Diagnostic accuracy of three commercially available one step RT-PCR assays for the detection of SARS-CoV-2 in resource limited settings

Background COVID-19 is an ongoing public health pandemic regardless of the countless efforts made by various actors. Quality diagnostic tests are important for early detection and control. Notably, several commercially available one step RT-PCR based assays have been recommended by the WHO. Yet, their analytic and diagnostic performances have not been well documented in resource-limited settings. Hence, this study aimed to evaluate the diagnostic sensitivities and specificities of three commercially available one step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in Ethiopia in clinical setting. Methods A cross-sectional study was conducted from April to June, 2021 on 279 respiratory swabs originating from community surveillance, contact cases and suspect cases. RNA was extracted using manual extraction method. Master-mix preparation, amplification and result interpretation was done as per the respective manufacturer. Agreements between RT-PCRs were analyzed using kappa values. Bayesian latent class models (BLCM) were fitted to obtain reliable estimates of diagnostic sensitivities, specificities of the three assays and prevalence in the absence of a true gold standard. Results Among the 279 respiratory samples, 50(18%), 59(21.2%), and 69(24.7%) were tested positive by TIB, Da An, and BGI assays, respectively. Moderate to substantial level of agreement was reported among the three assays with kappa value between 0 .55 and 0.72. Based on the BLCM relatively high specificities (95% CI) of 0.991(0.973–1.000), 0.961(0.930–0.991) and 0.916(0.875–0.952) and considerably lower sensitivities with 0.813(0.658–0.938), 0.836(0.712–0.940) and 0.810(0.687–0.920) for TIB MOLBIOL, Da An and BGI respectively were found. Conclusions While all the three RT-PCR assays displayed comparable sensitivities, the specificities of TIB MOLBIOL and Da An were considerably higher than BGI. These results help adjust the apparent prevalence determined by the three RT-PCRs and thus support public health decisions in resource limited settings and consider alternatives as per their prioritization matrix

Interrogating the Impact of Intestinal Parasite-Microbiome on Pathogenesis of COVID-19 in Sub-Saharan Africa

Intestinal parasitic infections affect more than 2 billion people throughout the world with disproportionately high prevalence rates in Low- and Middle-Income Countries (LMICs) (Herricks et al., 2017). Multicellular and highly complex parasites such as Ascaris, hook worm, Trichuris, Enterobius and Schistosoma, as well as unicellular organisms including Entamoeba, Giardia, Toxoplasma, Cyclospora, and Cryptosporidium are among major pathogens that contribute to the global intestinal parasitic disease burden. Parasites can cause persistent infection due to their ability to resist immune-mediated expulsion by modulating the host's immune response (McSorley and Maizels, 2012; Wammes et al., 2014; Chabé et al., 2017; Burrows et al., 2019; Ryan et al., 2020). There is a complex interaction between parasites and human microbiota which can triangulate with host's immune homeostasis and host responses to bystander antigens, vaccines or other unrelated diseases, both infectious and non-communicable diseases (McSorley and Maizels, 2012; Wammes et al., 2014). Recently, the world has grappled with an unprecedented pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) (WHO, 2020). The pathogenesis of severe disease in COVID-19 has been linked to the phenomenon of immune hyperactivation (Sinha et al., 2020; Tay et al., 2020). Here, we propose that the interplay between intestinal parasites and microbiome may have a potential direct or indirect effects on the pathogenesis of SARS-CoV-2 infection, in particular in the context of LMICs

First PCR Confirmed anthrax outbreaks in Ethiopia-Amhara region, 2018-2019.

