Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Development of a Psychometric Tool to Measure Community Solidarity Among Sexual Minorities: Evidence From a Pay-it-Forward Randomized Controlled Trial.

BACKGROUND: Community solidarity is increasingly important in public health. However, few studies have examined solidarity in relation to health outcomes. The purpose of this study was to develop a psychometric tool to measure solidarity among Chinese men who have sex with men (MSM) and assess whether community solidarity relates to differences in sexually transmitted infection testing. METHODS: We used data from the pay-it-forward randomized controlled trial of 301 men from Beijing and Guangzhou, China. Men who have sex with men were randomized into pay-it-forward (participants receive free gonorrhea/chlamydia testing as gifts and choose to donate toward subsequent MSM's tests), pay-what you-want, and standard payment arms. After testing decision, participants completed a cross-sectional questionnaire to assess community solidarity. Factor analysis was conducted to identify dimensions of solidarity. The solidarity factors were compared across study arms and assessed against gonorrhea/chlamydia test uptake in multivariable logistic regression. RESULTS: Two hundred eighty-eight participants responded to the survey. We identified 3 latent community solidarity factors: engagement, social network support, and sense of belonging. Several items related to belonging were significantly greater among participants in the pay-it-forward scenario compared with those assigned to other scenarios. Higher sense of belonging was associated with higher odds of gonorrhea and chlamydia test uptake. CONCLUSIONS: Community solidarity among MSM in China can be characterized by 3 factors: engagement, social network support, and sense of belonging. Sense of belonging was higher in the pay-it-forward intervention arm and may be associated with the uptake of gonorrhea/chlamydia test. Future studies are warranted to confirm the psychometric structure of community solidarity and further investigate behavioral mechanisms of pay it forward.

Amplification-Free Detection of SARS-CoV-2 and Respiratory Syncytial Virus Using CRISPR Cas13a and Graphene Field-Effect Transistors.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.

The Addition of High-Risk HPV Testing to Anal Cytology Increases the Identification of Anal Dysplasia in HIV-Infected Patients.

BACKGROUND: Anal dysplasia (AD) is prevalent in HIV-infected patients. Screening for AD is recommended for high-risk groups, including HIV-infected patients. We evaluated screening algorithms for AD using cytology, high-risk human papillomavirus (HRH) testing, or both. METHODS: HIV-infected patients were offered AD screening by both anal cytology and PCR-based detection of HRH. Patients with abnormal cytology (AC) or HRH genotypes were referred to the same oncologic surgeon for high-resolution anoscopy (HRA). RESULTS: Ninety patients underwent screening (84% men who have sex with men). Forty-four patients (52.6%) had abnormal screens (31.5% AC, 46% HRH). Twenty-six patients with AC and/or positive HRH had HRA. AC and nadir CD4+ cell count of < 200 cells/mm3 were predictors of abnormal histology on HRA by univariate analysis (OR 4.5 and 2.5, respectively). Using a log-linear model, we estimated that for every 49 cases with two normal screening tests, one case of AD would be missed. Conclusions: Universal screening for AD in an HIV+ population yielded a high percentage of abnormal findings. Addition of HRH to cytology screening increased positive screens by 24%. Larger studies are needed to determine the ideal screening method.

Proactive infectious disease approach to dermatologic patients who are taking tumor necrosis factor-alfa antagonists: Part I. Risks associated with tumor necrosis factor-alfa antagonists.

Tumor necrosis factor-alfa levels are linked to disease severity in patients with inflammatory conditions, such as psoriasis. Inhibitors of this cytokine are commonly used with significant success in the treatment of such inflammatory disorders. Their use, however, can be plagued by infectious complications. An awareness of potential infections associated with these therapies is critical in order to maximize preventive efforts both before and during therapy. This review provides a guide for dermatologists caring for patients in need of this type of biologic therapy to preemptively address the infectious risks. Part I of this continuing medical education article reviews background information on the various infectious risks associated with tumor necrosis factor inhibitor therapy and appropriate historical data to obtain in the context of pretherapy evaluations.

Proactive infectious disease approach to dermatologic patients who are taking tumor necrosis factor-alfa antagonists: Part II. Screening for patients on tumor necrosis factor-alfa antagonists.

