Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.

Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa.

The geographic and evolutionary origins of the SARS-CoV-2 Omicron variant (BA.1), which was first detected mid-November 2021 in Southern Africa, remain unknown. We tested 13,097 COVID-19 patients sampled between mid-2021 to early 2022 from 22 African countries for BA.1 by real-time RT-PCR. By November-December 2021, BA.1 had replaced the Delta variant in all African sub-regions following a South-North gradient, with a peak Rt of 4.1. Polymerase chain reaction and near-full genome sequencing data revealed genetically diverse Omicron ancestors already existed across Africa by August 2021. Mutations, altering viral tropism, replication and immune escape, gradually accumulated in the spike gene. Omicron ancestors were therefore present in several African countries months before Omicron dominated transmission. These data also indicate that travel bans are ineffective in the face of undetected and widespread infection.

Epidemiological Patterns of Seasonal Respiratory Viruses during the COVID-19 Pandemic in Madagascar, March 2020-May 2022.

Three epidemic waves of coronavirus disease-19 (COVID-19) occurred in Madagascar from March 2020 to May 2022, with a positivity rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 21% to 33%. Our study aimed to identify the impact of COVID-19 on the epidemiology of seasonal respiratory viruses (RVs) in Madagascar. We used two different specimen sources (SpS). First, 2987 nasopharyngeal (NP) specimens were randomly selected from symptomatic patients between March 2020 and May 2022 who tested negative for SARS-CoV-2 and were tested for 14 RVs by multiplex real-time PCR. Second, 6297 NP specimens were collected between March 2020 and May 2022 from patients visiting our sentinel sites of the influenza sentinel network. The samples were tested for influenza, respiratory syncytial virus (RSV), and SARS-CoV-2. From SpS-1, 19% (569/2987) of samples tested positive for at least one RV. Rhinovirus (6.3%, 187/2987) was the most frequently detected virus during the first two waves, whereas influenza predominated during the third. From SpS-2, influenza, SARS-CoV-2, and RSV accounted for 5.4%, 24.5%, and 39.4% of the detected viruses, respectively. During the study period, we observed three different RV circulation profiles. Certain viruses circulated sporadically, with increased activity in between waves of SARS-CoV-2. Other viruses continued to circulate regardless of the COVID-19 situation. Certain viruses were severely disrupted by the spread of SARS-CoV-2. Our findings underline the importance and necessity of maintaining an integrated disease surveillance system for the surveillance and monitoring of RVs of public health interest.

SARS-CoV-2 infection rate in Antananarivo frontline health care workers, Madagascar.

BACKGROUND: Health care workers (HCWs) represent a vulnerable population during epidemic periods. Our cohort study aimed to estimate the risk of infection and associated factors among HCWs during the first wave of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Madagascar. METHODS: A prospective cohort study was carried out in three hospitals that oversaw the first cases of COVID-19. Monthly ELISA-based serological tests were conducted, and nasopharyngeal swabs were collected in the case of symptoms linked to COVID-19 for RT-PCR analysis. Survival analyses were used to determine factors associated with SARS-CoV-2 infection. RESULTS: The study lasted 7 months from May 2020. We included 122 HCWs, 61.5% of whom were women. The median age was 31.9 years (IQR: 26.4-42.3). In total, 42 (34.4%) had SARS-CoV-2 infections, of which 20 were asymptomatic (47.6%). The incidence of SARS-CoV-2 infection was 9.3% (95% CI [6.5-13.2]) person-months. Sixty-five HCWs presented symptoms, of which 19 were positive by RT-PCR. When adjusted for exposure to deceased cases, infection was more frequent in HCWs younger than 30 years of age (RR = 4.9, 95% CI [1.4-17.2]). CONCLUSION: Our results indicate a high incidence of infection with SARS-CoV-2 among HCWs, with a high proportion of asymptomatic cases. Young HCWs are more likely to be at risk than others. Greater awareness among young people is necessary to reduce the threat of infection among HCWs.

Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2.

Introduction: Accurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility. Materials and Methods: We investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1-51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1). Results: Overall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1. Conclusion: In-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

Full Genome Nobecovirus Sequences From Malagasy Fruit Bats Define a Unique Evolutionary History for This Coronavirus Clade.