BackgroundAnthrax is a disease that affects humans and animals. In Ethiopia, anthrax is a reportable disease and assumed to be endemic, although laboratory confirmation has not been routinely performed until recently. We describe the findings from the investigation of two outbreaks in Amhara region.MethodsFollowing reports of suspected outbreaks in Wag Hamra zone (Outbreak 1) and South Gondar zone (Outbreak 2), multi-sectoral teams involving both animal and public health officials were deployed to investigate and establish control programs. A suspect case was defined as: sudden death with rapid bloating or bleeding from orifice(s) with unclotted blood (animals); and signs compatible with cutaneous, ingestion, or inhalation anthrax ≤7 days after exposure to a suspect animal (humans). Suspect human cases were interviewed using a standard questionnaire. Samples were collected from humans with suspected anthrax (Outbreak 1 and Outbreak 2) as well as dried meat of suspect animal cases (Outbreak 2). A case was confirmed if a positive test was returned using real-time polymerase chain reaction (qPCR).ResultsIn Outbreak 1, a total of 49 cows died due to suspected anthrax and 22 humans developed symptoms consistent with cutaneous anthrax (40% attack rate), two of whom died due to suspected ingestion anthrax. Three people were confirmed to have anthrax by qPCR. In Outbreak 2, anthrax was suspected to have caused the deaths of two livestock animals and one human. Subsequent investigation revealed 18 suspected cases of cutaneous anthrax in humans (27% attack rate). None of the 12 human samples collected tested positive, however, a swab taken from the dried meat of one animal case (goat) was positive by qPCR.ConclusionWe report the first qPCR-confirmed outbreaks of anthrax in Ethiopia. Both outbreaks were controlled through active case finding, carcass management, ring vaccination of livestock, training of health professionals and outreach with livestock owners. Human and animal health authorities should work together using a One Health approach to improve case reporting and vaccine coverage

Low-cost liquid medium for in vitro cultivation of Leishmania parasites in low-income countries

Background: Cutaneous Leishmaniasis (CL) induced by Leishmania aethiopica has two clinical manifestations: ulcerating, self-healing CL and non-ulcerating, non-healing CL. The grossly disfiguring multiple nodules on the face and exterior surface of limbs during non-ulcerative CL are sometimes misdiagnosed as other skin infections. Thus the need for definitive and prompt laboratory diagnosis will be required. Identifying Leishmania parasite by culture method is considered as a definitive method for initiation of treatment and as an effective component of leishmaniasis control methods. Recently the involvement of Fas (CD95) and Tumor Necrosis Factor (TNF) Related Apoptosis Inducing Ligand (TRAIL) induced apoptotic pathways were proposed to be involved in tissue destruction and ulceration during L. major induced CL. Aims: 1) to develop an alternative culture media that could minimize the cost for culturing Leishmania from patient lesions. 2) to investigate if the expression of FasL and TRAIL differs in ulcerating and non- ulcerative CL. Methods: GALF-1 media was formulated in our lab and compared to RPMI 1640 medium and conventional Locke s semi solid media (LSSM) which is one of the modifications of Novy-MacNeal-Nicolle (NNN) culture media. Amastigotes transformation, cryopreservation, recovery of parasites, cost and mass cultivation were analysed. Expression of Fas ligand (FasL), TRAIL and apoptosis were assessed by immunohistology in human skin biopsies from L. aethiopica induced ulcerative or non-ulcerative CL. FasL and TRAIL blocking experiments were performed in a murine model of CL. Results and discussion: GALF-1 is cheap and its ingredients available in a low income country such as Ethiopia. GALF-1 was able to transform amastigotes from Ethiopian patients samples and could be used to cultivate promastigotes in large quantities. Cost analysis showed 80% to 95 % decreased costs as compared to conventional media. Promastigotes cultured with GALF-1 could be cryopreserved in liquid nitrogen with comparable re-culture potential to conventional media. Affordability of diagnostic assays is a key issue for resource poor countries and the possibility to cut the cost of the efficient culture method for diagnosis through the use of inexpensive local formulated reagents could improve the diagnosis of leishmaniasis in low income endemic countries. More FasL expressing cells were detected in dermis of ulcerative CL as compared to non-ulcerative CL and controls. TRAIL expression was higher in ulcerative CL as compared to non-ulcerative CL and controls in both epidermis and dermis. Increased dermal expression of FasL and TRAIL was associated with ulcer formation during CL. This correlated with an inhibition of the ulcerative process in a murine CL model during FasL and TRAIL neutralisation.The mechanisms of the involvement of FasL and TRAIL in ulceration was not elucidated and putative reason(s) for the difference in dysregulation of apoptosis are discussed

Prognostic Value of C-Reactive Protein in SARS-CoV-2 Infection: A Simplified Biomarker of COVID-19 Severity in Northern Ethiopia

