Tatiana Elias Colombo

Detection of Mayaro virus infections during a dengue outbreak in Mato Grosso, Brazil

Arboviruses are common agents of human febrile illness worldwide. In dengue-endemic areas illness due to other arboviruses have been misdiagnosed as dengue based only on clinical-epidemiological data. In this study we investigated the presence of Brazilian arboviruses in sera of 200 patients presenting acute febrile illness, during a dengue outbreak in Sinop, MT, Brazil. The results showed that 38 samples were positive to Dengue virus (DENV) type 1, two samples to DENV type 4, and six to Mayaro virus. These results indicate that arboviruses others than DENV are circulating in Sinop and the surrounding region, which are going undiagnosed. In addition, molecular and evolutionary analyses indicate that two MAYV genotypes are co-circulating in Mato Grosso, Brazil. Thus, a strong surveillance program must be implemented to evaluate and monitor the distribution and the true importance of non-dengue arboviruses in the etiology of acute febrile illnesses.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum

A low-cost, equipment-free rapid antigen test distinguishes dengue virus serotypes and Zika virus in patient sera without detectable cross-reactivity. Distinguishing dengue from Zika More than mere summer pests, mosquitoes can transmit viruses, such as dengue and Zika. Diagnosing infections of these related flaviviruses can be difficult because of cross-reactivity in diagnostic tests. Bosch et al. developed monoclonal antibodies to detect viral nonstructural 1 (NS1) protein antigens specific to dengue and Zika. Incorporating the antibodies into an immunochromatography format yielded a rapid diagnostic assay that produces a visual readout in the presence of NS1. The assay identified the four dengue serotypes and Zika viral infections without cross-reaction when testing human serum samples from endemic areas in Central and South America and India. This approach could be useful for developing rapid diagnostics for other emerging pathogens. The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1–4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1–4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction–positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

Identification of fungal diseases at necropsy

The purpose of the Death Verification Service is to elucidate the causes of deaths that occur without medical assistance and of ill-defined deaths. In recent decades, the epidemiological reality of fungal infections has changed due to the rise in opportunistic infections chiefly in immunocompromised patients. A study of fungal diseases in autopsies performed in the Death Verification Service of the Medicine School in São José do Rio Preto between January 2000 and December 2009 was made. Sixty-seven cases of fungal disease, most involving men (70%), were found in 4824 autopsies. Cryptococcosis was the most prevalent (45%), followed by paracoccidioidomycosis, candidiasis, histoplasmosis, aspergillosis and mucormycosis. Associations between AIDS (n=14) and fungal diseases were identified for cryptococcosis (36%), candidiasis (28.5%) and histoplasmosis (28.5%). Pneumonia, AIDS and fungal diseases were evident in 26% of the cases, with the most prevalent etiologies being Cryptococcus neoformans (55.5%) and Histoplasma capsulatum (22%). Pneumonia alone occurred in 43% of cases, with cryptococcosis (53%) and paracoccidioidomycosis (33%) being the main infectious agents. Diabetes mellitus was associated with candidiasis in two cases and aspergillosis in one. One case of renal transplantation linked to paracoccidioidomycosis and one case of bone marrow aplasia with mucormycosis were reported. Despite the reduction in the number of autopsies over recent decades, these findings suggest that this procedure is useful to provide additional data on the etiology, underlying disease and specific risk factors, essential for quality control and to improve treatment protocols.

Clinical, laboratory and virological data from suspected ZIKV patients in an endemic arbovirus area

BACKGROUND The emergence of Zika virus (ZIKV) presents new challenges to both clinicians and public health authorities. Overlapping clinical features between the diseases caused by ZIKV, dengue (DENV) and chikungunya (CHIKV) and the lack of validated serological assays for ZIKV make accurate diagnosis difficult. Brazilian authorities largely rely on clinical and epidemiological data for the epidemiological and clinical classifications of most ZIKV cases. OBJECTIVE To report the laboratory and clinical profiles of patients diagnosed with Zika fever based only on clinical and epidemiological data. STUDY DESIGN We analyzed 433 suspected cases of ZIKV identified by the attending physician based on proposed clinical criteria. The samples were also screened for ZIKV, DENV and CHIKV using PCR. RESULTS Of the 433 patients analyzed, 168 (38.8%) were laboratory-confirmed for arboviruses: 96 were positive for ZIKV, 67 were positive for DENV (56 for DENV-2, 9 for DENV-1, and 2 for DENV-4), four were positive for co-infection with ZIKV/DENV-2, and one was positive for CHIKV. The most common signs or symptoms in the patients with laboratory-confirmed ZIKV were rash (100%), arthralgia (77.1%), fever (74.0%), myalgia (74.0%) and non-purulent conjunctivitis (69.8%). In patients with laboratory-confirmed DENV infections, the most frequently observed symptoms were rash (100%), fever (79.1%), myalgia (74.6%), headache (73.1%) and arthralgia (70.1%). The measure of association between clinical manifestations and laboratory manifestations among patients with ZIKV and DENV detected a statistically significant difference only in abdominal pain (p=0.04), leukopenia (p=0.003), and thrombocytopenia (p=0.01). CONCLUSION Our data suggests that clinical and epidemiological criteria alone are not a good tool for ZIKV and DENV differentiation, and that laboratory diagnosis should be mandatory.

