Healthcare Provider Perspectives on HIV Cure Research in Ghana.

Introduction: Antiretroviral therapy (ART) has reduced mortality and improved life expectancy among HIV patients but does not provide a cure. Patients must remain on lifelong medications and deal with drug resistance and side effects. This underscores the need for HIV cure research. However, participation in HIV cure research has risks without guaranteed benefits. We determined what HIV healthcare providers know about HIV cure research trials, the risks involved, and what kind of cure interventions they are likely to recommend for their patients. Methods: We conducted in-depth qualitative interviews with 39 HIV care providers consisting of 12 physicians, 8 counsellors, 14 nurses, 2 pharmacists, 2 laboratory scientists, and 1 community advocate from three hospitals. Interviews were transcribed verbatim and coded, and thematic analysis was performed independently by two investigators. Results: Participants were happy about the success of current treatments and hopeful that an HIV cure will be found in the near future, just as ART was discovered through research. They described cure as total eradication of the virus from the body and inability to test positive for HIV or transmit the virus. In terms of risk tolerance, respondents would recommend to their patients' studies with mild to moderate risks like what patients on antiretroviral therapy experience. Participants were reluctant to recommend treatment interruption to patients as part of a cure study and wished trials could be performed without stopping treatment. Healthcare providers categorically rejected death or permanent disability as an acceptable risk. The possibility of finding a cure that will benefit the individual or future generations was strong motivations for providers to recommend cure trials to their patients, as was transparency and adequate information on proposed trials. Overall, the participants were not actively seeking knowledge on cure research and lacked information on the various cure modalities under investigation. Conclusion: While hopeful for an HIV cure, healthcare providers in Ghana expect a cure to be definitive and pose minimal risk to their patients.

SARS-CoV-2 Molecular Evolutionary Dynamics in the Greater Accra Region, Ghana.

To assess dynamics of SARS-CoV-2 in Greater Accra Region, Ghana, we analyzed SARS-CoV-2 genomic sequences from persons in the community and returning from international travel. The Accra Metropolitan District was a major origin of virus spread to other districts and should be a primary focus for interventions against future infectious disease outbreaks.

Effect of antipyretics on performance of influenza surveillance in Ghana.

BACKGROUND: The World Health Organization's case definition for influenza-like illness (ILI) includes a measured temperature of ≥38°C. We conducted this study to assess the effect of antipyretics on performance of ILI surveillance in Ghana. METHODS: A cross-sectional study was conducted in two districts of Ghana from September 2013 to May 2014. We collected epidemiological data and respiratory specimens from an expanded ILI case definition, which included patients presenting to health facilities with measured temperature ≥38°C or reported fever (but afebrile at the time of evaluation), and cough, with onset in the last 10 days. Specimens were tested for influenza viruses by real time reverse-transcription polymerase chain reaction. RESULTS: Of 321 participants who met our expanded ILI case definition, 236 presented with temperature of <38°C but reported subjective fever. Of these, 17% (39/236) were positive for influenza virus; Of those with fever ≤38°C who took antipyretics, 21%(16/77) were positive for influenza, compared with 14%(23/159) of those who did not take antipyretics. The addition of subjective fever to the standard ILI case definition captured approximately an additional 57% influenza cases but also required testing of approximately four times as many patients. However, including those without fever on presentation that had taken antipyretics found an additional 23% of Influenza cases and only two times as much testing. CONCLUSION: Depending on the goals of surveillance (monitoring virus circulation or determining disease burden) and available resources, a more sensitive case definition including subjective fever and history of use of antipyretics may be warranted.

Delayed SARS-COV-2 clearance in infected persons in Ghana.

