Characterization and Investigation of Risk Factors for Late-Relapsing Hepatitis After Yellow Fever.

BACKGROUND: Late-relapsing hepatitis after yellow fever (LHep-YF) during the convalescent phase of the disease has been described during recent yellow fever (YF) outbreaks in Brazil. LHep-YF is marked by a rebound in liver enzymes and nonspecific clinical manifestations around 46-60 days after YF symptom onset. METHODS: Here we have characterized the clinical course and risk factors for LHep-YF using data from a representative cohort of patients who survived YF in Brazil, 2017-2018. A total of 221 YF-positive patients were discharged from the infectious disease reference hospital in Minas Gerais and were followed up at 30, 45, and 60 days post-symptom onset. RESULTS: From 46 to 60 days post-symptom onset, 16% of YF patients (n = 36/221) exhibited a rebound of aminotransferases (aspartate aminotransferase or alanine aminotransferase >500 IU/L), alkaline phosphatase, and total bilirubin levels. Other etiologies of liver inflammation such as infectious hepatitis, autoimmune hepatitis, and metabolic liver disease were ruled out. Jaundice, fatigue, headache, and low platelet levels were associated with LHep-YF. Demographic factors, clinical manifestations, laboratory tests, ultrasound findings, and viral load during the acute phase of YF were not associated with the occurrence of LHep-YF. CONCLUSIONS: These findings provide new data on the clinical course of Late-relapsing hepatitis during the convalescent phase of YF and highlight the need for extended patient follow-up after acute YF.

Wild-Type Yellow Fever Virus RNA in Cerebrospinal Fluid of Child.

We report a 3-year-old child who was hospitalized because of severe manifestations of the central nervous system. The child died after 6 days of hospitalization. Analysis of postmortem cerebrospinal fluid showed the presence of yellow fever virus RNA. Nucleotide sequencing confirmed that the virus was wild-type yellow fever virus.

Assessing the variability of Brazilian Vaccinia virus isolates from a horse exanthematic lesion: coinfection with distinct viruses.

During the last bovine vaccinia (BV) outbreaks, several Vaccinia virus (VACV) strains were isolated and characterised, revealing significant polymorphisms between strains, even within conserved genes. Although the epidemiology of VACV has been studied in BV outbreaks, there is little data about the circulation of the Brazilian VACV isolates. This study describes the genetic and biological characterisation of two VACV isolates, Pelotas 1 virus (P1V) and Pelotas 2 virus (P2V), which were obtained concomitantly from a horse affected by severe cutaneous disease. Despite being isolated from the same exanthematic clinical sample, P1V and P2V showed differences in their plaque phenotype and in one-step growth curves. Moreover, P1V and P2V presented distinct virulence profiles in a BALB/c mouse model, as observed with other Brazilian VACV isolates. Sequencing and phylogenetic analysis of four different genes demonstrated that the isolates are segregated in different VACV clusters. Our results raise interesting questions about the diversity of VACV isolates in Brazil.

The use of denaturing solution as collection and transport media to improve SARS-CoV-2 RNA detection and reduce infection of laboratory personnel.

Accurate testing to detect SARS-CoV-2 RNA is key to counteract the virus spread. Nonetheless, the number of diagnostic laboratories able to perform qPCR tests is limited, particularly in developing countries. We describe the use of a virus-inactivating, denaturing solution (DS) to decrease virus infectivity in clinical specimens without affecting RNA integrity. Swab samples were collected from infected patients and from laboratory personnel using a commercially available viral transport solution and the in-house DS. Samples were tested by RT-qPCR, and exposure to infective viruses was also accessed by ELISA. The DS used did not interfere with viral genome detection and was able to maintain RNA integrity for up to 16 days at room temperature. Furthermore, virus loaded onto DS were inactivated, as attested by attempts to grow SARS-CoV-2 in cell monolayers after DS desalt filtration to remove toxic residues. The DS described here provides a strategy to maintain diagnostic accuracy and protects diagnostic laboratory personnel from accidental infection, as it has helped to protect our lab crew.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Nested-multiplex PCR detection of Orthopoxvirus and Parapoxvirus directly from exanthematic clinical samples.

BACKGROUND: Orthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction. METHODS AND RESULTS: The OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability. CONCLUSION: These results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.

Occurrence of Pseudocowpox virus associated to Bovine viral diarrhea virus-1, Brazilian Amazon.

