Development of nanobodies against the coat protein of maize chlorotic mottle virus.

Maize lethal necrosis (MLN) is a maize disease caused by the maize chlorotic mottle virus (MCMV), a potyvirus which causes yield losses of 30-100%. The present study aimed to isolate nanobodies against the MCMV coat protein (CP) for the diagnosis of MLN. MCMV CP expressed in Escherichia coli was used for llama immunization. VHH (i.e. variable heavy domain of heavy chain) gene fragments were prepared from blood drawn from the immunized llama and used to generate a library in E. coli TG1 cells. MCMV specific nanobodies were selected by three rounds of phage display and panning against MCMV CP. The selected nanobodies were finally expressed in E. coli WK6 cells and purified. Eleven MCMV-specific nanobodies were identified and shown to detect MCMV in infected maize plants. Thus, our results show that nanobodies isolated from llama immunized with MCMV CP can distinguish infected and healthy maize plants, potentially enabling development of affordable MCMV detection protocols.

Effect of soybean and seaweed-based diets on growth performance, feed utilization, and gut microbiota of tilapia: A systematic review and meta-analysis.

Tilapia, a significant aquaculture species globally, relies heavily on feed for its production. While numerous studies have investigated the impact of soybean and seaweed-based diets on tilapia, a comprehensive understanding remains elusive. This review aimed at evaluating and synthesizing the existing literature on these diets' effects, focusing on growth performance, feed utilization, and gut microbiota. A systematic search of databases was conducted using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and a total of 57 studies were included in the qualitative analysis and 24 in the meta-analysis. The results indicated that soybean-based diets, at a 59.4% inclusion level improved the Specific Growth Rate (SGR) of tilapia with an effect size of -2.14 (95% CI: -2.92, -1.37; p < 0.00001; I2 = 99%) and did not improve the feed conversion rate (FCR), as the effect size was 1.80 (95% CI: 0.72, 2.89; p = 0.001; I2 = 100%). For seaweed-based diets, at a 15,9% inclusion level did not improve SGR, with an effect size of -0.74 (95% CI: -1.70, 0.22; p = 0.13; I2 = 99%), and the FCR with an effect size of -0.70 (95% CI: -1.94, 0.54; p = 0.27; I2 = 100%). Regarding the gut microbiota, was noted a lack of studies meeting the inclusion criteria for tilapia. However, findings from studies on other farmed fishes suggested that soybean and seaweed-based diets could have diverse effects on gut microbiota composition and promote the growth of beneficial microbiota. This study suggests that incorporating soybean-based diets at 59.4% inclusion can improve the SGR of tilapia. Seaweed-based diets, while not demonstrating improvement in the analyzed parameters with an inclusion level of 15.9%, have the potential to contribute to the sustainability of the aquaculture industry when incorporated at lower levels.

Schistosomiasis diagnosis: Challenges and opportunities for elimination.

OVERVIEW: The roadmap adopted by the World Health Organization (WHO) for eliminating neglected tropical diseases aims to eliminate schistosomiasis, as a public health concern, by 2030. While progress has been made towards reducing schistosomiasis morbidity control in several sub-Saharan African countries, there is still more that needs to be done. Proper surveillance using accurate diagnostics with acceptable sensitivity and specificity is essential for evaluating the success of all efforts against schistosomiasis. Microscopy, despite its low sensitivity, remains the gold standard approach for diagnosing the disease. Although many efforts have been made to develop new diagnostics based on circulating parasite proteins, genetic markers, schistosome egg morphology, and their paramagnetic properties, none has been robust enough to replace microscopy. This review highlights common diagnostic approaches for detecting schistosomiasis in field and clinical settings, major challenges, and provides new and novel opportunities and diagnosis pathways that will be critical in supporting elimination of schistosomiasis. METHODS: We searched for relevant and reliable published literature from PubMed, Scopus, google scholar, and Web of science. The search strategies were primarily determined by subtopic, and hence the following words were used (schistosom*, diagnosis, Kato-Katz, antibody test, circulating antigen, POC-CCA, UCP-LF-CAA, molecular diagnostics, nucleic acid amplification test, microfluidics, lab-on a disk, lab-on chip, recombinase polymerase amplification (RPA), LAMP, portable sequencer, nanobody test, identical multi-repeat sequences, diagnostic TPPs, REASSURED, extraction free), and Boolean operators AND and/OR were used to refine the searching capacity. Due to the global public health nature of schistosomiasis, we also searched for reliable documents, reports, and research papers published by international health organizations, World Health Organization (WHO), and Center for Disease control and Elimination.

