Duration of Time CD4/CD8 Ratio is Below 0.5 is Associated with Progression to Anal Cancer in Patients with HIV and High-Grade Dysplasia.

BACKGROUND: A CD4/CD8 ratio < 0.5 is associated with increased risk of advanced anal disease (AAD) but it is unknown if duration below 0.5 matters. The purpose of this study was to determine if duration of a CD4/CD8 ratio < 0.5 is associated with increased risk of invasive anal cancer (IC) in people living with HIV and high-grade dysplasia (HSIL). METHODS: This single institution, retrospective study used the University of Wisconsin Hospital and Clinics Anal Dysplasia and Anal Cancer Database. Patients with IC versus HSIL alone were compared. Independent variables were mean and percentage of time the CD4/CD8 ratio was < 0.5. Multivariate logistic regression was performed to estimate the adjusted odds of anal cancer. RESULTS: We identified 107 patients with HIV infection and AAD (87 with HSIL, 20 with IC). A history of smoking was significantly associated with the development of IC (95% in patients with IC vs. 64% in patients with HSIL; p = 0.015). Mean time the CD4/CD8 ratio was < 0.5 was significantly longer in patients with IC compared with patients with HSIL (7.7 years vs. 3.8 years; p = 0.002). Similarly, the mean percentage of time the CD4/CD8 ratio was < 0.5 was higher in those with IC versus those with HSIL (80% vs. 55%; p = 0.009). On multivariate analysis, duration CD4/CD8 ratio was < 0.5 was associated with increased odds of developing IC (odds ratio 1.25, 95% confidence interval 1.02-1.53; p = 0.034). CONCLUSIONS: In this retrospective, single-institution study of a cohort of people living with HIV and HSIL, increasing duration the CD4/CD8 ratio was < 0.5 was associated with increased odds of developing IC. Monitoring the number of years the CD4/CD8 ratio is < 0.5 could inform decision making in patients with HIV infection and HSIL.

Prevalence of High-Grade Anal Dysplasia and Anal Cancer in Veterans Living With HIV and CD4/CD8 Ratio as a Marker For Increased Risk: A Regional Retrospective Cohort Study.