In 2011, an outbreak of severe vesicular disease occurred in the state of Pará, Amazon region. Besides proliferative or verrucous lesions, cattle showed atypical clinical signs such as diarrhea and leading to death. The animals were submitted to clinical, pathological and molecular diagnosis, and laboratory tests have confirmed the presence of Pseudocowpox virus (PCPV), a Parapoxvirus genus member, and have also found Bovine viral diarrhea virus-1 (BVDV-1), probably causing persistent infection. The results of molecular diagnostics, followed by sequencing data demonstrated the circulation of both viruses (PCPV and BVDV-1) in an area previously affected by another poxvirus, as Vaccinia virus.The cocirculation between PCPV and BVDV-1 indicates a major concern for animal health because the clinical presentation can be a severe disease. This is the first detection of PCPV in the Brazilian Amazon.

Irinotecan/5-fluorouracil combination induces alterations in mitochondrial membrane potential and caspases on colon cancer cell lines.

The combination of irinotecan (CPT-11) and 5-fluorouracil (5-FU) is currently used in the treatment of advanced colorectal carcinoma. When compared to both agents alone, CPT-11 followed by 5-FU treatment demonstrated a synergistic effect. This observation can be related to increased in apoptosis induction after caspase activation. Several studies have demonstrated that changes in mitochondrial membrane potential occur earlier in apoptosis. In this study, we verified whether the collapse in mitochondrial membrane and the activation of caspases is responsible for increased apoptosis observed with CPT-11/5-FU treatment. Thus, HT-29 and SNU-C4 human colon carcinoma cell lines were exposed for 24 h to each drug alone, and to various combinations and treatment sequences, and assessed for colony formation, changes in the mitochondrial membrane potential, and the activities of caspase-3, -8, and -9. The CPT-11/5-FU treatment induced apoptosis in both cell lines; however, the most pronounced effect was observed in HT-29 cells. In these cells, both caspase-3 and -9 were involved in the activation of apoptosis after CPT-11/5-FU treatment. Moreover, in these cells, a reduction of 50% in mitochondrial membrane potential was observed with this treatment. On the other hand, in the SNU-C4 cell line in addition to caspase-3 and-9, caspase-8 seems to be important to apoptosis after CPT-11/5-FU treatment. Furthermore, in this cell line we did not observe alterations in mitochondrial membrane potential. In spite of the differences among the cell lines, these results indicated that the increase in apoptosis in HT-29 cells observed with CPT-11 followed by 5-FU treatment could be explained by a disruption in mitochondria membrane potential that induced caspases activation.

[Bovine parasitic otitis due to rhabditiform nematodes in Gyr cows from the State of Rio de Janeiro, Brazil]. / Otite parasitária bovina por nematóides rhabditiformes em vacas Gir no estado do Rio de Janeiro, Brasil.

The aim of this report was to describe the occurrence of the bovine parasitic otitis caused by rhabditiform nematodes in Gyr cows breed from the State of Rio de Janeiro, Brazil. Eighty six Gyr cows, from the same farm, presenting clinical otitis, were examined for the presence of rhabditiform nematodes with the aid of a swab. All the animals were positive for rhabditiform nematodes in both ear cannals.

The irinotecan/5-fluorouracil combination induces apoptosis and enhances manganese superoxide dismutase activity in HT-29 human colon carcinoma cells.

BACKGROUND: We examined whether induction of apoptosis and Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activities were involved in the greater cytotoxicity of the irinotecan (CPT-11)/5-fluorouracil (5-FU) combination for human colon cancer cells when compared to both drugs alone. METHODS: HT-29 and SNU-C4 human colon carcinoma cell lines were treated with 5-FU and CPT-11, then apoptosis was evaluated by flow cytometry and SOD activities were determined by polyacrylamide gel electrophoresis. RESULTS: Enhanced apoptosis of HT-29 cells was observed with all treatments containing 5-FU in SNU-C4 cells; however, in HT-29 cells, apoptosis was enhanced only with the CPT-11/5-FU combination. In the SNU-C4 cell line, none of the treatments exerted a significant effect on Cu,Zn-SOD or Mn-SOD activity. However, in HT-29 cells, the CPT-11/5-FU combination enhanced Mn-SOD activity when compared to cells treated with CPT-11 alone. Nevertheless, the combined treatment did not interfere with Cu,Zn-SOD activity. CONCLUSION: Treatment with the CPT-11/5-FU combination may promote in HT-29 cell apoptosis by enhancing Mn-SOD activity.