Occurrence of Klebsiella pneumoniae ST244 and ST11 extensively drug-resistant producing KPC, NDM, OXA-370 in wastewater, Brazil.

AIMS: To investigate the occurrence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and their clonal relationships from hospital and municipal wastewater treatment plants (WWTPs). METHODS AND RESULTS: Eighteen Kl. pneumoniae strains recovered from three WWTPs were identified by matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF). The antimicrobial susceptibility were evaluated by disk-diffusion and the carbapenemases production by Carbapenembac®. The carbapenemases genes were investigated by real-time PCR and the clonal relationship through multilocus sequence typing (MLST). Thirty nine % (7/18) of isolates were classified as multidrug-resistant (MDR), 61.1% (11/18) extensively drug-resistant (XDR), and 83.3% (15/18) showed carbapenemase activity. Three carbapenemase-encoding genes were found, blaKPC (55%), blaNDM (27.8%) and blaOXA-370 (11.1%) as well five sequencing types ST11, ST37, ST147, ST244, and ST281. ST11 and ST244, sharing four alleles were grouped into clonal complex 11 (CC11). CONCLUSIONS: Our results show the importance of monitoring antimicrobial resistance in WWTPs effluents to minimize the risk of spreading bacterial load and ARGs in aquatic ecosystems, using advanced treatment technologies to reduce these emerging pollutants at WWTPs.

Molecular Epidemiology of Haemophilus influenzae Strains with Reduced Susceptibility and Genetic Profiles of Resistance in the Postvaccination Period.

Haemophilus influenzae serotype b has been the main cause of invasive infections in children, during the prevaccination period. More than 20 years after the introduction of the conjugate vaccine against Hib, HiNT has emerged as the cause of localized infections in children and adults. The main objective of this work is to evaluate the susceptibility and resistance mechanisms of H. influenzae strains from carriers and describe the molecular epidemiology and their clonal relationships by multilocus sequence typing (MLST). Sixty-nine strains from clinical cases and asymptomatic carriers from 2009 to 2019 were analyzed, confirmed as H. influenzae, and serotyped by polymerase chain reaction. The susceptibility to antibiotics was evaluated by E-test strips. Genotyping was performed by MLST. HiNT was the most frequent in all age groups. Resistance to ampicillin, sulfamethoxazole+trimethoprim, and amoxicillin+clavulanic acid was detected, with the production of ß-lactamase being the main resistance mechanism. Among 21 HiNT strains with complete allelic MLST profiles, 19 new sequence types were described, reinforcing the already reported heterogeneity of nontypeable strains, and only one clonal complex (cc-1355) was observed. Our results show a high percentage of colonization regardless of age, increased antimicrobial resistance, and high genetic diversity, along with an increased number of cases caused by HiNT strains. These findings reinforce the need for continuous surveillance for HiNT strains as it has been reported worldwide after the introduction of the Hib conjugate vaccine.

Characterisation of Neisseria meningitidis cc11/ET-15 variant by whole genome sequencing.

BACKGROUND: Neisseria meningitidis strains belonging to clonal complex 11 is the cause of numerous outbreaks and epidemics in the United States, Canada and Europe, accounting for 49.5% of cases of meningococcal disease caused by serogroup C worldwide. In Brazil, it is the second most frequent clonal complex within this serogroup. The genetic characterisation of cc11/ET-15 variants is important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, to support specific actions of Health Surveillance aiming outbreaks control. OBJECTIVES: The objective of this study was to identify features in the genome of cc11/ET-15 clones through whole-genome sequencing (WGS), that differ from cc11/non-ET-15 strains that could explain their virulence. METHODS: The whole genome of three cc11/ET-15 representative strains were sequenced with a minimum coverage of 100X with the MiSeq System and compared to the genome of cc11/non-ET-15 strains. RESULTS: Genome analysis of cc11/ET-15 variants showed the presence of resistance factors, mobile genetic elements and virulence factors not found in cc11/non-ET-15 strains. MAIN CONCLUSIONS: Our results show that these strains carry virulence factors not identified in cc11/non-ET-15 strains, which could explain the high lethality rates attributed to this clone worldwide.

High-Resolution Melting (HRM) for rapid MLST analysis of Neisseria meningitidis.

