Persistence and risk factors of occult hepatitis B virus infections among antiretroviral therapy-naïve people living with HIV in Botswana.

Aim: This study aimed to determine the kinetics of occult hepatitis B virus infections (OBI) among people with HIV (PWH). Methods: The study used archived plasma samples from longitudinal HIV natural history studies. We identified new OBI cases and assessed risk factors for OBI using Cox proportional hazards regression analysis. Results: At baseline, 8 of 382 [(2.1%) (95% CI: 1.06-4.1)] samples tested positive for hepatitis B surface antigen (HBsAg+). Of the 374 HBsAg-negative samples, 76 had sufficient sample volume for HBV DNA screening. OBI positivity (OBI+) at baseline was reported in 11 of 76 [14.7 95% CI (8.3-24.1)] HBsAg-negative (HBsAg-) participants. Baseline HBsAg-negative samples with sufficient follow-up samples (n = 90) were used for analysis of newly identified OBI cases. Participants contributed 129.74 person-years to the study and were followed for a median of 1.02 years (IQR: 1.00-2.00). Cumulatively, there were 34 newly identified OBI cases from the 90 participants, at the rate of 26.2/100 person-years (95% CI: 18.7-36.7). Newly identified OBI cases were more common among men than women (61.1% vs. 31.9%) and among participants with CD4+ T-cell counts ≤450 cells/mL (p-value = 0.02). Most of the newly identified OBI cases [55.9% (19/34)] were possible reactivations as they were previously HBV core antibody positive. Conclusion: There was a high rate of newly identified OBI among young PWH in Botswana, especially in men and in participants with lower CD4+ T-cell counts. OBI screening in PWH should be considered because of the risk of transmission, possible reactivation, and risk factors for the development of chronic liver disease, including hepatocellular carcinoma.

Uncommon BK polyomaviruses detected in allogeneic hematopoietic cell transplant recipients.

The role of viral diversity in the pathogenesis of BK polyomavirus (BKPyV)-associated disease is poorly understood. Here, we report near full-length BKPyV genome sequences from two allogeneic hematopoietic cell transplant recipients infected with BKPyV genotype II, which is uncommon in the USA.

Vertical Transmission of Hepatitis C Virus Among Women with a History of Injection Opioid Use.

We evaluated vertical transmission and linkage to care in women with HCV and history of injection drug use employing co-localized testing and treatment. Transmission occurred in 1 of 23 infants, with mother-infant genetic distance of 1.26%. Rates for infant testing, maternal linkage and cure were 77%, 52%, and 100%, respectively.

Andragogy in Practice: Applying a Theoretical Framework to Team Science Training in Biomedical Research.

This study is the first to apply the theoretical principles of Malcolm Knowles' theory of andragogy to evaluate data collected from learners who participated in team science training workshops in a biomedical research setting. Briefly, andragogy includes six principles: the learner's self-concept, the role of experience, readiness to learn, orientation to learning, the learner's need to know, and intrinsic motivation. Using an embedded study design, the primary focus was on qualitative data, with quantitative data complementing the qualitative findings. The deductive analysis demonstrated that approximately 85% of the qualitative data could be connected to at least one andragogical principle. Participant responses to positive evaluation questions were largely related to two principles: readiness to learn and problem-based learning orientation. Participant responses to negative questions were largely connected to two different principles: the role of experience and self-direction. Inductive analysis found an additional theme: meeting biological needs. Quantitative survey results supported the qualitative findings. The study findings demonstrate that andragogy can serve as a valuable construct to integrate into the development of effective team science training for biomedical researchers.

Introducing trainees to research using an online, asynchronous course.

Introduction: Research is an important aspect of many students' training. However, formal research training is rarely included in curricula. Thus, we developed an online, asynchronous series of modules to introduce trainees to multiple topics that are relevant to the conduct of research. Methods: Research 101 was utilized by first-year medical students and undergraduate students conducting mentored research projects. Students' knowledge, confidence, and satisfaction were assessed using pre- and post-module surveys with five-point Likert scaled questions, open-ended text responses, and a final quiz. Results: Pre-module survey results showed that learners felt most confident with the Conducting a literature search and Race and racism in medicine modules and least confident with the Submitting an Institutional Review Board protocol at UC module. Post-module survey responses were significantly increased compared to pre-module results for all modules and questions (p < 0.0001). The response to "The content of this module met my needs" was endorsed across all modules (84.9% "yes" responses). A final quiz of 25 multiple-choice questions was completed by 92 participants who received a median score of 21. Content analysis of open-ended post-module survey responses identified several strengths and opportunities for improvement in course content and instructional methods. Conclusions: These data demonstrate that significant learning resulted from completion of Research 101, as post-module survey scores were significantly higher than pre-module survey scores for all modules and questions. Final quiz scores were positive but also highlighted opportunity for additional trainee learning and will guide evolution of future modules.

