Genotyping of dengue virus from infected tissue samples embedded in paraffin.

Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history.

Epidemiology of cytomegalovirus Infection among mothers and infants in Colombia.

We assessed maternal and infant cytomegalovirus (CMV) infection in Colombia. Maternal serum was tested for CMV immunoglobulin G antibodies at a median of 10 (interquartile range: 8-12) weeks gestation (n = 1501). CMV DNA polymerase chain reaction was performed on infant urine to diagnose congenital (≤21 days of life) and postnatal (>21 days) infection. Maternal CMV seroprevalence was 98.1% (95% confidence interval [CI]: 97.5%-98.8%). Congenital CMV prevalence was 8.4 (95% CI: 3.9%-18.3%; 6/711) per 1000 live births. Among 472 infants without confirmed congenital CMV infection subsequently tested at age 6 months, 258 (54.7%, 95% CI: 50.2%-59.1%) had postnatal infection.

Progressive Vaccinia Acquired through Zoonotic Transmission in a Patient with HIV/AIDS, Colombia.

In March 2015, a patient in Colombia with HIV/AIDS was hospitalized for disseminated ulcers after milking cows that had vesicular lesions on their udders. Vaccinia virus was detected, and the case met criteria for progressive vaccinia acquired by zoonotic transmission. Adherence to an optimized antiretroviral regimen resulted in recovery.

Genomic Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2, Colombia.

Coronavirus disease (COVID-19) in Colombia was first diagnosed in a traveler arriving from Italy on February 26, 2020. However, limited data are available on the origins and number of introductions of COVID-19 into the country. We sequenced the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from 43 clinical samples we collected, along with another 79 genome sequences available from Colombia. We investigated the emergence and importation routes for SARS-CoV-2 into Colombia by using epidemiologic, historical air travel, and phylogenetic observations. Our study provides evidence of multiple introductions, mostly from Europe, and documents >12 lineages. Phylogenetic findings validate the lineage diversity, support multiple importation events, and demonstrate the evolutionary relationship of epidemiologically linked transmission chains. Our results reconstruct the early evolutionary history of SARS-CoV-2 in Colombia and highlight the advantages of genome sequencing to complement COVID-19 outbreak investigations.

Molecular characterization of dengue virus reveals regional diversification of serotype 2 in Colombia.

Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013-2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification.

Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia.

BACKGROUND: Colombia was the second most affected country during the American Zika virus (ZIKV) epidemic, with over 109,000 reported cases. Despite the scale of the outbreak, limited genomic sequence data were available from Colombia. We sought to sequence additional samples and use genomic epidemiology to describe ZIKV dynamics in Colombia. METHODS: We sequenced ZIKV genomes directly from clinical diagnostic specimens and infected Aedes aegypti samples selected to cover the temporal and geographic breadth of the Colombian outbreak. We performed phylogeographic analysis of these genomes, along with other publicly-available ZIKV genomes from the Americas, to estimate the frequency and timing of ZIKV introductions to Colombia. RESULTS: We attempted PCR amplification on 184 samples; 19 samples amplified sufficiently to perform sequencing. Of these, 8 samples yielded sequences with at least 50% coverage. Our phylogeographic reconstruction indicates two separate introductions of ZIKV to Colombia, one of which was previously unrecognized. We find that ZIKV was first introduced to Colombia in February 2015 (95%CI: Jan 2015 - Apr 2015), corresponding to 5 to 8 months of cryptic ZIKV transmission prior to confirmation in September 2015. Despite the presence of multiple introductions, we find that the majority of Colombian ZIKV diversity descends from a single introduction. We find evidence for movement of ZIKV from Colombia into bordering countries, including Peru, Ecuador, Panama, and Venezuela. CONCLUSIONS: Similarly to genomic epidemiological studies of ZIKV dynamics in other countries, we find that ZIKV circulated cryptically in Colombia. More accurately dating when ZIKV was circulating refines our definition of the population at risk. Additionally, our finding that the majority of ZIKV transmission within Colombia was attributable to transmission between individuals, rather than repeated travel-related importations, indicates that improved detection and control might have succeeded in limiting the scale of the outbreak within Colombia.