Tumor necrosis factor-alfa levels are linked to disease severity in patients with inflammatory conditions, such as psoriasis. Inhibitors of this cytokine are commonly used with significant success in the treatment of such inflammatory disorders. Their use, however, can be plagued by infectious complications. An awareness of potential infections associated with these therapies is critical in order to maximize preventive efforts both before and during therapy. This review provides a guide for dermatologists caring for patients in need of this type of biologic therapy to preemptively address the infectious risks. Part II of this continuing medical education article reviews recommended screening methods for patients undergoing evaluations for tumor necrosis factor inhibitor therapy for psoriasis or other dermatologic diseases, and discusses possible prophylactic strategies to use, including the appropriate use of immunizations.

Absence of Anti-Babesia microti antibody in commercial intravenous immunoglobulin (IVIG).

BACKGROUND: Babesiosis is a worldwide emerging protozoan infection that is associated with a spectrum of disease severity from asymptomatic infection to severe organ damage and death. While effective treatment strategies are available, some immunocompromised patients experience severe acute and prolonged/relapsing illness due in part to an impaired host antibody response. Intravenous immunoglobulin (IVIG) has been used as an adjunctive therapy in some immunocompromised babesiosis patients, but its therapeutic effect is uncertain. We evaluated the presence of Babesia microti antibodies in commercial samples of IVIG. METHODS/PRINCIPLE FINDINGS: The presence of B. microti antibodies in commercial samples of IVIG were tested using an immunofluorescence assay. A subset of samples was then tested for B. microti antibodies using an enzyme linked immunosorbent assay. Out of 57 commercial IVIG samples tested using IFA, and 52 samples tested using ELISA, none were positive for B. microti antibodies. CONCLUSIONS: Commercially available IVIG may not be of therapeutic benefit for babesiosis patients. Additional sampling of IVIG for B. microti antibody and a clinical trial of babesiosis patients given IVIG compared with controls would provide further insight into the use of IVIG for the treatment of babesiosis.

A point-of-care microfluidic biosensing system for rapid and ultrasensitive nucleic acid detection from clinical samples.

Rapid and ultrasensitive point-of-care RNA detection plays a critical role in the diagnosis and management of various infectious diseases. The gold-standard detection method of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is ultrasensitive and accurate yet limited by the lengthy turnaround time (1-2 days). On the other hand, an antigen test offers rapid at-home detection (typically ~15 min) but suffers from low sensitivity and high false-negative rates. An ideal point-of-care diagnostic device would combine the merits of PCR-level sensitivity and rapid sample-to-result workflow comparable to antigen testing. However, the existing detection platforms typically possess superior sensitivity or rapid sample-to-result time, but not both. This paper reports a point-of-care microfluidic device that offers ultrasensitive yet rapid detection of viral RNA from clinical samples. The device consists of a microfluidic chip for precisely manipulating small volumes of samples, a miniaturized heater for viral lysis and ribonuclease inactivation, a Cas13a-electrochemical sensor for target preamplification-free and ultrasensitive RNA detection, and a smartphone-compatible potentiostat for data acquisition. As demonstrations, the devices achieve the detection of heat-inactivated SARS-CoV-2 samples with a limit of detection down to 10 aM within 25 minutes, which is comparable to the sensitivity of RT-PCR and rapidness of an antigen test. The platform also successfully distinguishes all nine positive unprocessed clinical SARS-CoV-2 nasopharyngeal swab samples from four negative samples within 25 minutes of sample-to-result time. Together, this device provides a point-of-care solution that can be deployed in diverse settings beyond laboratory environments for rapid and accurate detection of RNA from clinical samples. The device can potentially be expandable to detect other viral targets, such as human immunodeficiency virus self-testing and Zika virus, where rapid and ultrasensitive point-of-care detection is required.

SARS-CoV-2 Antibody Dynamics in Healthcare Workers after mRNA Vaccination.

Since the emergence of SARS-CoV-2, maintaining healthcare worker (HCW) health and safety has been fundamental to responding to the global pandemic. Vaccination with mRNA-base vaccines targeting SARS-CoV-2 spike protein has emerged as a key strategy in reducing HCW susceptibility to SARS-CoV-2, however, neutralizing antibody responses subside with time and may be influenced by many variables. We sought to understand the dynamics between vaccine products, prior clinical illness from SARS-CoV-2, and incidence of vaccine-associated adverse reactions on antibody decay over time in HCWs at a university medical center. A cohort of 296 HCWs received standard two-dose vaccination with either bnt162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) and were evaluated after two, six, and nine months. Subjects were grouped by antibody decay curve into steep antibody decliners gentle decliners. Vaccination with mRNA-1273 led to more sustained antibody responses compared to bnt162b2. Subjects experiencing vaccine-associated symptoms were more likely to experience a more prolonged neutralizing antibody response. Subjects with clinical SARS-CoV-2 infection prior to vaccination were more likely to experience vaccination-associated symptoms after first vaccination and were more likely to have a more blunted antibody decay. Understanding factors associated with vaccine efficacy may assist clinicians in determining appropriate vaccine strategies in HCWs.