Genomic Diversity and Geographic Distribution of Newcastle Disease Virus Genotypes in Africa: Implications for Diagnosis, Vaccination, and Regional Collaboration.

The emergence of new virulent genotypes and the continued genetic drift of Newcastle disease virus (NDV) implies that distinct genotypes of NDV are simultaneously evolving in different geographic locations across the globe, including throughout Africa, where NDV is an important veterinary pathogen. Expanding the genomic diversity of NDV increases the possibility of diagnostic and vaccine failures. In this review, we systematically analyzed the genetic diversity of NDV genotypes in Africa using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Information published between 1999 and 2022 were used to obtain the genetic background of different genotypes of NDV and their geographic distributions in Africa. The following genotypes were reported in Africa: I, II, III, IV, V, VI, VII, VIII, XI, XIII, XIV, XVII, XVIII, XX, and XXI. A new putative genotype has been detected in the Democratic Republic of the Congo. However, of 54 African countries, only 26 countries regularly report information on NDV outbreaks, suggesting that this number may be vastly underestimated. With eight different genotypes, Nigeria is the country with the greatest genotypic diversity of NDV among African countries. Genotype VII is the most prevalent group of NDV in Africa, which was reported in 15 countries. A phylogeographic analysis of NDV sequences revealed transboundary transmission of the virus in Eastern Africa, Western and Central Africa, and in Southern Africa. A regional and continental collaboration is recommended for improved NDV risk management in Africa.

African Swine Fever Diagnosis in Africa: Challenges and Opportunities.

The global spread of African swine fever (ASF) in recent decades has led to the need for technological advances in sampling and diagnostic techniques. The impetus for these has been the need to enable sampling by lay persons and to obtain at least a preliminary diagnosis in the field for early control measures to be put in place before final laboratory confirmation. In rural Africa, rapid diagnosis is hampered by challenges that include lack of infrastructure as well as human and financial resources. Lack of animal health personnel, access to affordable means to transport field samples to a laboratory, and lack of laboratories with the capacity to make the diagnosis result in severe under-reporting of ASF, especially in endemic areas. This review summarizes the challenges identified in gap analyses relevant to low- and middle-income countries, with a focus on Africa, and explore the opportunities provided by recent research to improve field diagnosis and quality of diagnostic samples used. Sampling techniques include invasive sampling techniques requiring trained personnel and non-invasive sampling requiring minimal training, sampling of decomposed carcass material, and preservation of samples in situations where cold chain maintenance cannot be guaranteed. Availability and efficacy of point-of-care (POC) tests for ASF has improved considerably in recent years and their application, as well as advantages and limitations, are discussed. The adequacy of existing laboratory diagnostic capacity is evaluated and opportunities for networking amongst reference and other laboratories offering diagnostic services are discussed. Maintaining laboratory diagnostic efficiency in the absence of samples during periods of quiescence is another issue that requires attention, and the role of improved laboratory networking is emphasized. Early diagnosis of ASF is key to managing the disease spread. Therefore, the establishment of the Africa Chapter of the Global African Swine Fever Research Alliance (GARA) increases opportunities for collaboration and networking among the veterinary diagnostic laboratories in the region.

Evaluation of Lab-on-a-Disc Technique Performance for Soil-Transmitted Helminth Diagnosis in Animals in Tanzania.