The genetic characterization of meningococcal isolates is extremely important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, with the purpose of supporting specific actions of Health Surveillance to contain outbreaks. The objective of this work is to determine a strategy for the epidemiological control of Neisseria meningitidis (Nm) through the detection of genetic signatures of Multilocus Sequence Typing (MLST) genes, by the method of high-resolution DNA melting analysis (qPCR-HRM), to identify the main hypervirulent clones circulating in the country. We analyzed 65 cc103 strains, 19 cc11, 38 cc32 and 8 cc41/44 and 17 were not associated to a specific cc. For the abcZ gene a total of 112 strains were tested, 79 for adk and gdh genes, 87 for aroE, 27 for fumC and 70 strains for pdhC gene. The results obtained were compared and validated with nucleotide sequencing. The percentage of correct allele detection for each clonal complex ranged between 77% and 100%. After an active search in PubMLST, it was found that by inserting results from at least 4 alleles in the MLST database, it is possible to determine the clonal complex of 99% to 100% of the deposited samples. The results obtained in this study suggest that it is possible to identify Nm clonal complexes by a combination analysis of melting curves (TM) of four constitutional genes included in the MLST scheme by qPCR-HRM.

Characterisation of Neisseria meningitidis cc11/ET-15 variant by whole genome sequencing

BACKGROUND Neisseria meningitidis strains belonging to clonal complex 11 is the cause of numerous outbreaks and epidemics in the United States, Canada and Europe, accounting for 49.5% of cases of meningococcal disease caused by serogroup C worldwide. In Brazil, it is the second most frequent clonal complex within this serogroup. The genetic characterisation of cc11/ET-15 variants is important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, to support specific actions of Health Surveillance aiming outbreaks control. OBJECTIVES The objective of this study was to identify features in the genome of cc11/ET-15 clones through whole-genome sequencing (WGS), that differ from cc11/non-ET-15 strains that could explain their virulence. METHODS The whole genome of three cc11/ET-15 representative strains were sequenced with a minimum coverage of 100X with the MiSeq System and compared to the genome of cc11/non-ET-15 strains. RESULTS Genome analysis of cc11/ET-15 variants showed the presence of resistance factors, mobile genetic elements and virulence factors not found in cc11/non-ET-15 strains. MAIN CONCLUSIONS Our results show that these strains carry virulence factors not identified in cc11/non-ET-15 strains, which could explain the high lethality rates attributed to this clone worldwide.

Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell-Culture-Contaminant Mollicutes.

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator's manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum's growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively. The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum - and potentially other species - viability and ultimately applied for reference material production improving the quality control of biological products.

Genetic Relatedness of NDM-Producing Klebsiella pneumoniae Co-Occurring VIM, KPC, and OXA-48 Enzymes from Surveillance Cultures from an Intensive Care Unit.

Klebsiella pneumoniae is an opportunistic bacterial pathogen most commonly associated with nosocomial infections, especially in intensive care unit (ICU) patients. Routine surveillance cultures for carbapenemase-producing Enterobacteriaceae have become a common practice for hospital infection prevention. The objective of this study was to investigate the genetic relatedness of carbapenem-resistant K. pneumoniae carrying blaNDM gene across multilocus sequence typing (MLST) scheme. Surveillance rectal swabs from 4,463 ICU patients admitted to the Rio de Janeiro hospital (March 2016-2017) were screened on CHROMagar mSuperCARBA. Of these, 631 isolates were subjected to VITEK 2 system for phenotypic microbial identification and antibiotic susceptibility testing. Out of 631 isolates, 108 were identified as K. pneumoniae, 103 of which were confirmed by PCR of 16S-23S rDNA internal transcribed spacer (ITS). Eleven blaNDM-positive isolates were subsequently screened for blaKPC, blaBKC, blaIMP, blaVIM, blaSPM, blaOXA-48, and mcr-1-8 genes. Twenty-seven percent (3/11) revealed co-occurrence with KPC, OXA-48, and VIM, 46% (5/11) with KPC and VIM, and 18% (2/11) with VIM type. No strains harbored the blaBKC, blaSPM, blaIMP, and mcr-1 to 8 resistance genes. All 11 isolates were resistant to ß-lactams, ciprofloxacin 90%, tigecycline 82%, gentamicin 73%, and amikacin 18%, and were classified as multidrug resistant (MDR), extensively drug resistant (XDR), and pandrug resistant (PDR). Molecular epidemiology data based on MLST revealed 11 different STs, 8 of which were novel and 3 were previously described. Six out of the eight new STs were associated with MDR and PDR strains and two clonal complexes were reported, including CC258 and CC15. The coexistence of NDM-producing K. pneumoniae and other carbapenemase has been frequently described worldwide. Moreover, we report for the first time K. pneumoniae co-harboring up to four carbapenemases from active surveillance cultures.