Computer-based intervention in HIV clinical care setting improves antiretroviral adherence: the LifeWindows Project.

We evaluated the efficacy of LifeWindows, a theory-based, computer-administered antiretroviral (ARV) therapy adherence support intervention, delivered to HIV + patients at routine clinical care visits. 594 HIV + adults receiving HIV care at five clinics were randomized to intervention or control arms. Intervention vs. control impact in the intent-to-treat sample (including participants whose ARVs had been entirely discontinued, who infrequently attended care, or infrequently used LifeWindows) did not reach significance. Intervention impact in the On Protocol sample (328 intervention and control arm participants whose ARVs were not discontinued, who attended care and were exposed to LifeWindows regularly) was significant. On Protocol intervention vs. control participants achieved significantly higher levels of perfect 3-day ACTG-assessed adherence over time, with sensitivity analyses maintaining this effect down to 70% adherence. This study supports the utility of LifeWindows and illustrates that patients on ARVs who persist in care at clinical care sites can benefit from adherence promotion software.

An analysis of electronically monitored adherence to antiretroviral medications.

Medication adherence studies increasingly collect data electronically, often using Medication Event Monitoring System (MEMS) caps. Analyses typically focus on summary adherence measures, although more complete analyses are possible using adaptive statistical methods. These methods were used to describe individual-subject adherence patterns for MEMS data from a clinical trial. Subjects were adaptively clustered into groups with similar adherence patterns and clusters were compared on a variety of subject characteristics. There were seven different adherence clusters: consistently high, consistently moderately high, consistently moderate, consistently moderately low, consistently low, deteriorating starting early, and deteriorating late. Compared to other subjects, subjects with consistently high and consistently moderately high adherence were more likely to be male, White, and older and to maintain during study participation a CD4 cell count over 500 and an HIV viral load of at most 400 copies/ml. These results demonstrate the effectiveness of adaptive methods for comprehensive analysis of MEMS data.

Art of prevention: Our approach to the measles-mumps-rubella vaccine in adult patients vaccinated against measles before 1968 on biologic therapy for the treatment of psoriasis.

BACKGROUND: In response to the evolving measles epidemic in the United States, the Centers for Disease Control and Prevention recommended that some adults be revaccinated against measles because they may have inadequate immunity against the virus. Patients receiving biologic medications for psoriasis face a clinical dilemma because they may be at an increased risk of developing severe measles; however, vaccination with the measles-mumps-rubella (MMR) vaccine is not recommended for those on biologic therapy according to the American Academy of Dermatology-National Psoriasis Foundation guidelines. OBJECTIVES: This study aimed to review available research on the safety and efficacy of live-attenuated vaccines in individuals receiving biologic therapy for psoriasis and to discuss our approach to vaccinating individuals on biologic agents for psoriasis with the MMR vaccine. METHODS: A review of the literature was performed via PubMed search. Our institution's anecdotal experiences are also discussed. RESULTS: Data, although limited, are available suggesting that live-attenuated vaccines may be safe for individuals on tumor necrosis factor-alpha inhibitors for psoriasis. Inadequate data are available for patients receiving other biologic medications. CONCLUSION: Providers should engage in shared decision-making to determine whether patients on tumor necrosis factor-alpha inhibitors for psoriasis should receive the MMR vaccine without an interruption in biologic therapy.

Modeling HIV Transmission from Sexually Active Alcohol-Consuming Men in ART Programs to Seronegative Wives.

