Evaluation of fluorescent in-situ hybridization technique for diagnosis of malaria in Ahero Sub-County hospital, Kenya.

BACKGROUND: Malaria is a major cause of morbidity and mortality. Treatment of malaria in a timely manner could avert deaths. Treatment ultimately relies on the rapid and accurate diagnosis. Fluorescence in situ hybridization (FISH), a cytogenetic technique based on detection of specific nucleic acid, has the potential to address the limitations of the current diagnostic approaches. This study investigates further the performance of FISH for the diagnosis of malaria in a rural setting in Western Kenya. METHODS: Blood samples from 302 patients presenting with fever (temperature ≥ 37.5 °C) were examined for malaria using the Giemsa microscopy (GM), rapid diagnostic test (RDT), polymerase chain reaction (PCR) and FISH. RESULTS: The sensitivity and specificity of FISH was 85.6% and 96.2% respectively, while the corresponding values for GM were 82.2% and 100% respectively. RDT and PCR had sensitivities of 91.1% and 98.9%, respectively with their specificities being 89.6 and 100%, respectively. The positive predictive values for RDT, GM, FISH and PCR were 78.8%, 100%, 90.6% and 100%, respectively. The negative predictive values for RDT, GM, FISH and PCR were 96.0%, 93.0%, 94.0% and 99.5%, respectively. Their respective diagnostic accuracies were 90.1%, 94.7% 93.0% and 99.7%. CONCLUSION: The present study demonstrates that the specificity and reproducibility of FISH assays are high, thus adding to the growing evidence on the potential of the technique as an effective tool for the detection of malaria parasites in remote settings.

A phased strengthening of laboratory capacity in the Eastern Mediterranean Region during the COVID-19 pandemic.

The Eastern Mediterranean Region (EMR) faces ongoing challenges in its public health system due to limited resources, logistical issues, and political disruptions. The COVID-19 pandemic accelerated the need for stronger laboratory capacities to handle the increased demand for testing. In a phased response, EMR countries utilized the National Influenza Centers to rapidly establish and scale molecular testing for SARS-CoV-2, the causative agent of COVID-19. The expansion of capacity included strong collaborations between public health bodies and private and academic sectors to decentralize and expand testing to the subnational level. To ensure that the quality of testing was not impacted by rapid expansion, national and subnational laboratories were enrolled in external quality assurance programs for the duration of the response. Implementation of genomic surveillance was prioritized for variant tracking, leading to the establishment of regional sequencing reference laboratories and the distribution of MinION sequencing platforms to complex emergency countries who previously had limited experience with pathogen sequencing. Challenges included a lack of technical expertise, including in implementing novel diagnostic assays and sequencing, a lack of bioinformatics expertise in the region, and significant logistical and procurement challenges. The collaborative approach, coordinated through the WHO Eastern Mediterranean Regional Office, enabled all 22 countries to achieve SARS-CoV-2 diagnostic capabilities, highlighting the pivotal role of laboratories in global health security.

Key aspects defining the development and implementation of a regional genomic surveillance strategy for the Eastern Mediterranean Region.

The COVID-19 pandemic highlighted the critical role of pathogen sequencing in making informed public health decisions. Initially, the Eastern Mediterranean Region faced limitations in sequencing capacity. However, with robust WHO and stakeholder support, the situation significantly improved. By 2022, COVID-19 sequencing was underway in 22 out of 23 regional countries, with varying throughput and capacity. Notably, three genomic hubs were established in Oman, UAE, and Morocco, playing a key role in providing expanded genomics training and support across the region. While primarily for COVID-19 surveillance, this sequencing capacity offers an opportunity to integrate genomic surveillance into existing networks. This integration can enable early detection and response to high-threat pathogens with pandemic potential. To advance this, WHO/EMRO collaborated with stakeholders to formulate the Eastern Mediterranean Regional Genomic Surveillance Strategy for Emerging Pathogens of Pandemic Concern. Consultative meetings with regional and international genomic surveillance experts identified strategy focal points, key partners, priority pathogens, and implementation steps. As the strategy awaits member states' ratification in Q4 2023, this manuscript outlines pivotal facets defined by member states and the strategic document's key deliverables and opportunities. These efforts aim to yield a substantial positive impact within the region.

Monitoring the quality of SARS-CoV-2 virus detection in molecular diagnostic laboratories in the Eastern Mediterranean Region during the COVID-19 pandemic.

