Yellow Fever Virus Genotyping Tool and Investigation of Suspected Adverse Events Following Yellow Fever Vaccination.

The yellow fever (YF) vaccine consists of an attenuated virus, and despite its relative safety, some adverse events following YF vaccination have been described. At the end of 2016, Brazil experienced the most massive sylvatic yellow fever outbreak over the last 70 years and an intense campaign of YF vaccination occurred in Minas Gerais state in Southeast Brazil from 2016 to 2018. The present study aimed to develop a genotyping tool and investigate 21 cases of suspected adverse events following YF vaccination. Initial in silico analyses were performed using partial NS5 nucleotide sequences to verify the discriminatory potential between wild-type and vaccine viruses. Samples from patients were screened for the presence of the YFV RNA, using 5'UTR as the target, and then used for amplification of partial NS5 gene amplification, sequencing, and phylogenetic analysis. Genotyping indicated that 17 suspected cases were infected by the wild-type yellow fever virus, but four cases remained inconclusive. The genotyping tool was efficient in distinguishing the vaccine from wild-type virus, and it has the potential to be used for the differentiation of all yellow fever virus genotypes.

Molecular profiling of drug resistant isolates ofMycobacterium tuberculosis in the state of Santa Catarina, southern Brazil

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoBin RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil.

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosis clinical isolates were analysed by spoligotyping and a partial region of the rpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations within rpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria.

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Recombinant envelope protein-based enzyme immunoassay for IgG antibodies is comparable to neutralization tests for epidemiological studies of dengue infection.

Dengue virus (DENV) is the most prevalent arbovirus in the world, found mainly in tropical regions. As clinical manifestations present frequently as nonspecific febrile illness, laboratory diagnosis is essential to confirm DENV infections and for epidemiological studies. Recombinant envelope (E) antigens of four serotypes of DENV were used to develop an immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA). To evaluate the IgG-ELISA, a panel of serum samples that had been tested previously by a plaque reduction neutralization test (PRNT) was investigated for the presence of anti-E antibodies against the four DENV serotypes. IgG-ELISA was found to have a sensitivity (91%) and specificity (98%) at a receiver-operating characteristic (ROC) optimized cutoff and demonstrated high performance as well as good indexes. A concordance of 97% was achieved between both assays, and only 21/704 (3%) samples were not concordant. The results of the present study demonstrate a moderate correlation between neutralizing antibody titers and IgG-ELISA values. These findings indicate that the recombinant protein-based IgG-ELISA is a suitable method for routine serodiagnosis, monitoring and seroepidemiological studies of DENV infections.