Hepatitis B Virus Genotype E/A Recombinants from Blood Donors in Beira, Mozambique.

Hepatitis B virus (HBV) is endemic in many parts of sub-Saharan Africa. Here, we present 5 full-length HBV recombinant genomes from blood donors in Beira, Mozambique. The genomes are recombinants between genotypes E and A and are distantly related to one another, based on their genetic distances.

BK Polyomavirus Diversity After Hematopoietic Stem Cell Transplantation.

BK polyomavirus (BKPyV) infection is common after hematopoietic stem cell transplantation (HSCT) and is associated with the development of hemorrhagic cystitis (HC). The role that BKPyV plays in the pathogenesis of HC is not well characterized. We investigated the impact of BKPyV diversity on the development of HC using a previously established cohort of pediatric HSCT patients. There were 147 urine samples with quantifiable BKPyV at month 1 after HSCT; 137 (93.2%) were amplified using our in-house polymerase chain reaction approach and sent for next-generation sequencing. Subtype Ia was most frequent (61.3%), followed by subtype Ib1 (31.4%). The median viral load of subtype Ia samples was higher than for subtype Ib1 at month 1. Across the protein coding regions, APOBEC-induced mutations and signature patterns associated with HC were identified. This is the largest sequencing study of a single cohort of HSCT patients, providing a vast resource of sequence data for future analyses.

Hepatitis C Virus Genotype 5 Variability in Treatment-Naïve Patients in South Africa.

INTRODUCTION: Hepatitis C virus (HCV) genotype 5 was originally identified in South Africa, where it represents 35-60% of all HCV infections. There are limited data on resistance-associated variants (RAVs) in South Africa. Thus, we investigated variability within the NS3/NS4A, NS5A, and NS5B genes of treatment-naïve individuals with HCV genotype 5 infection at the Dr. George Mukhari Academic Hospital (DGMAH) in Pretoria, South Africa. METHODS: Nested PCR was performed to amplify the NS3/4A, NS5A, and NS5B genes. RAVs were evaluated using the Geno2pheno tool. RESULTS: In the NS3/4A gene, F56S and T122A were detected in one sample each. The D168E mutation was detected in 7 samples. Within the NS5A gene, the T62M mutation was detected in 2 individuals. In the NS5B gene, 8 of 12 individuals (67%) had the A421V mutation, while all 12 individuals (100%) had the S486A mutation. DISCUSSION: RAVs were detected frequently among treatment-naïve individuals with HCV genotype 5 infection in South Africa. Thus, resistance testing may be prudent when initiating treatment of patients with genotype 5 infection. Additional population-based studies are needed to understand the prevalence of these RAVs during HCV genotype 5 infection.

The Synthetic Opioid Fentanyl Increases HIV Replication and Chemokine Co-Receptor Expression in Lymphocyte Cell Lines.

BACKGROUND: In the United States, the illicit use of synthetic opioids such as fentanyl has led to a serious public health crisis. Synthetic opioids are known to enhance viral replication and to suppress immunologic responses, but their effects on HIV pathogenesis remain unclear. Thus, we examined the impact of fentanyl on HIV-susceptible and HIV-infected cell types. METHODS: TZM-bl and HIV-infected lymphocyte cells were incubated with fentanyl at varying concentrations. Expression levels of the CXCR4 and CCR5 chemokine receptors and HIV p24 antigen were quantified with ELISA. HIV proviral DNA was quantified using SYBR RT-PCR. Cell viability was detected with the MTT assay. RNAseq was performed to characterize cellular gene regulation in the presence of fentanyl. RESULTS: Fentanyl enhanced expression of both chemokine receptor levels in a dose-dependent manner in HIV-susceptible and infected cell lines. Similarly, fentanyl induced viral expression in HIV-exposed TZM-bl cells and in HIV-infected lymphocyte cell lines. Multiple genes associated with apoptosis, antiviral/interferon response, chemokine signaling, and NFκB signaling were differentially regulated. CONCLUSIONS: Synthetic opioid fentanyl impacts HIV replication and chemokine co-receptor expression. Increased virus levels suggest that opioid use may increase the likelihood of transmission and accelerate disease progression.

Hepatitis B virus genotypes and drug resistance mutations circulating in blood donors in Beira, Mozambique.