BACKGROUND: The Department of Veterans Affairs cares for the largest population of patients with HIV of any healthcare system in the United States. Screening for anal dysplasia/cancer is recommended for all veterans with HIV. Exams are invasive, burdensome, and resource intensive. We currently lack markers of disease to tailor screening. OBJECTIVE: The purpose of this study was to establish the prevalence of advanced anal disease (high-grade dysplasia and anal cancer) and to determine whether CD4/CD8 ratio correlates with risk. DESIGN: This was a retrospective regional cohort study of veterans with HIV. SETTINGS: The study was conducted at eight medical centers between 2001 and 2019. PATIENTS: Patients with advanced disease were compared with patients with nonadvanced anal pathology. MAIN OUTCOME MEASURES: Logistic regression modeling was used to estimate adjusted odds of disease as a function of CD4/CD8. Lowest (nadir) CD4/CD8 and nearest CD4/CD8 ratio in each cohort were evaluated. RESULTS: A total of 2267 veterans were included. Fifteen percent had anal pathology (112 with advanced disease (37 cancer and 75 high-grade), 222 with nonadvanced disease). Nadir and nearest ratio were lower in patients with advanced disease versus nonadvanced (0.24 vs 0.45 (p < 0.001) and 0.50 vs 0.88 (p < 0.001)). In adjusted models, a 1-unit increase in nadir or nearest ratio conferred decreased risk of advanced disease (OR = 0.19 (95% CI, 0.07-0.53); p < 0.001; OR = 0.22 (95% CI, 0.12-0.43); p < 0.001). Using a minimum sensitivity analysis, a cutoff nadir ratio of 0.42 or nearest ratio of 0.76 could be used to risk stratify. LIMITATIONS: This was a retrospective analysis with a low screening rate. CONCLUSIONS: In a regional cohort of veterans with HIV, 15% were formally assessed for anal dysplasia. Advanced anal disease was present in 33% of those screened, 5% of the HIV-positive population. A strong predictor of advanced disease in this cohort is the CD4/CD8 ratio, which is a promising marker to stratify screening practices. Risk stratification using CD4/CD8 has the potential to decrease burdensome invasive examinations for low-risk patients and to intensify examinations for those at high risk. See Video Abstract at http://links.lww.com/DCR/B528. PREVALENCIA DE DISPLASIA ANAL DE ALTO GRADO Y CNCER ANAL EN VETERANOS QUE VIVEN CON EL VIH Y LA RELACIN CD / CD COMO MARCADOR DE MAYOR RIESGO UN ESTUDIO DE COHORTE REGIONAL RETROSPECTIVE: ANTECEDENTES:El Departamento de Asuntos de Veteranos atiende a la población más grande de pacientes con el virus de inmunodeficiencia humana (VIH) de cualquier sistema de salud en los Estados Unidos. Se recomienda la detección de displasia / cáncer anal para todos los veteranos con VIH. Los exámenes son invasivos, onerosos y requieren muchos recursos. Actualmente carecemos de marcadores de enfermedad para adaptar la detección.OBJETIVO:Establecer la prevalencia de enfermedad anal avanzada (displasia de alto grado y cáncer anal) y determinar si la relación CD4 / CD8 se correlaciona con el riesgo.DISEÑO:Estudio de cohorte regional retrospectivo de veteranos con VIH.AJUSTE:Ocho centros médicos entre 2001-2019.PACIENTES:Se comparó a pacientes con enfermedad avanzada con pacientes con patología anal no avanzada.PRINCIPALES MEDIDAS DE RESULTADO:Se utilizó un modelo de regresión logística para estimar las probabilidades ajustadas de enfermedad en función de CD4 / CD8. Se evaluó la relación CD4 / CD8 más baja (nadir) y la relación CD4 / CD8 más cercana en cada cohorte.RESULTADOS:Se incluyeron un total de 2267 veteranos. El 15% tenía patología anal (112 enfermedad avanzada (37 cáncer, 75 de alto grado), 222 enfermedad no avanzada). El nadir y el cociente más cercano fueron menores en los pacientes con enfermedad avanzada frente a los no avanzados (0,24 frente a 0,45 (p <0,001) y 0,50 frente a 0,88 (p <0,001)), respectivamente. En modelos ajustados, el aumento de una unidad en el nadir o el cociente más cercano confirió una disminución del riesgo de enfermedad avanzada (OR 0,19 (IC del 95%: 0,07, 0,53, p <0,001)) y (OR 0,22 (IC del 95%: 0,12, 0,43, p <0,001))), respectivamente. Utilizando un análisis de sensibilidad mínima, se podría utilizar un cociente del nadir de corte de 0,42 o el cociente más cercano de 0,76 para estratificar el riesgo.LIMITACIONES:Análisis retrospectivo con una tasa de detección baja.CONCLUSIONES:En una cohorte regional de veteranos con VIH, el 15% fueron evaluados formalmente por displasia anal. La enfermedad anal avanzada estuvo presente en el 33% de los examinados, el 5% de la población VIH +. Un fuerte predictor de enfermedad avanzada en esta cohorte es la relación CD4 / CD8, que es un marcador prometedor para estratificar las prácticas de detección. La estratificación del riesgo usando CD4 / CD8 tiene el potencial de disminuir los exámenes invasivos onerosos para los pacientes de bajo riesgo e intensificar los exámenes para los de alto riesgo. Consulte Video Resumen en http://links.lww.com/DCR/B528.

CD4/CD8 Ratio as a Novel Marker for Increased Risk of High-Grade Anal Dysplasia and Anal Cancer in HIV+ Patients: A Retrospective Cohort Study.