Molecular surveillance of brazilian meningococcal isolates serogroup c in the pre and post-men-c-vaccination period: Emergence of ST-3780.

Meningococcal disease is a devastating infection caused by Neisseria meningitidis (meningococcus), and it is classified into serogroups according to its polysaccharide capsule composition. In Brazil, serogroup C is the most frequently responsible for the majority of cases, representing a serious public health challenge. In 2010, the meningococcal serogroup C conjugate vaccine was included in the calendar of the National Immunization Program. We have evaluated 163 meningococcal isolates collected during the pre (2006-2010) and post (2011-2016) vaccination periods. Epidemiological data were determined through Multilocus Sequence Typing (MLST) analysis, vaccine antigens and Bexsero Antigen Sequence Typing (BAST) variant. Clonal complex 103 remains the most prevalent in the country with a high number of serogroup C strains to which CC103 is directly associated. A total of 42 different ST were found. The two most prevalent ST were ST-3780 (CC103) with 38 strains and ST-10781, which was not associated with a CC with nine strains. Allele abcZ-276 was reported among 98% of the strains analyzed and it was not found among other CC103 strains worldwide, makes this allele an important genetic marker for a specific new clone only assigned to Brazilian serogroup C strains, ST-3780. FHbp-25 and NHBA-42 peptides were the most prevalent among isolates in both periods studied. BAST-824 and BAST-3073 have been expressed only in CC103 over the studied years, however, it was not possible to associate a BAST variant to a specific CC. Serogroup C phenotype [P1.22,14-6,36-2: F3-9: ST-3780 (CC103)] was the most prevalent according to the antigenic profiles of circulating strains in Brazil (2007-2016). Our study suggests that CC103 is still a major hypervirulent CC circulating in Brazil and ST-3780 is currently spreading all over the country even after the introduction of MenC in 2010.

Molecular and phenotypical characterization of Cronobacter species isolated with high occurrence from oats and linseeds.

The aim of this study was to determine the prevalence Cronobacter from 30 samples of oats and 30 of linseeds commercially available in Brazil. The detection of Cronobacter was as according to the ISO 22964:2017. The isolates were characterized according to their phenotypically using Vitek 2.0 and antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene, PCR targeting rpoB gene, multiplex-PCR targeting cgcA gene and fusA allele sequencing. A total of 34 samples (56.7%) contained Cronobacter; 19 (63.3%) of linseeds and 15 (50.0%) of oats. The isolates were identified as C. sakazakii (n = 18, 52.9%), C. dublinensis (n = 7, 20.6%), C. turicensis (n = 6, 17.7%) and C. malonaticus (n = 3, 8.8%). Thirty-four Cronobacter isolates were assigned to 11 different fusA alleles of which 3 were new (169, 170 and 171). The PCR targeting rpoB gene and cgcA gene failed to identify 19 isolates. Seven (20.6%) strains showed resistance or intermediate/resistance to tetracycline, and one (2.9%) strain had intermediate resistance to piperacilin-tazobactam. The presence of Cronobacter in oats and linseeds indicate that these foods can be a potential threat to human health, particularly when preparing food for elderly or immunosuppressed persons. The incorrect use of this foods for feeding of neonates (<6 months) by careers should also be avoided.

Characterization of strains of Neisseria meningitidis causing meningococcal meningitis in Mozambique, 2014: Implications for vaccination against meningococcal meningitis.