Hepatitis B virus (HBV) infects nearly 300 million people and is the leading cause of hepatitis and hepatocellular carcinoma worldwide. Despite the high burden of HBV in sub-Saharan Africa, countries such as Mozambique have limited data available on circulating HBV genotypes and the presence of drug resistance mutations. Blood donors from Beira, Mozambique were tested for HBV surface antigen (HBsAg) and HBV DNA at the Instituto Nacional de Saúde in Maputo, Mozambique. Regardless of HBsAg status, donors with detectable HBV DNA were evaluated for HBV genotype. PCR was performed with primers amplifying a 2.1-2.2 kilobase fragment of the HBV genome. PCR products were submitted for next generation sequencing (NGS), and consensus sequences were evaluated for HBV genotype, recombination, and the presence or absence of drug resistance mutations. Of the 1281 blood donors tested, 74 had quantifiable HBV DNA. The polymerase gene could be amplified from 45 of 58 (77.6%) individuals with chronic HBV infection and 12 of 16 (75%) with occult HBV infection. Among these 57, 51 (89.5%) sequences belonged to HBV genotype A1, while 6 (10.5%) were HBV genotype E. All genotype E sequences were E/A recombinants, and clustered separately from other genotype E references. Genotype A samples had a median viral load of 637 IU/mL, while genotype E samples had a median viral load of 476,084 IU/mL. No drug resistance mutations were observed in the consensus sequences. The current study demonstrates the genotypic diversity of HBV in blood donors in Mozambique, but the absence of dominant (consensus) drug resistance mutations. Studies in other at-risk populations are essential for understanding the epidemiology, risk of liver disease, and likelihood of treatment resistance in resource-limited settings.

Replication and Injury Associated With SARS-CoV-2 in Cultured Hepatocytes.

Background: Liver dysfunction is one of the hallmarks of SARS-CoV-2 infection. The mechanism(s) of hepatic injury in SARS-CoV-2 infection remains controversial with some reporting viral replication and cellular injury and others suggesting lack of replication and injury due to non-cytopathogenic etiologies. To investigate this further, we evaluated SARS-CoV-2 replication in immortalized hepatic cell lines and primary hepatocytes, examined whether cell injury was associated with apoptotic pathways, and also determined the effect of the antiviral remdesivir on these processes. Methods: Immortalized hepatocyte cell lines (HepG2 and Huh7.5), as well as primary human hepatocytes, were exposed to SARS-CoV-2 at a multiplicity of infection of 0.1 PFU/mL. Viral replication was evaluated by plaque assays, immunohistochemical staining for the viral spike protein, and caspase-3 expression evaluated with and without exposure to remdesivir. Results: All hepatocyte cell lines and primary hepatocytes supported active replication of SARS-CoV-2. Significant cytopathic effect was observed by light microscopy, and caspase-3 staining supported activation of apoptotic pathways. Remdesivir abrogated infection in a dose-dependent fashion and was not independently associated with hepatocyte injury. Conclusion: Hepatocytes appear to be highly permissive of SARS-CoV-2 replication which leads to rapid cell death associated with activation of apoptotic pathways. Viral replication and hepatocytes injury are abrogated with remdesivir. We conclude that active viral replication is most likely a key contributor to liver enzyme abnormalities observed in the setting of acute SARS-CoV-2 infection.

Research 101: An online course introducing medical students to research.

Introduction: Research is an important aspect of many medical students' training. However, many medical students are not required to complete a scholarly project, and formal research training is often fragmented across the medical school curriculum. Thus, we developed an online, structured, asynchronous set of modules to introduce trainees to multiple topics relevant to the conduct of research. Methods: Research 101 was piloted by 27 first-year medical students at the University of Cincinnati College of Medicine. Students' knowledge, confidence, and satisfaction were assessed using a final quiz and pre- and post-module surveys with five-point Likert-scaled questions and open-ended text responses. Results: Pre-module survey results showed that learners felt most confident in Conducting a literature search and least confident in Submitting an Institutional Review Board (IRB) protocol at UC. Post-module mean scores were significantly increased compared to pre-module results for all modules and questions (P < 0.05). The response to "The content of this module met my needs" was high across all modules with 236 (84.0%) "yes" responses. Thematic analysis of open-ended text responses from post-module surveys identified several improvements to individual modules and to the overall structure of Research 101. A final quiz of 25 multiple choice questions covering content from all required modules was required. The median score was 21. Conclusions: Comparison of post-module to pre-module survey scores provided clear evidence of improved learning across all topics. The modules developed were responsive to the students' needs, and students provided additional improvements for subsequent iterations of Research 101.

The synthetic opioid fentanyl increases HIV replication and chemokine co-receptor expression in vitro.