BACKGROUND: People living with HIV are at risk for anal dysplasia/cancer. Screening/surveillance is costly and burdensome, and the frequency is not evidence based. Objective markers of increased risk of anal carcinogenesis are needed to tailor screening/surveillance. Low CD4/CD8 ratio is associated with increased overall cancer risk in people living with HIV but has yet to be examined for quantifying anal cancer risk. OBJECTIVE: We hypothesized that low CD4/CD8 ratios correlate with increased risk for high-grade anal dysplasia and cancer. DESIGN: This is a single-institution, retrospective review of people living with HIV from 2002 to 2018. SETTING: This study was conducted at the University of Wisconsin School of Medicine and Public Health. PATIENTS: Patients with advanced disease (high-grade anal dysplasia and/or anal cancer) were compared with patients with negative anal cytology. MAIN OUTCOME MEASURES: The independent variables were lowest (nadir) CD4/CD8 and CD4/CD8 nearest to screening/diagnosis. Logistic regression modeling was used to estimate the adjusted odds of advanced disease. RESULTS: A total of 377 people living with HIV were examined: 266 with negative cytology and 111 with advanced disease (16 cancer, 95 high-grade anal dysplasia). Mean nadir ratio and mean nearest ratio were lower in patients with advanced disease than in those with negative screening (0.26 vs 0.47 (p < 0.001) and 0.61 vs 0.87 (p < 0.001)). In adjusted analyses, increase in nadir ratio or nearest ratio of 1 unit conferred decreased risk of advanced disease (OR, 0.10; 95% CI, 0.02-0.45; p = 0.002) and (OR, 0.31; 95% CI, 0.12-0.83; p = 0.02). The optimal threshold for using CD4/CD8 ratio as a risk factor for advanced disease was 0.47 for nadir ratio (sensitivity 0.59 and specificity 0.91) and 0.95 for nearest ratio (sensitivity 0.56 and specificity 0.92). LIMITATIONS: This is a retrospective, single-institution study. CONCLUSIONS: Low CD4/CD8 ratio confers additional risk of high-grade anal dysplasia and anal cancer beyond the diagnosis of HIV, even when adjusting for known risks factors of anal cancer. Our data suggest that the CD4/CD8 ratio may be able to help identify people living with HIV who are at higher risk of anal cancer development. See Video Abstract at http://links.lww.com/DCR/B336. LA RELACIÓN CD4 / CD8 COMO UN MARCADOR NOVEDOSO PARA EL AUMENTO DEL RIESGO DE DISPLASIA ANAL DE ALTO GRADO Y CÁNCER ANAL EN PACIENTES VIH+: UN ESTUDIO DE COHORTE RETROSPECTIVO: Las personas que viven con el virus de la inmunodeficiencia humana están en riesgo de displasia / cáncer anal. La detección / vigilancia es costosa, laboriosa y la frecuencia no se basa en evidencias. Se necesitan marcadores objetivos de mayor riesgo de carcinogénesis anal para adaptar la detección / vigilancia. La relación baja de CD4 / CD8 se asocia con un mayor riesgo general de cáncer en personas que viven con el virus de inmunodeficiencia humana, pero aún no se ha examinado para cuantificar el riesgo de cáncer anal.Hicimos la hipotesis de que la relación baja de CD4 / CD8 se correlacionan con un mayor riesgo de displasia anal de alto grado y cáncer.Revisión retrospectiva de una sola institución de personas que viven con el virus de la inmunodeficiencia humana desde 2002 hasta 2018.Facultad de Medicina y Salud Pública de la Universidad de Wisconsin.Los pacientes con enfermedad avanzada (displasia anal de alto grado y / o cáncer anal) se compararon con pacientes con citología anal negativa.Las variables independientes más bajas fueron (nadir) CD4 / CD8 y la relación CD4 / CD8 más cercanas a la detección / diagnóstico. Se utilizó el modelo de regresión logística para estimar las probabilidades ajustadas de enfermedad avanzada.Se examinaron un total de 377 personas que viven con el virus de inmunodeficiencia humana, 266 con citología negativa y 111 con enfermedad avanzada (16 cáncer, 95 displasia anal de alto grado). La tasa nadir y la tasa media más cercana fueron más bajas en pacientes con enfermedad avanzada vs. aquellos con cribado negativo (0.26 v. 0.47 (p <0.001) y 0.61 v. 0.87 (p <0.001), respectivamente. En los análisis ajustados, el aumento en la tasa nadir o la tasa más cercana a una unidad confirió una disminución del riesgo de enfermedad avanzada (OR de 0,10 (IC del 95%: 0,02, 0,45, p = 0,002)) y (OR 0,31 (IC del 95%: 0,12, 0,83, p = 0.02)), respectivamente. El umbral óptimo para usar la relacion CD4 / CD8 como factor de riesgo de enfermedad avanzada fue 0,47 para la tasa nadir (sensibilidad 0,59 y especificidad 0,91) y 0,95 para la tasa más cercana (sensibilidad 0,56 y especificidad 0,92).Este es un estudio retrospectivo de una sola institución.La baja relación CD4 / CD8 confiere un riesgo adicional de displasia anal de alto grado y cáncer anal más allá del diagnóstico del virus de inmunodeficiencia humana, incluso cuando se ajustan los factores de riesgo conocidos de cáncer anal. Nuestros datos sugieren que la relación CD4/CD8 puede ayudar a identificar a las personas que viven con el virus de inmunodeficiencia humana que tienen un mayor riesgo de desarrollar cáncer anal. Consulte Video Resumen en http://links.lww.com/DCR/B336.