BACKGROUND: The rollout of antiviral therapy in Low and Middle Income Countries (LMICs) has reduced HIV transmission rates at the potential risk of resistant HIV transmission. We sought to predict the risk of wild type and antiviral resistance transmissions in these settings. METHODS: A predictive model utilizing viral load, ART adherence, genital ulcer disease, condom use, and sexual event histories was developed to predict risks of HIV transmission to wives of 233 HIV+ men in 4 antiretroviral treatment centers in Maharashtra, India. RESULTS: ARV Therapy predicted a 5.71-fold reduction in transmissions compared to a model of using condoms alone, with 79.9%, of remaining transmissions resulting in primary ART-resistance. CONCLUSIONS: ART programs reduce transmission of HIV to susceptible partners at a substantial increased risk for transmission of resistant virus. Enhanced vigilance in monitoring adherence, use of barrier protections, and viral load may reduce risks of resistant HIV transmissions in LMIC settings.

Outcomes of a multifaceted medication adherence intervention for HIV-positive patients.

Treatment of HIV infection with highly active antiretroviral therapy (HAART) requires high levels of adherence in order to obtain maximum benefit and minimize the development of antiviral resistance. Many patients in community clinical settings have imperfect adherence that may lead to poor clinical outcomes. The Connecticut HIV Medication Project (CHaMP) is a multidisciplinary program designed to evaluate patients receiving antiviral therapy. Based on results of a multifaceted assessment, a variety of targeted interventions and follow-up are offered. A retrospective analysis was performed on patients who were referred to the program over a 35-month period from March 2002 through January 2005. Two hundred forty-nine patients who were referred for adherence services had baseline and follow-up data available for analysis. Participants who maintained an unchanged antiretroviral regimen experienced a significant increase in self-reported adherence (89.1% to 96.9%, p < 0.001) and likelihood of reporting more than 95% adherence (36.6% to 73.1%, p < 0.001) by 7-day recall. Improvements in plasma HIV viremia (3.10 +/- 1.21 log copies to 2.78 +/- 0.98, p < 0.01) were also demonstrated. Limitations to this study included the unusually high level of baseline adherence, the large fraction of patients (28.6%) who were lost to follow-up, and follow-up that was limited to one time point at 12-16 weeks such that attrition of the intervention effect could not be assessed. The CHaMP experience demonstrates that the development of a multifaceted clinical program can have significant impact on medication adherence and viral burden in HIV infection.

Improved adherence with contingency management.

Contingency management (CM) based interventions that reinforce adherence to prescribed medications have shown promise in a variety of disadvantaged populations. Fifty-six participants with histories of illicit substance use who were prescribed antiretroviral medication but evidenced suboptimal adherence during a baseline assessment were randomly assigned to 16 weeks of weekly CM-based counseling or supportive counseling, followed by 16 additional weeks of data collection and adherence feedback to providers. The CM intervention involved review of data generated by electronic pill-bottle caps that record bottle opening (MEMS) and brief substance abuse counseling. CM participants were reinforced for MEMS-measured adherence with drawings from a bowl for prizes and bonus drawings for consecutive weeks of perfect adherence. Potential total earnings averaged $800. Mean MEMS-measured adherence to the reinforced medication increased from 61% at baseline to 76% during the 16-week treatment phase and was significantly increased relative to the supportive counseling group (p = 0.01). Furthermore, mean log-transformed viral load was significantly lower in the CM group. However, by the end of the 16-week follow-up phase, differences between groups in adherence and viral load were no longer significantly different. Proportions of positive urine toxicology tests did not differ significantly between the two groups at any phase. A brief CM-based intervention was associated with significantly higher adherence and lower viral loads. Future studies should evaluate methods to extend effects for longer term benefits.

Human Brucellosis in Rural Uganda: Clinical Manifestations, Diagnosis, and Comorbidities at Kabale Regional Referral Hospital, Kabale, Uganda.

BACKGROUND: Brucellosis is a zoonotic infection transmitted to humans through direct contact with infected animals, their products, or excreta such as urine or dung. Brucellosis is associated with significant morbidity in Southwestern Uganda, where cattle and goat rearing are a major economic industry. As in many settings in sub-Saharan Africa, diagnosis and management of brucellosis remain a challenge due to the presence of comorbidities and limitations in resources for diagnostic testing and therapy. METHODS: A chart review was conducted to characterize the clinical manifestations, diagnosis, comorbidities, and management of 101 patients treated for brucellosis at the Kabale Regional Referral Hospital from September 2002 to May 2010. RESULTS: Patients presented with substantial comorbidities. The most common manifestation of illness was osteoarticular, but disease manifestations were quite varied. A high rate of focal illness in this cohort (77%) was observed. CONCLUSIONS: Clinicians in this setting should be cognizant of the varied presentations, comorbidities, and treatment options for this disease.