Purpose To evaluate the role of C-reactive protein (CRP) in predicting severe COVID-19 patients. Methods A prospective observational cohort study was conducted from July 15 to October 28, 2020, at Kuyha COVID-19 isolation and treatment center hospital, Mekelle City, Northern Ethiopia. A total of 670 blood samples were collected serially. SARS-CoV-2 infection was confirmed by RT-PCR from nasopharyngeal swabs and CRP concentration was determined using Cobas Integra 400 Plus (Roche). Data were analyzed using STATA version 14. P-value < 0.05 was considered statistically significant. Results Overall, COVID-19 patients had significantly elevated CRP at baseline when compared to PCR-negative controls [median 11.1 (IQR: 2.0– 127.8) mg/L vs 0.9 (IQR: 0.5– 1.9) mg/L; p=0.0004)]. Those with severe COVID-19 clinical presentation had significantly higher median CRP levels compared to those with non-severe cases [166.1 (IQR: 48.6– 332.5) mg/L vs 2.4 (IQR: 1.2– 7.6) mg/L; p< 0.00001)]. Moreover, COVID-19 patients exhibited higher median CRP levels at baseline [58 (IQR: 2.0– 127.8) mg/L] that decreased significantly to 2.4 (IQR: 1.4– 3.9) mg/L after 40 days after symptom onset (p< 0.0001). Performance of CRP levels determined using ROC analysis distinguished severe from non-severe COVID-19 patients, with an AUC value of 0.83 (95% CI: 0.73– 0.91; p=0.001; 77.4% sensitivity and 89.4% specificity). In multivariable analysis, CRP levels above 30 mg/L were significantly associated with an increased risk of developing severe COVID-19 for those who have higher ages and comorbidities (ARR 3.99, 95% CI: 1.35– 11.82; p=0.013). Conclusion CRP was found to be an independent determinant factor for severe COVID-19 patients. Therefore, CRP levels in COVID-19 patients in African settings may provide a simple, prompt, and inexpensive assessment of the severity status at baseline and monitoring of treatment outcomes

Longitudinal profile of antibody response to SARS-CoV-2 in patients with COVID-19 in a setting from Sub-Saharan Africa: A prospective longitudinal study.

BACKGROUND Serological testing for SARS-CoV-2 plays an important role for epidemiological studies, in aiding the diagnosis of COVID-19, and assess vaccine responses. Little is known on dynamics of SARS-CoV-2 serology in African settings. Here, we aimed to characterize the longitudinal antibody response profile to SARS-CoV-2 in Ethiopia. METHODS In this prospective study, a total of 102 PCR-confirmed COVID-19 patients were enrolled. We obtained 802 plasma samples collected serially. SARS-CoV-2 antibodies were determined using four lateral flow immune-assays (LFIAs), and an electrochemiluminescent immunoassay. We determined longitudinal antibody response to SARS-CoV-2 as well as seroconversion dynamics. RESULTS Serological positivity rate ranged between 12%-91%, depending on timing after symptom onset. There was no difference in positivity rate between severe and non-severe COVID-19 cases. The specificity ranged between 90%-97%. Agreement between different assays ranged between 84%-92%. The estimated positive predictive value (PPV) for IgM or IgG in a scenario with seroprevalence at 5% varies from 33% to 58%. Nonetheless, when the population seroprevalence increases to 25% and 50%, there is a corresponding increases in the estimated PPVs. The estimated negative-predictive value (NPV) in a low seroprevalence scenario (5%) is high (>99%). However, the estimated NPV in a high seroprevalence scenario (50%) for IgM or IgG is reduced significantly to 80% to 85%. Overall, 28/102 (27.5%) seroconverted by one or more assays tested, within a median time of 11 (IQR: 9-15) days post symptom onset. The median seroconversion time among symptomatic cases tended to be shorter when compared to asymptomatic patients [9 (IQR: 6-11) vs. 15 (IQR: 13-21) days; p = 0.002]. Overall, seroconversion reached 100% 5.5 weeks after the onset of symptoms. Notably, of the remaining 74 COVID-19 patients included in the cohort, 64 (62.8%) were positive for antibody at the time of enrollment, and 10 (9.8%) patients failed to mount a detectable antibody response by any of the assays tested during follow-up. CONCLUSIONS Longitudinal assessment of antibody response in African COVID-19 patients revealed heterogeneous responses. This underscores the need for a comprehensive evaluation of seroassays before implementation. Factors associated with failure to seroconvert needs further research

Effect of co-infection with intestinal parasites on COVID-19 severity: A prospective observational cohort study

