Implementation of an HIV RNA Qualitative PCR Assay in an HIV Diagnostic Algorithm: A Single-Institution Experience.

OBJECTIVES: Boston Medical Center (BMC) is a private, not-for-profit 514-bed academic medical center and legacy safety net hospital serving a diverse global patient population. BMC recently implemented a new HIV-1/HIV-2 Qualitative RNA PCR (HIV RNA QUAL) cleared by the US Food and Drug Administration to (1) replace antibody discrimination follow-up testing after a reactive fourth-generation (4G) serology screen and (2) use as a stand-alone diagnostic for suspected seronegative acute HIV infection. METHODS: This report summarizes the results of a production monitor for the first 3 months postimplementation. RESULTS: The monitor characterized test utilization, diagnostic turnaround time, impact on send-out testing, results reflexed to HIV RNA discrimination follow-up, and discrepancies between screening and HIV RNA results that necessitated additional investigation. Another element was the novelty of using HIV RNA QUAL while awaiting the existing Centers for Disease Control and Prevention HIV testing algorithm update. The 4G screening components and the HIV RNA QUAL were also used to create an algorithm specific to and compliant with current guidelines for screening patients on HIV preexposure prophylaxis. CONCLUSIONS: Based on our findings, this new test algorithm may be reproducible and instructive at other institutions.

On-Site, On-Demand 3D-Printed Nasopharyngeal Swabs to Improve the Access of Coronavirus Disease-19 Testing.

Diagnostic testing that facilitates containment, surveillance, and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or future respiratory viruses, depends on a sample collection device that efficiently collects nasopharyngeal tissue and that can be manufactured on site when an outbreak or public health emergency is declared by a government. Here two novel stereolithography-based three-dimensional (3D)-printed nasopharyngeal swabs are reported which are made using a biocompatible and sterilizable photoresist. Such swabs are readily manufactured on-site and on-demand to ensure availability, if supply chain shortages emerge. Additionally, the 3D-printed swabs easily adapt to current workflow and testing procedures in hospital clinical laboratories to allow for effortless scaling up of test kits. Finally, the 3D-printed nasopharyngeal swabs demonstrate concordant SARS-CoV-2 testing results between the 3D-printed swabs and the COPAN commercial swabs, and enable detection of SARS-CoV-2 in clinical samples obtained from autopsies.

Pandemic Response Requires Research Samples: A U.S. Safety-Net Hospital's Experience and Call for National Action.

Biorepositories provide a critical resource for gaining knowledge of emerging infectious diseases and offer a mechanism to rapidly respond to outbreaks; the emergence of the novel coronavirus, SARS-CoV-2, has proved their importance. During the COVID-19 pandemic, the absence of centralized, national biorepository efforts meant that the onus fell on individual institutions to establish sample repositories. As a safety-net hospital, Boston Medical Center (BMC) recognized the importance of creating a COVID-19 biorepository to both support critical science at BMC and ensure representation in research for its urban patient population, most of whom are from underserved communities. This article offers a realistic overview of the authors' experience in establishing this biorepository at the onset of the COVID-19 pandemic during the height of the first surge of cases in Boston, Massachusetts, with the hope that the challenges and solutions described are useful to other institutions. Going forward, funders, policymakers, and infectious disease and public health communities must support biorepository implementation as an essential element of future pandemic preparedness.

Time to SARS-CoV-2 PCR Clearance in Immunocompromising Conditions: Is Test-Based Removal From Isolation Necessary in Severely Immunocompromised Individuals?

To determine the association between immunosuppression and time to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) clearance, we studied 3758 adults retested following initial SARS-CoV-2 infection. Cox proportional hazards models demonstrated delayed PCR clearance with older age, multiple comorbidities, and solid organ transplant but not by degree of immunocompromise. These findings challenge current retesting practices.

Recent endemic coronavirus infection is associated with less-severe COVID-19.

Four different endemic coronaviruses (eCoVs) are etiologic agents for the seasonal common cold, and these eCoVs share extensive sequence homology with human SARS coronavirus 2 (SARS-CoV-2). Here, we show that individuals with, as compared with those without, a recent documented infection with eCoV were tested at greater frequency for respiratory infections but had a similar rate of SARS-CoV-2 acquisition. Importantly, the patients with a previously detected eCoV had less-severe coronavirus disease 2019 (COVID-19) illness. Our observations suggest that preexisting immune responses against endemic human coronaviruses can mitigate disease manifestations from SARS-CoV-2 infection.

