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Project Summary
Abstract
Background
The SARS-CoV-2 pandemic is a global threat affecting 210 countries, with 2,177,469 confirmed cases and 6.67% mortality. In Africa, 17,243 cases have been confirmed, but many remain undiagnosed due to limited laboratory-capacity, suboptimal performance of used molecular-assays (~30% false negative, Yu et al. and Zhao et al., 2020) and lack of WHO-recommended rapid-tests.
Objectives
We aim to implement measures to minimize risks for COVID-19 in West/Central-Africa, putting together multidisciplinary EU-Africa highly-experienced virologists, immunologists, bioinformaticians and clinicians, to achieve the following objectives: (a) to integrate/improve available-infrastructure, methodologies, and expertise on COVID-19. For this purpose, we will create a platform enabling EU and African researchers/clinicians to better integrate and translate evidence into the COVID-19 clinical-practice; (b) to enhance capacities in Cameroon for screening/detecting individuals with high-risks of COVID-19, by setting-up effective core-facilities on-site; (c) to validate point-of-care SARS-CoV-2 molecular assays allowing same-day result delivery, thus permitting timely diagnosis, treatment, and retention in care of COVID-19 patients; (d) to implement SARS-CoV-2 diagnosis with innovative/highly sensitive ddPCR-based assays and viral genetic characterization; (e) to validate serological assays to identify COVID-19-exposed persons and follow-up of convalescents.
Methods
This capacity-building and implementation science is built on a prospective, observational and multi- country study conducted among COVID-19 suspects/contacts during 24 months in Cameroon. Following consecutive sampling of 1,536 COVID-19 suscepted patients/contacts, divided into 768 SARS-CoV2 positive and an equivalent control-arm, oro/nasopharyngeal swabs and sera will be collected. Well characterised biorepositories will be established locally; molecular testing will be performed on conventional real-time qPCR, point-of-care GeneXpert, antigen-tests and digital droplet PCR (ddPCR); SARS-CoV2 amplicons will be full-length sequenced; serological testing will be performed using ELISA, and antibody-based kits. Sensivity, specificity, positive- and negative-predictive values will be evaluated.
Expected outcomes
These efforts will contribute in creating the technical and clinical environment to facilitate earlier detection of Sars-CoV-2 in West/Central Africa. In particular, the goals will be: (a) to implement technology transfer for capacity-building on conventional and point-of-care molecular assays, achieving a desirable performance for clinical diagnosis of SARS-CoV2; (b) to integrate/improve the available infrastructure, methodologies, and expertise on Sars-CoV2 detection; (c) to improve the turn-around-time for diagnosing COVID- 19 infection with obvious advantage for patients/clinical management thanks to low-cost assays, thus permitting timely treatment and retention in care; (d) to assess the epidemiology of COVID-19 and circulating-variants in Cameroon (as well as in other nearby countries of Central Africa that can be added to the network).
Our frontline laboratory working group in Cameroon
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Annual Report
Activities Carried-out
The EDCTP PERFECT-Study was launched with a kick-off meeting on July 9, 2021, in the presence of partner institutions of which the “Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management” by Joseph Fokam (project coordinator), “Ospedale Pediatrico Bambino Gesu” by Dr Luna Colegrasso, the “Fondazione AVIRALIA Onlus” by Professor Carlo-Federico Perno, the “Association de Recherche en Virologie et Dermatologie” by Professor Anne Genevieve Marcelin, and the “Univesita degli studi di Roma Tor Vergata” by Professor Vittorio Colizzi). Logistics for field suppliers, and for laboratory materials and consumables were ordered. Ethical clearance was obtained (reference number 2022/01/1430/CE/CNERSH).
