INTRODUCTION: In 2020, the COVID-19 pandemic led to significant declines in cancer screening, including among women served by the National Breast and Cervical Cancer Early Detection Program (NBCCEDP). This study examined the spatial association between state-based COVID-19 test percent positivity and proportional change in NBCCEDP screening volume. METHODS: Using the COVID-19 Diagnostic Laboratory Testing dataset, we calculated state-based monthly COVID-19 test percent positivity from July through December 2020 and categorized rates into low, medium, and high groups. We used data from 48 NBCCEDP state awardees to calculate the state-based monthly proportional change in screening volume and compared data for July-December 2020 with the previous 5-year average for those months. We categorized changes in screening volume into large decrease, medium decrease, and minimal change and created maps of the associations between variable subgroups by using bivariate mapping in QGIS. RESULTS: Bivariate relationships between COVID-19 test percent positivity and proportional change in cancer screening volume varied over time and geography. In 5 of 6 months, 4 states had high COVID-19 test percent positivity and minimal change in breast or cervical cancer screening volume; 2 states had high COVID-19 test percent positivity and minimal change in breast and cervical cancer screening volume. CONCLUSION: Some states maintained pre-COVID-19 screening volumes despite high COVID-19 test percent positivity. Follow-up research will be conducted to determine how these states differ from those with consistent decreases in screening volume and identify factors that may have contributed to differences. This information could be useful for planning to maximize NBCCEDP awardees' ability to maintain screening volume during future public health emergencies.

Women from racial and ethnic minority groups face a disproportionate burden of cervical and breast cancers in the United States. The Coronavirus Disease 2019 (COVID-19) pandemic might exacerbate these disparities as supply and demand for screening services are reduced. The National Breast and Cervical Cancer Early Detection Program (NBCCEDP) provides cancer screening services to women with low income and inadequate health insurance. We examined COVID-19's impact on NBCCEDP screening services during January-June 2020. We found the total number of NBCCEDP-funded breast and cervical cancer screening tests declined by 87% and 84%, respectively, during April 2020 compared with the previous 5-year averages for that month. The extent of declines varied by geography, race/ethnicity, and rurality. In April 2020, screening test volume declined most severely in Health and Human Services Region 2 - New York (96% for breast, 95% for cervical cancer screening) compared to the previous 5-year averages. The greatest declines were among American Indian/Alaskan Native women for breast cancer screening (98%) and Asian Pacific Islander women for cervical cancer screening (92%). Test volume began to recover in May and, by June 2020, NBCCEDP breast and cervical cancer screening test volume was 39% and 40% below the 5-year average for that month, respectively. However, breast cancer screening remained over 50% below the 5-year average among women in rural areas. NBCCEDP programs reported assisting health care providers resume screening.

To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.

The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point-of-care. Here we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n=14) and a set of 90 seasonal influenza A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer recommended protocol and a modified version to attune to surveillance requirement. The generated IC50s were compared with the NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof-of-principal, clinical specimens (n=235) confirmed by real-time RT-PCR to contain influenza A(H1N1)pdm09 virus and pre-screened for the oseltamivir resistance marker H275Y using pyrosequencing, were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-types based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g. H3N2 with E119V) could only be detected among seasonal viruses using the FL assays. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays that required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza A and B virus isolates carrying established and potential NA inhibitor-resistance markers, and may become a useful tool for monitoring drug resistance in clinical specimens.

BACKGROUND: During 2010-2011, influenza B viruses with a novel neuraminidase substitution, denoted I221V, (B/I221V) associated with reduced in vitro oseltamivir susceptibility were detected in North Carolina (NC). METHODS: We determined the prevalence of I221V among B viruses submitted to CDC for antiviral resistance surveillance, including all B viruses submitted to NC and South Carolina (SC) state laboratories, during October 2010-September 2011.We conducted chart reviews and phone interviews to characterize NC and SC patients with B/I221V versus wild-type B virus infection (B/WT). RESULTS: We detected I221V in 45 (22%) of 209 B viruses from NC and 8 (10%) of 82 B viruses from SC. We detected I221V in three (0.3%) of 881 B viruses tested from 45 other states. B/I221V infection was not associated with differences in underlying conditions or illness severity compared with B/WT infection. No patients with B/I221V infection received oseltamivir prior to specimen collection. Among patients who completed oseltamivir, those with B/I221V infection reported a longer duration until illness resolution (5 versus 3 days); P=0.02. CONCLUSIONS: B/I221V co-circulated with B/WT in NC and SC during 2010-2011. I221V did not alter illness severity but may have reduced oseltamivir effectiveness. Thus, global surveillance for I221V is important.