INTRODUCTION: The COVID-19 pandemic placed unprecedented stress on laboratories in the Eastern Mediterranean Region. Building on existing capacity for influenza diagnostics, countries introduced COVID-19 diagnostic support to ~100% regional coverage. A key challenge during the expansion was maintaining quality testing in laboratories, ensuring that correct results were shared with medical facilities. METHODS: WHO organized two rounds of independently monitored severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) external quality assurance programs (EQAP). The Public Health Laboratory (PHL) division of WHO supplied external quality assurance (EQA) panels, from the Royal College of Pathologists of Australasia Quality Assurance Programme (RCPAQAP) Australia to laboratories not enrolled in recurring Global Influenza Surveillance and Response System (GISRS) quality assurance programs, in which national influenza centers routinely participate. RESULTS: Fifteen and 14 countries participated in PHL/EQAP for SARS-CoV-2 between 2020 and 2022. Concordance was consistent between rounds, reaching 96.4% and 89.9%. A separate assessment of GISRS/EQAP to national-level laboratories identified high levels of response and concordance for SARS-CoV-2 (100% response, 93% concordance), which was reduced for influenza (50% response rate, 80% concordance), reflecting the challenge of prioritizing pathogens during outbreaks. CONCLUSION: The proliferation of laboratories in response to COVID-19 was a success story from the pandemic. However, monitoring the quality of laboratories was challenging via existing EQAP. The addition of PHL/EQAP provided a mechanism to monitor performance of laboratories that were not designated as national influenza centers. While a high proportion of laboratories attained good results, continual emphasis on quality and enrollment in EQAP is key to ensuring sustainability of laboratory testing in future.

Strengthening pathogen genomic surveillance for health emergencies: insights from the World Health Organization's regional initiatives.

The onset of the COVID-19 pandemic triggered a rapid scale-up in the use of genomic surveillance as a pandemic preparedness and response tool. As a result, the number of countries with in-country SARS-CoV-2 genomic sequencing capability increased by 40% from February 2021 to July 2022. The Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032 was launched by the World Health Organization (WHO) in March 2022 to bring greater coherence to ongoing work to strengthen genomic surveillance. This paper describes how WHO's tailored regional approaches contribute to expanding and further institutionalizing the use of genomic surveillance to guide pandemic preparedness and response measures as part of a harmonized global undertaking. Challenges to achieving this vision include difficulties obtaining sequencing equipment and supplies, shortages of skilled staff, and obstacles to maximizing the utility of genomic data to inform risk assessment and public health action. WHO is helping to overcome these challenges in collaboration with partners. Through its global headquarters, six regional offices, and 153 country offices, WHO is providing support for country-driven efforts to strengthen genomic surveillance in its 194 Member States, with activities reflecting regional specificities. WHO's regional offices serve as platforms for those countries in their respective regions to share resources and knowledge, engage stakeholders in ways that reflect national and regional priorities, and develop regionally aligned approaches to implementing and sustaining genomic surveillance within public health systems.

Respiratory syncytial virus infection and disease in infants and young children observed from birth in Kilifi District, Kenya.

BACKGROUND: In developing countries, there are few data that characterize the disease burden attributable to respiratory syncytial virus (RSV) and clearly define which age group to target for vaccine intervention. METHODS: Six hundred thirty-five children, recruited during the period 2002-2003, were intensively monitored until each experienced 3 epidemics of RSV infection. RSV infection was diagnosed using immunofluorescence of nasal washing specimens collected at each episode of acute respiratory infection. Incidence estimates were adjusted for seasonality of RSV exposure. RESULTS: For 1187 child-years of observation (CYO), a total of 409 (365 primary and 82 repeat) episodes of RSV infection were identified. Adjusted incidence estimates of lower respiratory tract infection (LRTI), severe LRTI, and hospital admission were 90 cases per 1000 CYO, 43 cases per 1000 CYO, and 10 cases per 1000 CYO, respectively, and corresponding estimates among infants were 104 cases per 1000 CYO, 66 cases per 1000 CYO, and 13 cases per 1000 CYO, respectively. The proportion of cases of all-cause LRTI, and severe LRTI and hospitalizations attributable to RSV in the cohort was 13%, 19%, and 5%, respectively. Fifty-five percent to 65% of RSV-associated LRTI and severe LRTI occurred in children aged >6 months. The risk of RSV disease following primary symptomatic infection remained significant beyond the first year of life, and one-quarter of all reinfections were associated with LRTI. CONCLUSIONS: RSV accounts for a substantial proportion of the total respiratory disease in this rural population; we estimate that 85,000 cases of severe LRTI per year occur in infants in Kenya. The majority of this morbidity occurs during late infancy and early childhood--ages at which the risk of disease following infection remains significant. Disease resulting from reinfection is common. Our results inform the debate on the target age group and effectiveness of a vaccine.

The burden of influenza and RSV among inpatients and outpatients in rural western Kenya, 2009-2012.