Soil-transmitted helminth (STH) infections are caused by roundworms, hookworms, whipworms, and thread worms. Accurate diagnosis is essential for effective treatment, prevention, and control of these infections. This study evaluates a new diagnostic method called Single-image Parasite Quantification (SIMPAQ), which uses a lab-on-a-disc (LoD) technique to isolate STH eggs into a single imaging zone for digital analysis. The study evaluates the purification performance of the SIMPAQ technique for detecting STH eggs in animal samples. This was a cross-sectional study conducted among 237 pigs and 281 dogs in the Morogoro region in Tanzania. Faecal samples were collected and processed with the LoD technique, as well as flotation and McMaster (McM) methods for comparison purposes. The overall prevalence of STH infections was high as per the LoD technique (74%), followed by McM (65.44%) and flotation (65.04%). Moreover, the overall performance of the LoD technique, using McM as the gold standard, was 93.51% (sensitivity), 60.89% (specificity), 81.91% (PPV), and 83.21% (NPV). The LoD technique exhibited high prevalence, sensitivity, and NPV, which demonstrates its value for STH egg detection and its crucial role in the era of accurate STH diagnosis, promoting proper management of the infection.

Trends of measles in Tanzania: A 5-year review of case-based surveillance data, 2018-2022.

OBJECTIVES: Tanzania observed a gradual increase in the number of measles cases since 2019 with a large outbreak recorded during 2022. This study describes the trend of measles in Tanzania over a 5-year period from 2018-2022. METHODS: This was a descriptive study conducted using routine measles case-based surveillance system including 195 councils of the United Republic of Tanzania. RESULTS: Between 2018 and 2022 there were 12,253 measles cases reported. Out of 10,691 (87.25%) samples tested by enzyme-linked immunosorbent assay, 903 (8.4%) were measles immunoglobulin M positive. The highest number of laboratory-confirmed measles cases was in 2022 (64.8%), followed by 2020 (13.8%), and 2019 (13.5%). Out of 1279 unvaccinated cases, 213 (16.7%) were laboratory-confirmed measles cases compared to 77/723 (10.6%) who were partially vaccinated and 71/1121 (6.3%) who were fully vaccinated (P < 0.001). Children aged between 1-4 years constituted the most confirmed measles cases after laboratory testing, followed by those aged 5-9 years. There was a notable increase in the number of laboratory-confirmed measles cases in children <1 year and 10-14 years during 2022 compared to previous years. The vaccination coverage of the first dose of measles-containing vaccine (MCV1) was maintained >90% since 2013 while MCV2 increased gradually reaching 88% in 2022. CONCLUSIONS: Accumulation of susceptible children to measles due to suboptimal measles vaccination coverage over the years has resulted in an increase in the number of laboratory-confirmed measles cases in Tanzania with more cases recorded during the COVID-19 pandemic. Strengthening surveillance, routine immunization, and targeted strategies are key to achieving the immunity levels required to interrupt measles outbreaks.

Major biotic stresses affecting maize production in Kenya and their implications for food security.

Maize (Zea mays L.) is a staple food for many households in sub-Saharan Africa (SSA) and also contributes to the gross domestic product (GDP). However, the maize yields reported in most SSA countries are very low and this is mainly attributed to biotic and abiotic stresses. These stresses have been exacerbated by climate change which has led to long periods of drought or heavy flooding and the emergence of new biotic stresses. Few reports exist which compile the biotic stresses affecting maize production in SSA. Here, five major biotic stresses of maize in Kenya are presented which are attributed to high yield losses. They include Maize lethal necrosis, fall armyworm, gray leaf spot, turcicum leaf blight and desert locusts. Maize lethal necrosis and fall armyworm are new biotic stresses to the Kenyan maize farmer while gray leaf spot, and turcicum leaf blight are endemic to the region. The invasion by the desert locusts is speculated to be caused by climate change. The biotic stresses cause a reduction in maize yield of 30-100% threatening food security. Therefore, this review focuses on the cause, control measures employed to control these diseases and future prospective. There should be deliberate efforts from the government and researchers to control biotic stresses affecting maize yields as the effect of these stresses is being exacerbated by the changing climate.