Objective: This study aimed to determine the duration of SARS-CoV-2 clearance in persons in Ghana. The research question was whether the duration of virus clearance in Ghana matched the 14 days recommended by the World Health Organization (WHO); this had direct implications for transmission, which was key in managing the COVID-19 pandemic. Design: This was a retrospective analytical study. Setting: All facilities that submitted clinical specimens to Noguchi Memorial Institute for Medical Research (NMIMR) for SARS-CoV-2 diagnosis between March to June 2020 were included in the study. Interventions: Samples from 480 persons who tested positive for SARS-CoV-2 by RT-PCR from March to June 2020 at NMIMR and submitted at least two follow-up samples were retrospectively analysed. Individuals with two consecutive negative RT-PCR retesting results were considered to have cleared SARS-CoV-2. Results: The median time from the initial positive test to virus clearance was 20 days (IQR: 5-56 days). This was six days longer than the WHO-recommended 14 days, after which infected persons could be de-isolated. Sputum and nasopharyngeal swabs proved more sensitive for detecting viral RNA as the infection progressed. At a significance level of 0.05, age and sex did not seem to influence the time to SARS-CoV-2 clearance. Conclusions: The median time to SARS-CoV-2 clearance in this study was 20 days, suggesting that SARS-CoV-2 infected persons in Ghana take longer to clear the virus. This finding calls for further investigations into whether patients who remain PCR positive continue to be infectious and inform isolation practices in Ghana. Funding: The study was supported by the Ministry of Health/ Ghana Health Service through the provision of laboratory supplies, the US Naval Medical Research Unit #3, the World Health Organization, the Jack Ma Foundation and the Virology Department of Noguchi Memorial Institute for Medical Research, University of Ghana. Research projects within Noguchi Memorial Institute for Medical Research contributed reagents and laboratory consumables. However, the authors alone are responsible for the contents of this manuscript.

Delayed SARS-COV-2 clearance in infected persons in Ghana

Objective: This study aimed to determine the duration of SARS-CoV-2 clearance in persons in Ghana. The research question was whether the duration of virus clearance in Ghana matched the 14 days recommended by the World Health Organization (WHO); this had direct implications for transmission, which was key in managing the COVID-19 pandemic. Design: This was a retrospective analytical study. Setting: All facilities that submitted clinical specimens to Noguchi Memorial Institute for Medical Research (NMIMR) for SARS-CoV-2 diagnosis between March to June 2020 were included in the study. Interventions: Samples from 480 persons who tested positive for SARS-CoV-2 by RT-PCR from March to June 2020 at NMIMR and submitted at least two follow-up samples were retrospectively analysed. Individuals with two consecutive negative RT-PCR retesting results were considered to have cleared SARS-CoV-2. Results: The median time from the initial positive test to virus clearance was 20 days (IQR: 5-56 days). This was six days longer than the WHO-recommended 14 days, after which infected persons could be de-isolated. Sputum and nasopharyngeal swabs proved more sensitive for detecting viral RNA as the infection progressed. At a significance level of 0.05, age and sex did not seem to influence the time to SARS-CoV-2 clearance. Conclusions: The median time to SARS-CoV-2 clearance in this study was 20 days, suggesting that SARS-CoV-2 infected persons in Ghana take longer to clear the virus. This finding calls for further investigations into whether patients who remain PCR positive continue to be infectious and inform isolation practices in Ghana.

Occurrence of influenza and bacterial infections in cancer patients receiving radiotherapy in Ghana.