BACKGROUND: In Kenya, detailed data on the age-specific burden of influenza and RSV are essential to inform use of limited vaccination and treatment resources. METHODS: We analyzed surveillance data from August 2009 to July 2012 for hospitalized severe acute respiratory illness (SARI) and outpatient influenza-like illness (ILI) at two health facilities in western Kenya to estimate the burden of influenza and respiratory syncytial virus (RSV). Incidence rates were estimated by dividing the number of cases with laboratory-confirmed virus infections by the mid-year population. Rates were adjusted for healthcare-seeking behavior, and to account for patients who met the SARI/ILI case definitions but were not tested. RESULTS: The average annual incidence of influenza-associated SARI hospitalization per 1,000 persons was 2.7 (95% CI 1.8-3.9) among children <5 years and 0.3 (95% CI 0.2-0.4) among persons ≥5 years; for RSV-associated SARI hospitalization, it was 5.2 (95% CI 4.0-6.8) among children <5 years and 0.1 (95% CI 0.0-0.2) among persons ≥5 years. The incidence of influenza-associated medically-attended ILI per 1,000 was 24.0 (95% CI 16.6-34.7) among children <5 years and 3.8 (95% CI 2.6-5.7) among persons ≥5 years. The incidence of RSV-associated medically-attended ILI was 24.6 (95% CI 17.0-35.4) among children <5 years and 0.8 (95% CI 0.3-1.9) among persons ≥5 years. CONCLUSIONS: Influenza and RSV both exact an important burden in children. This highlights the possible value of influenza vaccines, and future RSV vaccines, for Kenyan children.

Demographic, socio-economic and geographic determinants of seasonal influenza vaccine uptake in rural western Kenya, 2011.

Influenza-associated acute lower respiratory infections cause a considerable burden of disease in rural and urban sub-Saharan Africa communities with the greatest burden among children. Currently, vaccination is the best way to prevent influenza infection and accompanying morbidities. We examined geographic, socio-economic and demographic factors that contributed to acceptance of childhood seasonal influenza vaccination among children living in a population-based morbidity surveillance system in rural western Kenya, where influenza vaccine was offered free-of-charge to children 6 months-10 years old from April to June, 2011. We evaluated associations between maternal and household demographic variables, socio-economic status, and distance from home to vaccination clinics with family vaccination status. 7249 children from 3735 households were eligible for vaccination. Of these, 2675 (36.9%) were fully vaccinated, 506 (7.0%) were partially vaccinated and 4068 (56.1%) were not vaccinated. Children living in households located >5km radius from the vaccination facilities were significantly less likely to be vaccinated (aOR=0.70; 95% CI 0.54-0.91; p=0.007). Children with mothers aged 25-34 and 35-44 years were more likely to be vaccinated than children with mothers less than 25 years of age (aOR=1.36; 95% CI 1.15-1.62; p<0.001; and aOR=1.35; 95% CI 1.10-1.64; p=0.003, respectively). Finally, children aged 2-5 years and >5 years of age (aOR=1.38; 95% CI 1.20-1.59; p<0.001; and aOR=1.41; 95% CI 1.23-1.63; p<0.001, respectively) and who had a sibling hospitalized within the past year (aOR=1.73; 95% CI 1.40-2.14; p<0.001) were more likely to be vaccinated. Shorter distance from the vaccination center, older maternal and child age, household administrator's occupation that did not require them to be away from the home, and having a sibling hospitalized during the past year were associated with increased likelihood of vaccination against influenza in western Kenya. These findings should inform the design of future childhood seasonal influenza vaccination campaigns in rural Kenya, and perhaps elsewhere in Africa.

The level and duration of RSV-specific maternal IgG in infants in Kilifi Kenya.

BACKGROUND: Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants. The rate of decay of RSV-specific maternal antibodies (RSV-matAb), the factors affecting cord blood levels, and the relationship between these levels and protection from infection are poorly defined. METHODS: A birth cohort (n = 635) in rural Kenya, was studied intensively to monitor infections and describe age-related serological characteristics. RSV specific IgG antibody (Ab) in serum was measured by the enzyme linked immunosorbent assay (ELISA) in cord blood, consecutive samples taken 3 monthly, and in paired acute and convalescent samples. A linear regression model was used to calculate the rate of RSV-matAb decline. The effect of risk factors on cord blood titres was investigated. RESULTS: The half-life of matAb in the Kenyan cohort was calculated to be 79 days (95% confidence limits (CL): 76-81 days). Ninety seven percent of infants were born with RSV-matAb. Infants who subsequently experienced an infection in early life had significantly lower cord titres of anti-RSV Ab in comparison to infants who did not have any incident infection in the first 6 months (P = 0.011). RSV infections were shown to have no effect on the rate of decay of RSV-matAb. CONCLUSION: Maternal-specific RSV Ab decline rapidly following birth. However, we provide evidence of protection against severe disease by RSV-matAb during the first 6-7 months. This suggests that boosting maternal-specific Ab by RSV vaccination may be a useful strategy to consider.