Anal Intraepithelial Neoplasia Screening With Anal Pap Tests: Follow-up and Corresponding Histology.

BACKGROUND: Anal cytology is used as a screening tool in the detection of precancerous anal squamous lesions. Follow-up clinical examination after abnormal anal cytology is recommended. The objective of this study was to determine how often abnormal cytology was followed by a clinical examination at our institution and how often cytology predicted histologic outcome. MATERIALS AND METHODS: A retrospective chart review was performed (2008-2018) on patients with anal cytology, demonstrating either low-grade or high-grade squamous intraepithelial lesion. Clinical examination within 1 y (digital rectal examination, anoscopy, or high-resolution anoscopy) was recorded. The probability of anal intraepithelial neoplasm on biopsy after dysplasia on cytology was calculated, and McNemar's test was used to determine if there was correspondence between cytology and histology. RESULTS: A total of 327 anal cytology results demonstrated dysplasia (75% low grade and 25% high grade) in 182 patients. Seventy-five percent of dysplastic anal cytology were followed by clinical examination within 1 y, and 50% were biopsied. The probability of dysplasia on histology after dysplasia on cytology was 72% (95% confidence interval: 64%-78.5%). Twenty-eight percent of low-grade cytology results were upgraded to advanced disease (high-grade or invasive cancer) on histology. A low-grade cytology result was unable to preclude high-grade histology in our population. CONCLUSIONS: There is room for improvement at our institution to consistently follow-up with clinical examination after abnormal anal cytology. Our data suggest this is especially important considering anal cytology is an imperfect predictor of histologic anal intraepithelial neoplasia and invasive disease. Clinical examination is a critical component of anal dysplasia screening and follow-up.

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to ß-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to ß-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various ß-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate ß-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection.

In Silico Screen and Structural Analysis Identifies Bacterial Kinase Inhibitors which Act with ß-Lactams To Inhibit Mycobacterial Growth.

New tools and concepts are needed to combat antimicrobial resistance. Actinomycetes and firmicutes share several eukaryotic-like Ser/Thr kinases (eSTK) that offer antibiotic development opportunities, including PknB, an essential mycobacterial eSTK. Despite successful development of potent biochemical PknB inhibitors by many groups, clinically useful microbiologic activity has been elusive. Additionally, PknB kinetics are not fully described, nor are structures with specific inhibitors available to inform inhibitor design. We used computational modeling with available structural information to identify human kinase inhibitors predicted to bind PknB, and we selected hits based on drug-like characteristics intended to increase the likelihood of cell entry. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We performed an in-depth characterization of PknB and found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel X-ray structure confirmed that the inhibitors bound as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity toward mycobacteria and nocardia. We demonstrated that the inhibitors are uniquely potentiated by ß-lactams but not antibiotics traditionally used to treat mycobacteria, consistent with PknB's role in sensing cell wall stress. This is the first demonstration in the phylum actinobacteria that some ß-lactam antibiotics could be more effective if paired with a PknB inhibitor. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can act with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

Sterol Carrier Protein 2, a Critical Host Factor for Dengue Virus Infection, Alters the Cholesterol Distribution in Mosquito Aag2 Cells.

