A pooled testing strategy for identifying SARS-CoV-2 at low prevalence.

Suppressing infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will probably require the rapid identification and isolation of individuals infected with the virus on an ongoing basis. Reverse-transcription polymerase chain reaction (RT-PCR) tests are accurate but costly, which makes the regular testing of every individual expensive. These costs are a challenge for all countries around the world, but particularly for low-to-middle-income countries. Cost reductions can be achieved by pooling (or combining) subsamples and testing them in groups1-7. A balance must be struck between increasing the group size and retaining test sensitivity, as sample dilution increases the likelihood of false-negative test results for individuals with a low viral load in the sampled region at the time of the test8. Similarly, minimizing the number of tests to reduce costs must be balanced against minimizing the time that testing takes, to reduce the spread of the infection. Here we propose an algorithm for pooling subsamples based on the geometry of a hypercube that, at low prevalence, accurately identifies individuals infected with SARS-CoV-2 in a small number of tests and few rounds of testing. We discuss the optimal group size and explain why, given the highly infectious nature of the disease, largely parallel searches are preferred. We report proof-of-concept experiments in which a positive subsample was detected even when diluted 100-fold with negative subsamples (compared with 30-48-fold dilutions described in previous studies9-11). We quantify the loss of sensitivity due to dilution and discuss how it may be mitigated by the frequent re-testing of groups, for example. With the use of these methods, the cost of mass testing could be reduced by a large factor. At low prevalence, the costs decrease in rough proportion to the prevalence. Field trials of our approach are under way in Rwanda and South Africa. The use of group testing on a massive scale to monitor infection rates closely and continually in a population, along with the rapid and effective isolation of people with SARS-CoV-2 infections, provides a promising pathway towards the long-term control of coronavirus disease 2019 (COVID-19).

Real-time PCR assay to detect the novel Clade Ib monkeypox virus, September 2023 to May 2024.

Monkeypox virus (MPXV) is an emerging zoonotic pathogen with complex epidemiology necessitating rapid diagnosis and distinguishing between clades and subclades. The emerging Clade Ib lacks the genomic region used in the Clade I-specific assay from the Centers for Disease Control and Prevention. We report an MPXV real-time PCR to specifically detect Clade Ib. The assay demonstrated proficient sensitivity and specificity in 92 samples and can be included along other TaqMan-based assays to detect MPXV and distinguish between clades and subclades.

Ongoing mpox outbreak in Kamituga, South Kivu province, associated with monkeypox virus of a novel Clade I sub-lineage, Democratic Republic of the Congo, 2024.

Since the beginning of 2023, the number of people with suspected monkeypox virus (MPXV) infection have sharply increased in the Democratic Republic of the Congo (DRC). We report near-to-complete MPXV genome sequences derived from six cases from the South Kivu province. Phylogenetic analyses reveal that the MPXV affecting the cases belongs to a novel Clade I sub-lineage. The outbreak strain genome lacks the target sequence of the probe and primers of a commonly used Clade I-specific real-time PCR.

Drug repurposing for Mpox: Discovery of small molecules as potential inhibitors against DNA-dependent RNA polymerase using molecular modeling approach.

Mpox (formerly Monkeypox), a zoonotic illness caused by the Mpox virus, belongs to the Orthopoxvirus genus in the family Poxviridae. To design and develop effective antiviral therapeutics against DNA viruses, the DNA-dependent RNA polymerase (DdRp) of poxviruses has emerged as a promising drug target. In the present study, we modeled the three-dimensional (3D) structure of DdRp using a template-based homology approach. After modeling, virtual screening was performed to probe the molecular interactions between 1755 Food and Drug Administration-approved small molecule drugs (≤500 molecular weight) and the DdRp of Mpox. Based on the binding affinity and molecular interaction patterns, five drugs, lumacaftor (-11.7 kcal/mol), conivaptan (-11.7 kcal/mol), betulinic acid (-11.6 kcal/mol), fluspirilene (-11.3 kcal/mol), and imatinib (-11.2 kcal/mol), have been ranked as the top drug compounds interacting with Mpox DdRp. Complexes of these shortlisted drugs with DdRp were further evaluated using state-of-the-art all-atoms molecular dynamics (MD) simulations on 200 nanoseconds followed by principal component analysis (PCA). MD simulations and PCA results revealed highly stable interactions of these small drugs with DdRp. After due validation in wet-lab using available in vitro and in vivo experiments, these repurposed drugs can be further utilized for the treatment of contagious Mpox virus. The outcome of this study may establish a solid foundation to screen repurposed and natural compounds as potential antiviral therapeutics against different highly pathogenic viruses.