BACKGROUND: Influenza co-infection with bacteria is a leading cause of influenza-related deaths and severe respiratory infections, especially among high-risk groups like cancer patients undergoing treatment. However, acute respiratory infection (ARI)-like symptoms developed by upper-torso cancer (UTC) patients receiving radiotherapy are considered as side-effects of the radiation. Hence influenza and bacterial pathogens implicated in ARI are not investigated. METHODS: This prospective cohort study examined 85 in-patients with upper-torso cancers undergoing radiotherapy at the National Radiotherapy, Oncology and Nuclear Medicine Centre (NRONMC) of Korle-Bu Teaching Hospital (KBTH) in Accra, Ghana. Eligible patients who consented were recruited into the study from September 2018 to April 2019. Influenza viruses A and B in addition to the following bacteria species Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitidis and Staphylococcus aureus were detected from oropharyngeal and nasopharyngeal swab specimens collected at three different time points. Presence of respiratory pathogens were investigated by influenza virus isolation in cell culture, bacterial culture, polymerase chain reaction (PCR) and next generation sequencing (NGS) assays. RESULTS: Of the 85 eligible participants enrolled into the study, 87% were females. Participants were 17 to 77 years old, with a median age of 49 years. Most of the participants (88%) enrolled had at least one pathogen present. The most prevalent pathogen was N. meningitidis (63.4%), followed by H. influenzae (48.8%), Influenza viruses A and B (32.9%), S. pneumoniae (32.9%) and S. aureus (12.2%). Approximately, 65% of these participants developed ARI-like symptoms. Participants with previous episodes of ARI, did not live alone, HNC and total radiation less than 50 Gy were significantly associated with ARI. All treatment forms were also significantly associated with ARI. CONCLUSION: Data generated from the study suggests that ARI-like symptoms observed among UTC patients receiving radiotherapy in Ghana, could be due to influenza and bacterial single and co-infections in addition to risk factors and not solely the side-effects of radiation as perceived. These findings will be prime importance for diagnosis, prevention, treatment and control for cancer patients who present with such episodes during treatment.

A qualitative study of COVID-19 vaccine intentions and mistrust in Black Americans: Recommendations for vaccine dissemination and uptake.

BACKGROUND: COVID-19 vaccination rates among Black Americans have been lower than White Americans and are disproportionate to their population size and COVID-19 impact. This study examined reasons for low vaccination intentions and preferred strategies to promote COVID-19 vaccination. METHODS: Between November 2020 and March 2021, we conducted semi-structured interviews with 24 participants who expressed low vaccination intentions in a RAND American Life Panel survey; we also interviewed five stakeholders who represent organizations or subgroups in Black communities that have been highly affected by COVID-19. RESULTS: Many interviewees discussed the "wait-and-see" approach, citing that more time and evidence for vaccine side effects and efficacy are needed. Perceived barriers to COVID-19 vaccination included structural barriers to access (e.g., transportation, technology) and medical mistrust (e.g., towards the vaccines themselves, the government, healthcare providers and healthcare systems, and pharmaceutical companies) stemming from historical and contemporary systematic racism against Black communities. Interviewees also discussed strategies to promote COVID-19 vaccines, including acknowledging systemic racism as the root cause for mistrust, preferred messaging content (e.g., transparent messages about side effects), modes, and access points (e.g., a variety of medical and non-medical sites), and trusted information sources (e.g., trusted leaders, Black doctors and researchers). CONCLUSIONS: These insights can inform ways to improve initial and booster vaccination uptake as the COVID-19 pandemic progresses.

Hepatitis C virus (HCV) infection among patients with sickle cell disease at the Korle-Bu teaching hospital.

BACKGROUND: Hepatitis C virus (HCV) infection is a blood borne infection that remains potentially transmissible through blood transfusions. Sickle cell disease (SCD) is a common inheritable haemoglobinopathy in Ghana that requires multiple blood transfusions as part of its management. The SCD patient is therefore at a high risk of HCV infection; however, data on the occurrence of HCV in SCD patients has not been documented in Ghana. This study sought to determine the prevalence and genotypes of HCV infection in SCD patients. MATERIALS AND METHODS: This was a cross-sectional study which enrolled 141 sickle-cell disease patients from the Ghana Institute for Clinical Genetics, Korle-Bu Teaching Hospital (KBTH). Patient information was obtained through a structured questionnaire. Aliquots of the plasma obtained was used for both serology with Advanced Quality Rapid Anti-HCV Test Strip and molecular testing by RT-PCR with primers targeting the HCV core gene. The amplified DNA were purified and subjected to phylogenetic analysis to characterize HCV genotypes. RESULTS: Twelve (9%) out of the 141 patients were sero-positive for HCV total antibodies. HCV RNA was amplified from 8 (6%) out of the total number of patients' samples. One of the 12 sero-positives was HCV RNA positive. Five (63%) out of the 8 HCV RNA positive samples were successfully sequenced. The phylogenetic tree constructed with the study and GenBank reference sequences, clustered all five study sequences into HCV genotype 1. CONCLUSION: The HCV seroprevalence of 9% among sickle cell disease patients is higher than reported for the general Ghanaian population which is 3%. Genotype 1 is the common HCV genotype infecting SCD patients. Sickle cell disease is likely to be a high-risk group for HCV inapparent infections in Ghana as seroprevalence does not correlate with viremia. However, even with higher seroprevalence, the group must be given priority in resource allocation for preventive, diagnostic and therapeutic strategies.

