Fast, cheap and sensitive: Homogenizer-based RNA extraction free method for SARS-CoV-2 detection by RT-qPCR.

Background: The SARS-CoV-2 gold standard detection method is an RT-qPCR with a previous step of viral RNA extraction from the patient sample either by using commercial automatized or manual extraction kits. This RNA extraction step is expensive and time demanding. Objective: The aim of our study was to evaluate the clinical performance of a simple SARS-CoV-2 detection protocol based on a fast and intense sample homogenization followed by direct RT-qPCR. Results: 388 nasopharyngeal swabs were analyzed in this study. 222 of them tested positive for SARS-CoV-2 by the gold standard RNA extraction and RT-qPCR method, while 166 tested negative. 197 of those 222 positive samples were also positive for the homogenization protocol, yielding a sensitivity of 88.74% (95% IC; 83.83 - 92.58). 166 of those negative samples were also negative for the homogenization protocol, so the specificity obtained was 97% (95% IC; 93.11 - 99.01). For Ct values below 30, meaning a viral load of 103 copies/uL, only 4 SARS-CoV-2 positive samples failed for the RNA extraction free method; for that limit of detection, the homogenizer-based method had a sensitivity of 97.92% (95% CI; 96.01 - 99.83). Conclusions: Our results show that this fast and cheap homogenization method for the SARS-CoV-2 detection by RT-qPCR is a reliable alternative of high sensitivity for potentially infectious SARS-CoV-2 positive patients. This RNA extraction free protocol would help to reduce diagnosis time and cost, and to overcome the RNA extraction kits shortage experienced during COVID-19 pandemic.

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.

High rates of SARS-CoV-2 infection in funeral home workers in Ecuador: Is it an occupational risk for COVID-19?

Aim The COVID-19 outbreak has already caused more than 6.5 million deaths, overwhelming health systems worldwide. The unusual demand for funeral home services could make these workers a potential risk group for occupational exposure to SARS-CoV-2 associated with corpses management for COVID-19 patients. Methodology This is a cross-sectional study aimed to describe the infection rate of SARS-CoV-2 in funeral home staff by testing them with RT-qPCR in Quito, Ecuador. A total of 232 funeral home workers, representing more than 40% of funeral home personnel in Quito, were included in the study, in June 2020, immediately after the population lockdown was lifted in Ecuador. Results A total of 48 individuals tested positive for SARS-CoV-2, yielding an infection rate of 20.7%. The SARS-CoV-2 infection rate was 18.1 and 20.0% among personnel managing corpses or not managing corpses, respectively. Among the SARS-CoV-2 positive patients, 81.3% reported no symptoms related to COVID-19, and 3 individuals had high viral loads over 108 copies/ml. Conclusion The high SARS-CoV-2 infection rate in funeral home staff suggested a potential occupational risk for COVID-19 but not related to corpses management. Public health guidelines for safe corpses management for COVID-19 victims and safe funeral services should be reinforced.

A comparative analysis of SARS-CoV-2 viral load across different altitudes.

SARS-CoV-2 has spread throughout the world, including areas located at high or very high altitudes. There is a debate about the role of high altitude hypoxia on viral transmission, incidence, and COVID-19 related mortality. This is the first comparison of SARS-CoV-2 viral load across elevations ranging from 0 to 4300 m. To describe the SARS-CoV-2 viral load across samples coming from 62 cities located at low, moderate, high, and very high altitudes in Ecuador. An observational analysis of viral loads among nasopharyngeal swap samples coming from a cohort of 4929 patients with a RT-qPCR test positive for SARS-CoV-2. The relationship between high and low altitude only considering our sample of 4929 persons is equal in both cases and not significative (p-value 0.19). In the case of low altitude, adding the sex variable to the analysis, it was possible to find a significative difference between men and women (p-value < 0.05). Considering initially sex and then altitude, it was possible to find a significative difference between high and low altitude for men (p-value 0.05). There is not enough evidence to state that viral load is affected directly by altitude range but adding a new variable as sex in the analysis shows that the presence of new variables influences the relationship of altitude range and viral load. There is no evidence that viral loads (Ct and copies/ml) differ at low or high altitude. Using sex as a co-factor, we found that men have higher viral loads than women at low and moderate altitude locations, while living at high altitude, no differences were found. When Ct values were aggregated by low, moderate, and high viral load, we found no significant differences when sex was excluded from the analysis. We conclude that viral load is not directly affected by altitude, but COVID-19 incidence and mortality are rather affected by socio-demographic and idiosyncratic dynamics.

Clinical Performance of Three Commercial SARS-CoV-2 Rapid Antigen Tests for Community-Dwelling Individuals in a Tropical Setting.

