Comparative analysis of SARS-CoV-2 detection methods using stool, blood, and nasopharyngeal swab samples.

Introduction: as a public health policy, the ongoing global coronavirus disease 2019 vaccination drives require continuous tracking, tracing, and testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic testing is important in virus detection and understanding its spread for timely intervention. This is especially important for low-income settings where the majority of the population remains untested. This is well supported by the fact that of about 9% of the Kenyan population had been tested for the virus. Methods: this was a cross-sectional study conducted at the Kisumu and Siaya Referral Hospitals in Kenya. Here we report on the sensitivity and specificity of the rapid antigen detection test (Ag-RDT) of SARS-CoV-2 compared with the quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) using stool and nasopharyngeal swab samples. Further, the mean Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody levels among symptomatic and asymptomatic individuals in western Kenya were evaluated. Results: the sensitivity and specificity of Ag-RDT were 76.3% (95% CI, 59.8-88.6%) and 96.3% (95% CI, 87.3-99.5%) with a negative and positive predictive value of 85% (95% CI, 73.8%-93.0%) and 93% (95% CI, 78.6%-99.2%) respectively. There was substantial agreement of 88% (Kappa value of 0.75, 95% CI, 0.74-0.77) between Ag-RDT and nasopharyngeal swab RT-qPCR, and between stool and nasopharyngeal swab RT-qPCR results (83.7% agreement, Kapa value 0.62, 95% CI 0.45-0.80). The mean IgM and IgG antibody response to SARS-CoV-2 were not different in asymptomatic individuals, 1.11 (95% CI, 0.78-1.44) and 0.88 (95% CI, 0.65-1.11) compared to symptomatic individuals 4.30 (95% CI 3.30-5.31) and 4.16 (95% CI 3.32 -5.00). Conclusion: the choice of an appropriate SARS-CoV-2 diagnostic, screening, and surveillance test should be guided by the specific study needs and a rational approach for optimal results.

Heterogenous transmission and seroprevalence of SARS-CoV-2 in two demographically diverse populations with low vaccination uptake in Kenya, March and June 2021.

Background: SARS-CoV-2 has extensively spread in cities and rural communities, and studies are needed to quantify exposure in the population. We report seroprevalence of SARS-CoV-2 in two well-characterized populations in Kenya at two time points. These data inform the design and delivery of public health mitigation measures. Methods: Leveraging on existing population based infectious disease surveillance (PBIDS) in two demographically diverse settings, a rural site in western Kenya in Asembo, Siaya County, and an urban informal settlement in Kibera, Nairobi County, we set up a longitudinal cohort of randomly selected households with serial sampling of all consenting household members in March and June/July 2021. Both sites included 1,794 and 1,638 participants in the March and June/July 2021, respectively. Individual seroprevalence of SARS-CoV-2 antibodies was expressed as a percentage of the seropositive among the individuals tested, accounting for household clustering and weighted by the PBIDS age and sex distribution. Results: Overall weighted individual seroprevalence increased from 56.2% (95%CI: 52.1, 60.2%) in March 2021 to 63.9% (95%CI: 59.5, 68.0%) in June 2021 in Kibera. For Asembo, the seroprevalence almost doubled from 26.0% (95%CI: 22.4, 30.0%) in March 2021 to 48.7% (95%CI: 44.3, 53.2%) in July 2021. Seroprevalence was highly heterogeneous by age and geography in these populations-higher seroprevalence was observed in the urban informal settlement (compared to the rural setting), and children aged <10 years had the lowest seroprevalence in both sites. Only 1.2% and 1.6% of the study participants reported receipt of at least one dose of the COVID-19 vaccine by the second round of serosurvey-none by the first round. Conclusions: In these two populations, SARS-CoV-2 seroprevalence increased in the first 16 months of the COVID-19 pandemic in Kenya. It is important to prioritize additional mitigation measures, such as vaccine distribution, in crowded and low socioeconomic settings.

High seroprevalence of Immunoglobulin G (IgG) and IgM antibodies to SARS-CoV-2 in asymptomatic and symptomatic individuals amidst vaccination roll-out in western Kenya.

