scarce. This analysis was conducted to evaluate patterns of respiratory pathogens in rural Zambia before and during the first year of the pandemic. METHODS: Surveillance was established in December 2018 at Macha Hospital in southern Zambia. Patients with respiratory symptoms in the outpatient and inpatient clinics were recruited. Nasopharyngeal samples were collected and tested for respiratory pathogens. The prevalence of respiratory symptoms and pathogens was evaluated and compared in the first (December 10, 2018-December 9, 2019) and second (December 10, 2019-November 30, 2020) years of surveillance. RESULTS: Outpatient visits and admissions for respiratory illness significantly decreased from the first to second year, especially among children. SARS-CoV-2 was not detected from any participants in Year 2. Among outpatients and inpatients with respiratory symptoms, the prevalence of respiratory syncytial virus and influenza viruses decreased from the first to second year. In contrast, the prevalence of rhinovirus/enterovirus, metapneumovirus and parainfluenza virus increased. CONCLUSIONS: The epidemiology of respiratory viruses in rural Zambia changed during the first year of the COVID-19 pandemic, suggesting that public health interventions may have had an impact on the introduction and circulation of respiratory pathogens in this area.

The NSEBA Demonstration Project: implementation of a point-of-care platform for early infant diagnosis of HIV in rural Zambia.

OBJECTIVES: To describe the experience and resource requirements of implementing point-of-care testing for early infant diagnosis of HIV in rural Zambia. METHODS: A demonstration project was conducted using a hub-and-spoke model in 2018-2019 at five clinics in rural Zambia. Two testing hubs were established, and all HIV-exposed infants were tested with the GeneXpert system. Data on costs, turnaround times and test results were collected. RESULTS: Seven hundred and eighty six tests were conducted. At the hubs, results were available a median of 2.4 (IQR: 2.1, 2.8) hours after sample collection and most mothers (84%) received same-day results. At the spoke facilities, results were available a median of 9 days (IQR: 7, 12) after sample collection and provided to the mother a median of 16 days (IQR: 10, 28) after sample collection. Eleven children tested positive, and 9 (82%) started treatment a median of 13 days (IQR: 7, 21) after sample collection and on the day mothers received results. In contrast, results from matching samples sent for routine testing were available a median of 38 days (IQR: 27, 61) after sample collection and provided to the mother a median of 91 days (IQR: 47, 135) after sample collection. CONCLUSIONS: Implementing point-of-care testing in a network of rural health centres in Zambia required significant initial and ongoing investment in infrastructure, training and supervision. However, point-of-care testing can rapidly diagnose HIV-infected infants, so they can benefit from early treatment.

Facility-based surveillance for influenza and respiratory syncytial virus in rural Zambia.

BACKGROUND: While southern Africa experiences among the highest mortality rates from respiratory infections, the burden of influenza and respiratory syncytial virus (RSV) in rural areas is poorly understood. METHODS: We implemented facility-based surveillance in Macha, Zambia. Outpatients and inpatients presenting with influenza-like illness (ILI) underwent testing for influenza A, influenza B, and RSV and were prospectively followed for 3 to 5 weeks to assess clinical course. Log-binomial models assessed correlates of infection and clinical severity. RESULTS: Between December 2018 and December 2019, 17% of all outpatients presented with ILI and 16% of inpatients were admitted with an acute respiratory complaint. Influenza viruses and RSV were detected in 17% and 11% of outpatient participants with ILI, and 23% and 16% of inpatient participants with ILI, respectively. Influenza (July-September) and RSV (January-April) prevalence peaks were temporally distinct. RSV (relative risk [RR]: 1.78; 95% confidence interval [CI] 1.51-2.11), but not influenza, infection was associated with severe disease among patients with ILI. Underweight patients with ILI were more likely to be infected with influenza A (prevalence ratio [PR]: 1.72; 95% CI 1.04-2.87) and to have severe influenza A infections (RR: 2.49; 95% CI 1.57-3.93). CONCLUSIONS: Populations in rural Zambia bear a sizeable burden of viral respiratory infections and severe disease. The epidemiology of infections in this rural area differs from that reported from urban areas in Zambia.

