Background and Objectives: Multisystem inflammatory syndrome of children (MISC) carries a high attributable morbidity. We describe children aged <16 years hospitalised with COVID-19 and/or MISC, April 2020 to June 2021. Method(s): All were tested for SARS-CoV-2, infectious disease consultations performed, modified CDC criteria for MISC applied, charts reviewed and data analyzed. Result(s): Among 79 consecutive children with SARS-CoV-2, 41(52%) were hospitalised; with median age 10.5 years; Afro-Caribbean ethnicity 40(98%); males 21(51%); SARS-CoV-2 RT-PCR positivity 26 (63%), IgG/IgM positivity 7(17%), community exposures 8 (20%). MISC-cases 18 (44%) vs. non-MISC 23(56%) had fever (94% vs. 30%; p<0.01), fatigue/lethargy (41% vs. 4%; p=0.004), rhinorrhoea (28% vs. 4%; p=0.035), elevated neutrophils (100% vs. 87%; p=0.024) and >=4 abnormal inflammatory biomarkers 13 (72%). MISC-cases had >2 organ/systems (100% vs. 35%; p<0.01), including gastrointestinal (72% vs. 17%; p<0.01), haematological/coagulopathic (67% vs. 4%; p<0.01); dermatologic (56% vs. 0%; p<0.01), cardiac (17% vs. 0%; p=0.042) with Kawasaki Syndrome (44% vs. 0%; p<0.01) and pleural effusions (17% vs. 0%; p=0.042). MISC-cases were treated with intravenous immune gammaglobulin (14, 78%), aspirin (12, 68%), steroids (9, 50%) and intensive care with non-invasive ventilation (2, 11%). One (6%) with pre-morbid illness died, the remainder recovered. Conclusion(s): MISC was treated successfully with intravenous gammaglobulin, steroids and/or aspirin in 94% before cardiopulmonary decompensation, or need for inotropes, vasopressors, or invasive ventilation. Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.

OBJECTIVES: COVID-19 in children was initially mild until the emergence of Multisystem Inflammatory Syndrome in Children (MIS-C). We describe pediatric COVID-19 in a developing country within the Caribbean. METHODS: Jamaican children who were hospitalized with SARS-CoV-2 infection, in one Caribbean regional academic referral center from April 2020 through June 2021 were included. Prospective surveillance and pediatric infectious disease consultations were performed using the CDC's MIS-C case definition. Data were extracted from patients' hospital charts using WHO's reporting form, entered into the RedCap database, and SPSS 28 was used for analysis. MIS-C and non-MIS-C patients were compared using independent sample t-tests for continuous variables and Fisher's exact test for categorical variables, p values < 0.05 were statistically significant. RESULTS: Seventy-nine children with COVID-19 with/without MIS-C presented to UHWI. Thirty-eight (48%) were mild ambulatory cases. Hospitalizations occurred in 41 (52%) children, with median age of 10 ½ years. SARS-CoV-2 RT-PCR positivity was present in 26 (63%), Immunoglobulin M, or Immunoglobulin G (IgM/IgG) positivity in 8 (20%), with community exposures in 7 (17%). Eighteen (44%) MIS-C positive patients were significantly more likely than 23 MIS-C negative patients (56%) to present with fever (94% vs. 30%; p < 0.001), fatigue/lethargy (41% vs. 4%; p = 0.006), lymphadenopathy (33% vs. 0%; p = 0.003), elevated neutrophils (100% vs. 87%; p = 0.024), and ESR (78% vs. 9%; p = 0.002). Involvement of > two organ systems occurred more frequently in MIS-C positive cases (100% vs. 34%; p < 0.001), including gastrointestinal (72% vs. 17%; p < 0.001); vomiting/nausea (39% vs. 9%; p < 0.028); hematological/coagulopathic (67% vs. 4%; p < 0.001); dermatologic involvement (56% vs. 0%; p < 0.001); and mucositis (28% vs. 0%; p = 0.001). MIS-C patients had Kawasaki syndrome (44%), cardiac involvement (17%), and pleural effusions (17%). MIS-C patients had >4 abnormal inflammatory biomarkers including D-dimers, C-reactive protein, ESR, ferritin, troponins, lactate dehydrogenase, neutrophils, platelets, lymphocytes, and albumen (72%). MIS-C patients were treated with intravenous immune gamma globulin (78%), aspirin (68%), steroids (50%), and non-invasive ventilation (11%). None required inotropes/vasopressors. MIS-C negative patients received standard care. All recovered except one child who was receiving renal replacement therapy and developed myocardial complications. CONCLUSIONS: In this first report of COVID-19 from the Caribbean, children and adolescents with and without MIS-C were not very severe. Critical care interventions were minimal and outcomes were excellent.