During the second year of the COVID-19 pandemic, the use of Rapid Diagnosis Antigen Tests (RDAgTs) for SARS-CoV-2 detection has substantially increased as some of the brands available in the market were certified for clinical use by international regulatory agencies. RDAgTs are a fast and cheap tool for SARS-CoV-2 surveillance with great potential to improve testing capacities in middle- and low-income countries compared to the gold standard RT-qPCR. However, as the clinical performance of RDAgTs has been shown to vary greatly between the commercial brands available, evaluation studies are necessary. Moreover, the available evaluation has been done in high-income countries while SARS-CoV-2 transmission is also actively happening in developing countries, many of which are located in tropical latitudes where cross-reactivity with other infectious agents is highly prevalent, which could compromise RDAgT specificity. Moreover, unreported mutations and/or new SARS-CoV-2 variants may compromise RDAgT sensitivity as genomic surveillance is limited in these settings. Here we describe a multicenter and manufacturer-independent evaluation of the clinical performance and analytical sensitivity of three different RDAgTs brands available in South America from three companies, Rapigen (South Korea), SD-Biosensor (South Korea), and Certest (Spain), compared to the gold standard RT-qPCR. A total number of 1,646 nasopharyngeal swabs from community-dwelling individuals were included in the study, and 379 of them were SARS-CoV-2 positive by RT-qPCR. The overall sensitivity for each RDAgT was 79% (IC95%: 72 - 86.2), 64.2% (IC95%: 56.7 - 71.6), and 45.8% (IC95%: 35.8 - 55.8) for SD-Biosensor, Certest, and Rapigen, respectively. The overall specificity for each RDAgT was 100%, 97.7% (IC95%: 96.8 - 98.6), and 100% for SD-Biosensor, Certest, and Rapigen, respectively. However, the limit of detection (LoD) to achieve a sensitivity over 90% was substantially lower for Certest RDAgT (102 copies/uL) compared to SD-Biosensor (103 copies/uL) or Rapigen (106 copies/uL) RDAgTs, considering that the gold standard RT-qPCR method used in this study has a high sensitivity of 97.7% and low LoD of 5 copies/uL. Additionally, the Certest RDAgT also showed an improved sensitivity up to 79.7% (IC95%: 70.2 - 89.2) for symptomatic individuals. Finally, the slight reduction in specificity for Certest RDAgTs was only associated with one of the laboratories performing this study, pointing out the need for locally assessed evaluation for RDAgTs like this one carried out in Ecuador. In conclusion, two of the three the RDAgTs tested in this study are a fast, cheap, and point of care tool for SARS-CoV-2 surveillance and reliable enough to detect SARS-CoV-2 infectious individuals.

Clinical Performance of Three Commercial SARS‐CoV‐2 Rapid Antigen Tests for Community-Dwelling Individuals in a Tropical Setting

During the second year of the COVID-19 pandemic, the use of Rapid Diagnosis Antigen Tests (RDAgTs) for SARS-CoV-2 detection has substantially increased as some of the brands available in the market were certified for clinical use by international regulatory agencies. RDAgTs are a fast and cheap tool for SARS-CoV-2 surveillance with great potential to improve testing capacities in middle- and low-income countries compared to the gold standard RT-qPCR. However, as the clinical performance of RDAgTs has been shown to vary greatly between the commercial brands available, evaluation studies are necessary. Moreover, the available evaluation has been done in high-income countries while SARS-CoV-2 transmission is also actively happening in developing countries, many of which are located in tropical latitudes where cross-reactivity with other infectious agents is highly prevalent, which could compromise RDAgT specificity. Moreover, unreported mutations and/or new SARS-CoV-2 variants may compromise RDAgT sensitivity as genomic surveillance is limited in these settings. Here we describe a multicenter and manufacturer‐independent evaluation of the clinical performance and analytical sensitivity of three different RDAgTs brands available in South America from three companies, Rapigen (South Korea), SD-Biosensor (South Korea), and Certest (Spain), compared to the gold standard RT-qPCR. A total number of 1,646 nasopharyngeal swabs from community-dwelling individuals were included in the study, and 379 of them were SARS-CoV-2 positive by RT-qPCR. The overall sensitivity for each RDAgT was 79% (IC95%: 72 - 86.2), 64.2% (IC95%: 56.7 - 71.6), and 45.8% (IC95%: 35.8 - 55.8) for SD-Biosensor, Certest, and Rapigen, respectively. The overall specificity for each RDAgT was 100%, 97.7% (IC95%: 96.8 - 98.6), and 100% for SD-Biosensor, Certest, and Rapigen, respectively. However, the limit of detection (LoD) to achieve a sensitivity over 90% was substantially lower for Certest RDAgT (102 copies/uL) compared to SD-Biosensor (103 copies/uL) or Rapigen (106 copies/uL) RDAgTs, considering that the gold standard RT-qPCR method used in this study has a high sensitivity of 97.7% and low LoD of 5 copies/uL. Additionally, the Certest RDAgT also showed an improved sensitivity up to 79.7% (IC95%: 70.2 – 89.2) for symptomatic individuals. Finally, the slight reduction in specificity for Certest RDAgTs was only associated with one of the laboratories performing this study, pointing out the need for locally assessed evaluation for RDAgTs like this one carried out in Ecuador. In conclusion, two of the three the RDAgTs tested in this study are a fast, cheap, and point of care tool for SARS-CoV-2 surveillance and reliable enough to detect SARS-CoV-2 infectious individuals.

