Genomewide Association Study of Severe Covid-19 with Respiratory Failure.

BACKGROUND: There is considerable variation in disease behavior among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (Covid-19). Genomewide association analysis may allow for the identification of potential genetic factors involved in the development of Covid-19. METHODS: We conducted a genomewide association study involving 1980 patients with Covid-19 and severe disease (defined as respiratory failure) at seven hospitals in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe. After quality control and the exclusion of population outliers, 835 patients and 1255 control participants from Italy and 775 patients and 950 control participants from Spain were included in the final analysis. In total, we analyzed 8,582,968 single-nucleotide polymorphisms and conducted a meta-analysis of the two case-control panels. RESULTS: We detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2, which were significant at the genomewide level (P<5×10-8) in the meta-analysis of the two case-control panels (odds ratio, 1.77; 95% confidence interval [CI], 1.48 to 2.11; P = 1.15×10-10; and odds ratio, 1.32; 95% CI, 1.20 to 1.47; P = 4.95×10-8, respectively). At locus 3p21.31, the association signal spanned the genes SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1. The association signal at locus 9q34.2 coincided with the ABO blood group locus; in this cohort, a blood-group-specific analysis showed a higher risk in blood group A than in other blood groups (odds ratio, 1.45; 95% CI, 1.20 to 1.75; P = 1.48×10-4) and a protective effect in blood group O as compared with other blood groups (odds ratio, 0.65; 95% CI, 0.53 to 0.79; P = 1.06×10-5). CONCLUSIONS: We identified a 3p21.31 gene cluster as a genetic susceptibility locus in patients with Covid-19 with respiratory failure and confirmed a potential involvement of the ABO blood-group system. (Funded by Stein Erik Hagen and others.).

Rectal colonization by resistant bacteria increases the risk of infection by the colonizing strain in critically ill patients with cirrhosis.

BACKGROUND & AIMS: It remains unclear whether rectal colonization with multidrug-resistant organisms (MDROs) is prevalent and predisposes to infections by the same pathogens in patients with cirrhosis. METHODS: Two series of critically ill patients were evaluated. In the Barcelona cohort, 486 consecutive patients were prospectively evaluated, 129 with and 357 without cirrhosis (2015-2016). Rectal swabs were performed at admission and weekly thereafter (until intensive care unit [ICU] discharge) to detect MDRO colonization. Risk factors for colonization and infection by MDROs were evaluated. A retrospective cohort from Frankfurt (421 patients with cirrhosis; 2010-2018) was investigated to evaluate MDRO rectal colonization in another epidemiological scenario. RESULTS: In the Barcelona cohort, 159 patients were colonized by MDROs (32.7%), 102 (64.2%) at admission and 57 (35.8%) during follow-up. Patients with cirrhosis showed higher rates of rectal colonization at admission than those without cirrhosis (28.7% vs. 18.2%, p = 0.01) but similar colonization rates during ICU stay. Extended-spectrum beta-lactamase-Enterobacterales were the most frequent MDROs isolated in both groups. Colonization by MDROs independently increased the risk of infection by MDROs at admission and during follow-up. Risk of new infection by the colonizing strain was also significantly increased in patients with (hazard ratio [HR] 7.41) and without (HR 5.65) cirrhosis. Rectal colonization by MDROs was also highly prevalent in Frankfurt (n = 198; 47%; 131 at admission [66.2%] and 67 [33.8%] during follow-up), with vancomycin-resistant enterococci being the most frequent colonizing organism. Rectal colonization by MDROs was also associated with an increased risk of infection by MDROs in this cohort. Infections occurring in MDR carriers were mainly caused by the colonizing strain. CONCLUSION: Rectal colonization by MDROs is extremely frequent in critically ill patients with cirrhosis. Colonization increases the risk of infection by the colonizing resistant strain. LAY SUMMARY: Rectal colonization by multidrug-resistant organisms (MDROs) is a prevalent problem in patients with cirrhosis requiring critical care. The pattern of colonizing bacteria is heterogeneous with relevant differences between centers. Colonization by MDROs is associated with increased risk of infection by the colonizing bacteria in the short term. This finding suggests that colonization data could be used to guide empirical antibiotic therapy and de-escalation policies in patients with cirrhosis.

Tigecycline population pharmacokinetics in critically ill patients with decompensated cirrhosis and severe infections.