INTRODUCTION: In sub Saharan Africa, the epidemiology, including the distribution of serogroups of strains of N. meningitidis is poorly investigated in countries outside "the meningitis belt". This study was conducted with the aim to determine the distribution of serogroups of strains of N. meningitidis causing meningococcal meningitis in children and adults in Mozambique. METHODS: A total of 106 PCR confirmed Neisseria meningitidis Cerebrospinal Fluid (CSF) samples or isolates were obtained from the biobank of acute bacterial meningitis (ABM) surveillance being implemented by the National Institute of Health, at three central hospitals in Mozambique, from January to December 2014. Serogroups of N. meningitidis were determined using conventional PCR, targeting siaD gene for Neisseria meningitidis. Outer Membrane Proteins (OMP) Genotyping was performed by amplifying porA gene in nine samples. RESULTS: Of the 106 PCR confirmed Neisseria meningitidis samples, the most frequent serotype was A (50.0%, 53/106), followed by W/Y (18.9%, 20/106), C (8.5%, 9/106), X (7.5%, 8/106) and B (0.9%, 1/106). We found non-groupable strains in a total of 15 (14.2%) samples. PorA genotypes from nine strains showed expected patterns with the exception of two serogroup C strains with P1.19,15,36 and P1.19-36,15 and one serogroup X with P1.19,15,36, variants frequently associated to serogroup B. CONCLUSION: Our data shows that the number of cases of meningococcal meningitis routinely reported in central hospitals in Mozambique is significant and the most dominant serogroup is A. In conclusion, although serogroup A has almost been eliminated from the "meningitis belt", this serogroup remains a major concern in countries outside the belt such as Mozambique.

Epidemiology and molecular characterization of Neisseria lactamica carried in 11-19 years old students in Salvador, Brazil.

Neisseria lactamica is a nonpathogenic commensal bacterium that is potentially associated with the development of natural immunity against N. meningitidis. However, the genetic variation present in natural populations of N. lactamica has not been fully investigated. To better understand its epidemiology and genetic variation, we studied N. lactamica carriage in 1200 students aged 11-19 years old in Salvador, Brazil. The carriage prevalence was 4.5% (54/1200), with no statistical difference among sex and age, although we observed a trend towards higher carriage prevalence among 11-year-old individuals. Whole genome sequence analysis revealed a high genetic diversity among the isolates, with the presence of 32 different STs, 28 (87.5%) of which were new. A total of 21/50 (42%) isolates belonged to three different clonal complexes. While none of the isolates contained nadA or fHpb alleles, we detected 21 FetA variants, 20 NhbA variants and two variants of PorB. The data provide detailed information on circulating N. lactamica isolates in adolescents in Brazil and are complementary to studies in other countries.

Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n=41, C. malonaticus n=10, C. dublinensis n=4, and C. muytjensii n=2) isolated from food (n=54) and clinical specimens (n=3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n=1) and ST440 (n=2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed ß-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, ß-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37°C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37°C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to >3.87Log10CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

The invasive MenC cc103 lineage with penicillin reduced susceptibility persisting in Brazil.

Penicillin is the antibiotic of choice for the treatment of meningococcal infections, and mutations in penA gene are involved with reduced susceptibility (penI) emergence to this antibiotic. This study aimed to characterize the penA allelic diversity, their association with penI phenotype and distribution among prevalent meningococci serogroups in Brazil. The entire penA from 49 invasive strains of distinct serogroups circulating in Brazil for more than two decades were obtained by PCR and sequencing. Additionally, the penA from 22 publicly available complete Neisseria meningitidis genomes from Brazil were included in the study. The allelic diversity was determined and a genetic tree was built using the penA sequence alignment. The penicillin MIC was obtained by the E-Test method. In general, the identified penA alleles correlated with the observed penI phenotype. The canonical penA1 was the most prevalent allele, however, several altered penA were also identified in strains presenting increased penicillin MICs. It was identified a new penA amino acid position (residue 480) that possibly influence the penicillin MIC in some strains. Interestingly, the altered penA14 was found in penI invasive MenC cc103 strains spread in Brazil and persisting since 2011, indicating that the biological cost imposed by penI phenotype can be ameliorated by particular features present in this lineage, which represents an additional public health threat.

The invasive Neisseria meningitidis MenC CC103 from Brazil is characterized by an accessory gene repertoire.

Neisseria meningitidis infections are a major issue for global health. The invasive MenC ST-103 clonal complex (CC103) has been the most prevalent in meningococcal outbreaks in Brazil, occurring also in several countries worldwide. Here we have analysed the population structure and accessory genome of MenC CC103 strains from a global perspective. An in-depth phylogenomic analysis revealed a lineage of N. meningitidis causing meningitis in Brazil and the United Kingdom. This lineage was also characterized as harbouring a particular accessory genome composed of CRISPR/Cas and restriction modification systems. This lineage was also characterized by a genomic island resembling an integrative and conjugative element. This island carried genes potentially associated with virulence and fitness. We propose this accessory gene repertoire could be contributing to the spatial-temporal persistence of the invasive MenC CC103 lineage.