SARS-CoV-2 infections among asymptomatic individuals contributed to COVID-19 cases: A cross-sectional study among prospective air travelers from Ghana.

Background: The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by asymptomatic individuals has been reported since the early stages of the coronavirus disease 2019 (COVID-19) outbreak in various parts of the world. However, there are limited data regarding SARS-CoV-2 among asymptomatic individuals in Ghana. The aim of the study was to use test data of prospective travelers from Ghana as a proxy to estimate the contribution of asymptomatic cases to the spread of COVID-19. Methods: The study analyzed the SARS-CoV-2 PCR test data of clients whose purpose for testing was classified as "Travel" at the COVID-19 walk-in test center of the Noguchi Memorial Institute for Medical Research (NMIMR) from July 2020 to July 2021. These individuals requesting tests for travel generally had no clinical symptoms of COVID-19 at the time of testing. Data were processed and analyzed using Microsoft Excel office 16 and STATA version 16. Descriptive statistics were used to summarize data on test and demographic characteristics. Results: Out of 42,997 samples tested at the center within that period, 28,384 (66.0%) were classified as "Travel" tests. Of these, 1,900 (6.7%) tested positive for SARS-CoV-2. The majority (64.8%) of the "Travel" tests were requested by men. The men recorded a SARS-CoV-2 positivity of 6.9% compared to the 6.4% observed among women. Test requests for SARS-CoV-2 were received from all regions of Ghana, with a majority (83.3%) received from the Greater Accra Region. Although the Eastern region recorded the highest SARS-CoV-2 positivity rate of 8.35%, the Greater Accra region contributed 81% to the total number of SARS-CoV-2 positive cases detected within the period of study. Conclusion: Our study found substantial SARS-CoV-2 positivity among asymptomatic individuals who, without the requirement for a negative SARS-CoV-2 result for travel, would have no reason to test. These asymptomatic SARS-CoV-2-infected individuals could have traveled to other countries and unintentionally spread the virus. Our findings call for enhanced tracing and testing of asymptomatic contacts of individuals who tested positive for SARS-CoV-2.

Disconnection in Information Exchange During Pediatric Trauma Transfers: A Qualitative Study.

Pediatric patients experiencing an emergency department (ED) visit for a traumatic injury often transfer from the referring ED to a pediatric trauma center. This qualitative study sought to evaluate the experience of information exchange during pediatric trauma visits to referring EDs from the perspectives of parents and referring and accepting clinicians through semi-structured interviews. Twenty-five interviews were conducted (10 parents and 15 clinicians) and analyzed through qualitative thematic analysis. A 4-person team collaboratively identified codes, wrote memos, developed major themes, and discussed theoretical concepts. Three interdependent themes emerged: (1) Parents' and clinicians' distinct experiences result in a disconnect of information exchange needs; (2) systems factors inhibit effective information exchange and amplify the disconnect; and (3) situational context disrupts the flow of information contributing to the disconnect. Individual-, situational-, and systems-level factors contribute to disconnects in the information exchanged between parents and clinicians. Understanding how these factors' influence information disconnect may offer avenues for improving patient-clinician communication in trauma transfers.

