Francisella salimarina infection in farmed cobia (Rachycentron canadum) and dusky grouper (Epinephelus marginatus) in Brazil: A case report.

This study aimed to identify and characterize isolates of Francisella salimarina associated with an outbreak on a marine fish farm in Brazil and to analyse their genetic variability and antimicrobial susceptibility. In 2021, diseased cobias (Rachycentron canadum, n = 10) and dusky groupers (Epinephelus marginatus, n = 10) were sampled and subjected to bacteriological and pathological examinations. The isolates obtained were morphologically and biochemically characterized and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) and 16S rRNA gene sequencing. The genetic diversity of these isolates was analysed using repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). Antimicrobial susceptibility was assessed using the disk diffusion technique. Macroscopically, the fish presented skin ulcerations, ocular lesions, hepatomegaly and splenomegaly. A pleomorphic, gram-negative, catalase- and oxidase-positive bacterium was isolated from seven cobias and two groupers. The 16S rRNA gene sequences showed >99% coverage and identity with other deposited sequences of F. salimarina. The results of the biochemical analysis corresponded to these bacterial species. Histologically, granulomas were observed in the spleen, liver and heart of the cobias (n = 6), and necrotizing and fibrinous dermatitis and myositis were identified in some groupers (n = 2). The isolates exhibited the same banding pattern when REP-PCR was performed, indicating that they were clonally related. Finally, the antibiogram test, no inhibition halo was observed for amoxicillin and trimethoprim-sulfamethoxazole. To our knowledge, this is the first report of F. salimarina infection in cobias and dusky groupers.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Delineation of the pan-proteome of fish-pathogenic Streptococcus agalactiae strains using a label-free shotgun approach.

BACKGROUND: Streptococcus agalactiae (GBS) is a major pathogen of Nile tilapia, a global commodity of the aquaculture sector. The aims of this study were to evaluate protein expression in the main genotypes of GBS isolated from diseased fishes in Brazil using a label-free shotgun nano-liquid chromatography-ultra definition mass spectrometry (nanoLC-UDMSE) approach and to compare the differential abundance of proteins identified in strains isolated from GBS-infected fishes and humans. RESULTS: A total of 1070 protein clusters were identified by nanoLC-UDMSE in 5 fish-adapted GBS strains belonging to sequence types ST-260 and ST-927 and the non-typeable (NT) lineage and 1 human GBS strain (ST-23). A total of 1065 protein clusters corresponded to the pan-proteome of fish-adapted GBS strains; 989 of these were identified in all fish-adapted GBS strains (core proteome), and 62 were shared by at least two strains (accessory proteome). Proteins involved in the stress response and in the regulation of gene expression, metabolism and virulence were detected, reflecting the adaptive ability of fish-adapted GBS strains in response to stressor factors that affect bacterial survival in the aquatic environment and bacterial survival and multiplication inside the host cell. Measurement of protein abundance among different hosts showed that 5 and 26 proteins were exclusively found in the human- and fish-adapted GBS strains, respectively; the proteins exclusively identified in fish isolates were mainly related to virulence factors. Furthermore, 215 and 269 proteins were up- and down-regulated, respectively, in the fish-adapted GBS strains in comparison to the human isolate. CONCLUSIONS: Our study showed that the core proteome of fish-adapted GBS strains is conserved and demonstrated high similarity of the proteins expressed by fish-adapted strains to the proteome of the human GBS strain. This high degree of proteome conservation of different STs suggests that, a monovalent vaccine may be effective against these variants.

Streptococcus agalactiae Sequence Type 283 in Farmed Fish, Brazil.

In 2016 and 2017, we characterized outbreaks caused by Streptococcus agalactiae serotype III sequence type (ST) 283 in Nile tilapia farms in Brazil. Whole-genome multilocus sequence typing clustered the fish isolates together with the zoonotic ST283 and other STs related to cases in humans, frogs, dogs, cattle, and dolphins.