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a spectrum of clinical presentations. Evidence from Africa indicates that significantly less COVID-19 patients suffer from serious symptoms than in the industrialized world. We and others previously postulated a partial explanation for this phenomenon, being a different, more activated immune system due to parasite infections. Here, we aimed to test this hypothesis by investigating a potential correlation of co-infection with parasites with COVID-19 severity in an endemic area in Africa. Methods: Ethiopian COVID-19 patients were enrolled and screened for intestinal parasites, between July 2020 and March 2021. The primary outcome was the proportion of patients with severe COVID-19. Ordinal logistic regression models were used to estimate the association between parasite infection, and COVID-19 severity. Models were adjusted for sex, age, residence, education level, occupation, body mass index, and comorbidities. Findings: 751 SARS-CoV-2 infected patients were enrolled, of whom 284 (37.8%) had intestinal parasitic infection. Only 27/255 (10.6%) severe COVID-19 patients were co-infected with intestinal parasites, while 257/496 (51.8%) non-severe COVID-19 patients were parasite positive (p<0.0001). Patients co-infected with parasites had lower odds of developing severe COVID-19, with an adjusted odds ratio (aOR) of 0.23 (95% CI 0.17–0.30; p<0.0001) for all parasites, aOR 0.37 ([95% CI 0.26–0.51]; p<0.0001) for protozoa, and aOR 0.26 ([95% CI 0.19–0.35]; p<0.0001) for helminths. When stratified by species, co-infection with Entamoeba spp., Hymenolepis nana, Schistosoma mansoni, and Trichuris trichiura implied lower probability of developing severe COVID-19. There were 11 deaths (1.5%), and all were among patients without parasites (p = 0.009). Interpretation: Parasite co-infection is associated with a reduced risk of severe COVID-19 in African patients. Parasite-driven immunomodulatory responses may mute hyper-inflammation associated with severe COVID-19. Funding: European and Developing Countries Clinical Trials Partnership (EDCTP) – European Union, and Joep Lange Institute (JLI), The Netherlands

Prevalence of intestinal parasites versus knowledge, attitude and practices (KAPs) with special emphasis to Schistosoma mansoni among individuals who have river water contact in Addiremets town, Western Tigray, Ethiopia.

BACKGROUND:Intestinal parasite infections are major public health problems in resource-limited countries that adversely affect the well-being of millions. Among these, intestinal schistosomiasis is a serious public health problem in tropical and sub-tropical countries. METHODS:A Community based cross sectional study was conducted from February to April 2017 in Addiremets town, Ethiopia. Socio-demographic associated risk factors and knowledge, attitude and practices of individuals regarding intestinal parasite infection including schistosomiasis were collected from 411 study participants using pretested structured questionnaires. From each study participant, a fresh stool sample was collected and direct microscopy, formol-ether concentration and Kato- Katz techniques were performed. Snails were checked and collected from the nearby study area river. The collected data was entered and analyzed using SPSS version 20. Bi-variant and multiple logistic regressions were used for correlation analysis. A P <0.05 was considered as statistically significant. RESULT:The overall intestinal parasite prevalence was 51.3% (211/411). The most prevalent parasites were S. mansoni 26.3%(108/411) and Hookworm 23.1%(95/411). The prevalence of intestinal parasites among males and females were 54.1%(131/242) and 47.3%(80/169) respectively. The highest proportion of parasite infection was reported among the age group of 5-9 year old participants, at 70.6%(36/51). The prevalence of S. mansoni was 26.3% (108/411) with mean infection intensity of 218 eggs per gram (range: 24 to 1728). Among study participants, 94.4% had good knowledge while 35.9% of them had poor practices towards intestinal parasite and Schistosomes infection prevention. CONCLUSION:High prevalence of intestinal parasitic infection was observed in Addiremets town and the most common parasites identified were S. mansoni and Hookworm. Most study participants had light infection intensity of Schistosomiasis, Ascariasis and Hookworm infection. Majority of the participants in the study area had good knowledge and positive attitude about intestinal parasitic infection and schistosomiasis control. Shells of Biomphalaria species, Bulinus species and Physa species were collected from the Mytsaeda river shore

Evaluation of microcapillary culture method for the isolation of Leishmania aethiopica parasites from patients with cutaneous lesions in Ethiopia