Assessment of Oropharyngeal Specimens for Discontinuation of Transmission-Based COVID-19 Precautions.

We compared oropharyngeal swab test performance with nasopharyngeal testing for discontinuation of transmission-based COVID-19 precautions. We performed a retrospective review of confirmed COVID-19-positive patients who received paired nasopharyngeal and oropharyngeal SARS-CoV-2 tests for clearance from isolation from May 4, 2020, to May 26, 2020. Using nasopharyngeal swabs as the reference standard, we calculated the sensitivity, specificity, and negative predictive value of oropharyngeal swabs. We also calculated the kappa between the 2 tests. A total of 189 paired samples were collected from 74 patients. Oropharyngeal swab sensitivity was 38%, specificity was 87%, and negative predictive value was 70%. The kappa was 0.25. Our study suggests that oropharyngeal swabs are inferior to nasopharyngeal swabs for test-based clearance from COVID-19 isolation.

qRT-PCR Platforms for Diagnosing and Reporting SARS-CoV-2 Infection in Human Samples.

The protocols herein outline the use of qRT-PCR to detect the presence of SARS-CoV-2 genomic RNA in patient samples. In order to cope with potential fluctuations in supply chain and testing demands and to enable expedient adaptation of reagents and assays on hand, we include details for three parallel methodologies (one- and two-step singleplex and one-step multiplex assays). The diagnostic platforms described can be easily adapted by basic science research laboratories for SARS-CoV-2 diagnostic testing with relatively short turnaround time. For complete details on the use and execution of this protocol, please refer to Vanuytsel et al. (2020).

The Clinical Microbiology Laboratory and the Opioid Epidemic: Challenges and Opportunities.

Increased infections from injection drug use harm patients and are costly to the health care system. The impact on clinical microbiology laboratories is less recognized. Microbiology laboratories face increased test volume and test complexity from the spectrum and burden of pathogens associated with injection drug use, which lead to diagnostic challenges and overtaxed resources. We describe stressed workflows, pathogens that defy protocols, and limits of current technologies. Laboratories may benefit from protocol revisions, additional resources, workflow oversight, and improved communication with clinical providers to optimally meet challenges associated with this public health crisis.

Rapid Implementation of a SARS-CoV-2 Diagnostic Quantitative Real-Time PCR Test with Emergency Use Authorization at a Large Academic Safety Net Hospital.

BACKGROUND: Significant delays in the rapid development and distribution of diagnostic testing for SARS-CoV-2 (COVID-19) infection have prevented adequate public health management of the disease, impacting the timely mapping of viral spread and the conservation of personal protective equipment. Furthermore, vulnerable populations, such as those served by the Boston Medical Center (BMC), the largest safety net hospital in New England, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions and substance use disorders, lower health maintenance, unstable housing, and a propensity for rapid community spread, highlighting the urgent need for expedient and reliable in-house testing. METHODS: We developed a SARS-CoV-2 diagnostic medium-throughput qRT-PCR assay with rapid turnaround time and utilized this Clinical Laboratory Improvement Amendments (CLIA)-certified assay for testing nasopharyngeal swab samples from BMC patients, with emergency authorization from the Food and Drug Administration (FDA) and the Massachusetts Department of Public Health. FINDINGS: The in-house testing platform displayed robust accuracy and reliability in validation studies and reduced institutional sample turnaround time from 5-7 days to less than 24 h. Of over 1,000 unique patient samples tested, 44.1% were positive for SARS-CoV-2 infection. CONCLUSIONS: This work provides a blueprint for academic centers and community hospitals lacking automated laboratory machinery to implement rapid in-house testing. FUNDING: This study was supported by funding from the Boston University School of Medicine, the National Institutes of Health, Boston Medical Center, and the Massachusetts Consortium on Pathogen Readiness (MASS CPR).

HCV screening, linkage to care, and treatment patterns at different sites across one academic medical center.

