Maximizing Reflexive HCV RNA Testing of HCV Antibody-Reactive Samples Within United States Public Health Laboratories.

Hepatitis C virus (HCV) infection can cause significant morbidity and mortality if left untreated and diagnosis is the first step in any treatment regimen. In the United States, a two-step algorithm is recommended for detection of current HCV infection. The algorithm consists of an HCV antibody screening test followed by a supplemental test for HCV RNA if the HCV antibody test is reactive. To assess HCV testing practices and identify associated challenges, the Association of Public Health Laboratories (APHL) conducted a survey on HCV diagnostics practices of US public health laboratories. Additionally, APHL and the Centers for Disease Control and Prevention's (CDC) Division of Viral Hepatitis (DVH) convened a two-day meeting of HCV subject matter experts (SMEs) to identify opportunities for improvement in diagnosis of current HCV infection. Automatic reflexive HCV RNA testing of HCV antibody-reactive specimens was identified as a high priority target area by HCV SMEs and as a gap in laboratory practice by the APHL survey, which found that only 54% of respondent laboratories always automatically reflexed or referred an Ab-reactive specimen to an HCV RNA test. To facilitate accurate diagnosis and ensure that patients are not lost to follow up, laboratories and public health programs should work to ensure that the entire HCV testing algorithm (i.e., antibody and nucleic acid testing) can be completed with a sample(s) collected during a single patient visit.

Evaluation of Three Automated Nontreponemal Rapid Plasma Reagin (RPR) Tests for the Laboratory Diagnosis of Syphilis.

Automated nontreponemal rapid plasma reagin (RPR) tests were recently introduced in the United States for syphilis testing and limited performance data are available. In collaboration with the Association of Public Health Laboratories, three public health laboratories (PHL) were chosen through a competitive selection process to evaluate the performance of three FDA-cleared automated RPR test systems: BioPlex 2200 Syphilis Total & RPR assay (Bio-Rad Laboratories), AIX 1000 (Gold Standard Diagnostics), and ASI Evolution (Arlington Scientific). Panels prepared at the CDC included: a qualitative panel comprised of 734 syphilis reactive/nonreactive sera; a quantitative panel of 50 syphilis reactive sera (RPR titer 1:64 to 1:1,024); and a reproducibility panel of 15 nonreactive and reactive sera (RPR titer 1:1 to 1:64). Panels were shipped frozen to the PHL and tested on the automated RPR systems following manufacturers' instructions. Prior test results were blinded to all laboratories. When compared to manual RPR (Arlington Scientific) performed at the CDC as a reference test, the qualitative panel results demonstrated an overall concordance of 95.9% for AIX 1000, 94.6% for ASI Evolution, and 92.6% for Bioplex RPR; quantitative panel showed within range titer of 2-fold for 94% of specimens for AIX 1000, 68% for ASI Evolution, and 64% for BioPlex RPR, and the reproducibility testing panel demonstrated point estimates ranging from 69 to 95%. Automated RPR instruments could reduce turnaround time and minimize interpretation errors. However, additional evaluations with more specimens could assist laboratories with implementing automated RPR tests and understanding their limitations.

Evaluation of a laboratory-developed multiplex real-time PCR assay for diagnosis of syphilis, herpes and chancroid genital ulcers in four public health laboratories in the USA.

OBJECTIVE: To evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens. METHODS: GUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated. RESULTS: HSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%-100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites. CONCLUSIONS: The GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

Detection of Lymphogranuloma Venereum-Associated Chlamydia trachomatis L2 Serovars in Remnant Rectal Specimens Collected from 7 US Public Health Laboratories.

ABSTRACT: The frequency of lymphogranuloma venereum or invasive Chlamydia trachomatis infection with serovar L1, L2, or L3 is unknown in the United States. While no diagnostic test is commercially available, we used a laboratory-developed test and detected lymphogranuloma venereum-associated serovar L2 in 14% of 132 remnant C. trachomatis-positive rectal swabs.

Direct-to-Consumer Sexually Transmitted Infection Testing Services: A Position Statement from the American Sexually Transmitted Diseases Association.

ABSTRACT: Direct-to-consumer test services have gained popularity for sexually transmitted infections in recent years, with substantially increased use as a result of the SARS-CoV-2 (CoVID-19) global pandemic. This method of access has been variously known as "self-testing," "home testing," and "direct access testing." Although these online services may be offered through different mechanisms, here we focus on those that are consumer-driven and require self-collected samples, and sample shipment to a centralized laboratory without involvement of health care providers and/or local health departments. We provide the American Sexually Transmitted Diseases Association's position on utilization of these services and recommendations for both consumers and health care providers.

Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network.

U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae AR-Ng testing, a subactivity of the CDC's AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC's national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

Three Years of Shared Service HIV Nucleic Acid Testing for Public Health Laboratories: Worthwhile for HIV-1 but Not for HIV-2.

