Development and Testing of the Method for the Detection of Lassa virus RNA, Based on real-Time Polymerase Chain reaction with reverse Transcription

Abstract. Objective of the study was the development of a method for the detection and quantitative analysis (realtime RT-PCR) to identify genetic markers of Lassa virus - LASV-Fl. Materials and methods. We utilized all the available in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) Lassa virus sequences that have been aligned to identify conservative sites applying the BioEdit 7.2.5 software package (IbisBiosciences, USA). To test the developed PCR kit, the control panel of Lassa virus RNA and pseudo-viral particles, 27 viral strains belonging to different fami­lies, as well as 37 serum samples from patients with feverish diseases selected in medical institutions of the Republic of Guinea in 2016-2018 and 55 samples of organ suspensions from multi-spiked mice were used. Results and discussion. The analytical sensitivity of the method varied from 103 copies/ml to 105 copies/ml and had 96.4 % diagnostic sensitivity, while the analytical and diagnostic specificity was 100 %. It is shown that the developed technique can be successfully introduced into practice for the detection of Lassa virus in the Republic of Guinea, using various types of material from small mammals, including whole blood and organ suspensions of M. natalensis, as well as samples of human blood sera collected 3-7 days after the onset of the disease. It is also suggested that this method can be used for strains of Lassa virus, common not only in Guinea but also in other endemic areas, but this fact must be confirmed in further studies

Detection of Crimean-Congo Hemorrhagic Fever Virus Markers in Samples of Ixodes Ticks Collected in the Territory of the Republic of Guinea

Objective of the study. This work was carried out to identify markers (antigen and RNA) of CrimeanCongo hemorrhagic fever (CCHF) virus in samples from ticks, collected in all landscape-geographical areas of Guinea: Lower, Middle, Upper and Forest, to obtain up-to-date data on the distribution of the pathogen in the country.Materials and methods. Total of 4276 specimens of 8 species of ticks collected in 2016–2019 in the territory of the Republic of Guinea were studied, which were compiled into 1406 samples. Ectoparasites were collected from livestock animals, dogs, and small mammals. Viral antigen was detected using enzyme immunoassay (ELISA). The presence of RNA of the CCHF virus was determined by reverse transcription polymerase chain reaction (RT-PCR).Results and discussion. As a result of the studies, the antigen of the CCHF virus was detected in 21 samples (1.5 %), and RNA – in 37 (2.6 %). All samples, in which the viral antigen was detected, contained RNA of the CCHF virus. Positive results were obtained in samples from all geographical areas of the country. The main vectors and reservoirs of the pathogen in Guinea are ticks of the species Rh. sanguineus, Rh. geigyi, Rh. annulatus and Am. variegatum. The data obtained confirm the previously available information on the possibility of the pathogen circulation in this region and determine the need for further study of the spread of the CCHF virus in the territory of the Republic of Guinea

Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study

Background Pneumonia is the leading cause of death among children younger than 5 years. In this study, we estimated causes of pneumonia in young African and Asian children, using novel analytical methods applied to clinical and microbiological findings. Methods We did a multi-site, international case-control study in nine study sites in seven countries: Bangladesh, The Gambia, Kenya, Mali, South Africa, Thailand, and Zambia. All sites enrolled in the study for 24 months. Cases were children aged 1–59 months admitted to hospital with severe pneumonia. Controls were age-group-matched children randomly selected from communities surrounding study sites. Nasopharyngeal and oropharyngeal (NP-OP), urine, blood, induced sputum, lung aspirate, pleural fluid, and gastric aspirates were tested with cultures, multiplex PCR, or both. Primary analyses were restricted to cases without HIV infection and with abnormal chest x-rays and to controls without HIV infection. We applied a Bayesian, partial latent class analysis to estimate probabilities of aetiological agents at the individual and population level, incorporating case and control data. Findings Between Aug 15, 2011, and Jan 30, 2014, we enrolled 4232 cases and 5119 community controls. The primary analysis group was comprised of 1769 (41·8% of 4232) cases without HIV infection and with positive chest x-rays and 5102 (99·7% of 5119) community controls without HIV infection. Wheezing was present in 555 (31·7%) of 1752 cases (range by site 10·6–97·3%). 30-day case-fatality ratio was 6·4% (114 of 1769 cases). Blood cultures were positive in 56 (3·2%) of 1749 cases, and Streptococcus pneumoniae was the most common bacteria isolated (19 [33·9%] of 56). Almost all cases (98·9%) and controls (98·0%) had at least one pathogen detected by PCR in the NP-OP specimen. The detection of respiratory syncytial virus (RSV), parainfluenza virus, human metapneumovirus, influenza virus, S pneumoniae, Haemophilus influenzae type b (Hib), H influenzae non-type b, and Pneumocystis jirovecii in NP-OP specimens was associated with case status. The aetiology analysis estimated that viruses accounted for 61·4% (95% credible interval [CrI] 57·3–65·6) of causes, whereas bacteria accounted for 27·3% (23·3–31·6) and Mycobacterium tuberculosis for 5·9% (3·9–8·3). Viruses were less common (54·5%, 95% CrI 47·4–61·5 vs 68·0%, 62·7–72·7) and bacteria more common (33·7%, 27·2–40·8 vs 22·8%, 18·3–27·6) in very severe pneumonia cases than in severe cases. RSV had the greatest aetiological fraction (31·1%, 95% CrI 28·4–34·2) of all pathogens. Human rhinovirus, human metapneumovirus A or B, human parainfluenza virus, S pneumoniae, M tuberculosis, and H influenzae each accounted for 5% or more of the aetiological distribution. We observed differences in aetiological fraction by age for Bordetella pertussis, parainfluenza types 1 and 3, parechovirus–enterovirus, P jirovecii, RSV, rhinovirus, Staphylococcus aureus, and S pneumoniae, and differences by severity for RSV, S aureus, S pneumoniae, and parainfluenza type 3. The leading ten pathogens of each site accounted for 79% or more of the site's aetiological fraction. Interpretation In our study, a small set of pathogens accounted for most cases of pneumonia requiring hospital admission. Preventing and treating a subset of pathogens could substantially affect childhood pneumonia outcomes

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century