The evaluation of five serological assays in determining seroconversion to peste des petits ruminants virus in typical and atypical hosts.

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015-2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0-88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4-62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania.

Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.

Serological and molecular evidence of chikungunya virus infection among febrile outpatients seeking healthcare in Northern Malawi.

Introduction: Despite global evidence of chikungunya fever (CHIKF) in humans that is caused by chikungunya virus (CHIKV), little is known about the occurrence of CHIKF in Malawi. This study was conducted to determine the seroprevalence of CHIKF and to molecularly confirm the presence of CHIKV ribonucleic acid (RNA) among febrile outpatients seeking health care at Mzuzu Central Hospital in the Northern Region of Malawi. Methods: Enzyme-immunosorbent assay (ELISA) was used to detect the presence or absence of specific antibodies against CHIKV. Reversetranscription polymerase chain reaction (RT-PCR) was conducted on randomly selected anti-CHIKV IgM-positive samples to detect CHIKV RNA. Results: Out of 119 CHIKF suspected samples analyzed, 73 tested positive for anti-CHIKV IgM antibodies, with an overall seroprevalence of 61.3%. Most of the CHIKV infected individuals presented with joint pain, abdominal pain, vomiting and nose bleeding with seroprevalence of 45.2%, 41.1%, 16.4% and 12.3%, respectively. All the randomly selected samples that were positive for CHIKV anti-IgM by ELISAhad detectable CHIKV RNA by RT-PCR. Conclusion: The presence of anti-CHIKV IgM antibodies suggests the presence of recent CHIKV infection. We therefore recommend for the inclusion of CHIKF as the differential diagnosis in febrile ill patients in Mzuzu city, Malawi.

Community knowledge, attitude and practices regarding zoonotic viral haemorrhagic fevers in five geo-ecological zones in Tanzania.

BACKGROUND: Viral haemorrhagic fevers (VHF) cause significant economic and public health impact in Sub-Saharan Africa. Community knowledge, awareness and practices regarding such outbreaks play a pivotal role in their management and prevention. This study was carried out to assess community knowledge, attitude and practices regarding VHF in five geo-ecological zones in Tanzania. METHODS: A cross-sectional study was conducted in Buhigwe, Kalambo, Kyela, Kinondoni, Kilindi, Mvomero, Kondoa and Ukerewe districts representing five geo-ecological zones in Tanzania. Study participants were selected by multistage cluster sampling design. A semi-structured questionnaire was used to collect socio-demographic and information related to knowledge, attitude and practices regarding VHFs. Descriptive statistics and logistic regression were used for the analysis. RESULTS: A total of 2,965 individuals were involved in the study. Their mean age was 35 (SD ± 18.9) years. Females accounted for 58.2% while males 41.8%. Most of the respondents (70.6%; n = 2093) had never heard of VHF, and those who heard, over three quarters (79%) mentioned the radio as their primary source of information. Slightly over a quarter (29.4%) of the respondents were knowledgeable, 25% had a positive attitude, and 17.9% had unfavourable practice habits. The level of knowledge varied between occupation and education levels (P < 0.005). Most participants were likely to interact with a VHF survivor or take care of a person suffering from VHF (75%) or visit areas with known VHF (73%). There were increased odds of having poor practice among participants aged 36-45 years (AOR: 3.566, 95% CI: 1.593-7.821) and those living in Western, North-Eastern and Lake Victoria zones (AOR: 2.529, 95% CI: 1.071-6.657; AOR: 2.639, 95% CI: 1.130-7.580 AOR: 2.248, 95% CI: 1.073-3.844, respectively). CONCLUSION: Overall, the knowledge on VHF among communities is low, while a large proportion of individuals in the community are involved in activities that expose them to the disease pathogens in Tanzania. These findings highlight the need for strengthening health educational and promotion efforts on VHF targeting specific populations.

Molecular Characterization and Phylogenetic Analysis of Dengue Fever Viruses in Three Outbreaks in Tanzania Between 2017 and 2019.

