Case Report: Fatal Viscerotropic Disease in a Young Woman Following Yellow Fever Vaccination.

Yellow fever is a viral hemorrhagic disease, and vaccination is the most effective way to minimize the impact of the disease. Serious adverse events after yellow fever vaccination are rare. We report the case of a young woman with an unusual presentation of yellow fever 17DD vaccine-associated acute viscerotropic disease, with severe hepatic impairment following a long incubation period. She died more than a month after yellow fever vaccination.

Coding-Complete Genome Sequence of a Yellow Fever Virus Isolated from a Baby Howler Monkey (Alouatta caraya) from São Paulo State, Brazil, in 2016.

We report a coding-complete sequence of a yellow fever virus, strain JabSPM02, containing the 3' untranslated region and all coding regions. The virus was recovered from an infected howler monkey from a rural area in São Paulo State, Brazil. Our findings show that it belongs to the South America 1E genotype.

Brefeldin A and Cytochalasin B reduce dengue virus replication in cell cultures but do not protect mice against viral challenge.

BACKGROUND: Dengue is an emerging arboviral disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae and genus Flavivirus. No specific anti-DENV drugs are currently available. METHODS: We investigated the antiviral activity of Brefeldin A (BFA) and Cytochalasin B (CB) against this infection. The drugs BFA and CB were used in the in vitro treatment of dengue-2 virus (DENV-2) infections in Vero cell cultures and in protection from lethality by post-challenge administration in Swiss mice. Viral load was quantified by qRT-PCR and plaque assay in Vero cell cultures, post-infection, treated or not with the drugs. Post-challenge drug levels were evaluated by survival analysis. RESULTS: Our results indicate that doses of 5 µg ml-1 of BFA and 10 µg ml-1 of CB are not toxic to the cells and induce a statistically significant inhibition of DENV-2 replication in Vero cells when compared to control. No BFA- or CB-treated mice survived the challenge with DENV-2. CONCLUSION: These data suggest that BFA and CB have an antiviral action against DENV-2 replication in Vero cell culture, but do not alter infected mice mortality.

A case presentation of a fatal dengue myocarditis showing evidence for dengue virus-induced lesion.

Dengue is a prevalent arthropod-borne viral disease in tropical and subtropical areas of the globe. Dengue clinical manifestations include asymptomatic infections; undifferentiated fever; dengue fever, which is characterized by fever, headache, retroorbital pain, myalgia, and arthralgia; and a severe form of the disease denominated dengue haemorrhagic fever/dengue shock syndrome, characterized by haemoconcentration, thrombocytopenia, and bleeding tendency. However, atypical manifestations, such as liver, central nervous system, and cardiac involvement, have been increasingly reported. We report an atypical and rare presentation of dengue disease marked by a dramatic and fatal cardiogenic shock due to acute myocarditis. Histopathological analysis of heart tissue showed several multifocal areas of muscle necrosis and intense interstitial oedema associated with clusters of virus particles inside the cardiomyocytes and in the interstitial space, providing evidence of a possible direct action of dengue virus on myocardium.

DENV-3 genotype III circulating in São Paulo, Brazil,from 2003 to 2008 is not associated with dengue haemorrhagic fever/dengue shock syndrome.

Dengue viruses (DENV) are the most important arboviruses of public health significance, and compriseof four distinct antigenic serotypes (DENV-1 to 4) that show substantial genetic diversity. These virusesusually cause dengue fever (DF) but some patients progress to a more severe form of the illness, i.e.dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). The first reports of DENV-3 cases inBrazil occurred in the year 2000 with co-circulation of DENV-1 and 2. Thereafter, DENV-3 spreadthroughout the country. DENV-3 phylogenetic analysis has revealed the existence of four to five DENV-3 genotypes. Genotype III of DENV-3 has been the main genotype circulating in Brazil, but recent studieshave indicated that DENV-3 genotype I and genotype V are also circulating in some states of Brazil. Inorder to evaluate DENV-3 genotypes circulating in São Paulo state from 2003 through 2008 we analyzedthe NS1 region of DENV-3 isolated from patients residing in Ribeirão Preto and presenting with differentclinical manifestations of dengue disease. Nucleotide sequences from 31 viruses were obtained andcompared to 105 DENV-3 corresponding sequences retrieved from GenBank. Phylogenetic analysisshowed that São Paulo DENV-3 sequences belong to genotype III and that Puerto Rico strains are closelyrelated to South American strains. There was no association between DENV-3 genotype and DHF/DSS.

SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections.

Dengue is the most important arthropod-borne viral disease in the world. A rapid diagnostic test for dengue is warranted, and real-time polymerase chain reaction may improve diagnosis. TaqMan and Sybr Green systems were evaluated for the diagnosis of dengue virus type 3 (DENV-3) infections. Out of 77 patients with clinical suspicion of dengue infection, specific IgM antibodies were detected in 40 patients. DENV-3 was detected and quantitated in 17 IgM-positive samples by both systems. These assays were shown to be rapid, and specific for detection of DENV-3, and that for diagnostic purposes, there is no difference between these two assays.

Identification of mycobacterium species in contaminated cultures by polymerase chain reaction.

STUDY OBJECTIVES: The detection of Mycobacterium sp on a culture remains the "gold standard" technique for the diagnosis of mycobacterial infections. A small percentage of these cultures, however, may be contaminated by other nonfastidious microorganisms, making accurate diagnosis difficult. We evaluated the use of a polymerase chain reaction (PCR) protocol that was specific for the genus Mycobacterium, and specifically for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare, for the identification of Mycobacterium sp growing on contaminated cultures. DESIGN: This prospective study was designed to identify Mycobacterium sp growing on mycobacterial cultures contaminated with other microorganisms. Samples and patients: Twenty-six samples, taken from 23 patients with probable mycobacterial disease, that resulted in Mycobacterium growth but were contaminated during their processing were evaluated in this study. Clinical data and the clinical status of each patient were used to ascertain the final diagnosis. RESULTS: All samples studied here exhibited Mycobacterium growth on solid media but were contaminated by nonfastidious bacteria, compromising the biochemical identification of the Mycobacterium sp. PCR correctly identified the genus Mycobacterium in all samples. M tuberculosis was identified in 14 samples, and M avium in 10 samples. No amplification of M intracellulare was obtained, and in two samples there was amplification only for the genus Mycobacterium. In the cultures of those patients in whom a mycobacterial infection was evident, PCR identified M avium and M tuberculosis in samples from 6 and 12 patients, respectively. However, PCR identified M avium (two patients) and M tuberculosis (two patients) in the cultures of four patients for whom a mycobacterial disease could not be confirmed by our case definition. Finally, in two samples from one patient only the genus Mycobacterium was amplified by PCR. CONCLUSION: PCR, with its advantages of greater speed and effectiveness than conventional detection methods, was successfully used to identify the Mycobacterium sp growing on contaminated cultures.

One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis.

Improved detection of dengue-1 virus from IgM-positive serum samples using C6/36 cell cultures in association with RT-PCR.

Dengue is the most important arboviral disease in the world, and its diagnosis is primarily made by serology. Virus isolation has been successful mainly in clinical samples obtained during the acute phase of illness, and is carried out through inoculation of clinical samples into C6/36 cell monolayers followed by the detection of infection by indirect immunofluorescence assay (IFA). We compared the efficiency of RT-PCR and IFA in the detection of dengue-1 virus after inoculation of C6/36 cells with samples obtained in the convalescent period of dengue infection. Out of 75 IgM-positive samples inoculated into C6/36 cells, 2 were positive by IFA while 17 were positive by RT-PCR. The 2 IFA-positive samples were collected during the acute phase of the illness; 17 positive samples were found by RT-PCR, including the 2 detected by IFA. For both methods, we also investigated the time necessary for viral detection using a fixed dose of 1 x 10(4) viruses/ml. RT-PCR and IFA detected the dengue virus 1 and 4 days after virus inoculation, respectively. The results obtained here indicate that RT-PCR is the most sensitive method in the detection of dengue viruses using C6/36 cells for viral isolation.

Optimizing dengue diagnosis by RT-PCR in IgM-positive samples: comparison of whole blood, buffy-coat and serum as clinical samples.

Dengue, a major public health problem in tropical and sub-tropical regions, is the most important arboviral disease of humans. Early diagnosis is very important on follow-up of infected patients, especially those at risk of the severe manifestations of this disease. Aiming at the improvement of the molecular diagnosis of these infections and due to the lack of studies that indicated the best sample for dengue virus detection by RT-PCR, viral detection by RT-PCR in blood, serum and buffy-coat of 75 IgM-positive serum samples for dengue was evaluated. Out of the 75 samples, 17 were positive for dengue using RT-PCR and from these samples, three were positive in the blood, 14 positive in the serum and eight positive in the buffy-coat. These results indicate that serum is the best clinical sample for RT-PCR amplification of dengue genomes.