Search

Background: Seasonal influenza leads to an increase in outpatient clinic visits. Timely, accurate, and affordable testing could facilitate improved treatment outcomes. Rapid influenza diagnostic tests (RIDTs) provide results in as little as 15 minutes and are relatively inexpensive, but have reduced sensitivity when compared to RT-PCR. The contributions of multiple factors related to test performance are not well defined for ambulatory care settings. We assessed clinical and laboratory factors that may affect the sensitivity and specificity of Sofia Influenza A+B Fluorescence Immunoassay. Study design: We performed a post-hoc assessment of surveillance data amassed over seven years from five primary care clinics. We analyzed 4,475 paired RIDT and RT-PCR results from specimens collected from patients presenting with respiratory symptoms and examined eleven potential factors with additional sub-categories that could affect RIDT sensitivity. Results: In an unadjusted analysis, greater sensitivity was associated with the presence of an influenza-like illness (ILI), no other virus detected, no seasonal influenza vaccination, younger age, lower cycle threshold value, fewer days since illness onset, nasal discharge, stuffy nose, and fever. After adjustment, presence of an ILI, younger age, fewer days from onset, no co-detection, and presence of a nasal discharge maintained significance. Conclusion: Clinical and laboratory factors may affect RIDT sensitivity. Identifying potential factors during point-of-care testing could aid clinicians in appropriately interpreting negative influenza RIDT results. [ABSTRACT FROM AUTHOR]

Background: Schools are primary venues of influenza amplification with secondary spread to communities. We assessed K-12 student absenteeism monitoring as a means for early detection of influenza activity in the community. Materials and methods: Between September 2014 and March 2020, we conducted a prospective observational study of all-cause (a-TOT), illness-associated (a-I), and influenza-like illness–associated (a-ILI) absenteeism within the Oregon School District (OSD), Dane County, Wisconsin. Absenteeism was reported through the electronic student information system. Students were visited at home where pharyngeal specimens were collected for influenza RT-PCR testing. Surveillance of medically-attended laboratory-confirmed influenza (MAI) occurred in five primary care clinics in and adjoining the OSD. Poisson general additive log linear regression models of daily counts of absenteeism and MAI were compared using correlation analysis. Findings: Influenza was detected in 723 of 2,378 visited students, and in 1,327 of 4,903 MAI patients. Over six influenza seasons, a-ILI was significantly correlated with MAI in the community (r = 0.57; 95% CI: 0.53–0.63) with a one-day lead time and a-I was significantly correlated with MAI in the community (r = 0.49; 0.44–0.54) with a 10-day lead time, while a-TOT performed poorly (r = 0.27; 0.21–0.33), following MAI by six days. Discussion: Surveillance using cause-specific absenteeism was feasible and performed well over a study period marked by diverse presentations of seasonal influenza. Monitoring a-I and a-ILI can provide early warning of seasonal influenza in time for community mitigation efforts. [ABSTRACT FROM AUTHOR]

Background: Influenza viruses pose significant disease burdens through seasonal outbreaks and unpredictable pandemics. Existing surveillance programs rely heavily on reporting of medically attended influenza (MAI). Continuously monitoring cause‐specific school absenteeism may identify local acceleration of seasonal influenza activity. The Oregon Child Absenteeism Due to Respiratory Disease Study (ORCHARDS; Oregon, WI) implements daily school‐based monitoring of influenza‐like illness‐specific student absenteeism (a‐ILI) in kindergarten through Grade 12 schools and assesses this approach for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities. Methods: Starting in September 2014, ORCHARDS combines automated reporting of daily absenteeism within six schools and home visits to school children with acute respiratory infection (ARI). Demographic, epidemiological, and symptom data are collected along with respiratory specimens. Specimens are tested for influenza and other respiratory viruses. Household members can opt into a supplementary household transmission study. Community comparisons are possible using a pre‐existing and highly effective influenza surveillance program, based on MAI at five family medicine clinics in the same geographical area. Results: Over the first 5 years, a‐ILI occurred on 6634 (0.20%) of 3,260,461 student school days. Viral pathogens were detected in 64.5% of 1728 children with ARI who received a home visit. Influenza was the most commonly detected virus, noted in 23.3% of ill students. Conclusion: ORCHARDS uses a community‐based design to detect influenza trends over multiple seasons and to evaluate the utility of absenteeism for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities. [ABSTRACT FROM AUTHOR]