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

Abstract Background Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. Methods A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. Results All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. Conclusions The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples.

BACKGROUND: Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. METHODS: A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. RESULTS: All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. CONCLUSIONS: The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study.

BACKGROUND: The incidence of microcephaly in Brazil in 2015 was 20 times higher than in previous years. Congenital microcephaly is associated with genetic factors and several causative agents. Epidemiological data suggest that microcephaly cases in Brazil might be associated with the introduction of Zika virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid samples of two pregnant women in Brazil whose fetuses were diagnosed with microcephaly. METHODS: In this case study, amniotic fluid samples from two pregnant women from the state of Paraíba in Brazil whose fetuses had been diagnosed with microcephaly were obtained, on the recommendation of the Brazilian health authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks' gestation. The women had presented at 18 weeks' and 10 weeks' gestation, respectively, with clinical manifestations that could have been symptoms of Zika virus infection, including fever, myalgia, and rash. After the amniotic fluid samples were centrifuged, DNA and RNA were extracted from the purified virus particles before the viral genome was identified by quantitative reverse transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic reconstruction and investigation of recombination events were done by comparing the Brazilian Zika virus genome with sequences from other Zika strains and from flaviviruses that occur in similar regions in Brazil. FINDINGS: We detected the Zika virus genome in the amniotic fluid of both pregnant women. The virus was not detected in their urine or serum. Tests for dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus B19 were all negative. After sequencing of the complete genome of the Brazilian Zika virus isolated from patient 1, phylogenetic analyses showed that the virus shares 97-100% of its genomic identity with lineages isolated during an outbreak in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, it clustered with sequences from North and South America, southeast Asia, and the Pacific. After assessing the possibility of recombination events between the Zika virus and other flaviviruses, we ruled out the hypothesis that the Brazilian Zika virus genome is a recombinant strain with other mosquito-borne flaviviruses. INTERPRETATION: These findings strengthen the putative association between Zika virus and cases of microcephaly in neonates in Brazil. Moreover, our results suggest that the virus can cross the placental barrier. As a result, Zika virus should be considered as a potential infectious agent for human fetuses. Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells are warranted. FUNDING: Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Fatal case of co-infection with dengue virus and Neisseria meningitidis during a dengue epidemic in the state of Rio de Janeiro, Brazil.

INTRODUCTION: Dengue and meningococcal disease are caused by two different agents: a flavivirus and a Gram-negative bacterium, respectively. The first symptoms of both diseases can be indistinct and a rapid and accurate diagnosis is crucial, considering that both diseases are associated with high morbidity and mortality, representing a major public-health problem in Brazil. CASE PRESENTATION: We report a fatal case of co-infection of dengue virus (DENV) and Neisseria meningitidis in a 54-year-old patient. The serum tested positive for DENV NS1 antigen, and N. meningitidis serogroup C was detected by nspA-PCR. Following the initial positive result for DENV infection, rRT-PCRwas performed and DENV-4 was confirmed. CONCLUSION: Our report highlights the importance of accurate differential diagnosis during periods of high circulation of DENV, in order to provide adequate management and an improved outcome.

HIV-1 Genetic Diversity and Transmitted Drug Resistance in Antiretroviral Treatment-Naive Individuals from Amapá State, Northern Brazil.

The pattern of HIV-1 subtype distribution and prevalence of transmitted drug resistance mutations (TDRM) is heterogeneous across different Brazilian regions. Little information is available about the molecular epidemiologic profile in Northern Brazil. HIV-1 protease (PR) and reverse transcriptase (RT) sequences were obtained from 97 drug-naive HIV-1-infected individuals from Amapá, one of the most isolated Northern Brazilian states, for subtype determination and analysis of drug resistance mutations. The most prevalent HIV-1 clade observed in Amapá was subtype B (74%), followed by subtype F1 (14%), BF1 recombinants (8%), subtype C (1%), CRF31_BC (1%), and CRF02_AG (1%). Only one TDRM (K103N) was detected in a single patient from our study population. This study reveals that the HIV-1 epidemic in Amapá is characterized by a high level of genetic diversity comparable to that observed in major Brazilian cities, but a much lower rate of TDRM (1%).