Diagnostic Assay Development for Poliovirus Eradication.

With poliovirus eradication nearing, few pockets of active wild poliovirus (WPV) transmission remain in the world. Intratypic differentiation (ITD) plays a crucial part in laboratory surveillance as the molecular detection method that can identify and distinguish wild and vaccine-like polioviruses isolated from acute flaccid paralysis cases or environmental sources. The need to detect new variants of WPV serotype 1 (WPV1) and the containment of all serotype 2 polioviruses (PV2) in 2015 required changes to the previous version of the method. The ITD version 5.0 is a set of six real-time reverse transcription-PCR (rRT-PCR) assays that serve as accurate diagnostic tools to easily detect and differentiate PV serotypes and genotypes. We describe the creation and properties of quantitation standards, including 16 control RNA transcripts and nine plaque-isolated viruses. All ITD rRT-PCR assays were validated using these standards, and the limits of detection were determined for each assay. We designed and pilot tested two new assays targeting recently circulating WPV1 genotypes and all PV2 viruses. The WPV1 assay had 99.1% specificity and 100% sensitivity, and the PV2 assay had 97.7% specificity and 92% sensitivity. Before proceeding to the next step in the global poliovirus eradication program, we needed to gain a better understanding of the performance of the ITD 5.0 suite of molecular assays and their limits of detection and specificities. The findings and conclusions in this evaluation serve as building blocks for future development work.

Population sensitivity of acute flaccid paralysis and environmental surveillance for serotype 1 poliovirus in Pakistan: an observational study.

BACKGROUND: To support poliomyelitis eradication in Pakistan, environmental surveillance (ES) of wastewater has been expanded alongside surveillance for acute flaccid paralysis (AFP). ES is a relatively new method of surveillance, and the population sensitivity of detecting poliovirus within endemic settings requires estimation. METHODS: Data for wild serotype 1 poliovirus from AFP and ES from January 2011 to September 2015 from 14 districts in Pakistan were analysed using a multi-state model framework. This framework was used to estimate the sensitivity of poliovirus detection from each surveillance source and parameters such as the duration of infection within a community. RESULTS: The location and timing of poliomyelitis cases showed spatial and temporal variability. The sensitivity of AFP surveillance to detect serotype 1 poliovirus infection in a district and its neighbours per month was on average 30.0% (95% CI 24.8-35.8) and increased with the incidence of poliomyelitis cases. The average population sensitivity of a single environmental sample was 59.4% (95% CI 55.4-63.0), with significant variation in site-specific estimates (median varied from 33.3-79.2%). The combined population sensitivity of environmental and AFP surveillance in a given month was on average 98.1% (95% CI 97.2-98.7), assuming four samples per month for each site. CONCLUSIONS: ES can be a highly sensitive supplement to AFP surveillance in areas with converging sewage systems. As ES for poliovirus is expanded, it will be important to identify factors associated with variation in site sensitivity, leading to improved site selection and surveillance system performance.

Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction.

An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.

Environmental surveillance for polioviruses in the Global Polio Eradication Initiative.

This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance.

Predictors of same day cancellation of elective surgery.

Same day cancellations of surgery have adverse effects on both patients and health care systems. To date, the majority of research has evaluated reasons for same day cancellation, and relatively little is known about risk factors for cancellation. The aim of this study is to develop and evaluate the accuracy of a model for preoperatively predicting which patients are at risk for experiencing same day cancellation. While accurately predicting which patients are likely to experience same day cancellation remains challenging, predictive models may aid in the early identification of patients at risk for cancellation. Future studies are required to assess whether the use of predictive analytics leads to reduced cancellation rates in practice.

Total Intravenous Anesthesia Compared to Inhalational Anesthesia in Patients Undergoing Arthroscopic Rotator Cuff Repair.