Effects of temperature changes in the transcriptional profile of the emerging fish pathogen Francisella noatunensis subsp. orientalis.

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD50) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD50 for Nile tilapia at 28 °C was ∼105.7, whereas at 22 °C, the LD50 was ∼102.2, showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island.

Severe outbreak of Centrocestus formosanus (Trematoda: Heterophyidae) in farm-raised ornamental platies Xiphophorus maculatus.

This report describes a severe outbreak of the gill fluke Centrocestus formosanus in farm-raised platies Xiphophorus maculatus in Brazil, with mortality rate approaching 95%. Typical clinical signs of infection were observed, with microscopic examinations of fresh gills revealing multiple cysts containing a once-folded metacercaria with an X-shaped excretory bladder. The 18S subunit of the metacercariae (BR1) was amplified by PCR, sequenced and analyzed by BLAST. Subsequent phylogenetic analyses revealed that the BR1 isolate was closely related to C. formosanus from Thailand. This is the first report of C. formosanus in ornamental fish in Brazil. Our observations suggest that platies are highly sensitive to this digenetic parasite. Controlling population densities of the parasite's intermediate host, the snail Melanoides tuberculata, would help to reduce outbreaks.

Complete genome sequencing of sixteen Francisella noatunensis subsp. orientalis isolates: A genomic approach for molecular characterization and spread dynamics of this clonal population.

Francisella noatunensis subsp. orientalis (FNO) is an important emerging pathogen associated with disease outbreaks in farm-raised Nile tilapia. FNO genetic diversity using PCR-based typing, no intra-species discrimination was achieved among isolates/strains from different countries, thus demonstrating a clonal behaviour pattern. In this study, we aimed to evaluate the population structure of FNO isolates by comparing whole-genome sequencing data. The analysis of recombination showed that Brazilian isolates group formed a clonal population; whereas other lineages are also supported by this analysis for isolates from foreign countries. The whole-genome multilocus sequence typing (wgMLST) analysis showed varying numbers of dissimilar alleles, suggesting that the Brazilian clonal population are in expansion. Each Brazilian isolate could be identified as a single node by high-resolution gene-by-gene approach, presenting slight genetic differences associated to mutational events. The common ancestry node suggests a single entry into the country before 2012, and the rapid dissemination of this infectious agent may be linked to market sales of infected fingerlings.

Molecular epidemiology of Clostridioides (previously Clostridium) difficile isolates from a university hospital in Minas Gerais, Brazil.

The molecular epidemiology of 38 non-duplicate toxigenic Clostridioides (previously Clostridium) difficile isolates from inpatients from a hospital in Brazil during a 6-year period (2012-2017) were investigated by multilocus sequence typing (MLST) and ribotyping. These isolates were classified into 20 sequence types (ST), six (30%) of which were novel, revealing a high diversity in a single hospital. Classic hypervirulent strains ST1/RT027 and ST11/RT078 were not identified, while ST42 (almost all RT106) was the most common type, being detected in 11 (28.9%) strains. Noteworthy, six (15.8%) isolates were classified into five STs from clade 2, four of which were new ST and RT. Our study suggests that possible hypervirulent strains other than ST1/RT027 might be inadvertently circulating in Brazilian hospitals and highlights the importance of permanent surveillance on circulating strains in a national scale.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMSE approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi.

BACKGROUND: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. METHODS: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. RESULTS: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)]. CONCLUSIONS: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.

Evaluating the efficacy of the new Ion PGM Hi-Q Sequencing Kit applied to bacterial genomes.