Understanding knowledge, attitudes and practices on Ebola Virus Disease: a multi-site mixed methods survey on preparedness in Rwanda.

BACKGROUND: The overall goal of this survey was to understand the knowledge, attitudes, and practices related to the Ebola Virus Disease (EVD) in Rwanda. METHODS: This mixed-method cross-sectional survey was conducted in five selected districts of Rwanda. Quantitative data were collected from 1,010 participants using Kobo Collect Software and the analysis was performed using SPSS and Python software. Qualitative data were specifically collected from 98 participants through Key Informant Interviews (KIIs) and Focus Group Discussion (FGDs). Interview transcripts were imported into NVIVO 8 for coding and subsequent analysis. RESULTS: As per our quantitative findings, we report that from the 1,010 respondents, 99.6% reported having previously heard of Ebola, 97.2% believed that vaccination is important in combatting the disease and 93.3% of individuals reported a willingness to receive vaccination should one become available. Around 54% of the respondents were correct in identifying that the disease is of a viral origin which originates from wild animals (42.1%). When asked if they believed that Rwanda is at risk of an EVD outbreak, 90% of the respondents believe that the country is at risk of an EVD outbreak, and the cofactors *gender* and *whether people dwell in Rubavu/Rusizi* were found to significantly impact their perception of threat. As per our qualitative findings, the respondents mentioned that both geographical proximity and relations with the Democratic Republic of Congo place Rwanda at risk of developing an internal outbreak. Although the respondents seemed to be aware of the Ebola prevention behaviours, it was noted that some of them will require significant time before reintegrating into the community an EVD survivor, as they will first need assurance that the patient has fully recovered. Therefore, the qualitative findings reinforce what we originally reported in the quantitative approach to this study. CONCLUSION: Our results show that there was high EVD-related knowledge and awareness among the general population in Rwanda. However, for strong public health awareness, preparedness, and protection, a massive investment should always be made in education about EVD with a special focus on districts neighboring countries where the disease is consistently being reported.

CD4+ regulatory T cells and CD4+ activated T cells in new active and relapse tuberculosis.

The aim of the present study was to examine characteristics of tuberculosis (TB) patients with different clinical forms and to study the frequency of Regulatory T cells (Treg cells) and Activated T cells in patients with new active and relapse TB. Forty-five pulmonary TB patients and a control group of 15 healthy individuals were enrolled in this study. Of the 45 TB patients, 15 were new cases with drug-susceptible active TB and 30 were relapsed cases (15 drug-susceptible and 15 multidrug resistant-TB). The age of study participants ranged from 21 to 68 years old. According to sex presentation, males were appreciably highly affected than females with a sex ratio of 2. The patients reported a mean recent weight loss of 8.9 kg. The Erythrocyte Sedimentation Rate was high in TB group, far exceeding the normal value. The results revealed that the number of CD3+ CD4+ T-cells significantly decreased whereas the level of blood Treg cells and expression of activation markers CD38 and HLA-DR on CD4+ T-cells significantly increased in TB group compared with the control group (p<0.05). The frequency of Treg cells was significantly higher in the TB group than the control group. Both the patients with new active TB and relapse TB demonstrated significantly higher levels of CD4+FoxP3+ Treg compared to healthy subjects (p<0.05). A high and significant percentage of Treg cells were found in patients with DS active TB than patients with MDR relapse TB. Interestingly, the frequency of CD4+FoxP3+ cells also differs according to the sputum smear microscopy status. The presence of high numbers of Treg cells and corresponding high immune activation may be an unfavourable factor that can predispose individuals to different clinical forms of TB, including relapse TB.

The impact of public transportation on the transmission of COVID-19 in Rwanda.