Background Inhalation anesthesia (IA) and total intravenous anesthesia (TIVA) are common general anesthesia techniques. During rotator cuff repair (RCR), an interscalene block is beneficial for intraoperative and early postoperative pain control. This study aimed to evaluate postoperative outcomes and opioid usage in patients undergoing arthroscopic RCR with an interscalene block and either IA or TIVA. Methodology A retrospective observational study was performed of 478 patients undergoing RCR at a single institution. Demographics, surgical details, intra and postoperative medications, and 90-day outcomes were collected. Univariate and multivariate analyses were performed to evaluate differences between groups. Results In total, 309 (64.6%) patients received IA and 169 (35.3%) received TIVA. Patients receiving IA were more likely to have comorbidities, such as diabetes (p = 0.002), sleep apnea (p = 0.006), gastroesophageal reflux disease (p < 0.001), and hypertension (p < 0.001). After adjusting for differences between groups in the multivariate analysis, patients who received TIVA had significantly shorter surgical time (ß = -14.85, p < 0.001) and perioperative time (ß = -21.01, p < 0.001) and significantly lower first post-anesthesia care unit Pasero opioid-induced sedation scores (ß = -0.022, p = 0.040). Patients who received TIVA were less likely to receive intraoperative narcotics (odds ratio = 0.38; p = 0.031). Conclusions TIVA appears to be a safe and effective anesthetic for patients undergoing arthroscopic RCR. TIVA is a potentially beneficial alternative to IA for this patient population.

Possible cross-species transmission of circoviruses and cycloviruses among farm animals.

Circoviruses consist of highly prevalent and genetically diverse porcine and avian pathogens. The genomes of cycloviruses, a proposed new genus in the family Circoviridae, were recently identified in human and chimpanzee faeces. Here, six cyclovirus and four circovirus genomes from the tissues of chickens, goats, cows, and a bat were amplified and sequenced using rolling-circle amplification and inverse PCR. A goat cyclovirus was nearly identical to a cyclovirus from a cow. USA beef contained circoviruses with >99% similarity to porcine circovirus 2b. Circoviruses in chicken were related to those of pigeons. The close genetic similarity of a subset of cycloviruses and circoviruses replicating in distinct animal species may reflect recent cross-species transmissions. Further studies will be required to determine the impact of these highly prevalent infections on the health of farm animals.

Multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces.

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named "Cyclovirus" whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

A highly prevalent and genetically diversified Picornaviridae genus in South Asian children.

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Conversion of PROMIS global health to EQ-5D health state values in patients undergoing lumbar spine surgery: A psychometric evaluation.

This study seeks to validate the conversion of PROMIS-GH scores to EQ-5D Health Utility Index (HUI) values. Patients undergoing lumbar spine surgery were prospectively surveyed using EQ-5D-3L and PROMIS-GH short form instruments. EQ-5D-HUI scores, and PROMIS scores converted to HUI were calculated. Neither instrument demonstrated any floor effects. The EQ-5D-HUI demonstrated significantly higher ceiling effects. Patients reported a significantly higher mean HUI score using the EQ-5D compared to PROMIS-GH. Strong positive correlation and agreement were observed. Conversion of the PROMIS-GH to the EQ-5D-HUI appears to be viable for evaluating the health status of patients undergoing lumbar spine surgery.

Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis.

We analyzed viral nucleic acids in stool samples collected from 35 South Asian children with nonpolio acute flaccid paralysis (AFP). Sequence-independent reverse transcription and PCR amplification of capsid-protected, nuclease-resistant viral nucleic acids were followed by DNA sequencing and sequence similarity searches. Limited Sanger sequencing (35 to 240 subclones per sample) identified an average of 1.4 distinct eukaryotic viruses per sample, while pyrosequencing yielded 2.6 viruses per sample. In addition to bacteriophage and plant viruses, we detected known enteric viruses, including rotavirus, adenovirus, picobirnavirus, and human enterovirus species A (HEV-A) to HEV-C, as well as numerous other members of the Picornaviridae family, including parechovirus, Aichi virus, rhinovirus, and human cardiovirus. The viruses with the most divergent sequences relative to those of previously reported viruses included members of a novel Picornaviridae genus and four new viral species (members of the Dicistroviridae, Nodaviridae, and Circoviridae families and the Bocavirus genus). Samples from six healthy contacts of AFP patients were similarly analyzed and also contained numerous viruses, particularly HEV-C, including a potentially novel Enterovirus genotype. Determining the prevalences and pathogenicities of the novel genotypes, species, genera, and potential new viral families identified in this study in different demographic groups will require further studies with different demographic and patient groups, now facilitated by knowledge of these viral genomes.