Benchtop NGS platforms are constantly evolving to follow new advances in genomics. Thus, the manufacturers are making improvements, such as the recent Ion PGM Hi-Q chemistry. We evaluate the efficacy of this new Hi-Q approach by comparing it with the former Ion PGM kit and the Illumina MiSEQ Nextera 3rd version. The Hi-Q chemistry showed improvement on mapping reads, with 49 errors for 10kbp mapped; in contrast, the former kit had 89 errors. Additionally, there was a reduction of 80% in erroneous variant detection with the Torrent Variant Caller. Also, an enhancement was observed in de novo assembly with a more confident result in whole-genome MLST, with up to 96% of the alleles assembled correctly for both tested microbial genomes. All of these advantages result in a final genome sequence closer to the performance with MiSEQ and will contribute to turn comparative genomic analysis a reliable task.

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa.

SIMBA: a web tool for managing bacterial genome assembly generated by Ion PGM sequencing technology.

BACKGROUND: The evolution of Next-Generation Sequencing (NGS) has considerably reduced the cost per sequenced-base, allowing a significant rise of sequencing projects, mainly in prokaryotes. However, the range of available NGS platforms requires different strategies and software to correctly assemble genomes. Different strategies are necessary to properly complete an assembly project, in addition to the installation or modification of various software. This requires users to have significant expertise in these software and command line scripting experience on Unix platforms, besides possessing the basic expertise on methodologies and techniques for genome assembly. These difficulties often delay the complete genome assembly projects. RESULTS: In order to overcome this, we developed SIMBA (SImple Manager for Bacterial Assemblies), a freely available web tool that integrates several component tools for assembling and finishing bacterial genomes. SIMBA provides a friendly and intuitive user interface so bioinformaticians, even with low computational expertise, can work under a centralized administrative control system of assemblies managed by the assembly center head. SIMBA guides the users to execute assembly process through simple and interactive pages. SIMBA workflow was divided in three modules: (i) projects: allows a general vision of genome sequencing projects, in addition to data quality analysis and data format conversions; (ii) assemblies: allows de novo assemblies with the software Mira, Minia, Newbler and SPAdes, also assembly quality validations using QUAST software; and (iii) curation: presents methods to finishing assemblies through tools for scaffolding contigs and close gaps. We also presented a case study that validated the efficacy of SIMBA to manage bacterial assemblies projects sequenced using Ion Torrent PGM. CONCLUSION: Besides to be a web tool for genome assembly, SIMBA is a complete genome assemblies project management system, which can be useful for managing of several projects in laboratories. SIMBA source code is available to download and install in local webservers at http://ufmg-simba.sourceforge.net .

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

Corynebacterium pseudotuberculosis may be under anagenesis and biovar Equi forms biovar Ovis: a phylogenic inference from sequence and structural analysis.

BACKGROUND: Corynebacterium pseudotuberculosis can be classified into two biovars or biovars based on their nitrate-reducing ability. Strains isolated from sheep and goats show negative nitrate reduction and are termed biovar Ovis, while strains from horse and cattle exhibit positive nitrate reduction and are called biovar Equi. However, molecular evidence has not been established so far to understand this difference, specifically if these C. pseudotuberculosis strains are under an evolutionary process. RESULTS: The ERIC 1 + 2 Minimum-spanning tree from 367 strains of C. pseudotuberculosis showed that the great majority of biovar Ovis strains clustered together, but separately from biovar Equi strains that also clustered amongst themselves. Using evolutionarily conserved genes (rpoB, gapA, fusA, and rsmE) and their corresponding amino acid sequences, we analyzed the phylogenetic relationship among eighteen strains of C. pseudotuberculosis belonging to both biovars Ovis and Equi. Additionally, conserved point mutation based on structural variation analysis was also carried out to elucidate the genotype-phenotype correlations and speciation. We observed that the biovars are different at the molecular phylogenetic level and a probable anagenesis is occurring slowly within the species C. pseudotuberculosis. CONCLUSIONS: Taken together the results suggest that biovar Equi is forming the biovar Ovis. However, additional analyses using other genes and other bacterial strains are required to further support our anagenesis hypothesis in C. pseudotuberculosis.

Comparative genome analysis of Weissella ceti, an emerging pathogen of farm-raised rainbow trout.