Comparison of strain-specific antibody responses during primary and secondary infections with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants. RSV repeatedly reinfects individuals: this may be due in part to the variability of the attachment (G) glycoprotein and changes in this protein have been shown to be under positive selection. Infants experiencing their primary infection show a genotype-specific antibody response with respect to the variable regions of the G protein. A prospective study of RSV infections in a birth cohort in rural Kenya identified infants experiencing repeat infections with RSV. The serum antibody responses of these infants were investigated with respect to their anti-RSV reactions in an enzyme-linked immunosorbent assay (ELISA) and the specificity of the response to a variable region of the G protein by ELISA and immunoblotting using bacterially expressed polypeptides representative of the currently circulating strains of RSV. The results presented here confirm that the primary antibody response to the variable regions of the G protein is generally genotype-specific, but show that the response may become cross-reactive (at least within group A viruses) during secondary infections even where the secondary infection is of the same genotype as the initial infection. Also, some infants who did not mount a detectable antibody response to whole RSV antigens during their primary infection nevertheless showed genotype-specific responses to the G protein. In conclusion, the strain-specific nature of the serum antibody response to the variable regions of the G protein of RSV observed in primary infections can become cross-reactive in subsequent reinfections.

Molecular analysis of respiratory syncytial virus reinfections in infants from coastal Kenya.

BACKGROUND: Individuals are reinfected with respiratory syncytial virus (RSV) repeatedly. The nature of reinfection, in relation to RSV genetic and antigenic diversity, is ill defined and has implications for persistence and vaccine control. METHODS: We examined the molecular relatedness of RSV causing primary and repeat infections, by phylogenetic analysis of the attachment (G) gene in 12 infants from a birth cohort in rural Kenya, using nasal wash samples collected during a 16-month period in 2002-2003, which spanned 2 successive epidemics. RESULTS: Six infants were infected during both epidemics, 4 with RSV-A in the first epidemic followed by RSV-B during the second epidemic and 2 with RSV-A during both epidemics, with no significant G gene sequence variability between samples. Two infants were infected and reinfected with different RSV-A strains during the same epidemic. Possible viral persistence was suspected in the remaining 4 infants, although reinfection with the same variant cannot be excluded. CONCLUSIONS: These are the first data that specifically address strain-specific reinfections in infancy in relation to the primary infecting variant. The data strongly suggest that, following primary infection, some infants lose strain-specific immunity within 7-9 months (between epidemics) and group-specific immunity within 2-4 months (during an epidemic period).

Respiratory syncytial virus epidemiology in a birth cohort from Kilifi district, Kenya: infection during the first year of life.

We report estimates of incidence of respiratory syncytial virus (RSV) infection during the first year of life for a birth cohort from rural, coastal Kenya. A total of 338 recruits born between 21 January 2002 and 30 May 2002 were monitored for symptoms of respiratory infection by home visits and hospital referrals. Nasal washings were screened by use of immunofluorescence. From 311 child-years of observation (cyo), 133 RSV infections were found, of which 48 were lower respiratory tract infections (LRTIs) and 31 were severe LRTIs, resulting in 4 hospital admissions. There were 121 primary RSV infections (248 cyo), of which 45 were LRTIs and 30 were severe LRTIs, resulting in 4 hospital admissions; there was no association with age. RSV contributed significantly to total LRTI disease in this vaccine-target group.

Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya.

Respiratory syncytial virus (RSV) causes significant burden of disease during infancy and childhood. This study examined the genetic relatedness of RSV positive samples from child inpatients and outpatients and a birth cohort from a rural coastal district of Kenya and also the distribution of strains between these three groups. Clinical samples were collected over a 4-year period in Kilifi District, Kenya from community and hospital surveillance. Three hundred ninety seven of 1,044 nasal specimens from children (under 5 years old) attending Kilifi District Hospital, and from community-monitored infants, were positive for RSV by multiplex RT-PCR. Of these, 376 samples were analysed further by restriction fragment length polymorphisms (RFLP) of the nucleocapsid (N) and attachment (G) protein genes. The G gene was sequenced for 109 samples and phylogenetic analysis carried out. The group A samples from Kilifi fell into two clusters based on G gene sequences, while only one group B cluster was observed. One RSV-B sample from 2003 demonstrated the presence of a 60-nucleotide duplication within the G gene, clustering with similar isolates from Buenos Aries from 1999. All had similar sequences to isolates from the UK, USA, Spain, or Uruguay. The Kilifi District samples showed greater than 97% homology to isolates from South Africa and Mozambique and 91-94% homology to isolates from The Gambia. Samples from different sources, clearly differing in disease severity, did not differ in genotype characteristics, suggesting that disease causing variants are a general reflection of infections within this community.