Preliminary Results
A total of 2656 participants were enrolled (56.6% males versus 43.4%), the median age was 40 [IQR: 31 – 49] years, only 2.1% were symptomatic and 9.6% had co-morbidities; 2.4% were known HIV-positive; and 19.69% had previous SARS-CoV-2 positivity; and 67.2% (1784) were vaccinated of whom 41.0% (674/1642) received boost doses. Overall positivity was 9.3% (246/2656) at PCR CT-value<40. Regarding the performance evaluation of molecular assays, the positivity was 30.0% (86/287) on Abbott and 37.6% (108/287) on DaAn gene, overall agreement was 82.6% (237/287), with k=0.82 (95%CI: 0.777-0.863), indicating an excellent diagnostic agreement. Regarding Viral Load (VL), positive agreement was 100% for samples with high VLs (CT<20), indicating an excellent agreement and interoperability between assays. In-depth assessment of clinical threshold of these RTPCR assays will be evaluated during the second year on digital droplet PCR. Regarding the use of antigen-rapid diagnostic tests, the AbbottTM Panbio COVID-19 Ag rapid test device (nasopharyngeal) had a positivity rate of 2/666 (0.3%); AbbottTM Panbio COVID-19 Ag rapid test device (nasal) showed zero positivity (0/544); Orient GeneTM Coronavirus Ag rapid test cassette (swab) also had zero positivity (0/425), and INDICAIDTM COVID 19 Rapid antigen test had a positivity rate of 5/524 (0.95%). These rapid antigen tests been analysed within the context of a low epidemiological burden, targeted sampling of positive cases are underway to better compare their diagnostic performance. Regarding the use of antibody diagnostic tests (RDT Ninonasal™, RDT Genrui Biotech and ELISA Diatheva), the overall antibody sero-positivity rate was 67.0% (254/379), with sero-prevalence of IgM of 2/379 (0.53%), sero-prevalence of IgG of 239/379 (63.06%), and the co-occurence of IgM/IgG of 13/379 (3.43%). Importantly, vaccination was associated to antibody to SARS-CoV-2 sero-positivity: OR 1.72 [1.09–2.713], p=0.023. Specifically, our data showed a significantly higher rate of antibody response among those who received the Pfizer vaccine as compared to the group of other vaccines (SARS-CoV-2 sero-positivity of 2.575 [1.451–4.567), p= 0.002. Among the 130 sequences generated so far, samples of the first wave were mainly the ancestral strain; sequences of the second wave partly the Alpha and Beta variants, followed by Delta variant during the third wave, and Omicron during the last wave (with first notification of Omicron case to the minister of public health for timely actions). Regarding sub-variants of the omicron, BA.1 was common (47.2%), followed by BA.5 (33.96%), BA.2 (13.21 %), BA.4 (5.66%). For a rapid variant detection Escaplex kit showed a good overall sensibility (84%), suggesting this point mutation assay for timeous variant surveillance in Africa.
Capacity Building and Networking
Following communication on the project, the project coordinator was called to join the government COVID-19 response strategy at the level of the national public emergency operations coordination. This prompted greater awareness and a higher community engagement on the project, marked by a higher sampling than the minimum expected. Preliminar. So far four students have been trained for their academic research works (one PhD, two Msc and one BMLs students).
Result Dissemination and Communication
Conference abstracts have been presented at AFRAMED 2021, ICASA 2021, and AFRAVIH 2022. Three scientific publications were done: one in PLoS ONE (doi: 10.1371/journal.pone.0273818) on the title: “Diagnostic Performance of Molecular and Serological Tests of SARS- CoV-2 on well-Characterised Specimens from COVID-19 Individuals : the EDCTP "PERFECT-Study protocol" (RIA2020EF-3000)’’; a second in the Journal for Public Health in Africa entitled: ‘’Epidemiological, virological and clinical features of SARS-CoV-2 among individuals during the first wave in Cameroon: Baseline analysis for the EDCTP PERFECT-Study RIA2020EF-3000” (JPHIA 2022; 13:2142); and the third paper entitled: ‘’High concordance in SARS- CoV-2 detection between automated (Abbott m2000) and manual (DaAn gene) RT-PCR systems: The EDCTP PERFECT-Study in Cameroon’’ (JPHIA 2022; 13:2163).
Conclusion and way forward
The expected outcomes were highly achieved, with excellent performance between molecular assays, detection of variants/subvariants and antibody response. In year-2, ddPCR, full-length sequencing using nanopore and Illumina, and targeted testing for additional positive cases, will be implemented
Participation to COVID-19 testing external quality assurance
Periodic Progress Reports
You can download the progress reports of the project.