BACKGROUND: In 2016, HIV-2 nucleic acid testing (NAT) was added to a shared service program that conducts HIV-1 NAT for public health laboratories performing the recommended algorithm for diagnosing HIV. Here, we evaluate the usefulness of HIV-2 NAT in this program as compared with HIV-1 NAT. METHODS: Specimens eligible for HIV-1 NAT were reactive on an HIV-1/2 antibody or antigen/antibody initial test and nonreactive or indeterminate on a supplemental antibody test or were reactive for HIV-1 antigen-only on an HIV-1/2 antigen/antibody initial test. Specimens eligible for HIV-2 NAT were reactive on an initial test, HIV-2 indeterminate or HIV indeterminate on a supplemental antibody test and had no detectable HIV-1 RNA or were reactive for HIV-2 antibody on an HIV-1/2 antigen/antibody test, and this reactivity was not confirmed with a supplemental antibody assay. All specimens were tested in a reference laboratory using APTIMA HIV-1 qualitative RNA and/or a validated qualitative HIV-2 RNA real-time PCR assay. RESULTS: During 2016 to 2019, HIV-1 RNA was detected in 234 (14%) of 1731 specimens tested. HIV-2 RNA was not detected in 52 specimens tested. Median time from specimen collection to reporting of HIV-1 and HIV-2 NAT results by year ranged from 9 to 10 days and from 22 to 27 days, respectively. Two specimens with HIV-2 indeterminate results on a supplemental antibody test had detectable HIV-1 RNA. CONCLUSIONS: A shared service model for HIV-1 NAT is both feasible and beneficial for public health laboratories. However, because no HIV-2 infections were detected, our data suggest that this program should reconsider the usefulness of HIV-2 NAT testing.

Performance of the Syphilis Health Check in Clinic and Laboratory-Based Settings.

BACKGROUND: In this study, we evaluate the performance of the Syphilis Health Check (SHC) in clinical and laboratory settings using fingerstick whole blood and serum. METHODS: Fingerstick whole blood and serum specimens from adult patients (n = 562) without prior syphilis history presenting at 2 county health department STD clinics in North Carolina were tested. Fingerstick specimens were tested with the SHC in clinic, and serum specimens were tested at the North Carolina State Laboratory of Public Health with: (1) qualitative rapid plasma reagin, (2) treponemal EIA, and (3) SHC. Sensitivity and specificity were calculated with 95% confidence intervals. RESULTS: The fingerstick whole blood had a sensitivity of 100% (7 of 7) and specificity of 95.7% (531 of 555), compared with consensus reference testing (CRT) (rapid plasma reagin and EIA reactive), but a sensitivity of 50% (8 of 16), and specificity of 95.9% (523 of 546), when compared with the treponemal EIA. Both laboratory-based SHC on serum and whole-blood SHC performed similarly, compared with CRT, and the treponemal EIA alone. Twenty-four specimens SHC reactive on whole blood were nonreactive by CRT. In 8 of these 24 cases, STD clinic staff reported difficulty reading the test line for the SHC. Of the fingerstick whole-blood SHC reactive specimens, only 14 of 31 were also serum SHC reactive. CONCLUSIONS: The SHC on whole blood appears to be sensitive at detecting patients likely to have syphilis and could be an option for testing among high-risk populations. However, given challenges in interpreting SHC test results, adequate training of persons performing testing and ongoing quality assurance measures are key.

A gyrovirus infecting a sea bird.

We characterized the genome of a highly divergent gyrovirus (GyV8) in the spleen and uropygial gland tissues of a diseased northern fulmar (Fulmarus glacialis), a pelagic bird beached in San Francisco, California. No other exogenous viral sequences could be identified using viral metagenomics. The small circular DNA genome shared no significant nucleotide sequence identity, and only 38-42 % amino acid sequence identity in VP1, with any of the previously identified gyroviruses. GyV8 is the first member of the third major phylogenetic clade of this viral genus and the first gyrovirus detected in an avian species other than chicken.

Molecular identification of adenoviruses associated with respiratory infection in Egypt from 2003 to 2010.