BACKGROUND: Dengue is a disease of public health interest, and Tanzania experienced major outbreaks in 2014 and 2019. Here, we report our findings on the molecular characterization of dengue viruses (DENV) that circulated during two smaller outbreaks (2017 and 2018) and one major epidemic (2019) in Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: We tested archived serum samples from 1,381 suspected dengue fever patients, with a median age of 29 (IQR:22-40) years, referred to the National Public Health Laboratory for confirmation of DENV infection. DENV serotypes were identified by reverse transcription polymerase chain reaction (RT-PCR), and specific genotypes were identified by sequencing the envelope glycoprotein gene and phylogenetic inference methods. DENV was confirmed in 823 (59.6%) cases. More than half (54.7%) of patients with dengue fever infection were males, and nearly three-quarters (73%) of the infected individuals were living in Kinondoni district, Dar es Salaam. DENV-3 Genotype III caused the two smaller outbreaks in 2017 and 2018, while DENV-1 Genotype V caused the 2019 epidemic. DENV-1 Genotype I was also detected in one patient in 2019. CONCLUSION/SIGNIFICANCE: This study has demonstrated the molecular diversity of dengue viruses circulating in Tanzania. We found that contemporary circulating serotypes did not cause the major epidemic of 2019 but rather due to a serotype shift from DENV-3 (2017/2018) to DENV-1 in 2019. Such a change increases the risk for patients previously infected with a particular serotype to develop severe symptoms upon potential re-infection with a heterologous serotype due to antibody-dependent enhancement of infection. Therefore, the circulation of serotypes emphasizes the need to strengthen the country's dengue surveillance system for better management of patients, early detection of outbreaks, and vaccine development.

Complete genome analysis of African swine fever virus genotypes II, IX and XV from domestic pigs in Tanzania.

African swine fever (ASF) caused by ASF virus (ASFV) is an infectious transboundary animal disease notifiable to the World Organization for Animal Health causing high mortality in domestic pigs and wild boars threatening the global domestic pig industry. To date, twenty-four ASFV genotypes have been described and currently genotypes II, IX, X, XV and XVI are known to be circulating in Tanzania. Despite the endemic status of ASF in Tanzania, only one complete genome of ASFV from the country has been described. This study describes the first complete genome sequence of ASFV genotype XV. In addition, the first Tanzanian complete genome of ASFV genotype IX and three ASFV strains belonging to genotype II collected during ASF outbreaks in domestic pigs in Tanzania were determined in this study using Illumina sequencing and comparative genomics analysis. The generated ASFV complete genome sequences ranged from 171,004 to 184,521 base pairs in length with an average GC content of 38.53% and encoded 152 to 187 open reading frames. The results of this study provide insights into the genomic structure of ASFV and can be used to monitor changes within the ASFV genome and improve our understanding of ASF transmission dynamics.

Genetic diversity and genomic analysis of G3P[6] and equine-like G3P[8] in children under-five from Southern Highlands and Eastern Tanzania.