Scientifics Publications
Diagnostic performance of molecular and serological tests of SARS-CoV-2 on well- characterised specimens from COVID-19 individuals: The EDCTP "PERFECT-study" protocol (RIA2020EF-3000)
Joseph Fokam 1,2,3,4, Claudia Alteri5,6, Luna Colagrossi7, Anne-Marie Genevieve8, Désiré Takou1, Alexis Ndjolo1,4, Vittorio Colizzi1,9,10, Nicaise Ndembi11, Carlo- Federico Perno1,7
1 Chantal BIYA International Reference Centre for Research on HIV/AIDS Prevention and Management (CIRCB), Yaoundé, Cameroon, 2 Faculty of Health Sciences (FHS), University of Buea, Buea, Cameroon, 3 National Public Health Emergency Operations Coordination Centre (NPHEOCC), Yaounde, Cameroon, 4 Faculty of Medicine and Biomedical Sciences (FMBS), University of Yaounde I, Yaounde, Cameroon, 5 University of Milan, Milan, Italy, 6 AVIRALIA Foundation Onlus, Rome, Italy, 7 Bambino Gesu Children’s Hospital, Rome, Italy, 8 Association de Recherche en Virologie et Dermatologie (ARVD), Paris, France, 9 University of Rome Tor Vergata, Rome, Italy, 10 Evangelical University of Cameroon, Bandjoun, Cameroon, 11 Africa Centres for Disease Control and Prevention, Abbis Ababa, Ethiopia
PLoS ONE 17(9): e0273818. https://doi.org/10.1371/journal. pone.0273818
Abstract
Background : The SARS-CoV-2 pandemic is a global threat affecting 210 countries, with 2,177,469 con- firmed cases and 6.67% case fatality rate as of April 16, 2020. In Africa, 17,243 cases have been confirmed, but many remain undiagnosed due to limited laboratory-capacity, subopti- mal performance of used molecular-assays (~30% false negative, Yu et al. and Zhao et al., 2020) and limited WHO-recommended rapid-tests. Objectives : We aim to implement measures to minimize risks for COVID-19 in Cameroon, putting together multidisciplinary highly-experienced virologists, immunologists, bioinformaticians and clinicians, to achieve the following objectives: (a) to integrate/improve available-infra- structure, methodologies, and expertise on COVID-19. For this purpose, we will create a platform enabling researchers/clinicians to better integrate and translate evidence into the COVID-19 clinical-practice; (b) to enhance capacities in Cameroon for screening/detecting individuals with high-risks of COVID-19, by setting-up effective core-facilities on-site; (c) to validate point-of-care SARS-CoV-2 molecular assays allowing same-day result delivery, thus permitting timely diagnosis, treatment, and retention in care of COVID-19 patients; (d) to implement SARS-CoV-2 diagnosis with innovative/highly sensitive ddPCR-based assays and viral genetic characterization; (e) to validate serological assays to identify COVID-19- exposed persons and follow-up of convalescents. Methods : This is a prospective, observational study conducted among COVID-19 suspects/contacts during 24 months in Cameroon. Following consecutive sampling of 1,536 individuals, oro/ nasopharyngeal swabs and sera will be collected. Well characterised biorepositories will be established locally; molecular testing will be performed on conventional real-time qPCR, point-of-care GeneXpert, antigen-tests and digital droplet PCR (ddPCR); SARS-CoV2 amplicons will be sequenced; serological testing will be performed using ELISA, and anti- body-based kits. Sensitivity, specificity, positive- and negative-predictive values will be evaluated. Expected outcomes : These efforts will contribute in creating the technical and clinical environment to facilitate earlier detection of Sars-CoV-2 in Africa in general and in Cameroon in particular. Specifi- cally, the goals will be: (a) to implement technology transfer for capacity-building on conven- tional and point-of-care molecular assays, achieving a desirable performance for clinical diagnosis of SARS-CoV2; (b) to integrate/improve the available infrastructure, methodolo- gies, and expertise on Sars-CoV2 detection; (c) to improve the turn-around-time for diag- nosing COVID-19 infection with obvious advantage for patients/clinical management thanks to low-cost assays, thus permitting timely treatment and retention in care; (d) to assess the epidemiology of COVID-19 and circulating-variants in Cameroon as compared to strains found in other countries.