BACKGROUND: The genus Weissella belongs to the lactic acid bacteria and includes 18 currently identified species, predominantly isolated from fermented food but rarely from cases of bacteremia in animals. Recently, a new species, designated Weissella ceti, has been correlated with hemorrhagic illness in farm-raised rainbow trout in China, Brazil, and the USA, with high transmission and mortality rates during outbreaks. Although W. ceti is an important emerging veterinary pathogen, little is known about its genomic features or virulence mechanisms. To better understand these and to characterize the species, we have previously sequenced the genomes of W. ceti strains WS08, WS74, and WS105, isolated from different rainbow trout farms in Brazil and displaying different pulsed-field gel electrophoresis patterns. Here, we present a comparative analysis of the three previously sequenced genomes of W. ceti strains from Brazil along with W. ceti NC36 from the USA and those of other Weissella species. RESULTS: Phylogenomic and orthology-based analyses both showed a high-similarity in the genetic structure of these W. ceti strains. This structure is corroborated by the highly syntenic order of their genes and the neutral evolution inferred from Tajima's D. A whole-genome multilocus sequence typing analysis distinguished strains WS08 and NC36 from strains WS74 and WS105. We predicted 10 putative genomic islands (GEI), among which PAIs 3a and 3b are phage sequences that occur only in WS105 and WS74, respectively, whereas PAI 1 is species specific. CONCLUSIONS: We identified several genes putatively involved in the basic processes of bacterial physiology and pathogenesis, including survival in aquatic environment, adherence in the host, spread inside the host, resistance to immune-system-mediated stresses, and antibiotic resistance. These data provide new insights in the molecular epidemiology and host adaptation for this emerging pathogen in aquaculture.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

BACKGROUND: The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. RESULTS: The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. CONCLUSION: The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

COST ANALYSIS OF MOTORCYCLE ACCIDENT VICTIMS AT A UNIVERSITY HOSPITAL: PERSPECTIVES FROM 2017 AND 2020.

Introduction: Motorcycle accidents constitute a public health problem that affects public and private health services due to the expenses of the victim's treatment and rehabilitation. Objective: Evaluate the impact of motorcycle accident costs in a university hospital in 2020. Method: Comparative analysis of the costs of motorcycle accident patients in 2020 and 2017. Results: Among 151 patients included in the study, the average cost was U$3,083.54, and the average days of hospitalization were 5.3 days. The patient with the highest cost to the hospital spent U$22,504.05, and the patient with the lowest cost spent U$356.72. The longest stay among these patients was 41 days, and the shortest was one day. The average cost per patient per day for the entire sample was U$581.80. Conclusion: The formulation and application of strategies that promote the reduction of motorcycle accidents in the city of Campinas are necessary. Level of evidence II, Retrospective study.

Introdução: Acidentes motociclísticos constituem um problema de saúde pública que atinge os serviços públicos e privados de saúde, em função dos gastos com o tratamento e com o processo de reabilitação da vítima. Objetivo: Avaliar o impacto dos custos dos acidentes motociclísticos em um hospital universitário em 2020. Método: Análise comparativa dos custos dos pacientes vítimas de acidente motociclístico no ano de 2020 e 2017. Resultados: Dentre 151 pacientes incluídos no estudo, o custo médio foi de U$3.083,54 e a média de dias de internação foi de 5,3 dias. O paciente que apresentou maior custo para o hospital, teve um gasto de U$22.504,05 e o que teve o menor custo, gastou U$356,72. O maior tempo de internação, entre estes pacientes, foi de 41 dias e o menor tempo foi de 1 dia. O custo médio por paciente por dia, em toda a amostra, foi de U$581,80. Conclusão: Faz-se necessário a formulação e aplicação de estratégias que promovam a redução dos acidentes motociclísticos na cidade de Campinas. Nível de evidência II; Estudo retrospectivo.