Rotavirus group A genomic characterization is critical for understanding the mechanisms of rotavirus diversity, such as reassortment events and possible interspecies transmission. However, little is known about the genetic diversity and genomic relationship of the rotavirus group A strains circulating in Tanzania. The genetic and genomic relationship of RVA genotypes was investigated in children under the age of five. A total of 169 Fecal samples were collected from under-five with diarrhea in Mbeya, Iringa and Morogoro regions of Tanzania. The RVA were screened in children under five with diarrhea using reverse transcription PCR for VP7 and VP4, and the G and P genotypes were determined using Sanger dideoxynucleotide cycle sequencing. Whole-genome sequencing was performed on selected genotypes. The overall RVA rate was 4.7% (8/169). The G genotypes were G3 (7/8) and G6 (1/8) among the 8 RVA positives, while the P genotypes were P[6] (4/8) and P[8] (2), and the other two were untypeable. G3P[6] and G3P[8] were the identified genotype combinations. The genomic analysis reveals that the circulating G3P[8] and G3P[6] isolates from children under the age of five with diarrhea had a DS-1-like genome configuration (I2-R2-C2-M2-axe-N2-T2-E2-H2). The phylogenic analysis revealed that all 11 segments of G3P[6] were closely related to human G3P[6] identified in neighboring countries such as Uganda, Kenya, and other African countries, implying that G3P[6] strains descended from a common ancestor. Whereas, G3P[8] were closely related to previously identified equine-like G3P[P8] from Kenya, Japan, Thailand, Brazil, and Taiwan, implying that this strain was introduced rather than reassortment events. We discovered amino acid differences at antigenic epitopes and N-linked glycosylation sites between the wild type G3 and P[8] compared to vaccine strains, implying that further research into the impact of these differences on vaccine effectiveness is warranted. The phylogenic analysis of VP7 also identified a bovine-like G6. For the first time in Tanzania, we report the emergence of novel equine-like G3 and bovine-like G6 RVA strains, highlighting the importance of rotavirus genotype monitoring and genomic analysis of representative genotypes.

Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance.

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.

Prevalence and genomic characterization of rotavirus group A genotypes in piglets from southern highlands and eastern Tanzania.

Animals have been identified as the potential reservoirs of rotavirus group A (RVA) for human infection. However, very little is known regarding the genotype and genomic profiles of circulating RVA in Tanzanian piglets. The rotavirus genetic diversity and genome analysis was assessed among piglets from Southern highlands and Eastern Tanzania. A total of 241 faecal samples were collected from piglets in the regions of Mbeya, Iringa, and Morogoro. RVA was detected and genotyped using reverse transcription polymerase chain reaction (RT-PCR). Sanger dideoxynucleotide cycle sequencing of the viral protein (VP) 4 and VP7 genes was afterwards performed to confirm the RT-PCR results. Selected genotypes were subjected to whole genome sequencing. The overall prevalence of RVA was 35.26% (85/241) in piglets (30.58% in Mbeya, 43.75% in Iringa and 31.16% in Morogoro). Upon genotyping, the G genotypes were G4 (26), G9 (10), G3 (6), G5 (3) and the remaining 40 were untypeable, while the P genotype, were P[6] (35), P[13] (3) and the remaining 47 were untypeable. The G4P[6] were the predominant genotype followed by G3P[6], G3P[13], G4P[13] and G5P[13] were most common genotypes combinations. On phylogenetic analysis, G4 was grouped to lineage V, sublineages VIIa and VIIc, G9 to lineage I, G5 to lineage II, G3 to lineage IV, P[6] to lineage V and sublineage Ic and the P[13] to lineage IV. We revealed amino acid differences between the circulating G4 and the G4 in the ProSystems RCE vaccine used in pigs. The whole genome reveals genomic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, G5-P[x]-I5-R1-C1-M1-A8-N1-Tx-E1-H1, G3/G4-P[13]/P[6]-Ix-R1-C1-M1-A8-N1-T1-E1-H1, G3-P[6]-Ix-R1-C1-M1-A8-N1-Tx-E1-H1 and G9-P[x]-Ix-R1-C1-M1-Ax-N1-Tx-E1-H1. The VP7 gene of G9, the VP4 gene of P[6] and NSP4 (E1) gene of some genotypes clustered together and closely related to humans origin or porcine-human reassortant strains with nucleotide similarities ranging from 97.90% to 99.74% from neighboring countries, implying possibility intragenogroup reassortment and interspecies transmission. The higher strain diversity observed within the gene segments highlight the importance of genomic analysis and continuous monitoring of RVA genotypes. Further research is needed to determine the risk factors associated with RVA infection in Tanzanian pigs in order to properly design a control program.

Pattern of Aedes aegypti and Aedes albopictus Associated with Human Exposure to Dengue Virus in Kinshasa, the Democratic Republic of the Congo.