Epidemiological, virological and clinical features of SARS-CoV-2 among individuals during the first wave in Cameroon: Baseline analysis for the EDCTP PERFECT-Study RIA2020EF-3000
Joseph Fokam,corresponding author 1-3 Désiré Takou, 1 Alex Durand Nka, 1 , 4 , 5 Aude Christelle Ka’e, 1 , 4 , 5 Bouba Yagai, 1 , 4 Collins Ambe Chenwi, 1 , 3 Ezechiel Ngoufack Jagni Semengue, 1 , 4 , 5 Grâce Angong Beloumou, 1 Sandrine Claire Djupsa Ndjeyep, 1 Aissatou Abba, 1 Willy Pabo, 1 Davy Gouissi, 1 Michel Carlos Tommo Tchouaket, 1 Laeticia Yatchou, 1 Krystel Zam, 1 Lucien Mama, 6 Regine Claudette Ekitti, 7 Nadine Fainguem, 1 , 4 , 5 Rachel Kamgaing, 1 Samuel Martin Sosso, 1 Nicaise Ndembi, 8 Vittorio Colizzi, 1 , 4 , 5 Carlo-Federico Perno, 1 , 9 and Alexis Ndjolo 1 , 3
1Chantal BIYA International Reference Centre for Research on HIV/AIDS Prevention and Management (CIRCB), Yaoundé, Cameroon 2Faculty of Health Sciences, University of Buea, Buea, Cameroon 3Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon 4University of Rome Tor Vergata, Rome, Italy 5Evangelical University of Cameroon, Bandjoun, Cameroon 6Cite Verte Health District, Delegation of Public Health, Yaoundé, Cameroon 7Police Medical Centre, Yaoundé, Cameroon 8Africa Centres for Disease Control and Prevention, Addis Ababa, Ethiopia 9IRCCS Bambino Gesù Children’s Hospital, Rome, Italy
J Public Health Afr. 2022 May 24; 13(1): 2142. Published online 2022 May 24. doi: 10.4081/jphia.2022.2142
Abstract
In Cameroon, COVID-19 infection spread rapidly and nationwide, with up to 721 deaths reported. To the best of our knowledge, no study reported the on-theground data using a large patients’ dataset to give a comprehensive knowledge on COVID-19 pandemic in Cameroon. The objective of this study was to shade lights on the epidemiological, virological and clinical features of COVID-19 in the Cameroonian context. An observational study was conducted among symptomatic and asymptomatic individuals tested for SARS-CoV-2 by PCR on nasopharyngeal samples from April 22nd, 2020 to January 5th, 2021. Out of 14119 individuals (59.8% male), overall SARS-CoV-2 positivity was 12.7% (from 7.9% in <10 years to 17.3% in >60 years, p<0.001). The positivity rate of symptomatic individuals was 36.1% versus 9.8% among asymptomatic ones, p<0.001. Age group ≤10 [aOR (95%CI): 0.515 (0.338-0.784), p=0.002] and being symptomatic [aOR (95% CI): 5.108 (4.521-5.771), p<0.001] were predictors of SARS-CoV-2 positivity. Regarding PCR Cycle Threshold (CT), 53.8% of positive individuals had a CT <30. According to age, compared to older individuals, those aged 21-40 years showed a higher proportion with high viraemia (CT<20; 21.3% versus 12.5% respectively, p=0.003). Similarly, symptomatic individuals showed a higher proportion with high viraemia (22.4%), when compared to asymptomatic (13.9%); p<0.001. During this first wave of the pandemic, overall SARS-CoV-2 positivity remained high (>10%) and was associated with the presence of symptoms and older age. Most of the infection is among young and asymptomatic individuals, suggesting the “track-and-test” strategy should target these potential transmitters.