Point-of-care p24 antigen detection for early infant diagnosis of HIV infection: cross-sectional and longitudinal studies in Zambia.

BACKGROUND: Early infant diagnosis of HIV infection is challenging in sub-Saharan Africa, particularly in rural areas, leading to delays in diagnosis and treatment. Use of a point-of-care test would overcome many challenges. This study evaluated the validity of a novel point-of-care p24 antigen detection test (LYNX) in rural and urban settings in southern Zambia. METHODS: Two studies were conducted: a cross-sectional study from 2014 to 2015 at Macha Hospital (LYNX Hospital study) and a longitudinal study from 2016 to 2018 at 12 health facilities in Southern Province, Zambia (NSEBA study). In both studies, children attending the facilities for early infant diagnosis were enrolled and a blood sample was collected for routine testing at the central lab and immediate on-site testing with the LYNX test. The performance of the LYNX test was measured in comparison to nucleic acid-based testing at the central lab. RESULTS: In the LYNX Hospital study, 210 tests were performed at a median age of 23.5 weeks (IQR: 8.9, 29.0). The sensitivity and specificity of the test were 70.0 and 100.0%, respectively. In the NSEBA study, 2608 tests were performed, including 1305 at birth and 1222 on children ≥4 weeks of age. For samples tested at birth, sensitivity was 13.6% (95% CI: 2.9, 34.9) and specificity was 99.6% (95% CI: 99.1, 99.9). While specificity was high for all ages, sensitivity increased with age and was higher for participants tested at ≥4 weeks of age (80.6%; 95% CI: 67.4, 93.7). Children with positive nucleic acid tests were more likely to be negative by the LYNX test if their mother received antiretroviral therapy during pregnancy (60.7% vs. 24.2%; p = 004). CONCLUSIONS: Considering the high specificity and moderate sensitivity that increased with age, the LYNX test could be of value for early infant diagnosis for infants ≥4 weeks of age, particularly in rural areas where centralized testing leads to long delays. Point-of-care tests with moderate sensitivity and high specificity that are affordable, easy-to-use, and easily implemented and maintained should be developed to expand access to testing and deliver same-day results to infants in areas where it is not feasible to implement nucleic acid-based point-of-care assays.

Treatment outcomes among children younger than five years living with HIV in rural Zambia, 2008-2018: a cohort study.

BACKGROUND: HIV testing and treatment guidelines for children in sub-Saharan Africa have evolved over time, such that children are now treated at younger ages. The objective of this study was to describe the treatment experience for immunologic, virologic, and growth outcomes among HIV-infected Zambian children younger than 5 years of age from 2008 to 2018. METHODS: Participants enrolled in a clinical cohort study in Macha, Zambia and initiating antiretroviral treatment before 5 years of age between 2008 and 2015 were included in the analysis and followed up to the end of 2018. Outcomes, including growth, CD4+ T-cell percentage, viral suppression, and mortality, were evaluated among all children using longitudinal and survival analyses. Comparisons by age at treatment initiation (< 1, 1 to < 2, and 2 to < 5 years) were also evaluated. RESULTS: Three hundred eighty-one children initiating treatment before 5 years of age between 2008 and 2015 were included in the analysis. Growth metrics and CD4+ T-cell percentage improved over time after treatment initiation. However, 20% of children remained underweight and 40% of children remained stunted after the first 36 months of treatment. 85% of children had a viral load < 400 copies/mL after 12 months of treatment. However, children < 1 year at treatment initiation were more likely to have a detectable viral load in the first 12 months of treatment and less likely to achieve viral suppression compared to older children. Mortality was highest in the first 12 months of treatment, among underweight children, and among children initiating treatment in 2008-2010 compared to 2011-2015. CONCLUSIONS: Most children initiating antiretroviral treatment from 2008 to 2015 in rural Zambia responded well to treatment. However, many children remained underweight and stunted, and experienced high mortality rates during the first few months of treatment. This supports continued efforts to improve early infant diagnosis, nutritional support, and pediatric drug formulations.

Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription.

Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.

Unreported alcohol use was common but did not impact hepatitis C cure in HIV-infected persons who use drugs.

We investigated the prevalence and impact of heavy alcohol use on the hepatitis C virus (HCV) care continuum amongst HIV/HCV co-infected persons who use drugs. In the CHAMPS study, 144 HIV/HCV co-infected persons were randomized to contingent cash incentives, peer mentors and usual care to evaluate the impact on HCV care. Alcohol use was ascertained using the 10-item AUDIT (hazardous: male ≥8, female ≥4) and phosphatidylethanol (PEth) (heavy: ≥50 ng/mL), an alcohol biomarker. Log binomial regression was used to evaluate the association between heavy alcohol use and failure to initiate treatment and to achieve sustained virologic response (SVR). Of the 135 participants with PEth data, median age was 55 years, 59% were male, 92% were Black, 91% reported a history of drug use, and 97% were on antiretroviral therapy. Hazardous drinking was reported on AUDIT by 28% of participants, and 35% had heavy alcohol use by PEth. Of the 47 individuals with a PEth ≥50 ng/mL, 23 (49%) reported no or minimal alcohol use by AUDIT. HCV treatment was initiated in 103 of 135 participants, and SVR was achieved in 92%. PEth ≥50 ng/mL (Relative Risk [RR] 0.72, 95% CI 0.35-1.48) was not significantly associated with failure to initiate HCV treatment or failure to achieve SVR (RR 0.85, 95% CI 0.46-1.57).In conclusion, alcohol use was common and frequently not detected by self-report. However, heavy alcohol use, even when measured objectively, was not associated with failure to initiate HCV treatment or to achieve cure.

Acceptability and feasibility of testing for HIV infection at birth and linkage to care in rural and urban Zambia: a cross-sectional study.

BACKGROUND: Early infant diagnosis is important for timely identification of HIV-infected infants and linkage to care. Testing at birth has been implemented to facilitate earlier diagnosis of HIV infection but may present new challenges. This study was conducted to understand the acceptability and feasibility of birth testing in urban and rural settings in southern Zambia. METHODS: This cross-sectional study was conducted at 11 hospitals and clinics in Livingstone, Choma, and Macha in Southern Province, Zambia from 2016 to 2018. Infants born to pregnant women living with HIV at the sites were eligible for enrollment. After enrollment, a questionnaire was administered to the mother and a dried blood spot card was collected from infants for testing at a central laboratory. When results were available, mothers were notified to return to the clinic. Acceptability of birth testing was evaluated based on the proportion of women who agreed to participate and the reasons for non-participation among women who declined. Feasibility of testing at birth was evaluated using turnaround times for returning results, the proportion of women receiving results, and linkage to care for infants testing positive. RESULTS: One thousand four hundred three women were approached for the study. A small proportion declined due to refusal of birth testing (0 to 8.2% across sites). One thousand two hundred ninety women agreed to have their infants tested. The proportion of mothers receiving results ranged from 51.6 to 92.1%, and was significantly lower at the hospital than clinics in Livingstone (51.6% vs. 69.8%; p < 0.0001) and Macha (69.5% vs. 85.7%; p < 0.0001) but not Choma (85.7% vs. 92.1%; p = 0.34). For mothers who received test results, the median turnaround time from sample collection was 67 days in Livingstone and 53 days in Macha and Choma. Overall, 23 (1.8%) infants tested positive for HIV but only 8 (34.8%) were linked to care a median of 68 days (range: 29, 784) after sample collection. CONCLUSIONS: While testing at birth was acceptable, this study highlights the operational challenges under a centralized laboratory testing system. Point-of-care platforms are needed for rapid testing and return of results so HIV-infected children can be identified, linked to care, and treated as early as possible.