We analyzed 4,352 participant- and staff-collected respiratory specimens from 2,796 subjects in the Oregon Child Absenteeism due to Respiratory Disease Study. Trained staff collected oropharyngeal specimens from school-aged children with acute respiratory illness while household participants of all ages collected their own midturbinate nasal specimens in year one and anterior nasal specimens in year two. Human ribonuclease P levels were measured using RT-PCR for all staff- and participant-collected specimens to determine adequacy, defined as Cycle threshold less than 38. Overall, staff- and participant-collected specimens were 99.9% and 96.4% adequate, respectively. Participant-collected midturbinate specimens were 95.2% adequate in year one, increasing to 97.2% in year two with anterior nasal collection. The mean human ribonuclease P Cycle threshold for participant-collected specimens was 31.18 in year one and 28.48 in year two. The results from this study suggest that community-based participant collection of respiratory specimens is comparable to staff-collected oropharyngeal specimens, is feasible, and may be optimal with anterior nasal collection. [ABSTRACT FROM AUTHOR]

Cocirculation of varying influenza types, strains, and lineages allows coinfection and intra‐season sequential infection, although a same‐strain sequential infection has not been previously described. This case report describes the first known case of sequential laboratory‐confirmed influenza A (H3N2) infections in a child within one season. [ABSTRACT FROM AUTHOR]

Focuses on the functions and capabilities of State Public Health Laboratories (SPHL) in the U.S. Role of SPHL in developing health policies; Importance of high quality laboratory services to health care systems; Information on the funding of SPHL; Details on the National Laboratory System.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses. [ABSTRACT FROM AUTHOR]

Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. [ABSTRACT FROM AUTHOR]

BACKGROUND. Large outbreaks of influenza A and B may occur in nursing homes despite high resident vaccination rates, even when the vaccine strain is matched to the circulating strain. This study reports the occurrence of separate influenza A and B outbreaks in a nursing home where more than 85% of residents were vaccinated. METHODS. Prospective surveillance was used to identify symptomatic residents in a rural Wisconsin nursing home with 680 residents. Viral cultures were obtained from all consenting residents identified with new respiratory symptoms even in the absence of temperature elevation. A "case" refers to a resident with a respiratory illness and an influenza isolate. RESULTS. During the 1992-93 season, 86% of 670 total residents were vaccinated, 104 (15.5%) were cases with influenza B. During the 1993-94 season, 89% of 690 total residents were vaccinated, 68 (9.8%) were cases with influenza A. The antigenic matches between vaccine and epidemic strains were characterized as "identical or minimal difference" by the Centers for Disease Control and Prevention. CONCLUSIONS. There is still a need to protect residents from infectious secretions and for contingency plans to permit the rapid use of antiviral agents. Future efforts are needed to develop vaccines that provide greater protection and to improve staff vaccination rates. [ABSTRACT FROM AUTHOR]

Rapid influenza diagnostic tests (RIDT) demonstrate varying sensitivities, often necessitating reverse transcriptase polymerase chain reaction (RT-PCR) to confirm results. The two methods generally require separate specimens. Using the same anterior nasal swab for both RIDT and molecular confirmation would reduce cost and waste and increase patient comfort. The aim of this study was to determine if RIDT residual nasal swab (rNS) specimens are adequate for RT-PCR and whole genome sequencing (WGS). We performed RT-PCR and WGS on paired rNS and nasopharyngeal or oropharyngeal (NP/OP) swab specimens that were collected from primary care patients across all ages. We randomly selected 199 and 40 paired specimens for RT-PCR and WGS, respectively, from the 962 paired surveillance specimens collected during the 2014-2015 influenza season. Sensitivity and specificity for rNS specimens were 81.3% and 96.7%, respectively, as compared to NP/OP specimens. The mean cycle threshold (Ct) value for the NP/OP specimen was significantly lower when the paired specimens were both positive than when the NP/OP swab was positive and the nasal swab was negative (25.5 vs 29.5; p<0.001). Genomic information was extracted from all 40 rNS specimens and 37 of the 40 NP/OP specimens. Complete WGS reads were available for 67.5% (14 influenza A; 13 influenza B) of the rNS specimens and 59.5% (14 influenza A; 8 influenza B) of the NP/OP specimens. It is feasible to use a single anterior nasal swab for RIDT followed by RT-PCR and/or WGS. This approach may be appropriate in situations where training and supplies are limited. Additional studies are needed to determine if residual nasal swabs from other rapid diagnostic tests produce similar results., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Quidel Corporation employs John Tamerius and Sushruth Reddy. Jonathan Temte has received in-kind material support and financial support from Quidel Corporation. No other authors have competing interests to declare., (Copyright: © 2023 Temte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Real-time PCR methodology can be applied to rapidly and accurately detect influenza viruses. During times of surge testing or enhanced pandemic surveillance, public health laboratories (PHLs) may experience overwhelming demand for testing, even while the prevalence of positive specimens remains low. To improve laboratory capacity and testing efficiency during surges, we evaluated whether nasopharyngeal (NP)/throat swab specimens can be pooled and tested for the presence of the 2009 H1N1 influenza virus without a reduction in sensitivity. Pools of 10 specimens were extracted and concentrated upon elution on the MagNA Pure LC instrument, and real-time PCR was performed on the Applied Biosystems 7500 Fast platform, using the CDC swine influenza virus real-time RT-PCR detection panel (rRT-PCR swine flu panel). Specimens in positive pools were singly re-extracted and retested by PCR to identify individual positive samples. Initial studies showed that spiking a pool of nine negative specimens (100 μl each) or 900 μl of virus transport medium with 100 μl of a positive clinical specimen caused no loss of sensitivity by rRT-PCR testing. Pools containing either multiple positive specimens or specimens positive for other respiratory viruses also showed no negative effect on crossing threshold (C(T)) values. To test the robustness of the pooling protocol, a panel of 50 blinded samples was sent to three PHLs and tested in five pools of 10. All PHLs correctly identified the positive specimens. This study demonstrates the feasibility of using a pooling strategy to increase capacity and conserve resources during surge testing and periods of enhanced influenza surveillance when the prevalence is low.