Key word: COVID-19, epidemiology, positivity, viraemia, symptoms, Cameroon
High concordance in SARSCoV-2 detection between automated ( Abbott m2000) and manual ( DaAn gene) RT-PCR systems: The EDCTP PERFECT-Study in Cameroon
Nadine Nguendjoung Fainguem, 1 , 2 , 3 Joseph Fokam, 1 , 4-6 Ezechiel Ngoufack Jagni Semengue, 1 , 2 , 3 Alex Durand Nka, 1 , 2 , 3 Désiré Takou, 1 , 2 Joshua Ageboh Nkembi-leke, 4 Claudia Alteri, 7 Luna Colagrossi, 8 Roméo Bouba Yagai, 1 , 2 Collins Ambe Chenwi, 1 Michel Carlos Tchouaket Tommo, 1 Grace Angong Beloumou, 1 Aude Christelle Ka’e, 1 , 2 , 3 Sandrine Claire Ndjeyep Djupsa, 1 Aissatou Abba, 1 Laeticia Grace Heunko Yatchou, 1 Krystel Nnomo Zam, 1 Rachel Kamgaing, 1 Samuel Martin Sosso, 1 Lucien Mama, 9 Nicaise Ndembi, 10 Vittorio Colizzi, 1 , 2 , 3 Carlo-Federico Perno, 1 , 8 Giulia Cappelli, 11 and Alexis Ndjolo 1 , 5
1Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management (CIRCB), Yaoundé, Cameroon 2University of Rome Tor Vergata, Rome, Italy 3Evangelical University of Cameroon, Bandjoun, Cameroon 4Faculty of Health Sciences, University of Buea, Buea, Cameroon 5Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon 6COVID-19 Laboratory Unit, Operations Section, National Public Health Emergency Operations Centre, Yaounde, Cameroon 7University of Milan, Milan, Italy 8Bambino Gesu Children’s Hospital, Rome, Italy 9Cite Verte Health District, Regional Delegation of Health, Yaounde, Cameroon 10Africa Centres for Disease Control and prevention, Abbis Ababa, Ethiopia 11Institute for Biological Systems, Italian National Research Council (CNR), Rome, Italy
J Public Health Afr. 2022 May 24; 13(1): 2163. Published online 2022 May 24. doi: 10.4081/jphia.2022.2163
Abstract
Molecular diagnosis of COVID-19 is critical to the control of the pandemic, which is a major threat to global health. Several molecular tests have been validated by WHO, but would require operational evaluation in the field to ensure their interoperability in diagnosis. In order to ensure field interoperability in molecular assays for detection of SARS-CoV-2 RNA, we evaluated the diagnostic concordance of SARS-CoV-2 between an automated (Abbott) and a manual (DaAn gene) realtime PCR (rRT-PCR), two commonly used assays in Africa. A comparative study was conducted on 287 nasopharyngeal specimens at the Chantal BIYA International Reference Centre (CIRCB) in Yaounde- Cameroon. Samples were tested in parallel with Abbott and DaAn gene rRT-PCR, and performance characteristics were evaluated by Cohen’s coefficient and Spearman’s correlation. A total of 273 participants [median age (IQR) 36 (26-46) years] and 14 EQA specimens were included in the study. Positivity was on 30.0% (86/287) Abbott and 37.6% (108/287) DaAn gene. Overall agreement was 82.6% (237/287), with k=0.82 (95%CI: 0.777-0.863), indicating an excellent diagnostic agreement. The positive and negative agreement was 66.67% (72/108) and 92.18 % (165/179) respectively. Regarding Viral Load (VL), positive agreement was 100% for samples with high VLs (CT<20). Among positive SARS-CoV- 2 cases, the mean difference in Cycle Threshold (CT) for the manual and Cycle Number (CN) for the automated was 6.75±0.3. The excellent agreement (>80%) between the Abbott and DaAn gene rRTPCR platforms supports interoperability between the two assays. Discordance occurs at low-VL, thus underscoring these tools as efficient weapons in limiting SARS-CoV-2 community transmission.
