Application of Hybridization Chain Reaction/CRISPR-Cas12a for the Detection of SARS-CoV-2 Infection.

Globally, the emergence of the coronavirus disease (COVID-19) has had a significant impact on life. The need for ongoing SARS-CoV-2 screening employing inexpensive and quick diagnostic approaches is undeniable, given the ongoing pandemic and variations in vaccine administration in resource-constrained regions. This study presents results as proof of concept to use hybridization chain reaction (HCR) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a complex for detecting SARS-CoV-2. HCR hairpin probes were designed using the NUPACK web-based program and further used to amplify the SARS-CoV-2 N gene in archived nasopharyngeal samples. The results were visualized using agarose gels and CRISPR Cas12a-based lateral flow strips. The assay was evaluated using the gold standard, real-time polymerase chain reaction (RT-PCR), as recommended by the World Health Organization (WHO). The results show the comparative efficiency of HCR to RT-PCR. This study shows that HCR and CRISPR are viable alternatives for diagnosing SARS-CoV-2 in samples.

Characterization of influenza infection in a high-income urban setting in Nairobi, Kenya.

BACKGROUND: Influenza viruses are an important cause of respiratory infections across all age groups. Information on occurrence and magnitude of influenza virus infections in different populations in Kenya however remains scanty, compromising estimation of influenza disease burden. This study examined influenza infection in an urban high-income setting in Nairobi to establish its prevalence and activity of influenza viruses, and evaluated diagnostic performance of a rapid influenza diagnostic test. METHODOLOGY: A cross-sectional hospital-based study was conducted in six private health facilities located within high-income residential areas in Nairobi from January 2019 to July 2020. Patients of all ages presenting with influenza-like illness (ILI) were recruited into the study. Detection of influenza virus was conducted using rapid diagnosis and reverse transcription-polymerase chain reaction (RT-PCR). Data were summarized using descriptive statistics and tests of association. Sensitivity, specificity and area under receiver operating characteristics curve was calculated to establish diagnostic accuracy of the rapid diagnosis test. RESULTS: The study recruited 125 participants with signs and symptoms of ILI, of whom 21 (16.8%) were positive for influenza viruses. Of all the influenza-positive cases, 17 (81.0%) were influenza type A of which 70.6% were pandemic H1N1 (A/H1N1 2009). Highest detection was observed among children aged 5-10 years. Influenza virus mostly circulated during the second half of the year, and fever, general fatigue and muscular and joint pain were significantly observed among participants with influenza virus. Sensitivity and specificity of the diagnostic test was 95% (95% confidence interval 75.1-99.9) and 100% (95% confidence interval 96.5-100.0), respectively. CONCLUSIONS: Findings of this study shows continuous but variable activity of influenza virus throughout the year in this population, with substantial disease burden. The findings highlight the need for continuous epidemiologic surveillance including genetic surveillance to monitor activity and generate data to inform vaccine introduction or development, and other interventions.

Impact of integrated water, sanitation, hygiene, health and nutritional interventions on diarrhoea disease epidemiology and microbial quality of water in a resource-constrained setting in Kenya: A controlled intervention study.

OBJECTIVES: We assessed the impact of water, hygiene and sanitation (WASH), maternal, new-born and child health (MNCH), nutrition and early childhood development (ECD) on diarrhoea and microbial quality of water in a resource-constrained rural setting in Kenya. METHODS: Through a controlled intervention study, we tested faecal and water samples collected from both the intervention and control sites before and after the interventions using microbiological, immunological and molecular assays to determine the prevalence of diarrhoeagenic agents and microbial quality of water. Data from the hospital registers were used to estimate all-cause diarrhoea prevalence. RESULTS: After the interventions, we observed a 58.2% (95% CI: 39.4-75.3) decline in all-cause diarrhoea in the intervention site versus a 22.2% (95% CI: 5.9-49.4) reduction of the same in the control site. Besides rotavirus and pathogenic Escherichia coli, the rate of isolation of other diarrhoea-causing bacteria declined substantially in the intervention site. The microbial quality of community and household water improved considerably in both the intervention (81.9%; 95% CI: 74.5%-87.8%) and control (72.5%; 95% CI: 64.2%-80.5%) sites with the relative improvements in the intervention site being slightly larger. CONCLUSIONS: The integrated WASH, MNCH, nutrition and ECD interventions resulted in notable decline in all-cause diarrhoea and improvements in water quality in the rural resource-limited population in Kenya. This indicates a direct public health impact of the interventions and provides early evidence for public health policy makers to support the sustained implementation of these interventions.

Analysis of Environmental and Pathogenic Bacteria Attached to Aerosol Particles Size-Separated with a Metal Mesh Device.