Host factors that enable dengue virus (DENV) to propagate in the mosquito host cells are unclear. It is known that cellular cholesterol plays an important role in the life cycle of DENV in human host cells but unknown if the lipid requirements differ for mosquito versus mammalian. In mosquito Aedes aegypti, sterol carrier protein 2 (SCP-2) is critical for cellular cholesterol homeostasis. In this study, we identified SCP-2 as a critical host factor for DENV production in mosquito Aag2 cells. Treatment with a small molecule commonly referred to as SCPI-1, (N-(4-{[4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl]amino}phenyl)acetamide hydrobromide, a known inhibitor of SCP-2, or knockdown of SCP-2 dramatically repressed the virus production in mosquito but not mammalian cells. We showed that the intracellular cholesterol distribution in mosquito cells was altered by SCP-2 inhibitor treatment, suggesting that SCP-2-mediated cholesterol trafficking pathway is important for DENV viral production. A comparison of the effect of SCP-2 on mosquito and human cells suggests that SCPI-1 treatment decreases cholesterol in both cell lines, but this decrease in cholesterol only leads to a decline in viral titer in mosquito host cells, perhaps, owing to a more drastic effect on perinuclear cholesterol storages in mosquito cells that was absent in human cells. SCP-2 had no inhibitory effect on another enveloped RNA virus grown in mosquito cells, suggesting that SCP-2 does not have a generalized anti-cellular or antiviral effect. Our cell culture results imply that SCP-2 may play a limiting role in mosquito-dengue vector competence.

Selective pharmacologic inhibition of a PASTA kinase increases Listeria monocytogenes susceptibility to ß-lactam antibiotics.

While ß-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the penicillin binding protein and serine/threonine kinase-associated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore ß-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to ß-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of ß-lactam antibiotics.

Pharmacological disruption of hepatitis C NS5A protein intra- and intermolecular conformations.

Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.

Human immunodeficiency virus testing pitfalls and clinical suspicion.

Universal human immunodeficiency virus (HIV) screening was recommended in 2012, and major improvements in HIV testing have occurred in the past decade, but identification of HIV infected individuals remains inadequate in the United States. We report the case of a seronegative HIV-infected man who despite clinical and laboratory findings of acquired immunodeficiency syndrome,repeatedly tested nonreactive to third-generation HIV enzyme immunoassays (EIAs) and Western blot testing. Serologic diagnosis in this case required fourth-generation EIA testing due to the seronegativity of standard testing. The fourth-generation HIV EIA was positive presumably because it detects p24 HIV antigen as well as antibodies, unlike rapid HIV tests and third-generation HIV EIAs.This case highlights not only the importance of frontline providers to understand the different testing methodologies for HIV screening and their limitations but the importance of clinical suspicion as well.

Analysis of >15 000 Solid Organ Transplant Recipients Reveals Nonanal Genitourinary HPV-related Disease as Highest Risk Predictor for Anal Squamous Intraepithelial Lesions/Anal Cancer.

