High SARS-CoV-2 infection rates and viral loads in community-dwelling individuals from rural indigenous and mestizo communities from the Andes during the first wave of the COVID-19 pandemic in Ecuador.

Background: Neglected indigenous groups and underserved rural populations in Latin America are highly vulnerable to COVID-19 due to poor health infrastructure and limited access to SARS-CoV-2 diagnosis. The Andean region in Ecuador includes a large number of isolated rural mestizo and indigenous communities living under poverty conditions. Objective: We herein present a retrospective analysis of the surveillance SARS-CoV-2 testing in community-dwelling populations from four provinces in the Ecuadorian Andes, carried out during the first weeks after the national lockdown was lifted in June 2020. Results: A total number of 1,021 people were tested for SARS-CoV-2 by RT-qPCR, resulting in an overall high infection rate of 26.2% (268/1,021, 95% CI: 23.6-29%), which was over 50% in several communities. Interestingly, community-dwelling super spreaders with viral loads over 108 copies/mL represented 7.46% (20/268, 95% CI: 4.8-11.1%) of the SARS-CoV-2 infected population. Conclusion: These results support that COVID-19 community transmission in rural communities from the Andean region was happening at the early stages of the COVID-19 pandemic in Ecuador and point out the weakness of the COVID-19 control program. Community-dwelling individuals in neglected rural and indigenous communities should be considered for a successful control and surveillance program in future pandemics in low- and middle-income countries.

Sustained COVID-19 community transmission and potential super spreading events at neglected afro-ecuadorian communities assessed by massive RT-qPCR and serological testing of community dwelling population.

Background: Neglected ethnic minorities from underserved rural populations in Latin America are highly vulnerable to coronavirus disease 2019 (COVID-19) due to poor health infrastructure and limited access to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. Esmeraldas is a mainly rural province of the Coastal Region of Ecuador characterized by a high presence of Afro-Ecuadorian population living under poverty conditions. Objective: We herein present a retrospective analysis of the surveillance SARS-CoV-2 testing in community-dwelling population from Esmeraldas carried out by our university laboratory in collaboration with regional health authorities during the first week of October 2020, in a region where no public SARS-CoV-2 detection laboratory was available at that time. Results: A total number of 1,259 people were tested for SARS-CoV-2 by Reverse Transcription quantitative Polimerasa Chain Reaction (RT-qPCR), resulting in an overall infection rate of 7.7% (97/1259, 95% CI: [6.32-9.35%]) for SARS-CoV-2, up to 12.1% in some communities. Interestingly, community-dwelling super spreaders with viral loads over 108 copies/ml represented 6.2% of the SARS-CoV-2-infected population. Furthermore, anti-SARS-CoV-2 IgG serological tests were applied to the same study group, yielding an overall seroprevalence of 11.68% (95% CI: [9.98-13.62%]) but as high as 24.47% at some communities. Conclusion: These results support active COVID-19 community transmission in Esmeraldas province during the first semester of the COVID-19 pandemic as it has been shown for other rural communities in the Ecuadorian Coastal Region.

High SARS-CoV-2 Infection Rates Among Special Forces Police Units During the Early Phase of the COVID-19 Pandemic in Ecuador.

Background: At the beginning of the COVID-19 pandemic, health workers and first-responders, such as police officers, were in charge of trying to contain a disease that was unknown at that time. The lack of information and the tremendous need to contain new outbreaks put police officers at higher risk. Methodology: A cross-sectional study was conducted to describe SARS-CoV-2 infection rates among Police Special Forces Officers in Quito, Ecuador. In this study, 163 community-dwelling police officers from elite divisions voluntarily participated in our SARS-CoV-2 detection program using reverse transcription quantitative real-time PCR (RT-qPCR). Results: A total of 20 out of 163 police officers tested positive for SARS-CoV-2, yielding an infection rate of 12.3%. Within this cohort, 10% (2/20) of SARS-CoV-2 positive individuals were potentially super spreaders with viral loads over 108 copies/ul. About 85% of the SARS-CoV-2 positive individuals were asymptomatic and 15% reported mild symptoms related to COVID-19. Conclusions: We found a high SARS-CoV-2 infection rate within the special forces police officers that, beyond a high health risk for themselves, their families, and coworkers. Our results point out the need for permanent SARS-CoV-2 testing among asymptomatic essential workers and first-responders to avoid local outbreaks and to prevent work-place absenteeism among police special units.

Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador)": High quality-low cost local SARS-CoV-2 tests for South America.

BACKGROUND: Dozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies. OBJECTIVE: We evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique. RESULTS: We report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction. CONCLUSIONS: "COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic.

SARS-CoV-2 infection in free roaming dogs from the Amazonian jungle.

During the ongoing COVID-19 pandemic, there were several reports of SARS-CoV-2 transmission from human to animals, mostly to companion cats and dogs but also to free ranging wild species like minks and deers. Under this scenario, SARS-CoV-2 surveillance in domestic animals to assess the risk of transmission between species have been suggested by the OIE. Here we present a case report of SARS-CoV-2 infection in free roaming dogs, found at a rural indigenous community from the Ecuadorian Amazonia. Oral and nasal swabs samples were collected from three dogs found during a COVID-19 surveillance intervention in Amazonian indigenous communities where severe COVID-19 outbreaks were suspected. Total RNA was extracted from dog samples and detection of SARS-CoV-2 gene targets N, ORF1ab and S was performed. The three dogs tested positive for at least two SARS-CoV-2 viral targets. Moreover, there was a high SARS-CoV-2 infection rate of 87.2% within this community. Given that 17.1% of SARS-CoV-2 positive individuals had an ultra high load greater than 108 copies/ml, transmission from humans to dogs likely occurred. To our knowledge, this study is the first report of SARS-CoV-2 positive free roaming dogs. Also, as those animals were found in the Amazonian forest, SARS-CoV-2 transmission to wild mammals is a potential concern. Given the high presence of free roaming dogs associated to rural and indigenous communities in South America, the potential role of these domestic animals on COVID-19 spread would deserve further surveillance studies involving SARS-CoV-2 detection by PCR and molecular epidemiology based on genome sequencing to confirm human to dog transmission.

Corrigendum to: "One health" inspired SARS-CoV-2 surveillance: The Galapagos Islands experience [One Health 11 (2020) 100185].

[This corrects the article DOI: 10.1016/j.onehlt.2020.100185.].

First Report of SARS-CoV-2 Lineage B.1.1.7 (Alpha Variant) in Ecuador, January 2021.

On January 5 2021, Ecuadorian COVID-19 genomic surveillance program detected a suspicious case of the B.1.1.7 lineage (alpha variant) of SARS-CoV-2 in Los Rios province, later confirmed by genome sequencing. The patient travelled from the UK by the end of December 2020. By contact tracing, several new cases were detected confirming B.1.1.7 transmission and spreading in Ecuador.

COVID-19 Community Transmission and Super Spreaders in Rural Villages from Manabi Province in the Coastal Region of Ecuador Assessed by Massive Testing of Community-Dwelling Population.

Neglected rural communities in Latin America are highly vulnerable to COVID-19 due to a poor health infrastructure and limited access to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. Manabí is a province of the Coastal Region of Ecuador characterized by a high prevalence of rural population living under poverty conditions. In the current study, we present the retrospective analysis of the results of a massive SARS-CoV-2 testing operation in nonhospitalized populations from Manabí carried out from August to September 2020. A total of 4,003 people from 15 cantons were tested for SARS-CoV-2 by reverse-transcriptase quantitative polymerase chain reaction, resulting in an overall infection rate of 16.13% for SARS-CoV-2, with several communities > 30%. Moreover, 29 SARS-CoV-2 super-spreader community-dwelling individuals with viral loads above 108 copies/mL were found. These results support that uncontrolled COVID-19 community transmission was happening in Manabí during the first semester of COVID-19 pandemic. This report endorses the utility of massive SARS-CoV-2 testing among asymptomatic population for control and surveillance of COVID-19.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization.

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.