Introduction: The onset of the COVID-19 pandemic has placed a significant burden on healthcare systems worldwide, particularly in sub-Saharan regions where healthcare resources are limited. The transmission of SARS-CoV-2 is facilitated by the movement of people from place to place. Therefore, implementing measures that restrict movement of people and contacts is crucial in controlling the spread of the disease. Following the identification of the first COVID-19 case in Rwanda, the government implemented stringent measures, including a complete nationwide lockdown, border closures, curfews, reduced capacity in public transportation and businesses, and mandatory testing. This study aims to assess epidemiological trends in COVID-19 cases in relation to changes in population mobility within the public transportation system. Methods: A descriptive analysis using publicly available data on COVID-19 epidemiological indicators (cases, deaths, vaccinations, and stringency index) and mobility data was conducted. Results: The results reveal a strong correlation between mobility in public transportation and other activities, underscoring Rwanda's reliance on its public transportation system. The study also identifies a pattern where increases in transit station mobility preceded spikes in COVID-19 cases, suggesting that the subsequent rise in public transportation usage may contribute to higher infection rates. Discussion: Therefore, this study emphasizes the importance of ongoing vigilance and regulatory measures regarding public transportation during infectious disease outbreaks.

Feasibility, Acceptability, Satisfaction, and Challenges of an mHealth App (e-ASCov) for Community-Based COVID-19 Screening by Community Health Workers in Rwanda: Mixed Methods Study.

Background: Although at the base of the pyramid-shaped organization of the Rwandan health system, community health workers (CHWs) are central to the community-based management of disease outbreaks. Objective: This mixed methods study aimed to explore the feasibility, acceptability, satisfaction, and challenges of a mobile health (mHealth) tool for community-based COVID-19 screening in Rwanda. Methods: Two urban (Gasabo and Nyarugenge) and 2 rural (Rusizi and Kirehe) districts in Rwanda participated in the project (smartphone app for COVID-19 screening). A mixed methods approach was used to inform the feasibility (awareness and expectation), acceptability (use and perceived benefits), satisfaction, and challenges of the mHealth intervention. At the end of the project, CHWs were asked to complete a quantitative questionnaire on the use of and satisfaction with the app. Then, in-depth interviews and focus group discussions were organized with CHWs. A content analysis was performed on the transcripts. Results: Overall, 383 CHWs were recruited and trained; 378 CHWs participated in the study. The mean age of CHWs was 36.7 (SD 6.6) to 45.3 (SD 9.9) years and most were women (237/378, 62.7%). More than 7000 people were registered with the use of the app and 20% were referred to a local COVID-19 testing facility. According to CHW reporting, the median number of people screened by each CHW ranged from 152 (IQR 70-276) for Nyarugenge to 24 (IQR 16-90) for Rusizi. COVID-19 positivity rates were higher in urban than rural districts: more than half of the CHWs in Gasabo reported a confirmed positive case versus only 2.4% for Kirehe and 15.4% for Rusizi. Despite the app being a novel tool, CHWs were well aware of the use of such a tool and had appropriate expectations. Acceptability and satisfaction were very high, with differences between urban and rural districts. Satisfaction was higher in Nyarugenge (72.8/100) and Gasabo (80.7/100) than in Kirehe (61.6/100) and Rusizi (64.5/100). More than 80% of the CHWs were willing to continue using the e-ASCov app, with the exception of CHWs in Kirehe (56.7%). The app was perceived as a tool to generate information on COVID-19, inform on the status of the pandemic, and help curb the spread of the pandemic in Rwanda. CHWs were satisfied with the app at all stages of its implementation in their districts. Conclusions: In this proof-of-concept study, a smartphone app for screening COVID-19 was useful as an mHealth tool to be used by CHWs, with the potential to increase health system efficiency in an epidemic context. The context should be analyzed for generalization on a country-wide scale, both in case of an epidemic and to take into account certain conditions at the community level. Information is needed on the conditions of generalization and transferability of this type of app to other health conditions so that CHWs can be given their full place in a pyramidal health system.

Evaluating the effectiveness of lockdowns and restrictions during SARS-CoV-2 variant waves in the Canadian province of Nova Scotia.