OBJECTIVES: Physiopathological changes in advanced cirrhosis could alter tigecycline pharmacokinetics (PK), thus affecting serum drug concentrations and compromising target attainment. We aimed to describe tigecycline PK in patients with decompensated cirrhosis and severe bacterial infections, identify the sources of PK variability and assess the performance of different dosing regimens to optimize the PK/pharmacodynamic (PD) target. METHODS: Serum concentrations and covariates were obtained from patients with severe infections under tigecycline treatment. A population PK analysis was performed using non-linear mixed-effects modelling and the final model was used to simulate tigecycline exposure to assess the PTA. RESULTS: Twenty critically ill patients were enrolled in the study. Data were best described by a two-compartment linear model. Mean ± SD parameter estimates for clearance (CL), intercompartmental clearance (Q), central and peripheral volumes of distribution (V1 and V2) were 14.8 ± 11 L/h, 38.4 ± 24 L/h, 63.7 ± 14 L and 233 ± 30 L, respectively. MELD score significantly influenced tigecycline CL, and total serum proteins significantly affected V1. Monte Carlo simulations showed that tigecycline elimination is hampered as MELD score values increase, consequently requiring lower drug doses. Patients with hypoproteinaemia would have lower peak tigecycline concentrations but similar steady-state concentrations compared with patients with normoproteinaemia. CONCLUSIONS: Our study confirms that tigecycline dose adjustment is needed in severe hepatic dysfunction and suggests using the MELD score for dose optimization since it is identified as a covariate that significantly influences tigecycline CL. Dosing regimens are recommended to reach several PK/PD targets considering this clinical variable and any MIC within the susceptibility range.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

BACKGROUND: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. METHODS: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. RESULTS: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score. CONCLUSIONS: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

Efficacy of Albumin Treatment for Patients with Cirrhosis and Infections Unrelated to Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: We performed a randomized trial to determine whether albumin should be administered to patients with infections unrelated to spontaneous bacterial peritonitis (SBP). METHODS: We performed a multicenter, open-label trial in which 118 patients with cirrhosis, non-SBP infections, and additional risk factors for poor outcome were randomly assigned to receive antibiotics plus albumin (study group; n = 61) or antibiotics alone (control group; n = 57). The primary outcome was in-hospital mortality; secondary outcomes were effect of albumin on disease course. RESULTS: There were no significant differences at baseline between groups in results from standard laboratory tests, serum markers of inflammation, circulatory dysfunction, or liver severity scores. However, the combined prevalence of acute on chronic liver failure (ACLF) and kidney dysfunction was significantly higher in the study group (44.3% vs 24.6% in the control group; P = .02), indicating greater baseline overall severity. There was no significant difference in the primary outcome between groups (13.1% in the study group vs 10.5% in the control group; P = .66). Circulatory and renal functions improved in only the study group. A significantly higher proportion of patients in the study group had resolution of ACLF (82.3% vs 33.3% in the control group; P = .03). A significantly lower proportion of patients in the study group developed nosocomial infections (6.6% vs 24.6% in the control group; P = .007). CONCLUSIONS: In a randomized trial of patients with advanced cirrhosis and non-SBP infections, in-hospital mortality was similar between those who received albumin plus antibiotics vs those who received only antibiotics (controls). However, patients given albumin were sicker at baseline and, during the follow-up period, a higher proportion had ACLF resolution and a lower proportion had nosocomial infections. ClinicalTrials.gov no: NCT02034279.

Meropenem population pharmacokinetics in patients with decompensated cirrhosis and severe infections.

OBJECTIVES: Meropenem pharmacokinetics (PK) may be altered in patients with cirrhosis, hampering target attainment. We aimed to describe meropenem PK in patients with decompensated cirrhosis and severe bacterial infections, identify the sources of PK variability and assess the performance of different dosing regimens to optimize the PK/pharmacodynamic (PD) target. METHODS: Serum concentrations and covariates were obtained from patients with severe infections under meropenem treatment. A population PK analysis was performed using non-linear mixed-effects modelling and the final model was used to simulate meropenem exposure to assess the PTA. RESULTS: Fifty-four patients were enrolled in the study. Data were best described by a one-compartment linear model. The estimated typical mean value for clearance (CL) was 8.35 L/h and the estimated volume of distribution (V) was 28.2 L. Creatinine clearance (CLCR) and MELD score significantly influenced meropenem CL, and acute-on-chronic liver failure (ACLF) significantly affected V. Monte Carlo simulations showed that a lower meropenem dose would be needed as CLCR decreases and as the MELD score increases. Patients with ACLF would have lower peak meropenem concentrations but similar steady-state concentrations compared with patients with no ACLF. CONCLUSIONS: Our study identified two new covariates that influence meropenem PK in patients with decompensated cirrhosis in addition to CLCR: MELD score and ACLF. Dosing regimens are recommended to reach several PK/PD targets considering these clinical variables and any MIC within the susceptibility range.