Analytical and clinical evaluation of a heat shock SARS-CoV-2 detection method without RNA extraction for N and E genes RT-qPCR.

BACKGROUND: The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. OBJECTIVE: The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. RESULTS: 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68.8% (232/337) and 89.4% (202/226) of SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98.6%, 93.3% and 84.8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. CONCLUSIONS: Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients at the time of testing. This affordable protocol can help overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

COVID-19 outbreaks at shelters for women who are victims of gender-based violence from Ecuador.

BACKGROUND: One of the constraints in containing the impact of the COVID-19 pandemic in Ecuador is limited testing capacity, especially in high-risk populations such as people living in humanitarian shelters. OBJECTIVES: The "United Nations High Commissioner for Refugees" office in Ecuador in collaboration with "Universidad de Las Américas" performed surveillance screening at shelters for women victims of gender-based violence. They had been granted access to RT-qPCR tests for SARS-CoV-2 diagnosis since July 2020, a few weeks after the general population lockdown was lifted. RESULTS: From 411 people tested, 52 tests were SARS-CoV-2 positive, yielding an overall high attack rate of 12.65%. Moreover, COVID-19 outbreaks were found in nine of 11 shelters that were included in the study. While attacks rates varied among shelters, no association was found with occupancy. CONCLUSION: This study is key to clarifying the epidemiological situation in this highly vulnerable population in Latin America. It highlights the importance of mass testing beyond the symptomatic population to prevent the spread of COVID-19.

Analytical and Clinical Evaluation of "AccuPower SARS-CoV-2 Multiplex RT-PCR kit (Bioneer, South Korea)" and "Allplex 2019-nCoV Assay (Seegene, South Korea)" for SARS-CoV-2 RT-PCR Diagnosis: Korean CDC EUA as a Quality Control Proxy for Developing Countries.

Background: Multiple RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA or their country of origin agency, but many of them lack of proper clinical evaluation. Objective: We evaluated the clinical performance of two Korean SARS-CoV-2 RT-PCR kits available in South America, AccuPower SARS-CoV-2 Multiplex RT-PCR kit (Bioneer, South Korea) and Allplex 2019-nCoV Assay (Seegene, South Korea), for RT-qPCR SARS-CoV-2 diagnosis using the CDC protocol as a gold standard. Results: We found strong differences among both kits clinical performance and analytical sensitivity; while the Allplex 2019-nCoV Assay has sensitivity of 96.5% and an estimated limit of detection of 4,000 copies/ml, the AccuPower SARS-CoV-2 Multiplex RT-PCR kit has a sensitivity of 75.5% and limit of detection estimated to be bigger than 20,000 copies/ml. Conclusions: AccuPower SARS-CoV-2 Multiplex RT-PCR kit and Allplex 2019-nCoV Assay are both made in South Korea but EUA by Korean CDC was only granted to the later. Our results support that Korean CDC EUA should be considered as a quality control proxy for Korean SARS-CoV-2 RT-PCR kits prior to importation by developing countries to guarantee high sensitivity diagnosis.

Crucial contribution of the universities to SARS-CoV-2 surveillance in Ecuador: Lessons for developing countries.

COVID-19 pandemic has challenged public health systems worldwide, particularly affecting developing countries in Latin America like Ecuador. In this report, we exposed the fundamental role of the Ecuadorian universities to improve COVID-19 surveillance in the country, with an overall contribution over 15% of the total SARS-CoV-2 RT-PCR tests done. We highlight the role of our university during the first semester of the COVID-19 pandemic, contributing to a massive free SARS-CoV-2 testing up to almost 10% of the total diagnosis completed in the country, mainly focus on underserved urban, rural and indigenous communities. Finally, we described our contribution to a high quality and low-cost SARS-CoV-2 RT-PCR diagnostic in Ecuador.

Analytical and Clinical Evaluation of Two RT-qPCR SARS-CoV-2 Diagnostic Tests with Emergency Use Authorization in Ecuador.