Evaluation of Aptima Zika Virus Assay

ABSTRACT The Zika virus (ZIKV) epidemic in the Americas poses a public health emergency that requires a swift response. Accurate and reliable ZIKV diagnostic tests serve as an important tool for limiting the spread of ZIKV infections. The Aptima Zika virus assay (Hologic, Marlborough, MA) performed on the automated Panther system is a rapid and high-throughput method for detecting ZIKV RNA using transcription-mediated amplification (TMA) technology. We evaluated the performance characteristics of the Aptima Zika virus assay on clinical serum and urine specimens (n = 124) from two different patient populations and samples spiked with ZIKV from three different lineages (n = 10). Compared to the real-time reverse transcription-PCR (rRT-PCR) reference method, the Aptima ZIKV assay detected ZIKV RNA with a diagnostic accuracy of 94.8% (95% confidence interval [CI], 89.4 to 97.6), a sensitivity of 94.7% (95% CI, 73.5 to 99.9), and a specificity of 94.8% (95% CI, 88.9 to 97.8). Similar results were obtained regardless of whether a serum or urine source was used. The limits of detection of the assay at a 95% detection probability were 11.5 genome copy equivalents (GCE)/ml (95% fiducial limits, 7.9 to 20.2) in serum and 17.9 GCE/ml (95% fiducial limits, 13.1 to 27.5) in urine. The Aptima Zika virus assay results were highly reproducible (99%), and no cross-reactivity was seen during the testing of a panel of 95 specimens with potentially interfering substances, such as clinically relevant bacteria, fungi, and viruses, including other flaviviruses. The excellent performance characteristics and the convenience of a fully automated testing system make the Aptima ZIKV assay an attractive choice for clinical laboratories detecting ZIKV RNA from serum and urine.

Dengue virus surveillance: Detection of DENV-4 in the city of São José do Rio Preto, SP, Brazil.

Dengue viruses are the most common arbovirus infection worldwide and are caused by four distinct serotypes of the dengue virus (DENV). In the present study, we assessed DENV transmission in São José do Rio Preto (SJRP) from 2010 to 2014. We analyzed blood samples from febrile patients who were attended at health care centers in SJRP. DENV detection was performed using multiplex RT-PCR, using flavivirus generic primers, based on the genes of the non-structural protein (NS5), followed by nested-PCR assay with species-specific primers. We analyzed 1549 samples, of which 1389 were positive for NS1 by rapid test. One thousand and eight-seven samples (78%) were confirmed as positive by multiplex RT-PCR: DENV-4, 48.5% (528/1087); DENV-1, 41.5% (449/1087); DENV-2, 9.5% (104/1087); and co-infection (5 DENV-1/DENV-4, 1 DENV-1/DENV-2), 0.5% (6/1087). Phylogenetic analysis of the DENV-4 grouped the isolates identified in this study with the American genotype and the showed a relationship between isolates from SJRP and isolates from the northern region of South America. Taken together, our data shows the detection and emergence of new dengue genotype in a new region and reiterate the importance of surveillance programs to detect and trace the evolution of DENV.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

INTRODUCTION The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

The role of lipids in the inception, maintenance and complications of dengue virus infection

Dengue fever is a viral condition that has become a recurrent issue for public health in tropical countries, common endemic areas. Although viral structure and composition have been widely studied, the infection phenotype in terms of small molecules remains poorly established. This contribution providing a comprehensive overview of the metabolic implications of the virus-host interaction using a lipidomic-based approach through direct-infusion high-resolution mass spectrometry. Our results provide further evidence that lipids are part of both the immune response upon Dengue virus infection and viral infection maintenance mechanism in the organism. Furthermore, the species described herein provide evidence that such lipids may be part of the mechanism that leads to blood-related complications such as hemorrhagic fever, the severe form of the disease.

Co-infection between Zika and different Dengue serotypes during DENV outbreak in Brazil

BACKGROUND The recent introduction of new arboviruses in the Americas, as Zika virus (ZIKV) and Chikungunya virus (CHIKV), increased the risk of outbreaks and arboviral co-infections. Herein, we report twelve cases of co-infection of ZIKV and different DENV serotypes in a city located in the northwest region of São Paulo State, Brazil, which is hyper-endemic to Dengue. METHODS Between January and November 2016, 1254 suspected cases of arboviral infection were available by our surveillance program in São José do Rio Preto. All suspected patients were examined and, when they were arboviral disease-suspectd, had sera separated and viral RNA analyzed by PCR/qPCR assays to determine the diagnosis of DENV 1-4, ZIKV, or CHIKV in the same samples. After the molecular results, twelve patients with ZIKV-DENV coinfection were identified and their clinical and laboratory characteristics were described. RESULTS The mean between symptoms onset and collected sample of 3 days. DENV-1 was identified in seven co-infected patients and DEN2 in other five. Two patients presented alarm signs of Dengue and no one was hospitalized. CONCLUSIONS The constant presence of co-circulating arboviruses increases the chance of co-infection and demonstrates the importance of the differential diagnosis, especially during periods of arboviral outbreaks. The impact of this co-infection is known individual and collectively.