Coagulation Failure in Patients With Acute-on-Chronic Liver Failure and Decompensated Cirrhosis: Beyond the International Normalized Ratio.

Balanced hemostasis with hypocoagulable and hypercoagulable features may occur in acute-on-chronic liver failure (ACLF). The characteristics and prognostic impact of the coagulation profile in ACLF are unknown. Consecutive patients with ACLF (n = 36) and acute decompensation (AD; n = 24) were included. Blood samples for thromboelastometry (TE) were obtained at admission and 72 hours thereafter. The coagulation profile was evaluated in patients with and without ACLF and in those with and without systemic inflammatory response syndrome. The impact of the coagulation profile on transfusion requirements, bleeding events, and short-term survival was assessed. At admission, patients with ACLF showed more hypocoagulable characteristics compared to AD subjects, with prolonged time to initial fibrin formation and clot formation time and decreased maximum clot firmness and alpha-angle values. TE parameters worsened at 72 hours in ACLF but improved in patients with AD. Prevalence of a hypocoagulable profile (three or more TE parameters outside range) was significantly higher in patients with ACLF either at admission (61% versus 29% in AD; P = 0.03) or during follow-up. Hypocoagulability correlated with systemic inflammation and was associated with higher 28-day (45% versus 16%; P = 0.02) and 90-day (52% versus 19%; P = 0.01) mortality rates but not with transfusion requirements or bleeding. Prolonged time to initial fibrin formation (extrinsic TE assay >80 seconds) and Model for End-Stage Liver Disease score at baseline were independent predictors of 28-day mortality. Conclusion: Patients with ACLF frequently show hypocoagulable features with prolonged time to initial fibrin formation and clot formation time and reduced clot firmness; these alterations worsen after admission, correlate with systemic inflammation, and translate into higher short-term mortality; hypofibrinolysis could contribute to organ failure in ACLF.

Human metapneumovirus in hospitalized children less than 5 years of age in Pakistan.

Acute respiratory infection (ARI) is the second leading cause of death in children less than 5 years of age worldwide. Human Metapneumovirus (hMPV) is associated with around 5-7% of the total pneumonia admissions in children. The aim of this study was to determine the magnitude of hMPV associated hospitalizations among children, in Karachi, Pakistan. A 3 years prospective study was conducted at the Aga Khan University Hospital (AKUH), from August 2009 to June 2012. Children less than 5 years of age, admitted with ARIs, were enrolled. Throat swabs were collected and tested for hMPV using real-time PCR. Multivariable log binomial regression analysis was performed. Out of 1150 children enrolled, hMPV was detected among 84/1150 (7%). About 87% of the enrolled children presented with cough, followed by fever (73%), nasal congestion (69%) and shortness of breath (68%). Of the hMPV positive subjects, most (56/84, 67%) were less than 12 months of age. The most common diagnosis in hMPV positive infants was pneumonia, followed by asthma and bronchiolitis. HMPV was identified year round, with peaks during February and August. Sore throat was found to be significantly associated with the hMPV infection (Adjusted RR 2.23; 95%CI 1.42-3.52). The proportion of hMPV was higher among hospitalized infants with ARI. Pneumonia was the primary discharge diagnoses of patients who tested positive for hMPV. hMPV could be a target for future vaccine to further decrease the burden of ARI morbidity and possibly mortality in developing countries.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

BACKGROUND: Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). METHODS: GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. FINDINGS: We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. INTERPRETATION: A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. FUNDING: Bill & Melinda Gates Foundation.

RSV associated hospitalizations in children in Karachi, Pakistan: Implications for vaccine prevention strategies.