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.

SARS-CoV-2 Viral Load Analysis at Low and High Altitude: A Case Study from Ecuador.

SARS-CoV-2 has spread throughout the world, including remote areas such as those located at high altitudes. There is a debate about the role of hypobaric hypoxia on viral transmission and COVID-19 incidence. A descriptive cross-sectional analysis of SARS-CoV-2 infection and viral load among patients living at low (230 m) and high altitude (3800 m) in Ecuador was completed. Within these two communities, the total number of infected people at the time of the study was 108 cases (40.3%). The COVID-19 incidence proportion at low altitude was 64% while at high altitude was 30.3%. The mean viral load from those patients who tested positive was 3,499,184 copies/mL (SD = 23,931,479 copies/mL). At low altitude (Limoncocha), the average viral load was 140,223.8 copies/mL (SD = 990,840.9 copies/mL), while for the high altitude group (Oyacachi), the mean viral load was 6,394,789 copies/mL (SD = 32,493,469 copies/mL). We found no statistically significant differences when both results were compared (p = 0.056). We found no significant differences across people living at low or high altitude; however, men and younger populations had higher viral load than women older populations, respectively.

SARS-CoV-2 transmission from infected owner to household dogs and cats is associated with food sharing.

OBJECTIVES: Several cases of reverse transmission of SARS-CoV-2 from human to pets were reported during the first year of the COVID-19 pandemic. Accordingly, the World Organization for Animal Health has recommended to improve SARS-CoV-2 surveillance on household animals to assess the risk of transmission between species. After such recommendation, we studied the potential SARS-CoV-2 infection in household dogs and cats in the city of Guayaquil, the most populated city in Ecuador. METHODS: Oral and nasal swab samples were collected from dogs and cats within 10 days of a positive SARS-CoV-2 test result of their owners. Total ribonucleic acid was extracted and detection of viral gene targets N and ORF1ab was performed by quantitative reverse transcription polymerase chain reaction. RESULTS: From the 50 cats and dogs tested, 12 were SARS-CoV-2 positive, giving a total positivity rate of 24%. A total of 1 of 8 cats tested positive, whereas 11 of 42 dogs were positive, yielding a positivity rate of 12.5% and 26.2%, respectively. SARS-CoV-2 was confirmed by whole genome sequencing. In addition, we also found a statistically significant association between SARS-CoV-2 pet positivity and food sharing with infected owners. CONCLUSION: This study is the second active surveillance of SARS-CoV-2 in household dogs and cats in Latin America. Moreover, it is the first study to address the risk factors associated with potential anthropogenic SARS-CoV-2 transmission to domestic cats and dogs. Given the high presence of free-roaming dogs and cats in rural and urban areas in Latin American countries and the high capacity shown by coronaviruses for interspecies transmission, our findings support the view that SARS-CoV-2 surveillance in pets is necessary to better understand the role that pet-human interaction plays in the COVID-19 spread.

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.

Diagnostic Performance of Seven Commercial COVID-19 Serology Tests Available in South America.

BACKGROUND: Although RT-qPCR remains the gold-standard for COVID-19 diagnosis, anti-SARS-CoV-2 serology-based assays have been widely used during 2020 as an alternative for individual and mass testing, and are currently used for seroprevalence studies. OBJECTIVE: To study the clinical performance of seven commercial serological tests for COVID-19 diagnosis available in South America. METHODS: We conducted a blind evaluation of five lateral-flow immunoassays (LFIA) and two enzyme-linked immunosorbent assays (ELISAs) for detecting anti-SARS-CoV-2 antibodies. RESULTS: We found no statistically significant differences among ELISA kits and LFIAs for anti-SARS-CoV-2 IgG sensitivity (values ranging from 76.4% to 83.5%) and specificity (100% for the seven serological assays). For anti-SARS-CoV-2 IgM, the five LFIAs have a significantly higher sensitivity for samples collected 15 days after the first time RT-qPCR positive test, with values ranging from 47.1% to 88.2%; moreover, the specificity varied from 85% to 100%, but the only LFIA brand with a 100% specificity had the lowest sensitivity. CONCLUSION: The diagnostic performance of the seven serological tests was acceptable for the seven brands tested for anti-SARS-CoV-2 IgG detection for seroprevalence screening purposes. On the other hand, our results show the lack of accuracy of anti-SARS-CoV-2 IgM detection in LFIAs as a tool for SARS-CoV-2 acute-phase infection diagnosis.

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.