Detection and Molecular Characterization of Zoonotic Poxviruses Circulating in the Amazon Region of Colombia, 2014.

During 2014, cutaneous lesions were reported in dairy cattle and farmworkers in the Amazon Region of western Colombia. Samples from 6 patients were analyzed by serologic and PCR testing, and results demonstrated the presence of vaccinia virus and pseudocowpox virus. These findings highlight the need for increased poxvirus surveillance in Colombia.

Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction.

INTRODUCTION: Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. OBJECTIVE: To develop and test a control RNA for YFV detection through real-time RT-PCR. METHODS: A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. RESULTS: A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). CONCLUSION: The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.

Multisystem Failure in Fatal Dengue: Associations between the Infectious Viral Serotype and Clinical and Histopathological Findings.

Dengue is the most important arthropod-borne viral infection of humans. However, its viral pathogenesis is still unknown. The information collected from dengue fatal cases is crucial for understanding the complex interactions between virulence and host factors. This study aimed to establish possible associations between the clinical characteristics, histopathological changes, replication, and tissue location of viral serotypes in dengue fatal cases. Clinical and histopathological characterizations, antigen localization in tissue, and detection of the infecting serotype and replication using real-time polymerase chain reaction were all performed on the dengue fatal cases. The majority of the cases involved people under the age of 20. Bleeding (48.3%), abdominal pain (44.8%), myalgia (52.9%), and headache (48.3%) were the most common clinical manifestations in the cases. There was multiorgan pathology, with histopathological changes primarily in the liver, spleen, and lung. Similarly, the viral antigen was found primarily in these organs; however, there were no associations between tissue changes, viral location, infecting serotypes, and replication processes. Dengue infection should be considered a multiorgan disease, the outcome of which is possibly not associated with the infecting viral serotype.

Novel Putative Tymoviridae-like Virus Isolated from Culex Mosquitoes in Colombia.

The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5'/3' RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3' end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

Clinical outcomes associated with Mu variant infection during the third epidemic peak of COVID-19 in Colombia.

BACKGROUND: The higher number of cases and deaths caused by COVID-19 in Colombia occurred during the third epidemic peak, where the Mu variant was associated with 50% of the cases. OBJECTIVE: To evaluate the association between the clinical outcome of COVID-19 with health conditions and SARS-CoV-2 lineages. METHODS: In this study, clinical metadata and SARS-CoV-2 lineages from 535 patients with different degrees of COVID-19 severity were obtained after the SARS-CoV-2 genomic surveillance in Colombia. Then, the associations between these variables were determined using a multidimensional unfolding analysis. RESULTS: Asymptomatic, symptomatic, severe, and deceased outcomes represented 15.2%, 29.7%, 7.3%, and 47.8% of the cases, respectively. Males tend to develop more serious COVID-19, and severe or fatal outcomes were typically observed in patients aged >60 years with comorbidities, including chronic obstructive pulmonary disease, heart disease, kidney disease, obesity, asthma, and smoking history. The SARS-CoV-2 Mu and Gamma variants dominated the third epidemic peak and accounted for most fatal cases with odd ratio values of 128.2 (CI 53.0-310.1) and 18.6 (CI 8.294-41.917). CONCLUSION: This study shows the high impact of SARS-CoV-2 lineages with higher prevalence on public health and the importance of monitoring COVID-19 risk factors to control the associated mortality.

Reduced levels of convalescent neutralizing antibodies against SARS-CoV-2 B.1+L249S+E484K lineage.

The E484K mutation at the SARS-CoV-2 Spike protein emerged independently in different variants around the world and has been widely associated with immune escape from neutralizing antibodies generated during previous infection or vaccination. In this work, the B.1 + L249S+E484K lineage was isolated along with A.1, B.1.420, and B.1.111 SARS-CoV-2 lineages without the E484K mutation and the neutralizing titer of convalescent sera was compared using microneutralization assays. While no significant differences in the neutralizing antibody titers were found between A.1 and B.lineages without the E484K mutation, the neutralizing titers against B.1 + L249S+E484K were 1.5, 1.9, 2.1, and 1.3-fold lower than against A.1, B.1.420, B.1.111-I, and B.1.111-II, respectively. However, molecular epidemiological data indicate that there is no increase in the transmissibility rate associated with this new lineage. This study supports the capability of new variants with the E484K mutation to be resistant to neutralization by humoral immunity, and therefore the need to intensify surveillance programs to determine if these lineages represent a risk for public health.

