Use of next-generation sequencing to detect mutations associated with antiviral drug resistance in cytomegalovirus.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54/UL97 associated with antiviral resistance. Plasma samples (n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

Assessment of Test Performance and Potential for Environmental Contamination Associated with a Point-of-Care Molecular Assay for Group A Streptococcus in an End User Setting.

Although U.S. Food and Drug Administration-approved and CLIA-waived point-of-care (POC) molecular systems are being implemented in routine clinical practice, instrument reliability, test performance in the hands of end users, and the potential for environmental contamination resulting from use of POC molecular systems have not been extensively evaluated. We performed a prospective evaluation of the Roche cobas Liat group A streptococcus (GAS) assay compared to routine real-time PCR. We evaluated test accuracy, instrument failure rate, and monitored for environmental contamination when testing was performed by minimally trained end users in an Express Care Clinic environment. The overall concordance of the Liat GAS assay with routine testing was 97.2% (455/468). The average Liat failure rate across three analyzers was 6.6% (33/501) (range, 3.7 to 11.6%), and no environmental contamination was detected during the course of the study. The cobas Liat platform and GAS assay demonstrated reliable performance in the end user setting and may serve as a rapid, POC option for routine diagnostic testing for certain infectious diseases, including GAS.

Testing for Herpes Simplex Virus in Low-Volume Cerebrospinal Fluid Samples: Comparison of Three Protocols To Optimize Detection.

Detection of herpes simplex virus 1 and 2 (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) is a medical emergency and requires rapid, sensitive testing. However, the volume of CSF received for microbiological studies may be limited, especially from young children. In this study, we compared three testing protocols to our routine real-time PCR method to determine the most sensitive approach for detecting HSV-1 and HSV-2 in low-volume (≤100 µl) CSF.

Direct Detection of Influenza A and B Viruses in Less Than 20 Minutes Using a Commercially Available Rapid PCR Assay.

We compared an FDA-cleared rapid (<20 min) PCR assay (Cobas Liat; Roche Diagnostics) to our routine influenza A and B real-time PCR assay (Simplexa Flu A/B & RSV Direct; Focus Diagnostics) using respiratory swabs (n = 197). The Cobas Liat influenza A and B assays demonstrated sensitivities of 99.2% (123/124) and 100% (23/23), respectively, while showing a specificity of 100% for each target.

Rapid and direct detection of herpes simplex virus in cerebrospinal fluid by use of a commercial real-time PCR assay.

Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.

Comparison of a transplant multiplex viral panel on the ICEPlex system with real-time PCR for detection of cytomegalovirus, Epstein-Barr virus, and BK virus in clinical specimens.

We compared a multiplex viral transplant panel on the ICEPlex system to real-time PCR for the detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). The sensitivities of the ICEPlex were 83.3%, 95.5%, and 65.5% for the detection of CMV, EBV, and BKV, respectively. Interestingly, the multiplex assay detected dual infections in 16/280 (5.7%) samples tested.

Characterization of a novel melt curve by use of the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay: frequencies of this novel (low) melt curve and commonly encountered (intermediate) melt curves.

We characterize a novel probe binding-site polymorphism detectable solely by melt curve analysis using the Roche LightCycler HSV 1/2 analyte-specific reagent real-time PCR assay. The frequencies of this novel (47°C) and previously described intermediate (60 to 62°C) melt curves were 0.016% and 4.9%, respectively.

Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens.

The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 µl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 µl raw stool; however, 100 µl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/µl for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10(-1) to 10(3) 50% tissue culture infective dose (TCID(50))/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs.

Quantitative real-time polymerase chain reaction for evaluating DNAemia due to cytomegalovirus, Epstein-Barr virus, and BK virus in solid-organ transplant recipients.