Unwillingness of patients in Ghana to interrupt antiretroviral therapy for HIV cure research.

OBJECTIVES: Though antiretroviral therapy (ART) has reduced HIV infection into a manageable chronic disease, it does not provide for a cure. HIV cure trials may carry risks for patients who are generally doing well on ART, making it imperative that their input is sought as various types of cure methods and trials are designed. Few studies have sought the views of African patients on HIV cure studies. The objective of this study was to determine the views and preferences of people living with HIV (PLWH) in Ghana on cure research. METHODS: We used a questionnaire to interview 251 PLWH in Ghana about their willingness to engage in HIV cure research. We investigated their motivations, the types of cure they would prefer and which risks were acceptable to them. RESULTS: Most participants were enthusiastic about participating in cure research and driven by both altruistic and personal motives. Patients preferred a cure where they would continue follow-up with their doctor (88%) compared to being assured that they have been completely cured and did not need further follow-up (11%). The vast majority of the respondents were risk averse. Most patients (67%) would decline to interrupt ART as part of a protocol for HIV cure research. In bivariate analysis, participants above the age of 40 years were more likely to agree to treatment interruption during cure studies (OR 2.77; 95% CI 1.21-.6.34. p â€‹= â€‹0.0159). CONCLUSIONS: Our results show that preferred cure modalities and risk tolerance for patients in Africa may be different from those of other parts of the world. Extensive social science and behavioural studies are needed on the continent to help inform future cure trials.

Circulation of hepatitis delta virus and occult hepatitis B virus infection amongst HIV/HBV co-infected patients in Korle-Bu, Ghana.

BACKGROUND: Within HIV/HBV infected patients, an increase in HDV infection has been observed; there is inadequate information on HDV prevalence as well as virologic profile in Ghana. This study sought to determine the presence of HDV in HIV/HBV co-infected patients in Ghana. METHODS: This was a longitudinal purposive study which enrolled 113 HIV/HBV co-infected patients attending clinic at Korle-Bu Teaching Hospital (KBTH) in Accra, Ghana. After consenting, 5 mL whole blood was collected at two-time points (baseline and 4-6 months afterwards). The sera obtained were tested to confirm the presence of HIV, HBV antibodies and/or antigens, and HBV DNA. Antibodies and viral RNA were also determined for HDV. Amplified HBV DNA and HDV RNA were sequenced and phylogenetic analysis carried out with reference sequences from the GenBank to establish the genotypes. RESULTS: Of the 113 samples tested 63 (55.7%) were females and 50 (44.25%) were males with a median age of 45 years. A total of 100 (88.5%) samples had detectable HBV surface antigen (HBsAg), and 32 out of the 113 had detectable HBV DNA. Nucleotide sequences were obtained for 15 and 2 samples of HBV and HDV, respectively. Phylogenetic analysis was predominantly genotype E for the HBVs and genotype 1 for the HDVs. Of the 13 samples that were HBsAg unreactive, 4 (30.8%) had detectable HBV DNA suggesting the incidence of occult HBV infections. The percentage occurrence of HDV in this study was observed to be 3.54. CONCLUSION: Our data suggest the presence and circulation of HDV and incidence of occult HBV infection in HIV/HBV co-infected patients in Ghana. This informs health staff and makes it imperative to look out for the presence of HDV and occult HBV in HIV/HBV co-infected patients presenting with potential risk of liver cancers and HBV transmission through haemodialysis and blood transfusions.

SARS-CoV-2 detection among international air travellers to Ghana during mandatory quarantine.