BACKGROUND: It is unclear whether sites that screen large numbers of patients for Hepatitis C Virus but achieve limited follow-up are more or less effective at having patients succeed through linkage and treatment than lower volume sites that have higher linkage percentages. The objective was to compare the rates of HCV identification, linkage to care, and treatment success between different study sites including the Emergency Department, 3 outpatient clinics with unique patients, and the inpatient setting at one medical center. METHODS: This is a descriptive analysis of 2 years of data from a protocol that integrated HCV screening and treatment into clinical services throughout multiple departments in one medical center. The program used a best practice advisory to prompt testing at all sites, with different triggers for it to fire at each site, and one central navigation program that attempted to link all patients diagnosed with hepatitis C virus to outpatient care. Outcomes included volume of tests performed in each site, Antibody and RNA rates at each site, demographic data, navigation and linkage outcomes, and post-linkage treatment completion. RESULTS: 28,435 patients were screened across 5 clinical locations. RNA+ rates and absolute numbers linked to MD (linkage rates among all RNA+) were: ED 7.2% RNA+, 224 (22.6%) linked; Inpatient 14.8% RNA+, 27 (17.6%) linked, General Internal Medicine 3.9% RNA+, 269 (65.8%) linked, Infectious Diseases 4.0% RNA+, 34(70.8%) linked, Family Medicine 2.0% RNA+, 28 (75.7%) linked. Demographics, linkage barriers, and treatment initiation rates were different at all sites. CONCLUSION: Among sites there were differences in the sociodemographic characteristics of patients diagnosed with HCV, as well as differences in the success linking patients to outpatient care. At this medical center, the ED screened the most patients, the inpatient area had the highest RNA positivity rate, the FM clinic had the highest linkage rate, GIM linked the most patients by absolute number, and GIM also had the highest number of patients start treatment.

A Laboratory Medicine Best Practices Systematic Review and Meta-analysis of Nucleic Acid Amplification Tests (NAATs) and Algorithms Including NAATs for the Diagnosis of Clostridioides (Clostridium) difficile in Adults.

The evidence base for the optimal laboratory diagnosis of Clostridioides (Clostridium) difficile in adults is currently unresolved due to the uncertain performance characteristics and various combinations of tests. This systematic review evaluates the diagnostic accuracy of laboratory testing algorithms that include nucleic acid amplification tests (NAATs) to detect the presence of C. difficile The systematic review and meta-analysis included eligible studies (those that had PICO [population, intervention, comparison, outcome] elements) that assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase (GDH) enzyme immunoassays (EIAs) or GDH EIAs plus C. difficile toxin EIAs (toxin). The diagnostic yield of NAAT for repeat testing after an initial negative result was also assessed. Two hundred thirty-eight studies met inclusion criteria. Seventy-two of these studies had sufficient data for meta-analysis. The strength of evidence ranged from high to insufficient. The uses of NAAT only, GDH-positive EIA followed by NAAT, and GDH-positive/toxin-negative EIA followed by NAAT are all recommended as American Society for Microbiology (ASM) best practices for the detection of the C. difficile toxin gene or organism. Meta-analysis of published evidence supports the use of testing algorithms that use NAAT alone or in combination with GDH or GDH plus toxin EIA to detect the presence of C. difficile in adults. There is insufficient evidence to recommend against repeat testing of the sample using NAAT after an initial negative result due to a lack of evidence of harm (i.e., financial, length of stay, or delay of treatment) as specified by the Laboratory Medicine Best Practices (LMBP) systematic review method in making such an assessment. Findings from this systematic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of GDH/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.

Further evidence of Mycobacterium tuberculosis in the sputum of culture-negative pulmonary tuberculosis suspects using an ultrasensitive molecular assay.

BACKGROUND: Rapid diagnosis of pulmonary tuberculosis (TB) is critical to TB control. However, many patients with paucibacillary TB disease remain undiagnosed. Current TB elimination goals require new tools to diagnose early disease. We evaluated performance of the Totally Optimized PCR (TOP) TB assay, a novel ultrasensitive molecular test. METHODS: We assessed analytical specificity against nontuberculous mycobacteria (NTM), and estimated the diagnostic accuracy of TOP in a pilot study in Brazil (n = 46) and a cross-sectional study in Boston (n = 60). We compared TOP results to culture and a composite reference standard (CRS). RESULTS: TOP exhibited no cross-reactivity against NTM. We tested 132 respiratory specimens from 106 patients with suspected pulmonary TB. The pilot demonstrated feasibility and 100% (95% CI 85-100) sensitivity in predominantly smear-positive specimens; TOP's specificity against solid media culture was low (58%, 37-77) but improved against a CRS (93%, 68-100). Similarly, when using the CRS in the Boston study, TOP (88%, 1-99) had greater sensitivity than solid or liquid media culture (25%, 3-65) and similar specificity (both 100%, 93-100). CONCLUSIONS: The TOP assay enables detection of M. tuberculosis in culture-negative paucibacillary disease. While the use of TOP for the diagnosis of paucibacillary disease will require further clinical validation, its high sensitivity indicate a more immediate utility as a rule out TB test.