Introduction: After the initial onset of the SARS-CoV-2 pandemic, the government of Canada and provincial health authorities imposed restrictive policies to limit virus transmission and mitigate disease burden. In this study, the pandemic implications in the Canadian province of Nova Scotia (NS) were evaluated as a function of the movement of people and governmental restrictions during successive SARS-CoV-2 variant waves (i.e., Alpha through Omicron). Methods: Publicly available data obtained from community mobility reports (Google), the Bank of Canada Stringency Index, the "COVID-19 Tracker" service, including cases, hospitalizations, deaths, and vaccines, population mobility trends, and governmental response data were used to relate the effectiveness of policies in controlling movement and containing multiple waves of SARS-CoV-2. Results: Our results indicate that the SARS-CoV-2 pandemic inflicted low burden in NS in the initial 2 years of the pandemic. In this period, we identified reduced mobility patterns in the population. We also observed a negative correlation between public transport (-0.78), workplace (-0.69), retail and recreation (-0.68) and governmental restrictions, indicating a tight governmental control of these movement patterns. During the initial 2 years, governmental restrictions were high and the movement of people low, characterizing a 'seek-and-destroy' approach. Following this phase, the highly transmissible Omicron (B.1.1.529) variant began circulating in NS at the end of the second year, leading to increased cases, hospitalizations, and deaths. During this Omicron period, unsustainable governmental restrictions and waning public adherence led to increased population mobility, despite increased transmissibility (26.41-fold increase) and lethality (9.62-fold increase) of the novel variant. Discussion: These findings suggest that the low initial burden caused by the SARS-CoV-2 pandemic was likely a result of enhanced restrictions to contain the movement of people and consequently, the spread of the disease. Easing public health restrictions (as measured by a decline in the BOC index) during periods of high transmissibility of circulating COVID-19 variants contributed to community spread, despite high levels of immunization in NS.

Third COVID-19 vaccine dose boosts antibody function in Rwandans with high HIV viral load.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing COVID-19 (coronavirus disease 2019) poses a greater health risk to immunocompromized individuals including people living with HIV (PLWH). However, most studies on PLWH have been conducted in higher-income countries. We investigated the post-vaccination antibody responses of PLWH in Rwanda by collecting peripheral blood from participants after receiving a second or third COVID-19 vaccine. Virus-binding antibodies as well as antibody neutralization ability against all major SARS-CoV-2 variants of concern were analyzed. We found that people with high HIV viral loads and two COVID-19 vaccine doses had lower levels of binding antibodies that were less virus neutralizing and less cross-reactive compared to control groups. A third vaccination increased neutralizing antibody titers. Our data suggest that people with high HIV viral loads require a third dose of vaccine to neutralize SARS-CoV-2 virus and new variants as they emerge.

Correlation of Malaria Rapid Test and Peripheral Blood Smear Microscopy among Patients attending Byumba Health Centre.

Background: Malaria presents a diagnostic challenge in most tropical countries including Rwanda. Microscopy remains the gold standard for diagnosing malaria, however, it is labour intensive and depends upon the skill of the examiner. Malaria rapid diagnostic tests (MRDTs) have been developed as an easy, convenient alternative to microscopy. Methods: A cross sectional study was conducted from October to November 2019 on 130 febrile patients who were directed to the laboratory department for blood screening for malaria parasites at Byumba Health centre. The main objective of this study was to correlate Microscopy and MRDTs in diagnosis of malaria. Results: After signing a consent form, blood samples were collected and screened for malaria parasites microscopically and by using MRDTs. Data collection forms were filled with relevant information and obtained results for MRDTs and for peripheral blood smear were recorded. The collected data were statistically analyzed using GraphPad Prism 9 software. The mean age found to be 16 years old. In this study peripheral blood smear microscopy was considered as a reference method. The sensitivity and specificity of RDT Histidine-Rich Protein 2 (HRP-2) were calculated and found to be 96.6% and 60% respectively. The negative predictive value was found to be 92.85% where positive predictive value was 73.3%. Conclusion: MRDTs should be used along with microscopy to avert complications associated with delayed diagnosis and similar studies are required to identify alternative techniques with high specificity for the diagnosis of malaria.

Effect of unmanned aerial vehicle (drone) delivery on blood product delivery time and wastage in Rwanda: a retrospective, cross-sectional study and time series analysis.