Dozens of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) or at least by a responsible agency of their country of origin, but many of them lack proper evaluation studies because of COVID-19 pandemic emergency. We evaluated the clinical performance of two commercially available kits in South America, the 2019-nCoV kit (Da An Gene, Guangzhou, China) and GenomeCoV19 kit (ABM, Richmond, Canada), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found striking differences among clinical performance and analytical sensitivity in both kits; whereas the 2019-nCoV kit (Da An Gene) has a limit of detection of 2,000 copies/mL and 100% of sensitivity, the GenomeCoV19 kit (ABM) has a poor sensitivity of 75% and a limit of detection estimated to be over 8.000 copies/mL. The GenomeCoV19 kit (ABM) lacks clinical use authorization in Canada; however, the 2019-nCoV kit (Da An Gene) is authorized by the Chinese CDC. Our results support that only SARS-CoV-2 diagnosis kits with clinical use authorization from their country of origin should be exported to developing countries lacking proper evaluation agencies to avoid a deep impact of the COVID-19 pandemic due to unreliable diagnosis.

Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests.

Hundreds of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with emergency use authorization (EUA) by the Food Drug Administration (FDA) or their country of origin agency, but also many of them without any independent clinical performance evaluation. We performed a clinical evaluation for two Chinese SARS-CoV-2 RT-PCR kits available in South America, COVID-19 Nucleic Acid Test Kit (eDiagnosis Biomedicine, Wuhan, China) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, Changsha, China), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found an excellent clinical performance and analytical sensitivity for both kits with sensitivity values of 100% and 95.3% and estimated limit of detection of 500 copies/mL and 1,000 copies/mL, for eDiagnosis and Sansure Biotech kits, respectively. COVID-19 Nucleic Acid Test Kit (eDiagnosis) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech) are both made in China and hold EUA by the Chinese CDC. Also, Sansure Biotech kit has EUA by the FDA. In conclusion, our results endorse the use of these two commercially available kits imported to Ecuador for SARS-CoV-2 diagnosis, as they had the similar clinical performance as the gold standard from the CDC.

Poor sensitivity of "AccuPower SARS-CoV-2 real time RT-PCR kit (Bioneer, South Korea)".

BACKGROUND: Several molecular kits are available for SARS-CoV-2 diagnosis, mostly lacking of proper clinical evaluation due to the emergency caused by COVID19 pandemia, particularly at developing countries like Ecuador. OBJECTIVE: We carried out an evaluation of the clinical performance of "AccuPower SARS-CoV-2 Real Time RT-PCR kit" (Bioneer, South Korea) for SARS-CoV-2 diagnosis using 2019-nCoV CDC EUA kit (IDT, USA) as a gold standard. RESULTS: 48 clinical specimens were included on the study, 38 tested SARS-CoV-2 positive and 10 SARS-CoV-2 negative for 2019-nCoV CDC EUA kit. For "AccuPower SARS-CoV-2 Real Time RT-PCR kit", only 30 were SARS-CoV-2 positive, indicating a low clinical performance with sensitivity of 78.9%. Moreover, the limit of detection for "AccuPower SARS-CoV-2 Real Time RT-PCR kit" was estimated to be higher than 40,000 viral RNA copies/mL of sample. CONCLUSIONS: Proper clinical performance evaluation studies from government agencies at developing countries should be mandatory prior to clinical use authorization of SARS-CoV-2 diagnosis kits, particularly when those kits lack of either FDA or its country of origin clinical use authorization, to prevent the distribution of low quality products that may have a negative impact of COVID19 surveillance at developing countries.

Testing for SARS-CoV-2 at the core of voluntary collective isolation: Lessons from the indigenous populations living in the Amazon region in Ecuador.

Voluntary collective isolation has been proposed to be the best response to COVID-19 for indigenous populations. While the potential value of voluntary collective isolation is appealing, the feasibility of this approach needs empirical evidence to support it as the best response to protect indigenous communities from COVID-19. This paper describes our experience during SARS-CoV-2 surveillance among Waorani communities in the Ecuadorian Amazonian region, from June to September 2020. We found that self-isolation strategies failed to contain the spread of SARS-CoV-2 from main urban areas to remote and isolated comunities.