Point-of-care influenza testing does not significantly shorten time to disposition among patients with an influenza-like illness.

INTRODUCTION: Availability of anti-viral agents and need to isolate infected patients increases the need to confirm the diagnosis of influenza before determining patient disposition. OBJECTIVES: We sought to determine if time-to-disposition (TTD) was shorter among patients tested for influenza using an Emergency Department (ED) Point-of-care (POC) test compared to core laboratory (lab) test and to determine difference in antibiotic use between groups. METHODS: We prospectively enrolled a convenience sample of ED patients for whom influenza testing was ordered during influenza season 2017. Participants were randomized to POC or lab. Data collected included demographics, chief complaint, influenza test results, turnaround time (TAT), whether antibiotics were given, and TTD. Descriptive statistics were calculated and group comparisons conducted using chi squared and Wilcoxon Rank Sum tests. RESULTS: Study population included 100 in the POC group and 97 in the lab group. Demographics were similar between POC and lab participants. More flu positive results were reported in the POC group compared to the lab group (51.0% vs. 33.0% p = 0.01). The median TTD was 146.5 min (IQR 98.5) for POC group and 165.5 min (IQR 127) for lab group (p = 0.26). The median TAT was 30.5 min (IQR 7.5) for POC group and 106.0 min (IQR 55) for core lab group (p = 0.001). Antibiotics were given to 14.0% of POC participants and 14.4% of lab participants (p = 0.93). CONCLUSIONS: Although use of a POC influenza test provided more rapid TAT than use of a core lab test, there was no significant difference in TTD or antibiotic use between groups.

Implementation and Preliminary Results of an Emergency Department Nontargeted, Opt-out Hepatitis C Virus Screening Program.

BACKGROUND: Emergency department (ED) visits provide an opportunity for hepatitis C virus (HCV) screening for patients who otherwise might not be tested. We report on a novel nontargeted, opt-out HCV screening and linkage-to-care (LTC) program implemented in an urban ED. METHODS: This is a descriptive analysis from 3 months (November 2016-January 2017) of a nontargeted, opt-out ED HCV screening and LTC program among patients at least 13 years old undergoing phlebotomy for clinical purposes. A multipurpose best practice advisory (BPA) alerted providers to the program and generated order labels. For patients who authorized testing, specimens were drawn in the ED for HCV antibody (Ab) and reflex confirmatory RNA tests. Public health navigators attempted to contact RNA-positive patients and arrange outpatient visits. RESULTS: HCV Ab tests were performed on 3,808 patients, a 6,950% increase from preprogram. The proportion of HCV Ab test positivity was 13.2% (504/3,808, 95% confidence interval [CI] = 12.2%-14.3%) and of those 97.8% (493/504) had a follow-up RNA test performed. A total of 292 were confirmed positive for active infection, for an overall RNA positivity rate of 7.7% (95% CI = 6.8%-8.5%). Of those with active infection, 155 (53%) were outside the Centers for Disease Control and Prevention birth cohort for increased risk for HCV including 46 (15.8%, 95% CI = 11.8%-20.4%) who also did not report injection drug use. Linkage attempts were documented on 223 (76.4%) patients and appointments were scheduled for 102 (38% of attempted). Sixty-six patients attended their LTC visit (22.5% of all RNA-positive patients, 30% of linkage-eligible patients). CONCLUSIONS: Nontargeted opt-out HCV testing can be successfully implemented in an ED setting. A number of patients diagnosed were outside traditional risk groups. Once diagnosed, an ED population may be difficult to engage in care, but a structured interdisciplinary program can successfully link patients to HCV care.

A Next-Generation Sequencing Primer-How Does It Work and What Can It Do?