The population's antibody response is a key factor in comprehending SARS-CoV-2 epidemiology. This is especially important in African settings where COVID-19 impact, and vaccination rates are relatively low. This study aimed at characterizing the Immunoglobulin G (IgG) and Immunoglobulin M (IgM) in both SARS-CoV-2 asymptomatic and symptomatic individuals in Kisumu and Siaya counties in western Kenya using enzyme linked immunosorbent assays. The IgG and IgM overall seroprevalence in 98 symptomatic and asymptomatic individuals in western Kenya between December 2021-March 2022 was 76.5% (95% CI = 66.9-84.5) and 29.6% (95% CI = 20.8-39.7) respectively. In terms of gender, males had slightly higher IgG positivity 87.5% (35/40) than females 68.9% (40/58). Amidst the ongoing vaccination roll-out during the study period, over half of the study participants (55.1%, 95% CI = 44.7-65.2) had not received any vaccine. About one third, (31.6%, 95% CI = 22.6-41.8) of the study participants had been fully vaccinated, with close to a quarter (13.3% 95% CI = 7.26-21.6) partially vaccinated. When considering the vaccination status and seroprevalence, out of the 31 fully vaccinated individuals, IgG seropositivity was 81.1% (95% CI = 70.2-96.3) and IgM seropositivity was 35.5% (95% CI = 19.22-54.6). Out of the participants that had not been vaccinated at all, IgG seroprevalence was 70.4% (95% CI 56.4-82.0) with 20.4% (95% CI 10.6-33.5) seropositivity for IgM antibodies. On PCR testing, 33.7% were positive, with 66.3% negative. The 32 positive individuals included 12(37.5%) fully vaccinated, 8(25%) partially vaccinated and 12(37.5%) unvaccinated. SARs-CoV-2 PCR positivity did not significantly predict IgG (p = 0.469 [95% CI 0.514-4.230]) and IgM (p = 0.964 [95% CI 0.380-2.516]) positivity. These data indicate a high seroprevalence of antibodies to SARS-CoV-2 in western Kenya. This suggests that a larger fraction of the population was infected with SARS-CoV-2 within the defined period than what PCR testing could cover.

The burden of influenza among Kenyan pregnant and postpartum women and their infants, 2015-2020.

BACKGROUND: In tropical Africa, data about influenza-associated illness burden are needed to assess potential benefits of influenza vaccination among pregnant women. We estimated the incidence of influenza among pregnant women and their infants in Siaya County, Kenya. METHODS: We enrolled women at <31 weeks of gestation and conducted weekly follow-up until 6-month postpartum to identify acute respiratory illnesses (ARIs). We defined ARI among mothers as reported cough, rhinorrhoea or sore throat and among infants as maternal-reported cough, difficulty breathing, rhinorrhoea or clinician diagnosis of respiratory illness. We collected nasal/nasopharyngeal and oropharyngeal swabs from mothers/infants with ARI and tested for influenza A and B using molecular assays. We calculated antenatal incidence of laboratory-confirmed influenza among mothers and postnatal incidence among mothers and infants. RESULTS: During June 2015 to May 2020, we analysed data from 3,026 pregnant women at a median gestational age of 16 weeks (interquartile range [IQR], 13, 18) and followed 2,550 infants. Incidence of laboratory-confirmed influenza during pregnancy (10.3 episodes per 1,000 person-months [95% confidence interval {CI} 8.6-11.8]) was twofold higher than in the postpartum period (4.0 [95% CI 2.6-5.5]; p < 0.01). Incidence was significantly higher among human immunodeficiency virus (HIV)-infected pregnant women (15.6 [95% CI 11.0-20.6] vs. 9.1 [95% CI 7.5-10.8]; p < 0.01). Incidence among young infants was 4.4 (95% CI 3.0-5.9) and similar among HIV-exposed and HIV-unexposed infants. CONCLUSION: Our findings suggest a substantial burden of influenza illnesses during pregnancy, with a higher burden among HIV-infected mothers. Kenyan authorities should consider the value of vaccinating pregnant women, especially if HIV infected.

The Impact of Maternal Human Immunodeficiency Virus Infection on the Burden of Respiratory Syncytial Virus Among Pregnant Women and Their Infants, Western Kenya.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of respiratory illness worldwide; however, burden data on mother-infant pairs remain sparse in sub-Saharan Africa, where human immunodeficiency virus (HIV) is prevalent. We evaluated the impact of maternal HIV infection on the burden of RSV among mothers and their infants in western Kenya. METHODS: We enrolled pregnant women (≤20 weeks' gestation) and followed them and their newborns weekly for up to 3-6 months postpartum, to document cases of acute respiratory illness (ARI). Nasal/oropharyngeal swabs were collected and tested for RSV using polymerase chain reaction. Analyses were stratified by maternal HIV status and incidence was computed per 1000 person-months. RESULTS: Compared to RSV-negative ARI cases, RSV-positive cases were associated with cough, apnea, and hospitalization among infants. RSV incidence per 1000 person-months among mothers was 4.0 (95% confidence interval [CI], 3.2-4.4), and was twice that among the HIV-infected mothers (8.4 [95% CI, 5.7-12.0]) compared to the HIV-uninfected mothers (3.1 [95% CI, 2.3-4.0]). Among infants, incidence per 1000 person-months was 15.4 (95% CI, 12.5-18.8); incidence did not differ by HIV exposure or prematurity. CONCLUSIONS: HIV infection may increase the risk of RSV illness among pregnant women. Future maternal RSV vaccines may have added benefit in areas with high HIV prevalence.