Neutralizing Antibody Responses Elicited by Inactivated Whole Virus and Genetic Vaccines against Dominant SARS-CoV-2 Variants during the Four Epidemic Peaks of COVID-19 in Colombia.

Several SARS-CoV-2 variants of concern (VOC) and interest (VOI) co-circulate in Colombia, and determining the neutralizing antibody (nAb) responses is useful to improve the efficacy of COVID-19 vaccination programs. Thus, nAb responses against SARS-CoV-2 isolates from the lineages B.1.111, P.1 (Gamma), B.1.621 (Mu), AY.25.1 (Delta), and BA.1 (Omicron), were evaluated in serum samples from immunologically naïve individuals between 9 and 13 weeks after receiving complete regimens of CoronaVac, BNT162b2, ChAdOx1, or Ad26.COV2.S, using microneutralization assays. An overall reduction of the nAb responses against Mu, Delta, and Omicron, relative to B.1.111 and Gamma was observed in sera from vaccinated individuals with BNT162b2, ChAdOx1, and Ad26.COV2.S. The seropositivity rate elicited by all the vaccines against B.1.111 and Gamma was 100%, while for Mu, Delta, and Omicron ranged between 32 to 87%, 65 to 96%, and 41 to 96%, respectively, depending on the vaccine tested. The significant reductions in the nAb responses against the last three dominant SARS-CoV-2 lineages in Colombia indicate that booster doses should be administered following complete vaccination schemes to increase the nAb titers against emerging SARS-CoV-2 lineages.

Monkeypox virus genome sequence from an imported human case in Colombia. / Secuencia genómica del virus de la viruela símica de un caso importado en Colombia

INTRODUCTION: Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus with a genome of approximately 197.209 bp. The current classification divides MPXV into three clades: Clade I (Central African or Congo Basin clade) and clades IIa and IIb (West African clades). OBJECTIVE: To report the complete genome and phylogenetic analysis of a human monkeypox case detected in Colombia. MATERIALS AND METHODS: Exudate from vesicular lesions was obtained from a male patient with recent travel history to Spain. A direct genomic approach was implemented in which total DNA from the sample was purified through a column-based method, followed by sequencing on the Nanopore GridION. Reads were aligned against the MPXV reference genome using minimap2 v.2.24 and phylogenetic inference was performed using maximum likelihood estimation. RESULTS: A total of 11.951 reads mapped directly to a reference genome with 96.8% of coverage (190.898 bp). CONCLUSION: Phylogenetic analysis of the MPXV circulating in Colombia demonstrated its close relationship to clade IIb responsible for the multi-country outbreak in 2022.

Introducción. El virus de la viruela del mono (MPXV) está compuesto por un genoma de ADN bicatenario, aproximadamente, de 197.209 pb. La clasificación actual agrupa el MPXV en tres clados: clado I (de la cuenca del Congo en África central), y clados IIa y IIb (de África occidental). Objetivo. Reportar el genoma completo y el análisis filogenético de un caso humano de viruela símica detectado en Colombia. Materiales y métodos. Se obtuvo exudado de lesiones vesiculares de un paciente varón con el antecedente de un viaje reciente a España. Se implementó un enfoque directo, en el cual se purificó el ADN total de la muestra mediante un método basado en columnas, seguido de la secuenciación directa en la plataforma Nanopore GridION. Las lecturas se alinearon con el genoma de referencia del MPXV, utilizando minimap2, v.2.24, y la inferencia filogenética fue realizada mediante la estimación por máxima verosimilitud. Resultados. Un total de 11.951 lecturas se alinearon directamente con el genoma de referencia con una cobertura del 96,8 % (190.898 pb). Conclusión. El análisis filogenético del MPXV circulante en Colombia demostró su estrecha relación con el clado de África occidental (clado IIb) responsable del brote en múltiples países en el 2022.

