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.

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus.

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications.

Inmunohistochemical detection of pandemic SARSCoV- 2 antigens in lung tissue. / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARSCoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

CISH and IHC for the Simultaneous Detection of ZIKV RNA and Antigens in Formalin-Fixed Paraffin-Embedded Cell Blocks and Tissues.

Zika virus is an arthropod-borne virus that has recently emerged as a significant public health emergency due to its association with congenital malformations. Serological and molecular tests are typically used to confirm Zika virus infection. These methods, however, have limitations when the interest is in localizing the virus within the tissue and identifying the specific cell types involved in viral dissemination. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) are common histological techniques used for intracellular localization of RNA and protein expression, respectively. The combined use of CISH and IHC is important to obtain information about RNA replication and the location of infected target cells involved in Zika virus neuropathogenesis. There are no reports, however, of detailed procedures for the simultaneous detection of Zika virus RNA and proteins in formalin-fixed paraffin-embedded (FFPE) samples. Furthermore, the chromogenic detection methods for Zika virus RNA published thus far use expensive commercial kits, limiting their widespread use. As an alternative, we describe here a detailed and cost-effective step-by-step procedure for the simultaneous detection of Zika virus RNA and proteins in FFPE samples. First, we describe how to synthesize and purify homemade RNA probes conjugated with digoxygenin. Then, we outline the steps to perform the chromogenic detection of Zika virus RNA using these probes, and how to combine this technique with the immunodetection of viral antigens. To illustrate the entire workflow, we use FFPE samples derived from infected Vero cells as well as from human and mouse brain tissues. These methods are highly adaptable and can be used to study Zika virus or even other viruses of public health relevance, providing an optimal and economical alternative for laboratories with limited resources. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of RNA probes conjugated with digoxigenin (DIG) Basic Protocol 2: Simultaneous detection of ZIKV RNA and proteins in FFPE cell blocks and tissues.

An updated RT-qPCR assay for the simultaneous detection and quantification of chikungunya, dengue and zika viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.

Complete Genome Sequence of a Colombian Zika Virus Strain Obtained from BALB/c Mouse Brain after Intraperitoneal Inoculation.

A Zika virus (ZIKV) strain was isolated from an acute febrile patient during the Zika epidemics in Colombia. The strain was intraperitoneally inoculated into BALB/c mice, and 7 days postinoculation, neurological manifestations and ZIKV infection in the brain were demonstrated. The reported genome sequence is highly related to strains circulating in the Americas.

Asymptomatic Plasmodium spp. infection in Tierralta, Colombia.

With the aim of determining the prevalence of asymptomatic Plasmodium spp. infection by thick smear and PCR and its association with demographic and epidemiological characteristics in the village of Nuevo Tay, Tierralta, Córdoba, Colombia, a cross-sectional population study was carried out, using random probabilistic sampling. Venous blood samples were taken from 212 people on day 0 for thick smear and PCR. Clinical follow-up and thick smears were carried out on days 14 and 28. The prevalence of Plasmodium spp. infection was 17.9% (38/212; 95% CI: 12.5-23.3%) and the prevalence of asymptomatic Plasmodiumspp. infection was 14.6% (31/212; 95% CI: 9.6-19.6%). Plasmodium vivax was found more frequently (20/31; 64.5%) than Plasmodium falciparum (9/31; 29%) and mixed infections (2/31; 6.5%). A significantly higher prevalence of asymptomatic infection was found in men (19.30%) than in women (9.18%) (prevalence ratio: 2.10; 95% CI: 1.01-4.34%; p = 0.02). People who developed symptoms had a significantly higher parasitemia on day 0 than those who remained asymptomatic, of 1,881.5 +/- 3,759 versus 79 +/- 106.9 (p = 0.008). PCR detected 50% more infections than the thick smears. The presence of asymptomatic Plasmodium spp. infection highlights the importance of carrying out active searches amongst asymptomatic populations residing in endemic areas.

[Nuclei ultrastructural changes of C6/36 cells infected with virus dengue type 2].

INTRODUCTION: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. OBJECTIVE: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. MATERIALS AND METHODS: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. RESULTS: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. CONCLUSIONS: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.

Comparison of asymptomatic Plasmodium spp. infection in two malaria-endemic Colombian locations.

BACKGROUND: Asymptomatic infection by Plasmodium spp. could help explain the dynamics of malaria transmission in low-transmission regions. We therefore compared the prevalence of asymptomatic infection by Plasmodium spp. in two Colombian locations, Tierralta and Tumaco, with different transmission patterns, different ecological and epidemiological characteristics and potentially different associated factors. METHOD: Two cross-sectional studies were carried out between September 2006 and November 2007. Infection by Plasmodium spp. was determined using polymerase chain reaction (PCR) and microscopy. RESULTS: Prevalence of asymptomatic infection at day 0 in Tierralta was 11.3% (95% CI 7.2-16.8) by microscopy and 16.5% (95% CI 11.5-22.9) by PCR, while in Tumaco, values were 2.4% (95% CI 0.7-5.5) and 5.8% (95% CI 2.3-9.2) respectively. General prevalence of asymptomatic plasmodium infection after 28 days of follow-up was 5% (95% CI 3.2-7.7), 6.6% (95% CI 3.8-10.6%) in Tierralta and 3.4% (95% CI 1.5-6.6%) in Tumaco. Plasmodium vivax was predominant in Tierralta, P. falciparum in Tumaco. The main associated factors were male sex (aOR 2.5; 95% CI 1.0-6.7) and two to five previous malaria episodes (aOR 3.0; 95% CI 1.0-9.4). PCR detected 61% more infections than microscopy. CONCLUSION: Measurement of the prevalence of asymptomatic Plasmodium spp. infection is suggested as part of the epidemiologic evaluation of malaria in low-transmission areas such as Colombia.

Asymptomatic Plasmodium spp. infection in Tierralta, Colombia

With the aim of determining the prevalence of asymptomatic Plasmodium spp. infection by thick smear and PCR and its association with demographic and epidemiological characteristics in the village of Nuevo Tay, Tierralta, Córdoba, Colombia, a cross-sectional population study was carried out, using random probabilistic sampling. Venous blood samples were taken from 212 people on day 0 for thick smear and PCR. Clinical follow-up and thick smears were carried out on days 14 and 28. The prevalence of Plasmodium spp. infection was 17.9 percent (38/212; 95 percent CI: 12.5-23.3 percent) and the prevalence of asymptomatic Plasmodiumspp. infection was 14.6 percent (31/212; 95 percent CI: 9.6-19.6 percent). Plasmodium vivax was found more frequently (20/31; 64.5 percent) than Plasmodium falciparum (9/31; 29 percent) and mixed infections (2/31; 6.5 percent). A significantly higher prevalence of asymptomatic infection was found in men (19.30 percent) than in women (9.18 percent) (prevalence ratio: 2.10; 95 percent CI: 1.01-4.34 percent; p = 0.02). People who developed symptoms had a significantly higher parasitemia on day 0 than those who remained asymptomatic, of 1,881.5 ± 3,759 versus 79 ± 106.9 (p = 0.008). PCR detected 50 percent more infections than the thick smears. The presence of asymptomatic Plasmodium spp. infection highlights the importance of carrying out active searches amongst asymptomatic populations residing in endemic areas.