Key words: Molecular diagnosis, SARS-CoV- 2, rRT-PCR, Concordance, Cameroon
Pre-existing immunity to SARS- CoV-2 before the COVID-19 pandemic era in Cameroon: A comparative analysis according to HIV-status
Abba Aissatou1,2*, Joseph Fokam1,3,4,5*, Ezechiel Ngoufack Jagni Semengue1,6,7, Désiré Takou1, Aude Christelle Ka’e1,6, Collins Chenwi Ambe1,8, Alex Durand Nka1,6,7, Sandrine Claire Djupsa1, Grâce Beloumou1, Laura Ciaffi 9, Michel Carlos Tommo Tchouaket 1,10, Audrey Rachel Mundo Nayang1, Willy Leroi Togna Pabo1, René Ghislain Essomba 4,5,11, Edie G. E. Halle 3, Marie-Claire Okomo4,5,11, Anne-Cecile ZK. Bissek12, Rose Leke1,13, Yap BoumII4, Georges Alain Etoundi Mballa 4,14, Carla Montesano6, Carlo-Federico Perno15, Vittorio Colizzi 1,6,7 and Alexis Ndjolo1,5
1 Laboratory of virology, Chantal BIYA International Reference Center for Research HIV/AIDS Prevention and Management, Yaounde ́ , Cameroon, 2Serology Unit, Garoua Regional Health Centre, Garoua, Cameroon, 3Faculty of Health Sciences, University of Buea, Buea, Cameroon, 4Laboratory Unit, Operations sections, National Public Health Emergency Operations Coordination Centre, Yaounde, Cameroon, 5Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon, 6Department of Biology, University of Rome “Tor Vergata”, Rome, Italy, 7Department of Science and Technology, Evangelical University of Cameroon, Bandjoun, Cameroon, 8Department of General Medicine, Mvangan District Hospital, Mvangan, Cameroon, 9Project Coordinator, National Agency for Research on AIDS and Viral Hepatitis, Yaounde, Cameroon, 10School of Health Sciences, Catholic University of Central Africa, Yaounde, Cameroon, 11National Public Health Laboratory, Ministry of Public Health, Yaounde, Cameroon, 12Division of Health Operational Research, Ministry of Public Health, Yaounde, Cameroon, 13The Biotechnology Center of the University of Yaounde I and the Ministry of Scientific Research, Yaounde, Cameroon, 14Division of Disease, Epidemic and Pandemic Control, Ministry of Public Health, Yaounde, Cameroon, 15Department of Microbilogy, Bambino Gesu Pediatric Hospital, Rome, Italy
J Public Health Afr. 2022 May 24; 13(1): 2163. Published online 2022 May 24. doi: 10.4081/jphia.2022.2163
Abstract
Background: The lower burden of COVID-19 in tropical settings may be due to preexisting cross-immunity, which might vary according to geographical locations and potential exposure to other pathogens. We sought to assess the overall prevalence of SARS-CoV-2 antibodies and determine SARS-CoV-2 seropositivity according to HIV-status before the COVID-19 pandemic era. Methods: A cross-sectional and comparative study was conducted at the Chantal BIYA International Reference Centre (CIRCB) on 288 stored plasma samples (163 HIV-positive versus 125 HIV-negative); all collected in 2017-2018, before the COVID-19 pandemic era. Abbott Panbio™ COVID-19 IgG/IgM assay was used for detecting SARS-CoV-2 immunoglobulin G (IgG) and M (IgM). Among people living with HIV (PLHIV), HIV-1 viral load and TCD4 cell count (LTCD4) were measured using Abbott Real Time PCR and BD FACSCalibur respectively. Statistical analyses were performed, with p<0.05 considered statistically significant. Results: The median [IQR] age was 25 [15-38] years. Overall seropositivity to SARS-CoV-2 antibodies was 13.5% (39/288) of which 7.3% (21) was IgG, 7.3% (21) IgM and 1.0% (3) IgG/IgM. According to HIV-status in the study population, SARSCoV-2 seropositivity was 11.0% (18/163) among HIV-positive versus 16.8% (21/ 125) among HIV-negative respectively, p=0.21. Specifically, IgG was 6.1% (10/ 163) versus 8.8% (11/125), p=0.26; IgM was 5.5% (9/163) versus 9.6%, (12/125), p=0.13 and IgG/IgM was 0.6% (1/163) versus 1.6% (2/125) respectively. Among PLHIV, SARS-CoV-2 seropositivity according to CD4 count was 9.2% (≥500 cells/ µL) versus 1.8% (200-499 cells/µL), (OR=3.5; p=0.04) and 0.6% (<200 cells/µL), (OR=17.7; p<0.01). According to viral load, SARS-CoV-2 seropositivity was 6.7% (≥40 copies/mL) versus 4.9% (<40 copies/mL), (OR= 3.8; p<0.01). Conclusion: Before COVID-19 in Cameroon, cross-reactive antibodies to SARSCoV-2 were in circulation, indicating COVID-19 preexisting immunity. This preexisting immunity may contribute in attenuating disease severity in tropical settings like Cameroon. Of relevance, COVID-19 preexisting immunity is lower with HIV-infection, specifically with viral replication and poor CD4-cell count. As poor CD4-count leads to lower cross-reactive antibodies (regardless of viral load), people living with HIV appear more vulnerable to COVID-19 and should be prioritized for vaccination.