Major progress is being made in vaccines against Respiratory Syncytial Virus (RSV), with multiple vaccine candidates currently in the clinical phase of development. Making an investment case for public sector financing of RSV vaccine will require estimation of burden, cost-effectiveness, and impact. The aim of this study is to determine the proportion, age distribution and clinical spectrum of RSV associated hospitalizations in children in Karachi, Pakistan. A three years prospective study was conducted at the Aga Khan University Hospital in Karachi, a city of 20 million in south Pakistan, from August 2009 to June 2012. Children less than five years old admitted with acute respiratory infections (ARI) were enrolled. Throat swabs were collected and tested for RSV using real-time PCR. Multivariable log binomial regression analysis was performed to identify the associated factors of RSV infection. Out of 1150 children enrolled, RSV was detected among 223 (19%). Highest rate of RSV detection was in young infants less than 3 months of age (48/168, 29%), which accounted for 22% of all RSV detected. Most common diagnosis in RSV positive infants (<12 months of age) was bronchiolitis followed by pneumonia, while in older children between the ages of one and 5 years of age, pneumonia and asthma were the most common diagnosis. Although identified year-round, RSV was most prevalent from August to October with peak in September, coinciding with the rainy season. This study identified RSV to be independently associated with younger age (P = 0.036), rainy season (P < 0.001), post-tussive emesis (P = 0.008), intubation (P = 0.003), and discharge diagnosis of bronchiolitis (P = 0.004). Vaccines against RSV that target this age group are likely to yield remarkable benefit.

Epidemiology of Pertussis Among Young Pakistani Infants: A Community-Based Prospective Surveillance Study.

BACKGROUND: Pertussis remains a cause of morbidity and mortality among young infants. There are limited data on the pertussis disease burden in this age group from low- and lower-middle-income countries, including in South Asia. METHODS: We conducted an active community-based surveillance study from February 2015 to April 2016 among 2 cohorts of young infants in 4 low-income settlements in Karachi, Pakistan. Infants were enrolled either at birth (closed cohort) or at ages up to 10 weeks (open cohort) and followed until 18 weeks of age. Nasopharyngeal swab specimens were obtained from infants who met a standardized syndromic case definition and tested for Bordetella pertussis using real-time polymerase chain reaction. We determined the incidence of pertussis using a protocol-defined case definition, as well as the US Centers for Disease Control and Prevention (CDC) definitions for confirmed and probable pertussis. RESULTS: Of 2021 infants enrolled into the study, 8 infants met the protocol-defined pertussis case definition, for an incidence of 3.96 (95% confidence interval [CI], 1.84-7.50) cases per 1000 infants. Seven of the pertussis cases met the CDC pertussis case definition (5 confirmed, 2 probable), for incidences of CDC-defined confirmed pertussis of 2.47 (95% CI, .90-5.48) cases per 1000 infants, and probable pertussis of 0.99 (95% CI, .17-3.27) cases per 1000 infants. Three of the pertussis cases were severe according to the Modified Preziosi Scale score. CONCLUSIONS: In one of the first prospective surveillance studies of infant pertussis in a developing country, we identified a moderate burden of pertussis disease in early infancy in Pakistan.

Respiratory viruses associated with severe pneumonia in children under 2 years old in a rural community in Pakistan.

The objective of this study was to determine the incidence of respiratory viruses associated with severe pneumonia among children less than 2 years of age in the rural district of Matiari in Sindh, Pakistan. This study was a community-based prospective cohort active surveillance of infants enrolled at birth and followed for 2 years. Cases were identified using the World Health Organization's Integrated Management of Childhood Illnesses' definition of severe pneumonia. Nasopharyngeal swabs were obtained for assessment by multiplex RT-PCR for eight viruses and their subtypes, including RSV, influenza virus, human metapneumovirus, enterovirus/rhinovirus, coronavirus, parainfluenza virus, adenovirus, and human bocavirus. Blood cultures were collected from febrile participants. A total of 817 newborns were enrolled and followed with fortnightly surveillance for 2 years, accounting for a total of 1,501 child-years of follow-up. Of the nasopharyngeal swabs collected, 77.8% (179/230) were positive for one or more of the above mentioned respiratory viruses. The incidence of laboratory confirmed viral-associated pneumonia was 11.9 per 100 child-years of follow-up. Enterovirus/rhinovirus was detected in 51.7% patients, followed by parainfluenza virus type III (8.3%), and RSV (5.7%). Of the uncontaminated blood cultures, 1.4% (5/356) were positive. Respiratory viruses are frequently detected during acute respiratory infection episodes in children under 2 years old in a rural community in Pakistan. However, causal association is yet to be established and the concomitant role of bacteria as a co-infection or super-infection needs further investigation. J. Med. Virol. 88:1882-1890, 2016. © 2016 Wiley Periodicals, Inc.

Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction.

BACKGROUND: The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection. METHODS: We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers. RESULTS: We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid. CONCLUSIONS: Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a "positive" sample.

BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.

BACKGROUND: Pakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses. OBJECTIVE: To assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections. STUDY DESIGN: In this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition. METHODS: BCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay. RESULTS: Significant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG. CONCLUSION: The presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.

Predictors of Respiratory Syncytial Virus, Influenza Virus, and Human Metapneumovirus Carriage in Children Under 5 Years With WHO-Defined Fast-Breathing Pneumonia in Pakistan.

BACKGROUND: Pneumonia is a leading cause of morbidity and mortality in children < 5 years. We describe nasopharyngeal carriage of respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and influenza virus among children with fast-breathing pneumonia in Karachi, Pakistan. METHODS: We performed a cross-sectional analysis of nasopharyngeal swabs from children aged 2-59 months with fast-breathing pneumonia, enrolled in the randomized trial of amoxicillin versus placebo for fast-breathing pneumonia (RETAPP) (NCT02372461) from 2014 to 2016. Swabs were collected using WHO standardized methods, processed at the Aga Khan University, Pakistan. Viral detection was performed using LUMINEX xTAG respiratory viral panel assay and logistic regression identified clinical and sociodemographic predictors. FINDINGS: Of the 1000 children tested, 92.2% (n = 922) were positive for viral carriage. RSV, hMPV, and influenza virus were detected in 59 (6.4%), 56 (6.1%), and 58 (6.3%) children and co-infections in three samples (two RSV-hMPV and one influenza-hMPV). RSV carriage was common in infants (56%), we observed a higher occurrence of fever in children with hMPV and influenza virus (80% and 88%, respectively) and fast breathing in RSV (80%) carriage. RSV carriage was positively associated with a history of fast/difficulty breathing (aOR: 1.96, 95% CI 1.02-3.76) and low oxygen saturation (aOR: 2.52, 95% CI 1.32-4.82), hMPV carriage was positively associated with a complete vaccination status (aOR: 2.22, 95% CI 1.23-4.00) and body temperature ≥ 37.5°C (aOR: 2.34, 95% CI 1.35-4.04) whereas influenza viral carriage was associated with body temperature ≥ 37.5°C (aOR: 4.48, 95% CI 2.53-7.93). CONCLUSION: We observed a high nasopharyngeal viral carriage among children with WHO-defined fast-breathing pneumonia in Pakistan. Fever, difficulty in breathing, hypoxia and vaccination status are important clinical predictors for viral nonsevere community-acquired pneumonia.

Environmental surveillance for COVID-19 using SARS-CoV-2 RNA concentration in wastewater - a study in District East, Karachi, Pakistan.

Background: Wastewater-based surveillance is used to track the temporal patterns of the SARS-CoV-2 virus in communities. Viral RNA particle detection in wastewater samples can indicate an outbreak within a catchment area. We describe the feasibility of using a sewage network to monitor SARS-CoV-2 trend and use of genomic sequencing to describe the viral variant abundance in an urban district in Karachi, Pakistan. This was among the first studies from Pakistan to demonstrate the surveillance for SARS-CoV-2 from a semi-formal sewage system. Methods: Four sites draining into the Lyari River in District East, Karachi, were identified and included in the current study. Raw sewage samples were collected early morning twice weekly from each site between June 10, 2021 and January 17, 2022, using Bag Mediated Filtration System (BMFS). Secondary concentration of filtered samples was achieved by ultracentrifugation and skim milk flocculation. SARS-CoV-2 RNA concentrations in the samples were estimated using PCR (Qiagen ProMega kits for N1 & N2 genes). A distributed-lag negative binomial regression model within a hierarchical Bayesian framework was used to describe the relationship between wastewater RNA concentration and COVID-19 cases from the catchment area. Genomic sequencing was performed using Illumina iSeq100. Findings: Among the 151 raw sewage samples included in the study, 123 samples (81.5%) tested positive for N1 or N2 genes. The average SARS-CoV-2 RNA concentrations in the sewage samples at each lag (1-14 days prior) were associated with the cases reported for the respective days, with a peak association observed on lag day 10 (RR: 1.15; 95% Credible Interval: 1.10-1.21). Genomic sequencing showed that the delta variant dominated till September 2022, while the omicron variant was identified in November 2022. Interpretation: Wastewater-based surveillance, together with genomic sequencing provides valuable information for monitoring the community temporal trend of SARS-CoV-2. Funding: PATH, Bill & Melinda Gates Foundation, and Global Innovation Fund.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Background: Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. Conclusions: TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