BACKGROUND: The accessibility of blood and blood products remains challenging in many countries because of the complex supply chain of short lifetime products, timely access, and demand fluctuation at the hospital level. In an effort to improve availability and delivery times, Rwanda launched the use of drones to deliver blood products to remote health facilities. We evaluated the effect of this intervention on blood product delivery times and wastage. METHODS: We studied data from 20 health facilities between Jan 1, 2015, and Dec 31, 2019, in Rwanda. First, we did a cross-sectional comparison of data on emergency delivery times from the drone operator collected between March 17, 2017, and Dec 31, 2019, with two sources of estimated driving times (Regional Centre for Blood Transfusion estimates and Google Maps). Second, we used interrupted time series analysis and monthly administrative data to assess changes in blood product expirations after the commencement of drone deliveries. FINDINGS: Between March 17, 2017, and Dec 31, 2019, 12 733 blood product orders were delivered by drones. 5517 (43%) of 12 733 were emergency orders. The mean drone delivery time was 49·6 min (95% CI 49·1 to 50·2), which was 79 min faster than existing road delivery methods based on estimated driving times (p<0·0001) and 98 min faster based on Google Maps estimates (p<0·0001). The decrease in mean delivery time ranged from 3 min to 211 min depending on the distance to the facility and road quality. We also found a decrease of 7·1 blood unit expirations per month after the start of drone delivery (95% CI -11·8 to -2·4), which translated to a 67% reduction at 12 months. INTERPRETATION: We found that drone delivery led to faster delivery times and less blood component wastage in health facilities. Future studies should investigate if these improvements are cost-effective, and whether drone delivery might be effective for other pharmaceutical and health supplies that cannot be easily stored at remote facilities. FUNDING: Canadian Institutes for Health Research.

A case control study of risk factors associated with pulmonary tuberculosis in romania: experience at a clinical hospital of pneumology.

BACKGROUND AND AIM: Tuberculosis (TB) remains a major public health issue in Romania. The aim of the present study was to evaluate the potential demographic, socioeconomic and behavioral risk factors for TB among hospitalized patients in Romania. METHODS: This is a case-control study conducted between March 1st 2014 and March 30th 2015 at Leon Daniello Clinical Hospital of Pneumology, Cluj Napoca. A total of 150 TB patients defined as "cases" were matched for age, sex and county of residence to 150 controls selected from patients attending the same hospital with respiratory diseases other than TB. Data collection was obtained through patient interviews using a structured questionnaire. Factors potentially associated with TB infection were analyzed using univariate and multivariate logistic regression. RESULTS: Factors independently associated with TB were illiteracy (OR=2.42, 95% CI 1.09-5.37), unemployment (OR=2.08, 95% CI 1.23-3.53), low household income (OR=4.12, 95% CI 2.53-6.71), smoking (more than 20 cigarettes per day) (OR=2.12, 95% CI 1.20-3.74), poor knowledge of TB (OR=3.46, 95% CI 1.97-6.07), presence of TB patient in household (OR=4.35, 95% CI 1.42-13.36), prior TB treatment (OR=2.2, 95% CI 1.93-2.5) and diabetes (OR=3.32, 95% CI 1.36-8.08). CONCLUSION: This study provided useful information that might help to develop and adapt effective policies for TB control in Romania.

Evaluation of interleukin-10 levels in the plasma of patients with various stages of tuberculosis.

UNLABELLED: Mycobacterium tuberculosis (Mtb) infection remains one of the world's major causes of illness and mortality. A clear understanding of the host defense against Mtb is imperatively needed forthe control of this epidemic. When tuberculosis (TB) infection occurs, a variety of pro and anti-inflammatory cytokines play a vital role in the pathogenesis of this disease. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines reported to suppress the protective immune response against tuberculosis. AIM: The aim of the present study was to evaluate the association of plasma IL-10 levels with various disease stages of TB and the possible effects of treatment on these levels. MATERIALS AND METHODS: A group of 30 patients with active pulmonary TB and a control group of 21 healthy individuals were enrolled in this study. The levels of IL-10 were measured before, during, and after treatment using commercially available enzyme-linked immune-sorbent assay (ELISA). Data were analyzed using GraphPad Prism version 5.0. RESULTS: The results showed that the levels of IL-10 had significant differences between the TB and control groups (p<0.05). The patients with abnormal chest X-Ray findings had higher IL-10 levels when compared to patients with normal X-Rays (p=0.03). A subgroup of 18 patients were followed during the treatment and the mean plasma concentration of IL-10 in patients before therapy was higher than in patients at 3 months of therapy and in patients after 6 months of therapy (p=0.01). However, the IL-10 level remained significantly higher in patients at the end of treatment compared with controls. These findings could be used in follow-up as clinical biomarker of the success of tuberculosis therapy.