Factors affecting patient presentation at a national dermatology referral clinic in Kampala, Uganda.

BACKGROUND: This study sought to gain a better understanding of the patient population in Kampala and was further designed to elucidate barriers that may delay individuals from receiving proper dermatologic care. METHODS: The study took place at the dermatovenereology clinic of a tertiary care hospital in Kampala. New adult patients were surveyed in July and August of 2013. The primary dependent variable was time from reported onset of symptoms to presentation to the clinic. Participant demographic characteristics, medical and treatment history, and perception of illness as measured by the dermatology life quality index (DLQI) were assessed. RESULTS: A total of 232 subjects participated in the study. The most common skin diseases were allergic (20.3%), infectious (15.1%), follicular (7.8%), and papulosquamous (7.8%) disorders. Greater home distance from the clinic correlated with later presentation times (r = 0.259, P < 0.001). DLQI score was not correlated with presentation time. HIV+ individuals presented earlier (mean 5 vs. 11 months, P = 0.043) and had higher DLQI scores (mean 12.6 vs. 9.3, P = 0.006) than HIV- individuals. The majority of participants (72.5%) had contact with at least one other healthcare worker (HCW) for management of their dermatologic symptoms; 65.8% reported that these previous treatments were ineffective. CONCLUSIONS: Efforts to educate HCWs should be focused on districts outside of Kampala and highlight recognition and proper treatment of allergic diseases. HCWs should aggressively treat skin problems in HIV+ individuals. HCWs practicing in Kampala without formal dermatological training should refer patients with skin disease to the clinic, as patients may receive care that is more appropriate.

Bladder Cancer versus Hemorrhagic Cystitis: A Case of Mistaken Identity in a 34-Year-Old Male Undergoing Therapy for Granulomatosis with Polyangiitis.

A 34-year-old male was referred for management of bladder cancer noted on workup for gross hematuria and new-onset irritative voiding symptoms. The patient's history was significant for recently diagnosed granulomatosis with polyangiitis for which he was undergoing treatment with oral cyclophosphamide and corticosteroids. Cystoscopy revealed lesions suspicious for malignancy, but the patient was diagnosed with hemorrhagic cystitis secondary to BK virus infection upon cytology review, and immunostaining confirmed a polyomavirus infection of the urothelium. The patient's symptoms resolved after a modification of his immunosuppressive regimen, and antiviral therapy was ultimately unnecessary. Though symptomatic BK virus infection of the genitourinary tract is common in immunosuppressed transplant patients, its occurrence in a patient undergoing immunomodulation for an autoimmune disease has not been reported yet. This case illustrates the potential for active BK virus infections in atypical patient populations and underscores the importance of rigorous hematuria workup, particularly in patients with multiple risk factors.

Clinical implications of elevated HIV-1 viral load results obtained from samples stored frozen in vacutainer plasma preparation tubes.

Studies have demonstrated that plasma samples collected and stored frozen using vacutainer plasma preparation tubes (PPT) may result in falsely elevated viral load (VL) values with the Roche COBAS TaqMan HIV-1 v1.0 test. At the University of Connecticut Health Center, a total of 349 samples from HIV-1-infected patients on HAART were collected and stored frozen in PPT. Viral load (VL) results were obtained using the Roche COBAS TaqMan HIV-1 v2.0 test (CTM v2.0) and Abbott RealTime HIV-1 assay (RealTime HIV-1). Of the 349 samples, 260 (74.5%) had VL values that differed by >0.5log10copies/mL; 64 of these were quantified by both assays. The remaining 196 samples were detected by CTM v2.0 but not detected in RealTime HIV-1: 62 of the most discordant samples in this category (CTM v2.0 detected/RealTime HIV-1 not detected) were further analyzed using two nested RT-PCR assays targeting pol integrase: full-length (864nt) and a highly conserved subregion (134nt). No HIV-1 RNA was detected in the discordant samples, confirming RealTime HIV-1 results. The increase in VL reactivity with the CTM v2.0 assay was presumably due to proviral DNA captured by the CTM total nucleic acid extraction chemistry but not the RNA-specific extraction procedure used in RealTime HIV-1. These results suggest that using CTM v2.0 with samples frozen in PPT could have significant clinical implications for HIV-1 patient management.