Bats are natural reservoirs for both Alpha- and Betacoronaviruses and the hypothesized original hosts of five of seven known zoonotic coronaviruses. To date, the vast majority of bat coronavirus research has been concentrated in Asia, though coronaviruses are globally distributed; indeed, SARS-CoV and SARS-CoV-2-related Betacoronaviruses in the subgenus Sarbecovirus have been identified circulating in Rhinolophid bats in both Africa and Europe, despite the relative dearth of surveillance in these regions. As part of a long-term study examining the dynamics of potentially zoonotic viruses in three species of endemic Madagascar fruit bat (Pteropus rufus, Eidolon dupreanum, Rousettus madagascariensis), we carried out metagenomic Next Generation Sequencing (mNGS) on urine, throat, and fecal samples obtained from wild-caught individuals. We report detection of RNA derived from Betacoronavirus subgenus Nobecovirus in fecal samples from all three species and describe full genome sequences of novel Nobecoviruses in P. rufus and R. madagascariensis. Phylogenetic analysis indicates the existence of five distinct Nobecovirus clades, one of which is defined by the highly divergent ancestral sequence reported here from P. rufus bats. Madagascar Nobecoviruses derived from P. rufus and R. madagascariensis demonstrate, respectively, Asian and African phylogeographic origins, mirroring those of their fruit bat hosts. Bootscan recombination analysis indicates significant selection has taken place in the spike, nucleocapsid, and NS7 accessory protein regions of the genome for viruses derived from both bat hosts. Madagascar offers a unique phylogeographic nexus of bats and viruses with both Asian and African phylogeographic origins, providing opportunities for unprecedented mixing of viral groups and, potentially, recombination. As fruit bats are handled and consumed widely across Madagascar for subsistence, understanding the landscape of potentially zoonotic coronavirus circulation is essential for mitigation of future zoonotic threats.

Robust and Functional Immune Memory Up to 9 Months After SARS-CoV-2 Infection: A Southeast Asian Longitudinal Cohort.

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

Longitudinal monitoring in Cambodia suggests higher circulation of alpha and betacoronaviruses in juvenile and immature bats of three species.

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46-7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar.

As the national reference laboratory for febrile illness in Madagascar, we processed samples from the first epidemic wave of COVID-19, between March and September 2020. We fit generalized additive models to cycle threshold (Ct) value data from our RT-qPCR platform, demonstrating a peak in high viral load, low-Ct value infections temporally coincident with peak epidemic growth rates estimated in real time from publicly-reported incidence data and retrospectively from our own laboratory testing data across three administrative regions. We additionally demonstrate a statistically significant effect of duration of time since infection onset on Ct value, suggesting that Ct value can be used as a biomarker of the stage at which an individual is sampled in the course of an infection trajectory. As an extension, the population-level Ct distribution at a given timepoint can be used to estimate population-level epidemiological dynamics. We illustrate this concept by adopting a recently-developed, nested modeling approach, embedding a within-host viral kinetics model within a population-level Susceptible-Exposed-Infectious-Recovered (SEIR) framework, to mechanistically estimate epidemic growth rates from cross-sectional Ct distributions across three regions in Madagascar. We find that Ct-derived epidemic growth estimates slightly precede those derived from incidence data across the first epidemic wave, suggesting delays in surveillance and case reporting. Our findings indicate that public reporting of Ct values could offer an important resource for epidemiological inference in low surveillance settings, enabling forecasts of impending incidence peaks in regions with limited case reporting.

A novel SARS-CoV-2 related coronavirus in bats from Cambodia.

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses.

Household transmission of COVID-19 among the earliest cases in Antananarivo, Madagascar.

BACKGROUND: Households are among the highest risk for the transmission of SARS-CoV-2. In sub-Saharan Africa, very few studies have described household transmission during the COVID-19 pandemic. Our work aimed to describe the epidemiologic parameters and analyze the secondary attack rate (SAR) in Antananarivo, Madagascar, following the introduction of SARS-CoV-2 in the country in March 2020. METHODS: A prospective case-ascertained study of all identified close contacts of laboratory-confirmed COVID-19 infections was conducted in Antananarivo from March to June 2020. Cases and household contacts were followed for 21 days. We estimated epidemic parameters of disease transmission by fitting parametric distributions based on infector-infected paired data. We assessed factors influencing transmission risk by analyzing the SAR. FINDINGS: Overall, we included 96 index cases and 179 household contacts. Adjusted with the best-fit normal distribution, the incubation period was 4.1 days (95% CI 0.7-7.5]). The serial interval was 6.0 days (95% CI [2.4-9.6]) after adjusting with the best-fit Weibull distribution. On average, each index case infected 1.6 family members (95%CI [0.9-2.3]). The mean SAR among close contacts was 38.8% (95% CI [19.5-58.2]) with the best-fit gamma distribution. Contacts older than 35 years old were more likely to be infected, and the highest SAR was found among them. CONCLUSION: The results of our study provide key insights into the epidemiology of the first wave of SARS-CoV-2 in Madagascar. High rates of household transmission were found in Antananarivo, emphasizing the need for preventive measures to reduce community transmission.

SARS-CoV-2 antibody seroprevalence follow-up in Malagasy blood donors during the 2020 COVID-19 Epidemic.

