Molecular and serological survey of paratuberculosis in cattle in selected districts of Western Uganda.

Knowledge of Mycobacterium avium subsp. paratuberculosis (MAP) herd infection status is important to plan appropriate control and prevention strategies for Paratuberculosis (PTB); however, in Uganda MAP infection status of most herds is unknown. This study aimed at determining the MAP infection status of cattle herds and the associated risk factors for MAP infection in six western districts of Uganda. The survey covered a total of 93 herds where faecal and blood samples were collected from 1814 cattle. A Recombinase Polymerase Amplification (RPA) and an antibody-based (ELISA) assays were used to test for the presence of MAP DNA in faeces and MAP antibodies in serum, respectively. The apparent cow-level prevalence of MAP infection was 3.2 and 2.7% using ELISA and RPA respectively and the true cow-level prevalence using ELISA and RPA was 4.9 and 3% respectively. A herd-level prevalence of 43% (ELISA) and 40.8% (RPA) and a within-herd prevalence of 3.8 ± 2.1% based on ELISA were obtained. Among the risk factors investigated, long dry spells were significantly associated with high MAP infection (p < 0.05). These results indicate that MAP is actively present in most areas where surveillance was carried out. This poses a serious threat to the livestock industry and potentially to public health as MAP is highly suspected to play a role in the pathogenesis of several diseases in humans. Other areas of the country are to be surveyed as well in order to establish full data on MAP infection status to enable interventions for the control and prevention of the disease.

A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field.

BACKGROUND: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. OBJECTIVES: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. STUDY DESIGN: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. RESULTS: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). CONCLUSION: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

Mycobacterium avium subsp. paratuberculosis Virulence: A Review.

To propose a solution for control of Mycobacterium avium subsp. paratuberculosis (MAP) infections in animals as well as in humans, and develop effective prevention, diagnostic and treatment strategies, it is essential to understand the molecular mechanisms of MAP pathogenesis. In the present review, we discuss the mechanisms utilised by MAP to overcome the host defense system to achieve the virulence status. Putative MAP virulence genes are mentioned and their probable roles in view of other mycobacteria are discussed. This review provides information on MAP strain diversity, putative MAP virulence factors and highlights the knowledge gaps regarding MAP virulence mechanisms that may be important in control and prevention of paratuberculosis.

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay.

African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay's analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.