BACKGROUND: Human adenoviruses of species B, C, and E (HAdV-B, -C, -E) are frequent causative agents of acute respiratory infections worldwide. As part of a surveillance program aimed at identifying the etiology of influenza-like illness (ILI) in Egypt, we characterized 105 adenovirus isolates from clinical samples collected between 2003 and 2010. METHODS: Identification of the isolates as HAdV was accomplished by an immunofluorescence assay (IFA) and confirmed by a set of species and type specific polymerase chain reactions (PCR). RESULTS: Of the 105 isolates, 42% were identified as belonging to HAdV-B, 60% as HAdV-C, and 1% as HAdV-E. We identified a total of six co-infections by PCR, of which five were HAdV-B/HAdV-C co-infections, and one was a co-infection of two HAdV-C types: HAdV-5/HAdV-6. Molecular typing by PCR enabled the identification of eight genotypes of human adenoviruses; HAdV-3 (n = 22), HAdV-7 (n = 14), HAdV-11 (n = 8), HAdV-1 (n = 22), HAdV-2 (20), HAdV-5 (n = 15), HAdV-6 (n = 3) and HAdV-4 (n = 1). The most abundant species in the characterized collection of isolates was HAdV-C, which is concordant with existing data for worldwide epidemiology of HAdV respiratory infections. CONCLUSIONS: We identified three species, HAdV-B, -C and -E, among patients with ILI over the course of 7 years in Egypt, with at least eight diverse types circulating.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

A molecular investigative approach to an outbreak of acute hemorrhagic conjunctivitis in Egypt, October 2010.

BACKGROUND: During October 2010, Egypt reported an outbreak of acute hemorrhagic conjunctivitis (AHC). A total of 1831 cases were reported from three governorates; 1703 cases in El Daqahliya, 92 cases in Port Said, and 36 in Damietta. The purpose of this study was to identify and characterize the causative agent associated with this outbreak. METHODS: The U.S. Naval Medical Research Unit No.3 (NAMRU-3) was contacted by the Egyptian Ministry of Health and Population to perform diagnostic laboratory testing on eighteen conjunctival swabs from patients with conjunctivitis from El Daqahliya Governorate. Conjunctival swabs were tested by molecular methods for human adenovirus (HAdV) and enteroviruses (EV). Virus isolation was performed; the isolated virus was further characterized by molecular typing and phylogenetic analysis. RESULTS: The majority of the samples (17/18) were positive for enterovirus and all were negative for HAdV. Molecular typing and sequencing of the isolated virus revealed the presence of coxsackievirus A24 variant. Phylogenetic analysis based on the VP1 and 3C regions demonstrated that the Egyptian viruses belonged to Genotype IV and are closely related to coxsackievirus A24 variant, reported in a similar outbreak in China in August 2010. CONCLUSIONS: This study strongly suggests that coxsackievirus A24 variant was associated with the acute hemorrhagic conjunctivitis outbreak reported in Egypt in October 2010. There is a possibility that the same strain of CV-A24v was implicated in the AHC outbreaks in both China and Egypt in 2010.

Detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness.

BACKGROUND: A specific diagnosis of a lower respiratory viral infection is often difficult despite frequent clinical suspicion. This low diagnostic yield may be improved by use of sensitive detection methods and biomarkers. METHODS: The prevalence, clinical predictors and inflammatory mediator profile of respiratory viral infection in serious acute respiratory illness were investigated. Sequential bronchoalveolar lavage (BAL) fluids from all patients hospitalised with acute respiratory illness over 12 months (n=283) were tested for the presence of 17 respiratory viruses by multiplex PCR assay and for newly discovered respiratory viruses (bocavirus, WU and KI polyomaviruses) by single-target PCR. BAL samples also underwent conventional testing (direct immunoflorescence and viral culture) for respiratory virus at the clinician's discretion. 27 inflammatory mediators were measured in a subset of the patients (n=64) using a multiplex immunoassay. RESULTS: 39 respiratory viruses were detected in 37 (13.1% of total) patients by molecular testing, including rhinovirus (n=13), influenza virus (n=8), respiratory syncytial virus (n=6), human metapneumovirus (n=3), coronavirus NL63 (n=2), parainfluenza virus (n=2), adenovirus (n=1) and newly discovered viruses (n=4). Molecular methods were 3.8-fold more sensitive than conventional methods. Clinical characteristics alone were insufficient to separate patients with and without respiratory virus. The presence of respiratory virus was associated with increased levels of interferon gamma-inducible protein 10 (IP-10) (p<0.001) and eotaxin-1 (p=0.017) in BAL. CONCLUSIONS: Respiratory viruses can be found in patients with serious acute respiratory illness by use of PCR assays more frequently than previously appreciated. IP-10 may be a useful biomarker for respiratory viral infection.

Identification of a novel polyomavirus from patients with acute respiratory tract infections.

We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus-specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen.

Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with algorithm-defined acute HIV-1 infection specimens.

BACKGROUND: The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. OBJECTIVE: To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. STUDY DESIGN: Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. RESULTS: Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. CONCLUSIONS: Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens.

Clinical and epidemiologic characterization of WU polyomavirus infection, St. Louis, Missouri.

WU polyomavirus is a recently described polyomavirus found in patients with respiratory infections. Of 2,637 respiratory samples tested in St. Louis, Missouri, 2.7% were positive for WU polyomavirus by PCR, and 71% were coinfected with other respiratory viruses. Persistent human infection with WU polyomavirus is described.