Metal mesh devices (MMDs) are novel materials that enable the precise separation of particles by size. Structurally, MMDs consist of a periodic arrangement of square apertures of characteristic shapes and sizes on a thin nickel membrane. The present study describes the separation of aerosol particles using palm-top-size collection devices equipped with three types of MMDs differing in pore size. Aerosols were collected at a farm located in the suburbs of Nairobi, Kenya; aerosol particles were isolated, and pathogenic bacteria were identified in this microflora by next-generation sequencing analysis. The composition of the microflora in aerosol particles was found to depend on particle size. Gene fragments were obtained from the collected aerosols by PCR using primers specific for the genus Mycobacterium. This analysis showed that Mycobacterium obuense, a non-tuberculous species of mycobacteria that causes lung diseases, was present in these aerosols. These findings showed that application of this MMD analytical protocol to aerosol particles can facilitate the investigation of airborne pathogenic bacteria.

Genomic characterization of an African G4P[6] human rotavirus strain identified in a diarrheic child in Kenya: Evidence for porcine-to-human interspecies transmission and reassortment.

Human rotavirus strains having the unconventional G4P[6] genotype have been sporadically identified in diarrheic patients in different parts of the world. However, the whole genome of only one human G4P[6] strain from Africa (central Africa) has been sequenced and analyzed, and thus the exact origin and evolutionary pattern of African G4P[6] strains remain to be elucidated. In this study, we characterized the full genome of an African G4P[6] strain (RVA/Human-wt/KEN/KCH148/2019/G4P[6]) identified in a stool specimen from a diarrheic child in Kenya. Full genome analysis of strain KCH148 revealed a unique Wa-like genogroup constellation: G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1. NSP3 genotype T7 is commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis showed that 10 of the 11 genes of strain KCH148 (VP7, VP4, VP6, VP1-VP3, NSP1, and NSP3-NSP5) appeared to be of porcine origin, the remaining NSP2 gene appearing to be of human origin. Therefore, strain KCH148 was found to have a porcine rotavirus backbone and thus is likely to be of porcine origin. Furthermore, strain KCH148 is assumed to have been derived through interspecies transmission and reassortment events involving porcine and human rotavirus strains. To our knowledge, this is the first report on full genome-based characterization of a human G4P[6] strain from east Africa. Our observations demonstrated the diversity of human G4P[6] strains in Africa, and provide important insights into the origin and evolutionary pattern of zoonotic G4P[6] strains on the African continent.

Rapid detection of Group B Streptococcus (GBS) from artificial urine samples based on IFAST and ATP bioluminescence assay: from development to practical challenges during protocol testing in Kenya.

We report the rapid detection (20 min) of Streptococcus agalactiae, Group B Streptococcus (GBS) employing on-chip magnetic isolation of GBS based on immiscible filtration assisted by surface tension (IFAST), followed by detection of the isolated GBS using an adenosine triphosphate (ATP) bioluminescence assay. Up to 80% GBS cells were isolated from spiked artificial urine samples with linear responses of bioluminescence signals from isolated cells at 2.3 × 102-9.1 × 105 CFU mL-1, demonstrating great promise for point-of-care detection of pathogenic bacteria in screening urine samples from pregnant women. Practical challenges during initial testing of the developed protocol with urine samples in Kenya are also described.

Genomic characterization of uncommon human G3P[6] rotavirus strains that have emerged in Kenya after rotavirus vaccine introduction, and pre-vaccine human G8P[4] rotavirus strains.

A monovalent rotavirus vaccine (RV1) was introduced to the national immunization program in Kenya in July 2014. There was increased detection of uncommon G3P[6] strains that coincided temporally with the timing of this vaccine introduction. Here, we sequenced and characterized the full genomes of two post-vaccine G3P[6] strains, RVA/Human-wt/KEN/KDH1951/2014/G3P[6] and RVA/Human-wt/KEN/KDH1968/2014/G3P[6], as representatives of these uncommon strains. On full-genomic analysis, both strains exhibited a DS-1-like genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that all 11 genes of strains KDH1951 and KDH1968 were very closely related to those of human G3P[6] strains isolated in Uganda in 2012-2013, indicating the derivation of these G3P[6] strains from a common ancestor. Because the uncommon G3P[6] strains that emerged in Kenya are fully heterotypic as to the introduced vaccine strain regarding the genotype constellation, vaccine effectiveness against these G3P[6] strains needs to be closely monitored.

Impact of rotavirus vaccination on rotavirus hospitalisation rates among a resource-limited rural population in Mbita, Western Kenya.