Abstract Background In addition to direct slide microscopy, traditional culture method (TCM) has long been considered as a gold standard method for the diagnosis of cutaneous leishmaniasis (CL). However, TCM is relatively expensive and time-consuming compared to the newly introduced microculture method (MCM), which has shown to be sensitive and rapid diagnostic method elsewhere for different Leishmania parasite species other than Leishmania (L.) aethiopica. The objective of this study was to evaluate the diagnostic performance of MCM for the diagnosis of CL caused by L. aethiopica. Methods One hundred forty-three lesion aspirates were collected from 124 suspected CL patients prospectively based on their consecutive series. Portion of the aspirates were cultured in duplicate in TCM with modified Novy-MacNeal-Nicolle (NNN) in tissue culture flask and microcapillary tubes containing RPMI 1640 with 10% fetal bovine serum (FBS) for MCM. Smears on glass slides from the remaining portion of the aspirate were used for direct microscopy to detect the parasite after stained with Giemsa staining solution. Up on a consensus, positive result in any two of the three tests was used as a reference standard to analyze sensitivity. Results As per consensus standard criteria, 52 of the lesions were qualified to evaluate MCM versus TCM. Forty-eight lesion samples were positive by MCM, 36 by TCM, and 37 by smear microscopy. The representative DNA from parasite culture isolates revealed the causative Leishmania parasite was L. aethiopica by ITS1 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Culturing L. aethiopica in vitro by MCM is more sensitive (92.3%) than by TCM (69.2%), P = 0.003. The median time for L. aethiopica promastigotes emergence in the culture was 3 days for MCM and 6 days for TCM, P < 0.001. Conclusions Our finding indicated that MCM is a sensitive and a rapid culturing method for the isolation of L. aethiopica than TCM and smear microscopy

Comprehensive competency assessment of malaria microscopists and laboratory diagnostic service capacity in districts stratified for malaria elimination in Ethiopia​.

BackgroundFederal Ministry of Health (FMoH) Ethiopia achieved significant declines in malaria mortality and incidence and has recently launched malaria elimination in selected low transmission settings. Successful malaria elimination calls for rapid and accurate diagnosis of cases so that the patients can promptly be treated before the occurrence of transmission. Therefore, this study assessed the competency of malaria microscopists using panal slides, and laboratory service availability and readiness in terms of supplies and equipments in malaria elimination targeted districts in Ethiopia.MethodA cross-sectional study was conducted from February to June 2018 in all hospitals, health centers and private clinics in 20 malaria elimination targeted districts, selected out of the 6 regional states in Ethiopia. All malaria microscopists available in the study health facilities during the study period were included in the study. Questionnaires were used for interviewing sociodemography of personnel and laboratory supplies. Per World Health Organization (WHO) criteria set for proficiency testing, 10 Giemsa stained malaria slide panels (8 positive low/high density pf/pv/Mixed and 2 negative slides) were administered to each study participant for performance assessment on malaria parasite detection, species identification and parasite count using light microscopy. The slide panels are PCR confirmed and WHO approved ones, which have been stored in the slide banks at the national reference laboratory in Ethiopian Public Health Institute.ResultIn this assessment, 17(16%) district hospitals, 71(67%) health centers (HCs) and 18(17%) private clinics (PCs) were included. Of the 18 PCs, only 10(55.6%) had license certificate. Of the study facilities, 91.5%(97) use light microscopy, 2.83%(3) use RDTs and 2.9%(3) use both microscopy and RDT to detect malaria. Accessible and appropriate storage of Giemsa was reported by 58.8%(10) hospitals, 81.7%(58) HCs & 72.2%(13) private clinics. Of the 1896 malaria positive & 474 negative slides administered to 237 study participants, 318(16.8%) slides reported falsely negative & 47(9.9%) reported falsely positive. The participants achieved "good" grade [Agreement(A): 84.6%, Kappa(K): 0.6] on parasite detection and "poor" agreement (A: 43.8%; K: 0.11) on every species identification. No or slight agreement seen on differentiation of P. falciparum from other species (A: 28.41%; K:0.29). Above 95%(201) of participants, did not count or used plus system of parasite estimation which is the least accurate and unreccomended method per WHO guideline.ConclusionIn the current study, low performance of malaria microscopists particularly in species identification & poor to moderate capacity of laboratories observed. This is really a great obstacle to malaria elimination strategy of the country. Therefore, national malaria control and elimination program in collaboration with partners is supposed to provide comprehensive training for professionals giving laboratory service and to fulfill laboratory supplies to have the gold standard service