Close monitoring of drug susceptibility among human influenza viruses was necessitated by widespread resistance to M2 inhibitors in influenza H1N1 (pre-pandemic and 2009 pandemic) and H3N2 viruses, and of oseltamivir resistance in pre-pandemic H1N1 viruses. The FDA-approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir, as well as investigational NAIs, peramivir and laninamivir, are currently the principal treatment options for managing influenza infection. However, there are challenges associated with assessing virus susceptibility to this class of drugs. Traditional cell culture-based assays are not reliable for phenotypic testing of NAI susceptibility due to complexity in interpretation. Two types of laboratory assays are currently available for monitoring NAI susceptibility, phenotypic such as the neuraminidase inhibition (NI) assay and genotypic. The NI assay's requirement for propagated virus lengthens testing turnaround; therefore, the need for timely detection of molecular markers associated with NAI resistance (e.g., H275Y in H1N1) has spurred the development of rapid, high-throughput assays, such as real-time RT-PCR and pyrosequencing. The high sensitivity of genotypic assays allows testing of clinical specimens thus eliminating the need for virus propagation in cell culture. The NI assays are especially valuable when a novel virus emerges or a new NAI becomes available. Modifications continue to be introduced into NI assays, including optimization and data analysis criteria. The optimal assay of choice for monitoring influenza drug susceptibility varies widely depending on the needs of laboratories (e.g., surveillance purposes, clinical settings). Optimally, it is desirable to combine functional and genetic analyses of virus isolates and, when possible, the respective clinical specimens.

During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, peramivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wildtype-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrosequencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates containing H275Y variant at 50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38-52- and 33-97-fold enhancement in oseltamivir- and peramivir-resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions.

Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.

For influenza viruses, pyrosequencing has been successfully applied to the high throughput detection of resistance markers in genes encoding the drug-targeted M2 protein and neuraminidase. In this study, we expanded the utility of this assay to the detection of multiple receptor-binding variants of the hemagglutinin protein of influenza viruses directly in clinical specimens. Specifically, a customized pyrosequencing protocol that permits detection of virus variants with the D, G, N or E amino acid at position 222 in the hemagglutinin of the 2009 pandemic influenza A (H1N1) virus was developed. This customized pyrosequencing protocol was applied to the analysis of 241 clinical specimens. The use of the optimized nucleotide dispensation order allowed detection of mixtures of variants in ten samples (4.1%) which the standard cyclic nucleotide dispensation protocol failed to detect. The optimized pyrosequencing protocol is expected to provide a more accurate tool in the analysis of virus variant composition.

Two distinct genetic clades of seasonal influenza A(H1N1) viruses have cocirculated in the recent seasons: clade 2B oseltamivir-resistant and adamantane-susceptible viruses, and clade 2C viruses that are resistant to adamantanes and susceptible to oseltamivir. We tested seasonal influenza A(H1N1) viruses collected in 2008-2010 from the United States and globally for resistance to antivirals approved by the Food and Drug Administration. We report 28 viruses with both adamantane and oseltamivir (dual) resistance from 5 countries belonging to 4 distinct genotypes. Because of limited options for antiviral treatment, emergence of dual-resistant influenza viruses poses a public health concern, and their circulation needs to be closely monitored.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean +/-sd 50% inhibitory concentration [IC(50)] 0.25 +/-0.12 nM) and zanamivir (mean IC(50) 0.29 +/-0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n=5540) collected during the 2008-2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n=2259) were resistant to oseltamivir. All influenza A(H3N2) (n=834) and B (n=914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n=22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n=1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance.

Neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only effective antiviral drugs for treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. IC50 values are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In this study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivity of viruses was assessed against zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675). All isolates were sensitive to all five NAIs by all three NI assays, with the exception of H275Y variants which showed substantially elevated IC50 values against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages when compared to the others: CL required less virus volume, FL provided the greatest difference in the IC50 values between wild type and variant, whereas the IC50 values obtained from CM may be most predictive of drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but lacks their shortcomings.