In 2014, Ebola virus disease (EVD) in West Africa was first reported during March in 3 southeastern prefectures in Guinea; from there, the disease rapidly spread across West Africa. We describe the epidemiology of EVD cases reported in Guinea's capital, Conakry, and 4 surrounding prefectures (Coyah, Dubreka, Forecariah, and Kindia), encompassing a full year of the epidemic. A total of 1,355 EVD cases, representing approximately 40% of cases reported in Guinea, originated from these areas. Overall, Forecariah had the highest cumulative incidence (4x higher than that in Conakry). Case-fatality percentage ranged from 40% in Conakry to 60% in Kindia. Cumulative incidence was slightly higher among male than female residents, although incidences by prefecture and commune differed by sex. Over the course of the year, Conakry and neighboring prefectures became the EVD epicenter in Guinea.

BACKGROUND: The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts. OBJECTIVES: To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity. STUDY DESIGN: Two sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI). RESULTS: Strain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001). CONCLUSIONS: This comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay's ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance.

BACKGROUND: Oseltamivir resistance among 2009 pandemic influenza A (H1N1) viruses (pH1N1) is rare. We investigated a cluster of oseltamivir-resistant pH1N1 infections in a hospital ward. METHODS: We reviewed patient records and infection control measures and interviewed health care personnel (HCP) and visitors. Oseltamivir-resistant pH1N1 infections were found with real-time reverse-transcription polymerase chain reaction and pyrosequencing for the H275Y neuraminidase (NA) mutation. We compared hemagglutinin (HA) sequences from clinical samples from the outbreak with those of other surveillance viruses. RESULTS: During the period 6-11 October 2009, 4 immunocompromised patients within a hematology-oncology ward exhibited symptoms of pH1N1 infection. The likely index patient became febrile 8 days after completing a course of oseltamivir; isolation was instituted 9 days after symptom onset. Three other case patients developed symptoms 1, 3, and 5 days after the index patient. Three case patients were located in adjacent rooms. HA and NA sequences from case patients were identical. Twelve HCP and 6 visitors reported influenza symptoms during the study period. No other pH1N1 isolates from the hospital or from throughout the state carried the H275Y mutation. CONCLUSIONS: Geographic proximity, temporal clustering, presence of H275Y mutation, and viral sequence homology confirmed nosocomial transmission of oseltamivir-resistant pH1N1. Diagnostic vigilance and prompt isolation may prevent nosocomial transmission of influenza.

Reverse-transcription PCR (RT-PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) is a high-throughput nucleic acid-based technology that relies on the accurate measurement of the molecular weight of PCR amplicons that can be used to deduce the base counts (number of As, Gs, Cs and Ts) of DNA. These amplicons represent highly variable regions with information-rich sequences, which are flanked by broad-range primers designed based on highly conserved loci. This technology was first introduced in 2005 for microbial identification and subtyping, and was later applied to influenza virus detection and identification. The influenza RT-PCR/ESI-MS assay allows analysis of approximately 300 samples per 24 h, and aids in the characterization of influenza viruses based on their 'core' gene signatures. Notably, this assay was used to identify one of the first cases of the 2009 H1N1 pandemic viruses. One of the main advantages of the RT-PCR/ESI-MS technology is its universality and adaptability for pathogen characterization. Efforts are being made to customize the currently used influenza surveillance assay for use in the diagnosis of the H1N1 pandemic virus. In this article, we provide a summary of known applications of the RT-PCR/ESI-MS assay in the field of influenza.

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.

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: The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus. METHODOLOGY/PRINCIPAL FINDINGS: Here, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with "core" genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between "core" genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir. CONCLUSIONS/SIGNIFICANCE: The RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains and animal viruses with pandemic potential (swine triple-reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay in MDCK cells, and a subset was also tested in both yield reduction and focus inhibition assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 muM (0.5mug/ml) (EC50s 0.19 - 22.48 muM, 0.03 - 3.53 mug/ml), and for all viruses, with the exception of a single dual resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 muM (0.5mug/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.

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.

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.