BACKGROUND: The incidence of the 2020 COVID-19 epidemic in Africa seems to be different from that of the rest of the world, however its true extent is probably underestimated. Conducting population based sero-surveys during the epidemic has moreover been extremely challenging, driving our group and others to study blood donor samples. METHODS: We collected regional epidemiological COVID-19 surveillance data, and simultaneously monitored anti-SARS-CoV-2 antibody seroprevalences monthly throughout the epidemic in 5 major Region-associated Blood Transfusion Centres of Madagascar over a period of 9 months. FINDINGS: Soon after attaining the first epidemic peaks between May and August 2020, both crude and population-weighted test-performance-adjusted seroprevalences of anti-SARS-CoV-2 antibodies was in Malagasy blood donors rapidly increased up to over 40% positivity. INTERPRETATION: These findings suggest a high cumulative incidence of infection and seroconversion, which may have contributed to the observed deceleration of infection rates, but was not sufficient to prevent the second epidemic wave that struck Madagascar in Spring 2021. FUNDING: This project was funded by the United States Agency for International Development.

The COVID-19 epidemic in Madagascar: clinical description and laboratory results of the first wave, march-september 2020.

BACKGROUND: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country. METHODS: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PCR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory-confirmed cases were mapped, and six genomes of viruses were fully sequenced. RESULTS: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2-positive patients, the median age was 39 years (IQR: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A and 20B indicative of several independent introduction of viruses. CONCLUSIONS: Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave.

Assessment of inactivation procedures for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), presents a challenge to laboratorians and healthcare workers around the world. Handling of biological samples from individuals infected with the SARS-CoV-2 virus requires strict biosafety measures. Within the laboratory, non-propagative work with samples containing the virus requires, at minimum, Biosafety Level-2 (BSL-2) techniques and facilities. Therefore, handling of SARS-CoV-2 samples remains a major concern in areas and conditions where biosafety for specimen handling is difficult to maintain, such as in rural laboratories or austere field testing sites. Inactivation through physical or chemical means can reduce the risk of handling live virus and increase testing ability especially in low-resource settings due to easier and faster sample processing. Herein we assess several chemical and physical inactivation techniques employed against SARS-CoV-2 isolates from Cambodia. This data demonstrates that all chemical (AVL, inactivating sample buffer and formaldehyde) and heat-treatment (56 and 98 °C) methods tested completely inactivated viral loads of up to 5 log10.

Nipah virus circulation at human-bat interfaces, Cambodia.

OBJECTIVE: To better understand the potential risks of Nipah virus emergence in Cambodia by studying different components of the interface between humans and bats. METHODS: From 2012 to 2016, we conducted a study at two sites in Kandal and Battambang provinces where fruit bats (Pteropus lylei) roost. We combined research on: bat ecology (reproductive phenology, population dynamics and diet); human practices and perceptions (ethnographic research and a knowledge, attitude and practice study); and Nipah virus circulation in bat and human populations (virus monitoring in bat urine and anti-Nipah-virus antibody detection in human serum). FINDINGS: Our results confirmed circulation of Nipah virus in fruit bats (28 of 3930 urine samples positive by polymerase chain reaction testing). We identified clear potential routes for virus transmission to humans through local practices, including fruit consumed by bats and harvested by humans when Nipah virus is circulating, and palm juice production. Nevertheless, in the serological survey of 418 potentially exposed people, none of them were seropositive to Nipah virus. Differences in agricultural practices among the regions where Nipah virus has emerged may explain the situation in Cambodia and point to actions to limit the risks of virus transmission to humans. CONCLUSION: Human practices are key to understanding transmission risks associated with emerging infectious diseases. Social science disciplines such as anthropology need to be integrated in health programmes targeting emerging infectious diseases. As bats are hosts of major zoonotic pathogens, such integrated studies would likely also help to reduce the risk of emergence of other bat-borne diseases.

Perception of health risks in Lao market vendors.

Wet markets are a critical part of South-East Asian culture and economy. However, their role in circulation and transmission of both endemic and emerging disease is a source of concern in a region considered a hotspot of disease emergence. In the Lao People's Democratic Republic (Lao PDR, Laos), live and dead wild animals are frequently found in wet markets, despite legislation against the bushmeat trade. This is generally considered to increase the risk of disease transmission and emergence, although whether or not wildlife vendors themselves have indeed increased incidence of zoonotic disease has rarely been assessed. In preparation for a future longitudinal study of market vendors investigating vendors' exposure to zoonotic pathogens, we conducted a pilot survey of Lao market vendors of wildlife meat, livestock meat and vegetables, to identify demographic characteristics and potential control groups within markets. We also investigated baseline risk perception for infectious diseases among market vendors and assessed the association between risk perception and risk mitigation behaviours. The surveys conducted with 177 vendors revealed similar age, sex, ethnic background and geographical origin between vendor types, but differences in professional background and work history for livestock meat vendors. The perception of disease risk was very low across all vendors, as was the reported use of personal protective equipment, and the two appeared unrelated. Personal risk discounting and assumptions about transmission routes may explain this lack of association. This information will help inform the development of future research, risk communication and risk mitigation policy, especially in the light of the COVID-19 pandemic.