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay ( approximately 60-fold increase in IC50 compared to a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays ( approximately 50- and 350-fold increases in IC50, respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA (E119V/I). Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and investigative NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients is previously reported; however, the E119I detected here is a novel mutation which reduces susceptibility to several NAIs. Neither mutation was not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multi-drug resistant influenza viruses in oseltamivir-treated immunocompromised subjects, and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

BACKGROUND: M2 blockers, amantadine and rimantadine, and neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir, are FDA-approved for control of influenza A virus infections. The 2009 pandemic viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane resistant swine virus. NAI-resistance in the H1N1pdm viruses has been rare and is mainly limited to oseltamivir exposed patients. The pyrosequencing assay has been proven a useful tool in surveillance for drug resistance in seasonal influenza A viruses. METHOD: Here we provide a protocol which allows detection of adamantane resistance markers as well as the I43T change- which is unique to the H1N1pdm M2 protein. The protocol also allows detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. RESULTS: We report here detection of the first cases of the oseltamivir resistance mutation H275Y and the change I223V in viruses from the US using the approach described in this study. Moreover, the assay permits a quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. CONCLUSIONS: pyrosequencing is well suited for detection of drug resistance markers and signature mutations in the M and NA of the pandemic H1N1 influenza viruses.

The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007-2008 and 2008-2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC(50) approximately 100-3000 time>those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n=725) showed that 53 (7.3%) had mutations at residue D151 (D-->E/G/N), while 9 (1.2%) had mutations at Q136 (Q-->K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC(50) for oseltamivir and peramivir, and elevated IC(50) for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI-resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC(50) for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK-grown viruses but not in matching original clinical specimens (n=33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.

Please cite this paper as: Deyde et al. (2009). Genomic events underlying the changes in adamantane resistance among influenza A(H3N2) viruses during 2006-2008. Influenza and Other Respiratory Viruses 3(6), 297-314.Background Adamantanes resistance in H3N2 viruses has been increasing since 2000, and in 2005-2006 reached nearly 100% in most countries, with the circulation of the N-lineage. In 2006-2007, however, a significant decrease in resistance was observed in many regions. Objectives To explore potential links between adamantane resistance and the A(H3N2) viruses that circulated between 2006 and 2008. Methods A total of 1451 Influenza A (H3N2) viruses collected globally in 2001-2008 were screened for the presence of adamantane resistance markers. A subset of 100 viruses representing the broad genetic and geographic spectrum of these viruses was selected for complete genome sequencing and phylogenetic analyses. Results Full genome sequence analysis of 2006-2007 viruses revealed co-circulation of four distinct genotypes, designated A-D. Phylogenetic analyses demonstrated reassortment between viruses from the N-lineage and other viruses that had circulated in prior seasons, including those bearing an adamantane sensitive marker. Genotype D viruses became dominant in late 2006-2007 and continued to be the main H3N2 genotype in 2007-2008. Viruses of this genotype retained all N-lineage genome segments except PB2 and NP, which were acquired through reassortment. Conclusions The decrease in adamantane resistance at that time was due to transient co-circulation of genotypes that emerged through reassortment. Our findings emphasize the importance of complete genome sequencing in understanding the complex nature of the relationship between influenza virus evolution and antiviral resistance. The recent emergence of the pandemic multi-reassortant H1N1 virus underscores the importance of whole genome sequence monitoring for rapid detection of such unusual and novel strains.

We report here the design of a pyrosequencing approach for the detection of molecular markers of resistance to the neuraminidase inhibitors zanamivir and oseltamivir in influenza viruses of type B. Primers were designed to analyze the sequences at eight amino acid positions E119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering) in the neuraminidase (NA) which have been previously found to be associated with resistance or reduced susceptibility to oseltamivir and/or zanamivir in the NA inhibition assay. In addition, the designed primers could be utilized to the distinguish between the NAs of influenza B viruses from the two major lineages (Victoria and Yamagata) that have co-circulated globally in recent years, thus providing a valuable tool for virus strain surveillance.