Testing for cytomegalovirus-, Epstein-Barr virus-, and BK virus-specific gene targets in specimens from solid-organ transplant recipients for DNA by quantitative real-time polymerase chain reaction has been implemented in many diagnostic facilities. This technology provides rapid, accurate, and reproducible results for early detection, monitoring, and medical management of patients with these infections. Because these assays are becoming commonly used in clinical practice, the technical variables associated with specimen processing (e.g., nucleic acid extraction, gene target, and result reporting), amplification, and unique patient characteristics (e.g., age, sex, underlying diseases, immune status, and immunosuppressive regimens received) are factors that may influence the understanding and interpretation of test results. We emphasize the need for standardization of existing variables through parallel comparative and proficiency testing, uniform units for expressing results, to provide for clinical correlation with the results of these molecular assays.

Polyomavirus polymerase chain reaction as a surrogate marker of polyomavirus-associated nephropathy.

BACKGROUND: Polyomavirus-associated nephropathy (PVAN) is a significant cause of allograft loss after renal transplantation. A noninvasive assay that can guide the evaluation of PVAN would be of clinical value. We compared the utility of BK virus (BKV) polymerase chain reaction (PCR) and urine cytology in screening for concurrent PVAN. METHODS: We used PCR to test urine and plasma samples from renal recipients simultaneously for BKV DNA. Additionally, we tested urine samples for decoy cells. Sample results were correlated with biopsy-proven PVAN. Receiver-operator characteristic curves were used to determine viral load thresholds associated with concurrent PVAN. RESULTS: In this cross-sectional study, BKV viruria, viremia, and urinary decoy cells were detected in 24%, 9%, and 13% of renal recipients, respectively. Among 114 patients who had renal allograft biopsy, four (3.5%) were diagnosed with PVAN. Using pathology as gold standard for the diagnosis of PVAN, BKV viremia threshold of >1.6E+04 copies/mL had 100% sensitivity, 96% specificity, 50% positive predictive value, and 100% negative predictive value. A BKV viruria threshold of >2.5E+07 copies/mL had 100% sensitivity, 92% specificity, 31% positive predictive value, and 100% negative predictive value. In contrast, urine decoy cells had 25% sensitivity, 84% specificity, 5% positive predictive value, and 97% negative predictive value for the diagnosis of concurrent PVAN. CONCLUSION: BKV PCR may be a clinically useful noninvasive test to identify renal recipients with concurrent PVAN. BKV DNA >1.6E+04 copies/mL of plasma and >2.5E+07 copies/mL of urine were highly associated with concurrent PVAN whereas a negative PCR test makes the diagnosis of PVAN highly unlikely.

Duration of influenza A virus shedding in hospitalized patients and implications for infection control.

OBJECTIVE: To assess the duration of shedding of influenza A virus detected by polymerase chain reaction (PCR) and cell culture among patients hospitalized with influenza A virus infection. SETTING: Mayo Clinic (Rochester, Minnesota) hospitals that cater to both the community and referral populations. METHODS: Patients 18 years old and older who were hospitalized between December 1, 2004, and March 15, 2005, with a laboratory-confirmed (ie, PCR-based) diagnosis of influenza A virus infection were consecutively enrolled. Additional throat swab specimens were collected at 2, 3, 5, and 7 days after the initial specimen (if the patient was still hospitalized). All specimens were tested by PCR and culture (both conventional tube culture and shell vial assay). Information on demographic characteristics, date of symptom onset, comorbidities, immunosuppression, influenza vaccination status, and receipt of antiviral treatment was obtained by interview and medical record review. Patients were excluded if informed consent could not be obtained or if the date of symptom onset could not be ascertained. RESULTS: Of 149 patients hospitalized with influenza A virus infection, 50 patients were enrolled in the study. Most patients were older (median age, 76 years), and almost all (96%) had underlying chronic medical conditions. Of 41 patients included in the final analysis, influenza A virus was detected in 22 (54%) by PCR and in 12 (29%) by culture methods at or beyond 7 days after symptom onset. All 12 patients identified by culture also had PCR results positive for influenza A virus. CONCLUSION: Hospitalized patients with influenza A virus infection can shed detectable virus beyond the 5- to 7-day period traditionally considered the duration of infectivity. Additional research is needed to assess whether prolonging the duration of patient isolation is warranted to prevent nosocomial outbreaks during the influenza season.