OBJECTIVES: To determine the prevalence of SARS-CoV-2 detection among international travellers to Ghana during mandatory quarantine. DESIGN: A retrospective cross-sectional study. SETTING: Air travellers to Ghana on 21st and 22nd March 2020. PARTICIPANTS: On 21st and 22nd March 2020, a total of 1,030 returning international travellers were mandatorily quarantined in 15 different hotels in Accra and tested for SARS-CoV-2. All of these persons were included in the study. MAIN OUTCOME MEASURE: Positivity for SARS-CoV-2 by polymerase chain reaction. RESULTS: The initial testing at the beginning of quarantine found 79 (7.7%) individuals to be positive for SARS-CoV-2. In the exit screening after 12 to 13 days of quarantine, it was discovered that 26 of those who tested negative for SARS-CoV-2 in the initial screening subsequently tested positive. CONCLUSIONS: Ghana likely averted an early community spread of COVID-19 through the proactive approach to quarantine international travellers during the early phase of the pandemic. FUNDING: None.

Data management during COVID-19 outbreak response in Ghana: a reference laboratory perspective on key issues and measures.

The COVID-19 pandemic caused by SARS-CoV-2 is an important subject for global health. Ghana experienced low-moderate transmission of the disease when the first case was detected in March 12, 2020 until the middle of July when the number of cases begun to drop. By August 24, 2020, the country's total number of confirmed cases stood at 43,622, with 263 deaths. By the same time, the Noguchi Memorial Institute for Medical Research (NMIMR) of the University of Ghana, the primary testing centre for COVID-19, had tested 285,501 with 28,878 confirmed cases. Due to database gaps, there were initial challenges with timely reporting and feedback to stakeholders during the peak surveillance period. The gaps resulted from mismatches between samples and their accompanying case investigation forms, samples without case investigation forms and vice versa, huge data entry requirements, and delayed test results. However, a revamp in data management procedures, and systems helped to improve the turnaround time for reporting results to all interested parties and partners. Additionally, inconsistencies such as multiple entries and discrepant patient-sample information were resolved by introducing a barcoding electronic capture system. Here, we describe the main challenges with COVID-19 data management and analysis in the laboratory and recommend measures for improvement. FUNDING: The work was supported by the Government of Ghana.

Data management during COVID-19 outbreak response in Ghana: a reference laboratory perspective on key issues and measures

The COVID-19 pandemic caused by SARS-CoV-2 is an important subject for global health. Ghana experienced lowmoderate transmission of the disease when the first case was detected in March 12, 2020 until the middle of July when the number of cases begun to drop. By August 24, 2020, the country's total number of confirmed cases stood at 43,622, with 263 deaths. By the same time, the Noguchi Memorial Institute for Medical Research (NMIMR) of the University of Ghana, the primary testing centre for COVID-19, had tested 285,501 with 28,878 confirmed cases. Due to database gaps, there were initial challenges with timely reporting and feedback to stakeholders during the peak surveillance period. The gaps resulted from mismatches between samples and their accompanying case investigation forms, samples without case investigation forms and vice versa, huge data entry requirements, and delayed test results. However, a revamp in data management procedures, and systems helped to improve the turnaround time for reporting results to all interested parties and partners. Additionally, inconsistencies such as multiple entries and discrepant patient-sample information were resolved by introducing a barcoding electronic capture system. Here, we describe the main challenges with COVID-19 data management and analysis in the laboratory and recommend measures for improvement

SARS-CoV-2 detection among international air travellers to Ghana during mandatory quarantine

Objectives: To determine the prevalence of SARS-CoV-2 detection among international travellers to Ghana during mandatory quarantine. Design: A retrospective cross-sectional study. Setting: Air travellers to Ghana on 21st and 22nd March 2020. Participants: On 21st and 22nd March 2020, a total of 1,030 returning international travellers were mandatorily quarantined in 15 different hotels in Accra and tested for SARS-CoV-2. All of these persons were included in the study. Main outcome measure: Positivity for SARS-CoV-2 by polymerase chain reaction. Results: The initial testing at the beginning of quarantine found 79 (7.7%) individuals to be positive for SARS-CoV2. In the exit screening after 12 to 13 days of quarantine, it was discovered that 26 of those who tested negative for SARS-CoV-2 in the initial screening subsequently tested positive. Conclusions: Ghana likely averted an early community spread of COVID-19 through the proactive approach to quarantine international travellers during the early phase of the pandemic

Molecular diagnosis for the novel coronavirus SARS-CoV-2: lessons learnt from the Ghana experience.

