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.

First Report on Ovine Paratuberculosis in the Sudan: Diagnosis Using Different Techniques.

Paratuberculosis (PTB) has been reported in the Sudan in cattle and goats for more than 50 years but has never been reported in sheep. However, suspicion of the disease in a breeding flock of sheep in Khartoum North locality was made due to a history of unknown cause of loss of weight. Blood and faecal samples were collected from all animals (N = 59): harvested sera were tested for anti-Mycobacterium avium subsp. paratuberculosis (MAP) antibodies by Enzyme Linked Immunosorbent Assay (ELISA); faeces were screened for acid-fast bacilli by Ziehl-Neelsen staining, tested for MAP DNA by recombinase polymerase amplification (RPA) and some faecal samples were cultured for MAP isolation. Typical MAP acid-fast bacilli were seen in 10.2% (6/59) of the faecal smears, 37.5% of the tested faecal samples (12/32) were positive for MAP DNA and only 3 (5.1%) animals were seropositive for MAP. MAP positive cultures were obtained from 2 out the 6 samples showing typical MAP acid-fast bacilli; the isolates were confirmed by real-time PCR and sequencing. As sheep are animals of utmost economic importance as the main export animals for the country, this first report of ovine PTB warrants special considerations and more investigations for planning control programmes of the disease.

Mycobacterium avium subsp. paratuberculosis and microbiome profile of patients in a referral gastrointestinal diseases centre in the Sudan.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in animals with zoonotic potential; it has been linked to many chronic diseases in humans, especially gastrointestinal diseases (GID). MAP has been extensively studied in Europe and America, but little reports were published from Africa. Sudan is a unique country with close contact between humans and livestock. Despite such interaction, the one health concept is neglected in dealing with cases of humans with GID. In this study, patients admitted to the reference GID hospital in the Sudan over a period of 8 months were screened for presence of MAP in their faeces or colonic biopsies. A total of 86 patients were recruited for this study, but only 67 were screened for MAP, as 19 did not provide the necessary samples for analysis. Both real-time PCR and culture were used to detect MAP in the collected samples and the microbial diversity in patients´ faecal samples was investigated using 16S rDNA nanopore sequencing. In total, 27 (40.3%) patients were MAP positive: they were 15 males and 12 females, of ages between 21 and 80 years. Logistic regression analysis revealed no statistical significance for all tested variables in MAP positive patients (occupation, gender, contact with animal, milk consumption, chronic disease, etc.). A unique microbiome profile of MAP-positive patients in comparison to MAP-negative was found. These findings suggest that a considerable proportion of the population could be MAP infected or carriers. Therefore, increase awareness at community level is urgently needed to decrease the risk of MAP at human/animal interface. This study represents the first report of MAP in humans in the Sudan; nevertheless, a better view of the situation of MAP in humans in the country requires a larger study including patients with other conditions.

Paratuberculosis: The Hidden Killer of Small Ruminants.

Paratuberculosis (PTB) is a contagious and chronic enteric disease of ruminants and many non-ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), and is characterised by diarrhoea and progressive emaciation with consequent serious economic losses due to death, early culling, and reduced productivity. In addition, indirect economic losses may arise from trade restrictions. Besides being a production limiting disease, PTB is a potential zoonosis; MAP has been isolated from Crohn's disease patients and was associated with other human diseases, such as rheumatoid arthritis, Hashimoto's thyroiditis, Type 1 diabetes, and multiple sclerosis. Paratuberculosis in sheep and goats may be globally distributed though information on the prevalence and economic impact in many developing countries seem to be scanty. Goats are more susceptible to infection than sheep and both species are likely to develop the clinical disease. Ingestion of feed and water contaminated with faeces of MAP-positive animals is the common route of infection, which then spreads horizontally and vertically. In African countries, PTB has been described as a "neglected disease", and in small ruminants, which support the livelihood of people in rural areas and poor communities, the disease was rarely reported. Prevention and control of small ruminants' PTB is difficult because diagnostic assays demonstrate poor sensitivity early in the disease process, in addition to the difficulties in identifying subclinically infected animals. Further studies are needed to provide more insight on molecular epidemiology, transmission, and impact on other animals or humans, socio-economic aspects, prevention and control of small ruminant PTB.

Seroprevalence of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle in Khartoum State, Sudan.

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease mainly of domestic and wild ruminants. It occurs worldwide, causing significant economic losses through decreased productivity, low fertility, increased cull rates and mortality. It is listed by the OIE (World Organization for Animal Health) as a disease of concern to trade in animals. Prevalence of this disease can be studied by detecting anti-MAP antibodies by Enzyme linked immunosorbent Assay (ELISA). The aim of this study was to investigate the current prevalence of MAP infection in cattle in Khartoum State. The overall apparent prevalence of MAP infection was found to be 6.3% and 18.9% at animal and herd levels, respectively. All seropositive animals were cross-bred females of good body condition; most of them (>90%) were >3 years old and >50% were from medium-sized herds in Omdurman. No significant association (p > 0.05) was found between seropositivity and animal herd size. The prevalence of MAP infection in Khartoum State is still low to medium compared to other parts of the world, but it is comparable to those reported from other African countries. Further studies with the view of designing nationwide surveys in domestic ruminants and camels in other states of the country are needed for establishing control programmes.