Low Neutralizing Antibody Titers against the Mu Variant of SARS-CoV-2 in 31 BNT162b2 Vaccinated Individuals in Colombia.

Global surveillance programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are showing the emergence of variants with mutations in the spike protein. Genomic and laboratory surveillance are important to determine if these variants may be more infectious or less susceptible to antiviral treatments and vaccine-induced antibodies. Three of the most predominant SARS-CoV-2 variants in Colombia during the epidemiological peaks of 2021 were isolated: Mu, a variant of interest; Gamma, a variant of concern; B.1.111, which lacks genetic markers associated with greater virulence. Microneutralization assays were performed by incubating 120 mean tissue culture infectious doses (TCID50) of each SARS-CoV-2 isolate with five two-fold serial dilutions of sera from 31 BNT162b2-vaccinated volunteers. The mean neutralization titer (MN50) was calculated by the Reed-Muench method. At the end of August, Mu represented 49% of coronavirus disease 2019 (COVID-19) cases in Colombia, followed by 25% of Gamma. In contrast, B.1.111 became almost undetectable. The evaluation of neutralizing antibodies suggests that patients vaccinated with BNT162b2 generate neutralizing antibody titers against the Mu variant at significantly lower concentrations relative to B.1.111 and Gamma. This study shows the importance of continuing surveillance programs of emerging variants, as well as the need to evaluate the neutralizing antibody response induced by other vaccines.

Prolonged SARS-CoV-2 nucleic acid conversion time in military personnel outbreaks with presence of specific IgG antibodies.

Coronavirus disease 2019 (COVID-19) is transmitted person-to-person mainly by close contact or droplets from respiratory tract. However, the actual time of viral shedding is still uncertain as well as the different routes of transmission. We aimed to characterize RNA shedding from nasopharyngeal and rectal samples in prolonged cases of mild COVID-19 in young male soldiers. Seventy patients from three different military locations were monitored after recommending to follow more strict isolation measures to prevent the spread of the virus. Then, nasopharyngeal, rectal, and blood samples were taken. SARS-CoV-2 RNA was detected by RT-PCR and specific antibodies by chemiluminescent immunoassays. The median nucleic acid conversion time (NACT) was 60 days (IQR: 7-85 days). Rectal swabs were taken in 60 % of patients. Seven patients (10 %) were positive in nasopharyngeal and rectal swabs, and five (7.14 %) remained positive in rectal swabs, but negative in nasopharyngeal samples. Four patients (5.71 %) that had been discharged, were positive again after 15 days. No significant difference was found in nucleic acid conversion time between age groups nor clinical classification. Maintaining distancing among different positive patients is essential as a possible re-exposure to the virus could cause a longer nucleic acid conversion time in SARS-COV-2 infections.

Molecular characterization of mosquitoes (Diptera: Culicidae) from the Colombian rainforest.

A few studies have carried out the taxonomic and molecular characterization of sylvatic mosquito species in Latin America, where some species have been incriminated as vectors for arboviruses and parasites transmission. The present study reports the molecular characterization of mosquito species in the Sierra Nevada de Santa Marta, a natural ecosystem in the Northern coast of Colombia. Manual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. The COI marker was used for species confirmation, and phylogenetic analysis was performed using the neighbor-joining method, with the Kimura-2-Parameters model. Aedes serratus , Psorophora ferox , Johnbelkinia ulopus , Sabethes chloropterus , Sabethes cyaneus , Wyeomyia aporonoma , Wyeomyia pseudopecten , Wyeomyia ulocoma and Wyeomyia luteoventralis were identified. We assessed the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed the identification at the species level. Classical and molecular taxonomy demonstrated to be useful and complementary when morphological characteristics are not well preserved, or the taxonomic group is not represented in public molecular databases.

Novel Highly Divergent SARS-CoV-2 Lineage With the Spike Substitutions L249S and E484K.