Key words: HIV, SARS-CoV-2, immunoglobulin G/M, T-CD4 lymphocytes, HIV viral load
Development of a Sequencing Protocol for SARS-CoV-2 Genotyping: An alternative Approach for Genomic Surveillance in low and middle-income settings
Davy-Hyacinthe Anguechia Gouissia,b,,Desire Takoua, Ezechiel Ngoufack Jagni Semenguea,c, Grace Beloumoua, Sandrine Djupsaa , Collins Chenwia,d, Alex Durand Nkaa,c, Aissatou Abbaa, Aude Christelle Ka'ea,c, Willy Le Roi Togna Paboa, Aurelie Kengnia, Naomi Karell Etamea, Larissa Gaelle Mokoa,b, Evariste Molimboua, Derrick Tambe Ayuk Ngwesea,b, Rachel Audrey Nayang Mundoa, Michel Tommoa, Nadine Fainguema,c, Vittorio Colizzia,c,e, Carlo-Federico Pernoa,c,f, Joseph Fokama,b,g*
a. Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management, Yaounde, Cameroon; b. Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon; c. University of Rome “Tor Vergata”, Rome, Italy; d. Mvangan District Hospital, Mvangan, Cameroon e. Faculty of Science and Technology, Evangelic University of Cameroon, Bandjoun, Cameroon; f. Bambino Gesu Pediatric Hospital, Rome, Italy; g. Faculty of Health Sciences, University of Buea, Buea, Cameroon.
Abstract
Timely detection of SARS-CoV-2 variants of concerns (VOCs) is key for an efficient response against COVID-19 and any future pandemic. However, access to Next Generation Sequencing (reference tool) remains challenging for resource-limited settings. Following experience with the HIV-1 Sanger-sequencing, we developed an in-house SARS-CoV-2 sequencing protocol using specific primers targeting a fragment of SARS-CoV-2 Spike protein region. Using the developed Sanger-sequencing protocol on 163 nasopharyngeal specimens, 130 Spike sequences were successfully generated, giving an overall performance of 79.75% (130/163) [72.76-85.64] (95% CI), effective on SARS-COv-2 lineages of origin and all variants (Alpha, Beta, Gamma, Delta, Omicron) referenced under accession numbers OQ248255 – OQ248384. Thus, in the frame of limited access to NGS, adapting this targeted sequencing protocol would contribute timeously in the genomic surveillance of pathogens with epidemic/pandemic potential. The protocol for sanger sequencing of SARS-CoV-2 was designed using new primers to amplify and sequence a fragment of the part of the Spike protein gene. This sequencing assay were successfully generated, giving an overall performance of 79.75% (130/163). Sanger-sequencing could represent a good alternative to population-based genotyping.
Key word: SARS-CoV-2; Variants of concern; Sanger sequencing; Resource-limited settings.