BACKGROUND: A novel coronavirus, SARS-CoV-2 is currently causing a worldwide pandemic. The first cases of SARS-CoV-2 infection were recorded in Ghana on March 12, 2020. Since then, the country has been combatting countrywide community spread. This report describes how the Virology Department, Noguchi Memorial Institute for Medical Research (NMIMR) is supporting the Ghana Health Service (GHS) to diagnose infections with this virus in Ghana. METHODS: The National Influenza Centre (NIC) in the Virology Department of the NMIMR, adopted real-time Polymerase Chain Reaction (rRT-PCR) assays for the diagnosis of the SARS-CoV-2 in January 2020. Samples from suspected cases and contact tracing across Ghana were received and processed for SARS-CoV-2. Samples were 'pooled' to enable simultaneous batch testing of samples without reduced sensitivity. OUTCOMES: From February 3 to August 21, the NMIMR processed 283 946 (10%) samples. Highest number of cases were reported in June when the GHS embarked on targeted contact tracing which led to an increase in number of samples processed daily, peaking at over 7,000 samples daily. There were several issues to overcome including rapid consumption of reagents and consumables. Testing however continued successfully due to revised procedures, additional equipment and improved pipeline of laboratory supplies. Test results are now provided within 24 to 48 hours of sample submission enabling more effective response and containment. CONCLUSION: Following the identification of the first cases of SARS-CoV-2infection by the NMIMR, the Institute has trained other centres and supported the ramping up of molecular testing capacity in Ghana. This provides a blueprint to enable Ghana to mitigate further epidemics and pandemics. FUNDING: The laboratory work was supported with materials from the Ghana Health Service Ministry of Health, the US Naval Medical Research Unit #3, the World Health Organization, the Jack Ma Foundation and the University of Ghana Noguchi Memorial Institute for Medical Research. Other research projects hosted by the Noguchi Memorial Institute for Medical Research contributed reagents and laboratory consumables. The funders had no role in the preparation of this manuscript.

Overexpression of the nucleocapsid protein of Middle East respiratory syndrome coronavirus up-regulates CXCL10.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory diseases in humans and has a high mortality rate. During infection, MERS-CoV regulates several host cellular processes including antiviral response genes. In order to determine if the nucleocapsid protein of MERS-CoV (MERS-N) plays a role in viral-host interactions, a murine monoclonal antibody was generated so as to allow detection of the protein in infected cells as well as in overexpression system. Then, MERS-N was stably overexpressed in A549 cells, and a PCR array containing 84 genes was used to screen for genes transcriptionally regulated by it. Several up-regulated antiviral genes, namely TNF, IL6, IL8, and CXCL10, were selected for independent validation in transiently transfected 293FT cells. Out of these, the overexpression of MERS-N was found to up-regulate CXCL10 at both transcriptional and translational levels. Interestingly, CXCL10 has been reported to be up-regulated in MERS-CoV infected airway epithelial cells and lung fibroblast cells, as well as monocyte-derived macrophages and dendritic cells. High secretions and persistent increase of CXCL10 in MERS-CoV patients have been also associated with severity of disease. To our knowledge, this is the first report showing that the MERS-N protein is one of the contributing factors for CXCL10 up-regulation during infection. In addition, our results showed that a fragment consisting of residues 196-413 in MERS-N is sufficient to up-regulate CXCL10, while the N-terminal domain and serine-arginine (SR)-rich motif of MERS-N do not play a role in this up-regulation.

A cross-clade H5N1 influenza A virus neutralizing monoclonal antibody binds to a novel epitope within the vestigial esterase domain of hemagglutinin.