BACKGROUND: Solid organ transplantation is a risk predictor for virally-mediated anal squamous intraepithelial lesions and cancer (anal disease). Precancerous squamous intraepithelial lesions can be detected by screening, and treatment may prevent cancer progression. Screening recommendations are not well defined. We aim to define prevalence and describe risk predictors for anal disease in a large population of solid organ transplant recipients. METHODS: Retrospective single-center cohort analysis included solid organ transplant recipients cared for between 2001 and 2022 (N = 15 362). The cohort of recipients who developed anal disease was compared with those who did not. Greedy propensity score matching was performed for organ-specific recipients, and time-to-event analysis for the development of anal disease was performed in those with genitourinary human papilloma virus (HPV) disease versus those without. RESULTS: Prevalence of anal disease was 0.6% (cancer 0.2%). The average years from transplant to the diagnosis of anal disease was 11.67. Anal disease was more common in women (68.5% versus 31.5%, P  < 0.001), patients who had other HPV-related genitourinary diseases (40.4% versus 0.6%, P  < 0.001), who were of younger age at transplant (39.62 versus 46.58, P  < 0.001), and had increased years from transplant (17.06 versus 12.57, P  < 0.001). In multivariate analysis, the odds of anal disease increased by 4% each year posttransplant. History of genitourinary HPV disease (odds ratio 69.63) and female sex (odds ratio 1.96) were the most significant risk predictors for anal disease. CONCLUSIONS: The prevalence of anal cancer among solid organ transplant recipients was equal to the general population (0.2%). Due to the low prevalence of overall disease, these data suggest that anal screenings in transplant recipients should be targeted to higher-risk subsets: female recipients farther out from transplant and patients with genitourinary HPV-related diseases.

Telaprevir to boceprevir switch highlights lack of cross-reactivity.

Hepatitis C viral protease inhibitors increase sustained virologic response rates compared to interferon and ribavirin but also add side effects. Telaprevir and boceprevir are structurally similar, and share cross-resistant mutations. This case report highlights successful management of telaprevir skin rash and anal discomfort by switching to boceprevir.

Fluorescence resonance energy transfer-based intracellular assay for the conformation of hepatitis C virus drug target NS5A.

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests that large portions of the carboxy terminus of NS5A are in an unfolded and disordered state. Quercetin, a natural product known to disrupt NS5A function in cells, specifically disrupted a conformationally specific domain 3 FRET signal. Intermolecular FRET indicated that the NS5A amino termini, but not other regions, are in close proximity in multimeric complexes. Overall, this assay provides a new window on the intracellular conformation(s) of NS5A and how the conformation changes in response to cellular and viral components of the replication and assembly complex as well as antiviral drugs.

Analysis of hepatitis C virus intrahost diversity across the coding region by ultradeep pyrosequencing.

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naïve patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Comparison of hepatitis C virus treatment between incarcerated and community patients.

UNLABELLED: The prevalence of chronic hepatitis C virus (HCV) infection among incarcerated individuals in the United States is estimated to be between 12% and 31%. HCV treatment during incarceration is an attractive option because of improved access to health care and directly observed therapy. We compared incarcerated and nonincarcerated HCV-infected patients evaluated for treatment at a single academic center between January 1, 2002 and December 31, 2007. During this period, 521 nonincarcerated and 388 incarcerated patients were evaluated for HCV treatment. Three hundred and nineteen (61.2%) nonincarcerated patients and 234 (60.3%) incarcerated patients underwent treatment with pegylated interferon and ribavirin. Incarcerated patients were more likely to be male, African-American race, and have a history of alcohol or intravenous drug use. Treated incarcerated patients were less likely to have genotype 1 virus and were less likely to have undergone previous treatment. There was a similar prevalence of coinfection with human immunodeficiency virus (HIV) in both groups. A sustained viral response (SVR) was achieved in 97 (42.9%) incarcerated patients, compared to 115 (38.0%) nonincarcerated patients (P = 0.304). Both groups had a similar proportion of patients that completed a full treatment course. Stepwise logistic regression was conducted, and the final model included full treatment course, non-genotype 1 virus, younger age at treatment start, and negative HIV status. Incarceration status was not a significant predictor when added to this model (P = 0.075). CONCLUSION: In a cohort of HCV-infected patients managed in an academic medical center ambulatory clinic, incarcerated patients were as likely to be treated for HCV and as likely to achieve an SVR as nonincarcerated patients.