Effect of Time Since Death on Multipathogen Molecular Test Results of Postmortem Specimens Collected Using Minimally Invasive Tissue Sampling Techniques.

BACKGROUND: We used postmortem minimally invasive tissue sampling (MITS) to assess the effect of time since death on molecular detection of pathogens among respiratory illness-associated deaths. METHODS: Samples were collected from 20 deceased children (aged 1-59 months) hospitalized with respiratory illness from May 2018 through February 2019. Serial lung and/or liver and blood samples were collected using MITS starting soon after death and every 6 hours thereafter for up to 72 hours. Bodies were stored in the mortuary refrigerator for the duration of the study. All specimens were analyzed using customized multipathogen TaqMan® array cards (TACs). RESULTS: We identified a median of 3 pathogens in each child's lung tissue (range, 1-8; n = 20), 3 pathogens in each child's liver tissue (range, 1-4; n = 5), and 2 pathogens in each child's blood specimen (range, 0-4; n = 5). Pathogens were not consistently detected across all collection time points; there was no association between postmortem interval and the number of pathogens detected (P = .43) and no change in TAC cycle threshold value over time for pathogens detected in lung tissue. Human ribonucleoprotein values indicated that specimens collected were suitable for testing throughout the study period. CONCLUSIONS: Results suggest that lung, liver, and blood specimens can be collected using MITS procedures up to 4 days after death in adequately preserved bodies. However, inconsistent pathogen detection in samples needs careful consideration before drawing definitive conclusions on the etiologic causes of death.

Detection of pandemic influenza A/H1N1/pdm09 virus among pigs but not in humans in slaughterhouses in Kenya, 2013-2014.

OBJECTIVE: We conducted four cross-sectional studies over 1 year among humans and pigs in three slaughterhouses in Central and Western Kenya (> 350 km apart) to determine infection and exposure to influenza A viruses. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from participants who reported acute respiratory illness (ARI) defined as fever, cough or running nose. Nasal swabs and blood samples were collected from pigs. Human NP/OP and pig nasal swabs were tested for influenza A virus by real-time reverse transcriptase polymerase chain reaction (PCR) and pig serum was tested for anti-influenza A antibodies by ELISA. RESULTS: A total of 288 participants were sampled, 91.3% of them being male. Fifteen (5.2%) participants had ARI but the nine swabs collected from them were negative for influenza A virus by PCR. Of the 1128 pigs sampled, five (0.4%) nasal swabs tested positive for influenza A/H1N1/pdm09 by PCR whereas 214 of 1082 (19.8%) serum samples tested for Influenza A virus antibodies. There was higher seroprevalence in colder months and among pigs reared as free-range. These findings indicate circulation of influenza A/H1N1/pdm09 among pigs perhaps associated with good adaptation of the virus to the pig population after initial transmission from humans to pigs.

Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya.

Molecular diagnostic methods are becoming increasingly available for assessment of acute lower respiratory illnesses (ALRI). However, nasopharyngeal/oropharyngeal (NP/OP) swabs may not accurately reflect etiologic agents from the lower respiratory tract where sputum specimens are considered as a more representative sample. The pathogen yields from NP/OP against sputum specimens have not been extensively explored, especially in tropical countries. We compared pathogen yields from NP/OP swabs and sputum specimens from patients ≥18 years hospitalized with ALRI in rural Western Kenya. Specimens were tested for 30 pathogens using TaqMan Array Cards (TAC) and results compared using McNemar's test. The agreement for pathogen detection between NP/OP and sputum specimens ranged between 85-100%. More viruses were detected from NP/OP specimens whereas Klebsiella pneumoniae and Mycobacterium tuberculosis were more common in sputum specimens. There was no clear advantage in using sputum over NP/OP specimens to detect pathogens of ALRI in adults using TAC in the context of this tropical setting.

Seroprevalence of influenza A virus in pigs and low risk of acute respiratory illness among pig workers in Kenya.