Association of Laboratory Methods, Colonization Density, and Age With Detection of Streptococcus pneumoniae in the Nasopharynx.

Culture-based methods for detecting Streptococcus pneumoniae in the nasopharynx lack sensitivity. In this study, we aimed to compare the performance of culture and molecular methods in detecting pneumococcus in the nasopharynx of healthy individuals and to evaluate the associations of age and colonization density with detection. Between 2010 and 2012, nasopharyngeal specimens were collected from healthy individuals living on Navajo Nation and White Mountain Apache Tribal lands in the United States. Pneumococci were detected by means of broth-enrichment culture and autolysin-encoding gene (lytA) quantitative polymerase chain reaction (qPCR). Among 982 persons evaluated (median age, 18.7 years; 47% male), 35% were culture-positive and an additional 27% were qPCR-positive. Agreement between culture and qPCR was 70.9% but was higher among children (age <18 years) (75.9%-84.4%) than among adults (age ≥18 years) (61.0%-74.6%). The mean density of colonization was lower for culture-negative samples (3.14 log10 copies/mL) than for culture-positive samples (5.02 log10 copies/mL), overall and for all age groups. The percent culture-positive increased with increasing density, exceeding 80% at densities of ≥10,000 copies/mL. Mean colonization density decreased with age. Use of qPCR improved detection of pneumococcus in the nasopharynx of healthy individuals. This finding was most notable among adults, probably because of improved detection of low-density colonization.

Nasopharyngeal carriage of Streptococcus pneumoniae serotypes among children in India prior to the introduction of pneumococcal conjugate vaccines: a cross-sectional study.

BACKGROUND: Streptococcus pneumoniae is a major cause of pneumonia, meningitis, and other serious infections among children in India. India introduced the 13-valent pneumococcal conjugate vaccine (PCV) in several states in 2017, and is expected to expand to nationwide coverage in the near future. To establish a baseline for measuring the impact of PCV in India, we assessed overall and serotype-specific nasopharyngeal carriage in two pediatric populations. METHODS: A cross-sectional study was conducted in Palwal District, Haryana, from December 2016 to July 2017, prior to vaccine introduction. Children 2-59 months of age with clinical pneumonia seeking healthcare and those in the community with no clear illness were targeted for enrollment. A nasopharyngeal swab was collected and tested for pneumococcus using conventional culture and sequential multiplex PCR. Isolates were tested for antimicrobial resistance using an E test. Children were considered colonized if pneumococcus was isolated by culture or PCR. The prevalence of pneumococcal and serotype-specific colonization was compared between groups of children using log-binomial regression. RESULTS: Among 601 children enrolled, 91 had clinical pneumonia and 510 were community children. The proportion colonized with S. pneumoniae was 74.7 and 54.5% among children with clinical pneumonia and community children, respectively (adjusted prevalence ratio: 1.38; 95% confidence interval: 1.19, 1.60). The prevalence of PCV13 vaccine-type colonization was similar between children with clinical pneumonia (31.9%) and community children (28.0%; p = 0.46). The most common colonizing serotypes were 6A, 6B, 14, 19A, 19F, and 23F, all of which are included in the PCV13 vaccine product. Antimicrobial resistance to at least one drug was similar between isolates from children with clinical pneumonia (66.1%) and community children (61.5%; p = 0.49); while resistance to at least two drugs was more common among isolates from children with clinical pneumonia (25.8% vs. 16.4%; p = 0.08). Resistance for all drugs was consistently higher for PCV13 vaccine-type serotypes compared to non-vaccine serotypes in both groups. CONCLUSION: This study provides baseline information on the prevalence of serotype-specific pneumococcal colonization among children prior to the introduction of PCV in India. Our results suggest a role for pneumococcal vaccines in reducing pneumococcal colonization and antimicrobial resistant isolates circulating in India.

Regulation of the BMP Signaling-Responsive Transcriptional Network in the Drosophila Embryo.

The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.

Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development.

Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development.