Pandemic influenza A(H1N1)pdm09: an unrecognized cause of mortality in children in Pakistan.

The role of influenza virus as a cause of child mortality in South Asia is under-recognized. We aimed to determine the incidence and case fatality rate of influenza A(H1N1)pdm09 infections in hospitalized children in Karachi, Pakistan. Children less than 5 y old admitted with respiratory illnesses to the Aga Khan University Hospital, Karachi, from 17 August 2009 to 16 September 2011, were tested for influenza A(H1N1)pdm09 using a real-time reverse transcriptase polymerase chain reaction. Out of 2650 children less than 5 y old admitted with a respiratory illness during the study period, 812 (31%) were enrolled. Influenza A(H1N1)pdm09 virus was detected in 27 (3.3%) children. There were 4 deaths in children who tested positive for influenza A(H1N1)pdm09 (case fatality rate of 15%). Children with influenza A(H1N1)pdm09 were 5 times more likely to be admitted or transferred to the intensive care unit, 5.5 times more likely to be intubated, and 12.9 times more likely to die as compared to children testing negative for influenza A(H1N1)pdm09.

Promoting Science Literacy and Awareness across the Globe: the Role of Scientists as Science Ambassadors.

Science literacy has many personal and societal benefits that allows for better informed decision-making. Although the importance of science literacy is recognized globally, there are many challenges associated with its promotion. Scientists are more frequently engaging with nonscientific audiences through public outreach activities and with increasing support from institutions and professional societies. This is especially true regarding microbiologists and other related professionals since the start of the global 2019 coronavirus disease pandemic heightened the need to convey novel and rapidly evolving scientific information to lay audiences. The means by which professionals engage with these audiences affect the efficacy of the relay of scientific information. One method of engagement is the "ambassador approach," which aims to establish dialogue among different groups of people and scientists. In this perspective article, we discuss this approach, highlighting activities for the promotion of science literacy organized by the American Society for Microbiology Ambassador Program and similar programs of other scientific societies. We discuss the benefits and challenges of implementing an ambassador approach, propose potential improvements that could be made to existing programs promoting science literacy, and ultimately advocate for increased implementation of science ambassador programs.

Building up a genomic surveillance platform for SARS-CoV-2 in the middle of a pandemic: a true North-South collaboration.

Next-generation sequencing technology has revolutionised pathogen surveillance over the last two decades. However, the benefits are not equitably distributed, with developing countries lagging far behind in acquiring the required technology and analytical capacity. Recent declines in the cost associated with sequencing-equipment and running consumables have created an opportunity for broader adoption. During the COVID-19 pandemic, rapid diagnostics development and DNA sequencing revolutionised the ability to diagnose and sequence SARS-CoV-2 rapidly. Socioeconomic inequalities substantially impact the ability to sequence SARS-CoV-2 strains and undermine a developing country's pandemic preparedness. Low- and middle-income countries face additional challenges in establishing, maintaining and expanding genomic surveillance. We present our experience of establishing a genomic surveillance system at the Aga Khan University, Karachi, Pakistan. Despite being at a leading health sciences research institute in the country, we encountered significant challenges. These were related to collecting standardised contextual data for SARS-CoV-2 samples, procuring sequencing reagents and consumables, and challenges with library preparation, sequencing and submission of high-quality SARS-CoV-2 genomes. Several technical roadblocks ensued during the implementation of the genomic surveillance framework, which were resolved in collaboration with our partners. High-quality genome sequences were then deposited on open-access platforms per the best practices. Subsequently, these efforts culminated in deploying Pakistan's first SARS-CoV-2 phyllo surveillance map as a Nextstrain build. Our experience offers lessons for the successful development of Genomic Surveillance Infrastructure in resource-limited settings struck by a pandemic.