BACKGROUND: Influenza A viruses pose a significant risk to human health because of their wide host range and ability to reassort into novel viruses that can cause serious disease and pandemics. Since transmission of these viruses between humans and pigs can be associated with occupational and environmental exposures, we investigated the association between occupational exposure to pigs, occurrence of acute respiratory illness (ARI), and influenza A virus infection. METHODS: The study was conducted in Kiambu County, the county with the highest level of intensive small-scale pig farming in Kenya. Up to 3 participants (> 2 years old) per household from pig-keeping and non-pig-keeping households were randomly recruited and followed up in 2013 (Sept-Dec) and 2014 (Apr-Aug). Oropharyngeal (OP) and nasopharyngeal (NP) swabs were collected from participants with ARI at the time of study visit. For the animal study, nasal and oropharyngeal swabs, and serum samples were collected from pigs and poultry present in enrolled households. The human and animal swab samples were tested for viral nucleic acid by RT-PCR and sera by ELISA for antibodies. A Poisson generalized linear mixed-effects model was developed to assess the association between pig exposure and occurrence of ARI. RESULTS: Of 1137 human participants enrolled, 625 (55%) completed follow-up visits including 172 (27.5%) pig workers and 453 (72.5%) non-pig workers. Of 130 human NP/OP swabs tested, four (3.1%) were positive for influenza A virus, one pig worker, and three among non-pig workers. Whereas none of the 4462 swabs collected from pig and poultry tested positive for influenza A virus by RT-PCR, 265 of 4273 (6.2%) of the sera tested positive for virus antibodies by ELISA, including 11.6% (230/1990) of the pigs and 1.5% (35/2,283) of poultry. The cumulative incidence of ARI was 16.9% among pig workers and 26.9% among the non-pig workers. The adjusted risk ratio for the association between being a pig worker and experiencing an episode of ARI was 0.56 (95% CI [0.33, 0.93]), after adjusting for potential confounders. CONCLUSIONS: Our findings demonstrate moderate seropositivity for influenza A virus among pigs, suggesting the circulation of swine influenza virus and a potential for interspecies transmission.

A diagnostic and epidemiologic investigation of acute febrile illness (AFI) in Kilombero, Tanzania.

INTRODUCTION: In low-resource settings, empiric case management of febrile illness is routine as a result of limited access to laboratory diagnostics. The use of comprehensive fever syndromic surveillance, with enhanced clinical microbiology, advanced diagnostics and more robust epidemiologic investigation, could enable healthcare providers to offer a differential diagnosis of fever syndrome and more appropriate care and treatment. METHODS: We conducted a year-long exploratory study of fever syndrome among patients ≥ 1 year if age, presenting to clinical settings with an axillary temperature of ≥37.5°C and symptomatic onset of ≤5 days. Blood and naso-pharyngeal/oral-pharyngeal (NP/OP) specimens were collected and analyzed, respectively, using AFI and respiratory TaqMan Array Cards (TAC) for multi-pathogen detection of 57 potential causative agents. Furthermore, we examined numerous epidemiologic correlates of febrile illness, and conducted demographic, clinical, and behavioral domain-specific multivariate regression to statistically establish associations with agent detection. RESULTS: From 15 September 2014-13 September 2015, 1007 febrile patients were enrolled, and 997 contributed an epidemiologic survey, including: 14% (n = 139) 1<5yrs, 19% (n = 186) 5-14yrs, and 67% (n = 672) ≥15yrs. AFI TAC and respiratory TAC were performed on 842 whole blood specimens and 385 NP/OP specimens, respectively. Of the 57 agents surveyed, Plasmodium was the most common agent detected. AFI TAC detected nucleic acid for one or more of seven microbial agents in 49% of AFI blood samples, including: Plasmodium (47%), Leptospira (3%), Bartonella (1%), Salmonella enterica (1%), Coxiella burnetii (1%), Rickettsia (1%), and West Nile virus (1%). Respiratory TAC detected nucleic acid for 24 different microbial agents, including 12 viruses and 12 bacteria. The most common agents detected among our surveyed population were: Haemophilus influenzae (67%), Streptococcus pneumoniae (55%), Moraxella catarrhalis (39%), Staphylococcus aureus (37%), Pseudomonas aeruginosa (36%), Human Rhinovirus (25%), influenza A (24%), Klebsiella pneumoniae (14%), Enterovirus (15%) and group A Streptococcus (12%). Our epidemiologic investigation demonstrated both age and symptomatic presentation to be associated with a number of detected agents, including, but not limited to, influenza A and Plasmodium. Linear regression of fully-adjusted mean cycle threshold (Ct) values for Plasmodium also identified statistically significant lower mean Ct values for older children (20.8), patients presenting with severe fever (21.1) and headache (21.5), as well as patients admitted for in-patient care and treatment (22.4). CONCLUSIONS: This study is the first to employ two syndromic TaqMan Array Cards for the simultaneous survey of 57 different organisms to better characterize the type and prevalence of detected agents among febrile patients. Additionally, we provide an analysis of the association between adjusted mean Ct values for Plasmodium and key clinical and demographic variables, which may further inform clinical decision-making based upon intensity of infection, as observed across endemic settings of sub-Saharan Africa.

Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites (Balamuthia mandrillaris and Acanthamoeba), six bacterial pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Mycoplasma pneumoniae, Mycobacterium tuberculosis, and Bartonella), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.