Dengue is a worldwide public health concern. The current study assessed the extent of human exposure to the dengue virus in relation to the distribution pattern of Aedes aegypti and Ae. albopictus in Kinshasa. Cross-sectional surveys were carried out in 2021 and 2022. The baseline entomological survey involved 19 municipalities using a grid cell sampling approach. All containers holding water were inspected for the presence of larvae in each grid. The collected larvae were kept in an insectary until the adult emergence for morphological identification. Four hundred febrile patients attending the hospital were screened for the presence of dengue antibodies (IgG, IgM) and NS1 antigen using a rapid diagnostic test (RDT) Biosynex®. Residences of positive cases were geo-referenced. We evaluated 1850 grid cells, of which 19.5% were positive for Aedes larvae. The positive grid cells were identified in the Ndjili (44.0%), Mont Ngafula (32.0%) and Ngaliema (26.0%), and Limete (32.0%) municipalities. The Ae. aegypti (11.2%) predominated in the northwestern, and Ae. albopictus (9.1%) appeared in the high vegetation coverage areas. Of 61 (15.3%) participants exposed to dengue, 8.3% presented acute dengue. Young, (6-17 years), male, and Mont Amba district participants were most exposed to dengue. In conclusion, dengue occurrence in Kinshasa overlaps somewhat the geographical and ecological distributions of Ae. aegypti and Ae. albopictus. Both species are not homogenously distributed, likely due to environmental factors. These findings can assist the targeted control activities.

Estimating Risk of Introduction of Ebola Virus Disease from the Democratic Republic of Congo to Tanzania: A Qualitative Assessment.

Between April 2018 and November 2020, the Democratic Republic of Congo (DRC) experienced its 11th Ebola virus disease (EVD) outbreak. Tanzania's cross-border interactions with DRC through regular visitors, traders, and refugees are of concern, given the potential for further spread to neighboring countries. This study aimed to estimate the risk of introducing EVD to Tanzania from DRC. National data for flights, boats, and car transport schedules from DRC to Tanzania covering the period of May 2018 to June 2019 were analyzed to describe population movement via land, port, and air travel and coupled with available surveillance data to model the risk of EVD entry. The land border crossing was considered the most frequently used means of travel and the most likely pathway of introducing EVD from DRC to Tanzania. High probabilities of introducing EVD from DRC to Tanzania through the assessed pathways were associated with the viability of the pathogen and low detection capacity at the ports of entry. This study provides important information regarding the elements contributing to the risk associated with the introduction of EBV in Tanzania. It also indicates that infected humans arriving via land are the most likely pathway of EBV entry, and therefore, mitigation strategies including land border surveillance should be strengthened.

Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles.

In recent decades "saliva" has emerged as an important non-invasive biofluid for diagnostic purposes in both human and animal health sectors. However, with the rapid evolution of molecular detection technologies, the limitation has been the lack of an efficient method for the facile amplification of target RNA from such a complex matrix. Herein, we demonstrate the novel application of hydrogel microparticles of primer-immobilized networks (PIN) for direct quantitative reverse transcription PCR (dirRT-qPCR) of viral RNA from saliva samples without prior RNA purification. Each of these highly porous PIN particles operates as an independent reactor. They filter in micro-volumes of the analyte solution. Viral RNA is captured and converted to complementary DNA (cDNA) through the RT step using covalently incorporated RT primers. The PIN with cDNA of the viral target will be ready for subsequent highly specific qPCR. Preceded by heat-treatment for viral lysis, we were able to conduct PIN dirRT-qPCR with 95% efficiency of the matrix (M) gene for influenza A virus (IAV) and 5' untranslated region (5' UTR) for chicken coronavirus spiked into saliva samples. The addition of reverse transcriptase enzyme (RTase) and 10% dilution of the matrix improved the assay sensitivity considerably. PIN particles' compatibility with microfluidic PCR chip technology has significantly reduced total sample processing time to 50 min, instead of an average of 120 min that are normally used by other assays. We anticipate this technology will be useful for other viral RNA targets by changing the incorporated RT primer sequences and can be adapted for onsite diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00065-0.