OBJECTIVES: A two-dose oral monovalent rotavirus vaccine (RV1) was introduced into the Kenyan National Immunization Program in July 2014. We assessed trends in hospitalisation for rotavirus-specific acute gastroenteritis (AGE) and strain distribution among children <5 years in a rural, resource-limited setting in Kenya before and after the nationwide implementation of the vaccine. METHODS: Data on rotavirus AGE and strain distribution were derived from a 5-year hospital-based surveillance. We compared rotavirus-related hospitalisations and strain distribution in the 2-year post-vaccine period with the 3-year pre-vaccine baseline. Vaccine administrative data from the Unit of Vaccines and Immunization Services (UVIS) for Mbita sub-county were used to estimate rotavirus immunisation coverage in the study area. RESULTS: We observed a 48% (95% CI: 27-64%) overall decline in rotavirus-related hospitalisations among children aged <5 years in the post-vaccine period. Coverage with the last dose of rotavirus vaccine increased from 51% in year 1% to 72% in year 2 of the vaccine implementation. Concurrently, reductions in rotavirus hospitalisations increased from 40% in the first year to 53% in the second year of vaccine use. The reductions were most pronounced among the vaccine-eligible group, with the proportion of cases in this age group dropping to 14% in post-vaccine years from a high of 51% in the pre-vaccine period. A diversity of rotavirus strains circulated before the introduction of the vaccine with G1P[8] being the most dominant strain. G2P[4] replaced G1P[8] as the dominant strain after the vaccine was introduced. CONCLUSIONS: Rotavirus vaccination has resulted in a notable decline in hospital admissions for rotavirus infections in a rural resource-limited population in Kenya. This provides early evidence for continued use of rotavirus vaccines in routine childhood immunisations in Kenya. Our data also underscore the need for expanding coverage on second dose so as to maximise the impact of the vaccine.

Impact of rotavirus vaccination on rotavirus and all-cause gastroenteritis in peri-urban Kenyan children.

A monovalent rotavirus vaccine (RV1) was introduced into the National Immunization Program in Kenya in July 2014. We examined the impact of the vaccine on hospitalization for all-cause acute gastroenteritis (AGE) and rotavirus-specific AGE and strain distribution at a large referral hospital which serves a predominantly peri-urban population in Central Kenya. Data on rotavirus AGE and strain distribution were derived from ongoing hospital-based AGE surveillance. Hospital administrative data were used to compare trends in all-cause AGE. Pre-vaccine (July 2009-June 2014) and post-vaccine (July 2014-June 2016) periods were compared for changes in hospitalization for all-cause AGE and rotavirus AGE and strain distribution. Following the vaccine introduction, the proportion of children aged <5years hospitalized for rotavirus declined by 30% (95% CI: 19-45%) in the first year and 64% (95% CI: 49-77%) in the second year. Reductions in rotavirus positivity were most pronounced among the vaccine-eligible group (<12months) in the first year post-vaccination at 42% (95% CI: 28-56%). Greater reductions of 67% (95% CI: 51-79%) were seen in the second year in the 12-23months age group. Similarly, hospitalizations for all-cause AGE among children <5years of age decreased by 31% (95% CI: 24-40%) in the first year and 58% (95% CI: 49-67%) in the second year of vaccine introduction. Seasonal peaks of rotavirus and all-cause AGE were reduced substantially. There was an increased detection of G2P[4], G3P[6] and G3P[8], which coincided temporally with the timing of the vaccine introduction. Thus, introducing the rotavirus vaccine into the routine immunization program in Kenya has resulted in a notable decline in rotavirus and all-cause AGE hospitalizations in Central Kenya. This provides early evidence for public health policy makers in Kenya to support the sustained use of the rotavirus vaccine in routine immunizations.

Detection of rotavirus in clinical specimens using an immunosensor prototype based on the photon burst counting technique.

In this study, a sensitive fluorescence sensor was developed for the detection of small, fluorescence-labeled particles dispersed in a solution. The prototype system comprises of a laser confocal optical system and a mechanical sample stage to detect photon bursting of fluorescence-labeled small particles in sample volumes less than 5 µL within 3 minutes. To examine the feasibility of the prototype system as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. The advantages and disadvantages of this immunosensor with respect to ELISA and RT-PCR, the current gold standards for virus detection, are discussed.

Variation in rotavirus vaccine coverage by sub-counties in Kenya.

Rotavirus gastroenteritis is an important cause of childhood morbidity and mortality in Kenya. In July 2014, Kenya introduced the rotavirus vaccine into her national immunization program. Although immunization coverage is crucial in assessing the real-world impact of this vaccine, variability in the vaccine coverage across the country is likely to occur. In view of this, we estimated the extent of coverage for the rotavirus vaccine at two socio-economically different sub-counties using the administrative data. The findings indicate disparities in vaccine coverage and access between the sub-counties and, thus, underscore the need to strengthen immunization systems to facilitate timely, accessible, and equitable vaccine delivery across the country. Both sub-counties recorded high vaccine dropout, suggestive of poor utilization of the vaccine. In this regard, increased social mobilization is needed to encourage vaccine compliance and to enhance tracking of vaccine defaulters. While efforts to improve the accuracy of the administrative coverage estimates are crucial, vaccination coverage surveys will be needed to verify the administrative coverage data and help identify specific factors relating to rotavirus vaccine coverage in the country.