The need for effective influenza antiviral susceptibility surveillance methods has increased due to the emergence of near-universal adamantane resistance in influenza A/H3N2 viruses during the 2005-2006 season and the appearance of oseltamivir resistance in the influenza A/H1N1 virus subtype during the 2007-2008 season. The two classes of influenza antivirals, the neuraminidase inhibitors (NAIs) and the adamantanes, are well characterized, as are many mutations that can confer resistance to these drugs. Adamantane resistance is imparted mainly by a S31N mutation in the matrix gene, while NAI resistance can result from a number of mutations in the neuraminidase gene. During the 2007-2008 season, a neuraminidase mutation (H274Y) conferring resistance to the NAI oseltamivir emerged worldwide in the A/H1N1 virus subtype. Surveillance methodology and data from New York (NY) and Wisconsin (WI) for the 2006-2007 and 2007-2008 influenza seasons are presented. We used an existing pyrosequencing method (R. A. Bright et al., Lancet 366:1175-1181, 2005) and a modified version of this method for detection of adamantane resistance mutations. For NAI resistance mutation detection, we used a mutation-specific pyrosequencing technique and developed a neuraminidase gene dideoxy sequencing method. Adamantane resistance in the A/H3N2 virus samples was 100% for 2007-2008, similar to the 99.8% resistance nationwide as reported by the CDC. Adamantane resistance was found in only 1.2% of NY and WI A/H1N1 virus samples, compared to that found in 10.8% of samples tested nationwide as reported by the CDC. Influenza A/H1N1 virus H274Y mutants were found in 11.1% of NY samples for 2007-2008, a level comparable to the 10.9% nationwide level reported by the CDC; in contrast, mutants were found in 17.4% of WI samples. These results indicate the need for regional influenza antiviral surveillance.

Background: Human rhinoviruses (HRVs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious., Methods and Findings: To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples). Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group ("C"). None of these new strains could be cultured in traditional cell lines., Conclusions: By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group.

Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

During implementation of the Centers for Disease Control and Prevention's Mycobacterium tuberculosis nucleic acid amplification (NAA) evaluation program, 27.1% of participants used the same biological safety cabinet for NAA and specimen processing; 28.8% reported not using unidirectional workflow. An association between false positives and adverse responses to quality assurance questions (P = 0.04) illustrated the need for following NCCLS recommendations.

Emerging natural and man-made threats to the health of the nations population require development of a seamless laboratory network to address preventable health risks; this can be achieved only by defining the role of public health laboratories in public and private laboratory service delivery. Establishing defined core functions and capabilities for state public health laboratories will provide a basis for assessing and improving quality laboratory activities. Defining public health laboratory functions in support of public health programs is the beginning of the process of developing performance standards for laboratories, against which state public health laboratories, and eventually local public health and clinical laboratories, will establish and implement best laboratory practices. Public health is changing, and as apart of that change, public health laboratories must advocate for and implement improvements for public health testing and surveillance. These changes are outlined also in the Association of Public Health Laboratories consensus report (Association of Public Health Laboratories. Core functions and capabilities of state public health laboratories: a white paper for use in understanding the role and value of public health laboratories in protecting our nation's health. Washington, DC: Association of Public Health Laboratories, 2000).