Automated processing, extraction and detection of herpes simplex virus types 1 and 2: A comparative evaluation of three commercial platforms using clinical specimens.

BACKGROUND: Recently, automated platforms have been developed that can perform processing, extraction and testing for herpes simplex virus (HSV) nucleic acid on a single instrument. OBJECTIVES: In this study, we compared three commercially-available systems; Aptima®/Panther (Hologic, San Diego, CA), ARIES® (Luminex Corporation, Austin, TX), and cobas® 4800 (Roche Molecular Systems Inc, Pleasanton, CA) for the qualitative detection of HSV-1/2 in clinical samples. STUDY DESIGN: Two-hundred seventy-seven specimens (genital [n=193], dermal [n=84]) were submitted for routine HSV-1/2 real-time PCR by a laboratory developed test. Following routine testing, samples were also tested by the Aptima, ARIES, and cobas HSV-1/2 assays per the manufacturer's recommendations. Results were compared to a "consensus standard" defined as the result obtained from ≥3 of the 4 assays. RESULTS: Following testing of 277 specimens, the cobas and ARIES assays demonstrated a sensitivity of 100% for HSV-1 (61/61) and HSV-2 (55/55). The Aptima assays showed a sensitivity of 91.8% (56/61) for HSV-1 and 90.9% (50/55) for HSV-2. Percent specificities for HSV-1 were 96.2% (202/210) by cobas, 99.5% (209/210) by ARIES and 100% (236/236) by Aptima. For HSV-2, the specificities were 98.1% (211/215) by cobas, 99.5% (215/216) by ARIES and 100% (216/216) by Aptima. The turnaround time for testing 24 samples was 2.5h by the cobas 4800, 3.1h by Aptima/Panther, and 3.9h by ARIES. CONCLUSIONS: The three commercial systems can perform all current functions on a single platform, thereby improving workflow and potentially reducing errors associated with manual processing of samples.

An Assessment of the AdenoPlus Point-of-Care Test for Diagnosing Adenoviral Conjunctivitis and Its Effect on Antibiotic Stewardship.

OBJECTIVE: To determine the sensitivity and specificity of the AdenoPlus test compared with real-time polymerase chain reaction (PCR) and to determine whether there was a reduction in antibiotic prescriptions with the use of AdenoPlus compared with the previous year. PATIENTS AND METHODS: A total of 125 patients with suspected infectious conjunctivitis were accrued from June 4, 2015, through September 27, 2015. Forty-six participants from the prospective cohort completed both AdenoPlus and PCR testing. Two hundred fifty age-matched individuals were in the retrospective cohort. RESULTS: There was a significant reduction in the percentage of patients who received an antibiotic ophthalmic prescription in the prospective cohort vs the retrospective cohort (32% vs 45%; χ2P=.01). AdenoPlus test sensitivity was 50% (5 of 10) and specificity was 92% (33 of 36) compared with real-time PCR testing. CONCLUSION: The AdenoPlus test has high specificity for diagnosing adenoviral conjunctivitis but lower sensitivity than has been previously published. These data suggest that negative AdenoPlus results should be confirmed by real-time PCR owing to the low overall sensitivity of AdenoPlus observed.

Detection of high-risk human papillomavirus infection in tonsillar specimens using 2 commercially available assays.

THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled.

Evaluation of 2 multiplex real-time PCR assays for the detection of HSV-1/2 and Varicella zoster virus directly from clinical samples.

We compared the performance of 2 multiplex assays (Focus Simplexa and Quidel Lyra) to individual real-time PCR for the detection of herpes simplex virus-1 (HSV-1), HSV-2, and Varicella zoster virus (VZV) from clinical specimens. Results were compared to a consensus standard, defined as the result obtained by at least 2 of the 3 molecular methods. The sensitivity of the Quidel assay ranged from 92.0% for HSV-1 to 97.7% for HSV-2, while the specificity for all targets was 100%. The Focus assay demonstrated 100% sensitivity for all targets, and the percent specificity ranged from 96.8% for HSV-1 to 100% for HSV-2 and VZV.