Next-generation sequencing refers to a high-throughput technology that determines the nucleic acid sequences and identifies variants in a sample. The technology has been introduced into clinical laboratory testing and produces test results for precision medicine. Since next-generation sequencing is relatively new, graduate students, medical students, pathology residents, and other physicians may benefit from a primer to provide a foundation about basic next-generation sequencing methods and applications, as well as specific examples where it has had diagnostic and prognostic utility. Next-generation sequencing technology grew out of advances in multiple fields to produce a sophisticated laboratory test with tremendous potential. Next-generation sequencing may be used in the clinical setting to look for specific genetic alterations in patients with cancer, diagnose inherited conditions such as cystic fibrosis, and detect and profile microbial organisms. This primer will review DNA sequencing technology, the commercialization of next-generation sequencing, and clinical uses of next-generation sequencing. Specific applications where next-generation sequencing has demonstrated utility in oncology are provided.

Prevalence and risk factors for MRSA nasal colonization among persons experiencing homelessness in Boston, MA.

Homeless individuals face an elevated risk of methicillin-resistant Staphylococcus aureus (MRSA) infection. Identifying the prevalence and risk factors for MRSA nasal colonization may reduce infection risk. A cross-sectional study was conducted at a health clinic for homeless persons in Boston, MA, USA (n=194). In-person interviews and nasal swab specimens were collected. MRSA isolates were genotyped using pulse-field gel electrophoresis (PFGE) and assessed for antibiotic susceptibility. The prevalence of MRSA nasal colonization was 8.3 %. Seventy-five percent of isolates reflected clonal similarity to USA300. USA100 (18.8 %) and USA500 (6.3 %) were also recovered. Resistance to erythromycin (81.3 %), levofloxacin (31.3 %) and clindamycin (23.1 %) was identified. Recent inpatient status, endocarditis, haemodialysis, heavy drinking, not showering daily and transience were positively associated with MRSA nasal colonization. Carriage of community-acquired MRSA strains predominated in this population, although nosocomial strains co-circulate. Attention to behavioural and hygiene-related risk factors, not typically included in MRSA prevention efforts, may reduce risk.

Laboratory Testing of Donors and Stool Samples for Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection.

Fecal microbiota transplantation is an efficacious and inexpensive therapy for recurrent Clostridium difficile infection, yet its safety is thought to depend on appropriate fecal donor screening. FDA guidance for regulation of this procedure is in flux, but screening and manufacture of fecal material from asymptomatic donors present many challenges to clinical laboratories. This minireview summarizes FDA regulatory changes, principles of donor selection, and recommended laboratory screening practices for fecal microbiota transplantation.

Detection and Quantification of Mycobacterium tuberculosis in the Sputum of Culture-Negative HIV-infected Pulmonary Tuberculosis Suspects: A Proof-of-Concept Study.

RATIONALE: Rapid diagnosis of pulmonary tuberculosis (TB) is critical for timely initiation of treatment and interruption of transmission. Yet, despite recent advances, many patients remain undiagnosed. Culture, usually considered the most sensitive diagnostic method, is sub-optimal for paucibacillary disease. METHODS: We evaluated the Totally Optimized PCR (TOP) TB assay, a new molecular test that we hypothesize is more sensitive than culture. After pre-clinical studies, we estimated TOP's per-patient sensitivity and specificity in a convenience sample of 261 HIV-infected pulmonary TB suspects enrolled into a TB diagnostic study in Mbarara, Uganda against MGIT culture, Xpert MTB/RIF and a composite reference standard. We validated results with a confirmatory PCR used for sequencing M. tuberculosis. MEASUREMENTS AND RESULTS: Using culture as reference, TOP had 100% sensitivity but 35% specificity. Against a composite reference standard, the sensitivity of culture (27%) and Xpert MTB/RIF (27%) was lower than TOP (99%), with similar specificity (100%, 98% and 87%, respectively). In unadjusted analyses, culture-negative/TOP-positive patients were more likely to be older (P<0·001), female (P<0·001), have salivary sputum (P = 0·05), sputum smear-negative (P<0.001) and less advanced disease on chest radiograph (P = 0.05). M. tuberculosis genotypes identified in sputum by DNA sequencing exhibit differential growth in culture. CONCLUSIONS: These findings suggest that the TOP TB assay is accurately detecting M. tuberculosis DNA in the sputum of culture-negative tuberculosis suspects. Our results require prospective validation with clinical outcomes. If the operating characteristics of the TOP assay are confirmed in future studies, it will be justified as a "TB rule out" test.