The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised public health concerns. Monoclonal antibodies (MAbs) against hemagglutinin (HA) have been increasingly used successfully for therapeutic purposes. Previously, MAb 9F4, generated against clade 1 H5N1 HA, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pH-mediated conformational change of HA. Furthermore, mouse-human chimeric MAb 9F4 was found to retain high degrees of neutralizing activity. In this study, through escape mutant generation and in-silico prediction, it was revealed that MAb 9F4 binds to a novel epitope in the vestigial esterase sub-domain of HA comprising at least three non-continuous amino acid residues, arginine (R) at position 62, tryptophan (W) at position 69 and phenylalanine (F) at position 79, which interacted with MAb 9F4 in a conformation-dependent manner. Binding and neutralization studies suggested that R62 is the critical residue for MAb 9F4 binding whereas W69 and F79 seem to cooperate with R62 to stabilize the epitope. Mutation of either R62 or W69 did not affect replicative fitness of the virus in vitro. Interestingly, MAb 9F4 retained neutralizing efficacy against a clade 2.3.2.1a H5N1 virus consisting of an arginine to lysine substitution at position 62 in HA.

Implanted Muscle-Derived Stem Cells Ameliorate Erectile Dysfunction in a Rat Model of Type 2 Diabetes, but Their Repair Capacity Is Impaired by Their Prior Exposure to the Diabetic Milieu.

INTRODUCTION: Muscle-derived stem cells (MDSCs) and other SCs implanted into the penile corpora cavernosa ameliorate erectile dysfunction in type 1 diabetic rat models by replenishing lost corporal smooth muscle cells (SMCs) and decreasing fibrosis. However, there are no conclusive data from models of type 2 diabetes (T2D) and obesity. AIM: To determine whether MDSCs from obese Zucker (OZ) rats with T2D at an early stage of diabetes (early diabetic SCs isolated and cultured in low-glucose medium [ED-SCs]) counteract corporal veno-occlusive dysfunction and corporal SMC loss or lipo-fibrosis when implanted in OZ rats at a late stage of diabetes and whether MDSCs from these OZ rats with late diabetes (late diabetic SCs isolated and cultured in high-glucose medium [LD-SC]) differ from ED-SCs in gene transcriptional phenotype and repair capacity. METHODS: ED-SCs and LD-SCs were compared by DNA microarray assays, and ED-SCs were incubated in vitro under high-glucose conditions (ED-HG-SC). These three MDSC types were injected into the corpora cavernosa of OZ rats with late diabetes (OZ/ED, OZ/LD, and OZ/ED-HG rats, respectively). Untreated OZ and non-diabetic lean Zucker rats functioned as controls. Two months later, rats were subjected to cavernosometry and the penile shaft and corporal tissues were subjected to histopathology and DNA microarray assays. MAIN OUTCOME MEASURES: In vivo erectile dysfunction assessment by Dynamic Infusion Cavernosometry followed by histopathology marker analysis of the penile tissues. RESULTS: Implanted ED-SCs and ED-HG-SCs improved corporal veno-occlusive dysfunction, counteracted corporal decreases in the ratio of SMCs to collagen and fat infiltration in rats with long-term T2D, and upregulated neuronal and endothelial nitric oxide. LD-SCs acquired an inflammatory, pro-fibrotic, oxidative, and dyslipidemic transcriptional phenotype and failed to repair the corporal tissue. CONCLUSION: MDSCs from pre-diabetic rats injected into the corpora cavernosa of rats with long-term T2D improve corporal veno-occlusive dysfunction and the underlying histopathology. In contrast, MDSCs from rats with long-term uncontrolled T2D are imprinted by the hyperglycemic and dyslipidemic milieu with a noxious phenotype associated with an impaired tissue repair capacity. SCs affected by diabetes could lack tissue repair efficacy as autografts and should be reprogrammed in vitro or substituted by SCs from allogenic non-diabetic sources.