SARS-CoV-1 Genomic Surveillance and Reliability of PCR Single Point Mutation Assay (SNPsig®EscapePLEX) for the Rapid Detection of Variants of Concern in Cameroon
Joseph Fokama,b,c,d*, Davy-Hyacinthe Anguechia Gouissia,d,, Desire Takoua, Ezechiel Ngoufack Jagni Semenguea,e,f, Collins Chenwia,g, Grace Beloumoua, Sandrine Djupsaa, Alex Durand Nkaa,e,f, Willy Paboa, Aissatou Abbaa, Aude Christelle Ka’ea,e, Aurelie Kengnia, Naomi Karell Etamea, Larissa Gaelle Mokoa,d, Evariste Molimboua,f, Rachel Audrey Nayang Mundoa, Michel Tommoa, Nadine Fainguema,e,f, Lionele Mba Fotsinga , Luna Colagrossih, Claudia Alterii, Dorine Ngonoj, John Otokoye Otshudiemaj, Clement Ndongmok, Yap Boum IIc, Georges Mballa Etoundic, Edie G.E Halleb, Emmanuel Eben-Moussia, Carla Montesanoe, Anne-Genevieve Marcelinl, Vittorio Colizzia,e, Carlo-Federico Pernoh, Alexis Ndjoloa,b,, Nicaise Ndembim,
Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management, Yaounde, Cameroon; Faculty of health sciences, University of Buea, Buea, Cameroon; National Public Health Emergency Operations Centre, Ministry of Public Health, Yaounde, Cameroon; Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon; University of Rome “Tor Vergata”, Rome, Italy; Faculty of Science and Technology, Evangelic University of Cameroon, Bandjoun, Cameroon; Mvangan District Hospital, Mvangan, Cameroon; Bambino Gesu Pediatric Hospital, Rome, Italy; University of Milan, Milan, Italy; World Health Organisation Afro, country office, Yaoundé, Cameroon; Centres for Disease Control and prevention, Yaoundé, Cameroon; Association de Recherche en Virologie et Dermatologie, Paris, France; Africa Centres for Disease Control and Prevention, Abbis Ababa, Ethiopia.
Abstract
Background: Surveillance of SARS-CoV-2 variants of concern (VOC) and lineages is crucial for decision-making.Our objective was to study the SARS-CoV-2 clade dynamics across epidemiological waves and evaluate the reliability of SNP EscapePLEX-kit in detecting VOC in Cameroon. Material and Methods: A laboratory-based study was conducted on SARS-CoV-2 positive nasopharyngeal specimens (Ct-value<30) at the Chantal BIYA International Reference Centre in Yaoundé-Cameroon, between April-2020 to August-2022. For each sample, Sanger42 sequencing and SNP-EscapePLEX-kit were performed, using sequencing as gold standard to evaluate the performance of SNP-EscapePLEX. Results: Of the 130 sequences generated, SARS-CoV-2 clades during wave-1 (April- November 2020) showed 97%(30/31) wild-type lineages and 3%(1/31) Gamma-variant; wave-2 (December-2020 to May-2021), 25%(4/16) Alpha-variant, 25%(4/16) Beta-variant, 44%(7/16) wild-type and 6%(1/16) mu; wave-3 (June-October 2021), 94%(27/29) Delta48 variant, 3%(1/29) Alpha-variant, 3%(1/29) wild-type; wave-4 (November-2021 to August- 2022), 98%(53/54) Omicron-variant and 2%(1/54) Delta-variant. Omicron sub-variants were BA.1(47%), BA.5(34%), BA.2(13%) and BA.4(6%). Overall sensitivity and specificity of SNP-Escaplex was 84%[78-87] and 89%[76-95] respectively, with 75%[63-76] and 100%[96- 100] respectively for Delta-variant; and 96%[90-96] and 100%[93-100] for Omicron-variants. Conclusion: Genomic surveillance reveals a rapid dynamic in SARS-CoV-2 strains between epidemiological waves in Cameroon. For wide variant surveillance in resource-limited settings, EscapePLEX-kit represents a suitable tool, pending upgrading for distinguishing Omicron sub-lineages.
Key word: SARS-CoV-2, variants of concern, SNPsig®EscapePLEX , Cameroon
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