The US is experiencing a major public health crisis that is fueled by the illicit use of synthetic opioids including fentanyl. While several drugs of abuse can enhance viral replication and/or antagonize immune responses, the impact of specific synthetic opioids on HIV pathogenesis is poorly understood. Thus, we evaluated the effects of fentanyl on HIV replication in vitro. HIV-susceptible or HIV-expressing cell lines were incubated with fentanyl. HIV p24 synthesis and chemokine receptor levels were quantified by ELISA in culture supernatants and cell lysates, respectively. Addition of fentanyl resulted in a dose-dependent increase in HIV replication. Fentanyl enhanced expression of the HIV chemokine co-receptors CXCR4 and CCR5 and caused a dose-dependent decrease in cell viability. The opioid antagonist naltrexone blocked the effect of fentanyl on HIV replication and CCR5 receptor levels but not CXCR4 receptor levels. TLR9 expression was induced by HIV; however, fentanyl inhibited TLR9 expression in a dose-dependent manner. These data demonstrate that the synthetic opioid fentanyl can promote HIV replication in vitro. As increased HIV levels are associated with accelerated disease progression and higher likelihood of transmission, additional research is required to enhance the understanding of opioid-virus interactions and to develop new and/or optimized treatment strategies for persons with HIV and opioid use disorder.

BK Polyomavirus Subtypes II and IV in Hematopoietic Cell Transplant Recipients.

Symptomatic BK polyomavirus (BKPyV) infections are common and relevant in immunocompromised patients. Here, we present full-length BKPyV genomes from samples from patients who received hematopoietic cell transplants in the United States. These individuals had non-subtype I BKPyV, as determined by amplification, next-generation sequencing, and phylogenetic analysis.

Human pegivirus type 1 infection in Asia-A review of the literature.

The human pegivirus type 1 (HPgV-1)-as known as hepatitis G virus and GB virus C-is a common single-stranded RNA flavivirus. Because few studies have demonstrated an association between HPgV-1 infection and disease, screening for HPgV-1 is not performed routinely. Nonetheless, a beneficial impact of HPgV-1 infection on HIV disease progression has been reported in multiple studies. Given the burden of HIV in Asia and the complex interactions between viral co-infections and the host, we provide a comprehensive overview of the existing data from Asia on HPgV-1 infection, including the prevalence and circulating genotypes in all Asian countries with data reported. This review highlights the research conducted thus far and emphasizes the need for additional studies on HPgV-1 across the Asian continent.

Optimized amplification of BK polyomavirus in urine.

BK polyomavirus (BKPyV) is a ubiquitous pathogen that typically results in asymptomatic infection. However, in immunocompromised individuals, BKPyV viral shedding in the urine can reach 109 copies per mL. These high viral levels within urine provide ideal samples for next-generation sequencing to accurately determine BKPyV genotype and identify mutations associated with pathogenesis. Sequencing data obtained can be further analyzed to better understand and characterize the genetic diversity present in BKPyV. Here, methods are described for the successful extraction of viral DNA from urine and the subsequent amplification methods to prepare a sample for next-generation sequencing.

Drugs of Abuse and Their Impact on Viral Pathogenesis.

Commonly misused substances such as alcohol, cocaine, heroin, methamphetamine, and opioids suppress immune responses and may impact viral pathogenesis. In recent years, illicit use of opioids has fueled outbreaks of several viral pathogens, including the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). This review focuses on the myriad of mechanisms by which drugs of abuse impact viral replication and disease progression. Virus-drug interactions can accelerate viral disease progression and lead to increased risk of virus transmission.

In silico analysis of mutations associated with occult hepatitis B virus (HBV) infection in South Africa.

Occult hepatitis B virus (OBI) infection is defined by the presence of viral DNA in the liver and/or serum in absence of hepatitis B surface antigen (HBsAg). While multiple studies have identified mutations that are associated with OBI, only a small portion of these mutations have been functionally characterized in vitro. Using complementary in silico approaches, the effects of OBI-associated amino acid mutations on HBV protein function in HBV/HIV-positive ART-naïve South Africans were evaluated. Two OBI-associated mutations in the PreS1 region, one in the PreS2 region, and seven in the surface region of subgenotype A1 sequences were identified as deleterious. In subgenotype A2 sequences, 11 OBI-associated mutations in the PreS1 region, seven in the PreS2 region, and 31 in the surface region were identified as deleterious. In the polymerase region, 14 OBI-associated mutations in subgenotype A1 and 71 OBI-associated mutations in subgenotype A2 were identified as deleterious. This study utilized in silico approaches to characterize the likely impact of OBI-associated mutations on viral function, thereby identifying and prioritizing candidates and reducing the significant cost associated with functional studies that are essential for mechanistic studies of the OBI phenotype.