COVID-19 pandemics has led to genetic diversification of SARS-CoV-2 and the appearance of variants with potential impact in transmissibility and viral escape from acquired immunity. We report a new and highly divergent lineage containing 21 distinctive mutations (10 non-synonymous, eight synonymous, and three substitutions in non-coding regions). The amino acid changes L249S and E484K located at the CTD and RBD of the Spike protein could be of special interest due to their potential biological role in the virus-host relationship. Further studies are required for monitoring the epidemiologic impact of this new lineage.

Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity has the potential to impact the virus transmissibility and the escape from natural infection- or vaccine-elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in Colombia between January and April 2021, were processed for genome sequencing and lineage determination following the nanopore amplicon ARTIC network protocol and PANGOLIN pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a variant of interest (VOI) with the accumulation of several substitutions affecting the Spike protein, including the amino acid changes I95I, Y144T, Y145S and the insertion 146 N in the N-terminal domain, R346K, E484K and N501Y in the Receptor binding Domain (RBD) and P681H in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and fixation in a relatively short time in Magdalena, Atlantico, Bolivar, Bogotá D.C, and Santander that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies will be required to assess the biological and epidemiologic roles of the substitution pattern found in the B.1.621 lineage.

SARS-CoV-2 sequencing: The technological initiative to strengthen early warning systems for public health emergencies in Latin America and the Caribbean / [Secuenciación del SARS-CoV-2: la iniciativa tecnológica para fortalecer los sistemas de alerta temprana ante emergencias de salud pública en Latinoamérica y el Caribe].

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem on a scale unprecedented in the last 100 years, as has been the response focused on the rapid genomic characterization of SARS-CoV-2 in virtually all regions of the planet. This pandemic emerged during the era of genomic epidemiology, a science fueled by continued advances in next-generation sequencing. Since its recent appearance, genomic epidemiology included the precise identification of new lineages or species of pathogens and the reconstruction of their genetic variability in real time, evidenced in past outbreaks of influenza H1N1, MERS, and SARS. However, the global and uncontrolled scale of this pandemic created a scenario where genomic epidemiology was put into practice en masse, from the rapid identification of SARS-CoV-2 to the registration of new lineages and their active surveillance throughout the world. Prior to the COVID-19 pandemic, the availability of genomic data on circulating pathogens in several Latin America and the Caribbean countries was scarce or nil. With the arrival of SARS-CoV-2, this scenario changed significantly, although the amount of available information remains scarce and, in countries such as Colombia, Brazil, Argentina, and Chile, the genomic information of SARS-CoV-2 was obtained mainly by research groups in genomic epidemiology rather than the product of a public health surveillance policy or program. This indicates the need to establish public health policies aimed at implementing genomic epidemiology as a tool to strengthen surveillance and early warning systems against threats to public health in the region.

La pandemia de COVID-19 causada por el SARS-CoV-2 es un problema de salud pública sin precedentes en los últimos 100 años, así como la respuesta centrada en la caracterización genómica del SARS-CoV-2 prácticamente en todas las regiones del planeta. Esta pandemia surgió durante la era de la epidemiología genómica impulsada por los continuos avances en la secuenciación de próxima generación. Desde su reciente aparición, la epidemiología genómica permitió la identificación precisa de nuevos linajes o especies de agentes patógenos y la reconstrucción de su variabilidad genética en tiempo real, lo que se hizo evidente en los brotes de influenza H1N1, MERS y SARS. Sin embargo, la escala global y descontrolada de esta pandemia ha generado una situación que obligó a utilizar de forma masiva herramientas de la epidemiología genómica como la rápida identificación del SARS-CoV-2 y el registro de nuevos linajes y su vigilancia activa en todo el mundo. Antes de la pandemia de COVID-19 la disponibilidad e datos genómicos de agentes patógenos circulantes en varios países de Latinoamérica y el Caribe era escasa o nula. Con la llegada del SARS-CoV-2 dicha situación cambió significativamente, aunque la cantidad de información disponible sigue siendo escasa y, en países como Colombia, Brasil, Argentina y Chile, la información genómica del SARS-CoV-2 provino principalmente de grupos de investigación en epidemiología genómica más que como producto de una política o programa de vigilancia en salud pública.