Cardioviruses are genetically diverse and cause common enteric infections in South Asian children.

Cardioviruses cause enteric infections in mice and rats which when disseminated have been associated with myocarditis, type 1 diabetes, encephalitis, and multiple sclerosis-like symptoms. Cardioviruses have also been detected at lower frequencies in other mammals. The Cardiovirus genus within the Picornaviridae family is currently made up of two viral species, Theilovirus and Encephalomyocarditis virus. Until recently, only a single strain of cardioviruses (Vilyuisk virus within the Theilovirus species) associated with a geographically restricted and prevalent encephalitis-like condition had been reported to occur in humans. A second theilovirus-related cardiovirus (Saffold virus [SAFV]) was reported in 2007 and subsequently found in respiratory secretions from children with respiratory problems and in stools of both healthy and diarrheic children. Using viral metagenomics, we identified RNA fragments related to SAFV in the stools of Pakistani and Afghani children with nonpolio acute flaccid paralysis (AFP). We sequenced three near-full-length genomes, showing the presence of divergent strains of SAFV and preliminary evidence of a distant recombination event between the ancestors of the Theiler-like viruses of rats and those of human SAFV. Further VP1 sequencing showed the presence of five new SAFV genotypes, doubling the reported genetic diversity of human and animal theiloviruses combined. Both AFP patients and healthy children in Pakistan were found to be excreting SAFV at high frequencies of 9 and 12%, respectively. Further studies are needed to examine the roles of these highly common and diverse SAFV genotypes in nonpolio AFP and other human diseases.

Early postoperative pain and opioid consumption after arthroscopic shoulder surgery with or without open subpectoral biceps tenodesis and interscalene block.

OBJECTIVE: The addition of open subpectoral biceps tenodesis to arthroscopic shoulder surgery with interscalene block has been anecdotally observed to result in increased postoperative pain. This study aims to evaluate the impact of tenodesis on early postoperative pain and recovery. METHODS: A retrospective review of patients undergoing arthroscopic shoulder surgery with general anesthesia and interscalene block was conducted. RESULTS: Patients undergoing tenodesis experienced longer OR time, pain numeric rating scale (NRS), and consumed more morphine milligram equivalents (MME) in PACU. After controlling for confounding factors, tenodesis was significantly associated with increased opioid MME consumption in the PACU (ß = 1.045, p = .028) and last PACU pain NRS (ß = 0.541, p = .009). CONCLUSION: Overall, pain scores and narcotic consumption were low after surgery, making these differences potentially clinically insignificant. Further study is required to evaluate whether these trends are consistent among this population.

Genomic characterization of novel human parechovirus type.

Using a simple metagenomic approach, we identified a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.

An outbreak of acute hemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant in Pakistan.

Acute hemorrhagic conjunctivitis (AHC) is a self-limiting viral infection of the eyes but having epidemic potential. In winter 2004-2005, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Islamabad, Pakistan. The etiological agent was confirmed as coxsackievirus A24 variant (CA24v) by virus isolation and sequencing of a part of the VP1/VP3 gene. Phylogenetic analysis in VP1 region showed that Pakistan isolates has closest matches both in Asia and Europe while in VP1/VP3 region they were more closely related to Chinese strains, suggesting their common source in Asia which is constantly evolving to cause AHC outbreaks in susceptible hosts in different parts of the world.

High prevalence of G3 rotavirus in hospitalized children in Rawalpindi, Pakistan during 2014.

Rotavirus A species (RVA) is the leading cause of severe diarrhea among children in both developed and developing countries. Among different RVA G types, humans are most commonly infected with G1, G2, G3, G4 and G9. During 2003-2004, G3 rotavirus termed as "new variant G3" emerged in Japan that later disseminated to multiple countries across the world. Although G3 rotaviruses are now commonly detected globally, they have been rarely reported from Pakistan. We investigated the genetic diversity of G3 strains responsible RVA gastroenteritis in children hospitalized in Rawalpindi, Pakistan during 2014. G3P[8] (18.3%; n = 24) was detected as the most common genotype causing majority of infections in children less than 06 months. Phylogenetic analysis of Pakistani G3 strains showed high amino acid similarity to "new variant G3" and G3 strains reported from China, Russia, USA, Japan, Belgium and Hungary during 2007-2012. Pakistani G3 strains belonged to lineage 3 within sub-lineage 3d, containing an extra N-linked glycosylation site compared to the G3 strain of RotaTeqTM. To our knowledge, this is the first report on the molecular epidemiology of G3 rotavirus strains from Pakistan and calls for immediate response measures to introduce RV vaccine in the routine immunization program of the country on priority.

Evaluation of the bag-mediated filtration system as a novel tool for poliovirus environmental surveillance: Results from a comparative field study in Pakistan.

Poliovirus (PV) environmental surveillance (ES) plays an important role in the global eradication program and is crucial for monitoring silent PV circulation especially as clinical cases decrease. This study compared ES results using the novel bag-mediated filtration system (BMFS) with the current two-phase separation method. From February to November 2016, BMFS and two-phase samples were collected concurrently from twelve sites in Pakistan (n = 117). Detection was higher in BMFS than two-phase samples for each Sabin-like (SL) PV serotype (p<0.001) and wild PV type 1 (WPV1) (p = 0.065). Seventeen sampling events were positive for WPV1, with eight discordant in favor of BMFS and two in favor of two-phase. A vaccine-derived PV type 2 was detected in one BMFS sample but not the matched two-phase. After the removal of SL PV type 2 (SL2) from the oral polio vaccine in April 2016, BMFS samples detected SL2 more frequently than two-phase (p = 0.016), with the last detection by either method occurring June 12, 2016. More frequent PV detection in BMFS compared to two-phase samples is likely due to the greater effective volume assayed (1620 mL vs. 150 mL). This study demonstrated that the BMFS achieves enhanced ES for all PV serotypes in an endemic country.

Common genotypes of Hepatitis B virus prevalent in injecting drug abusers (addicts) of North West Frontier Province of Pakistan.

BACKGROUND: The epidemiological significance of Hepatitis B virus genotypes has been well established and becoming an essential concern day by day however, much little is known about the mixed infection with more than one Hepatitis B virus genotypes and their clinical relevance. METHODS: Intravenous drug abusers are considered as a major risk group for the acquisition and transmission of blood borne infections like hepatitis B, however, in Pakistan, no such data has ever been reported about the epidemiology of HBV and its genotypes in Injecting Drug Users. 250 individuals were analyzed for hepatitis B virus genotypes after prior screening with serological assay for the detection of HBsAg. RESULTS: 56 (22.4%) individuals were found positive on ELSIA for HBsAg. The genotype distribution was found to be as: genotype D, 62.5%; genotype A, 8.92% while 28.57% individuals were found to be infected with a mixture of genotype A and D. CONCLUSION: There is an urgent need of the time to develop public health care policies with special emphasis towards the control of HBV transmission through high risk groups especially Injecting Drug Users.

Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan.

BACKGROUND: Enteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder. METHODS: Stool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases. RESULTS: NPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers. CONCLUSION: The present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.