Tenofovir Disoproxil Fumarate/Emtricitabine and Baricitinib for Patients at High Risk of Severe Coronavirus Disease 2019: The PANCOVID Randomized Clinical Trial.

BACKGROUND: This study was designed to evaluate if patients with high risk for severe coronavirus disease 2019 (COVID-19) would benefit from treatment with tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) followed by baricitinib in case of hypoxemia and systemic inflammation. METHODS: PANCOVID is an open-label, double-randomized, phase 3 pragmatic clinical trial including adults with symptomatic COVID-19 with ≥2 comorbidities or aged ≥60 years and was conducted between 10 October 2020 and 23 September 2021. In the first randomization, patients received TDF/FTC or no TDF/FTC. In the second randomization, patients with room air oxygen saturation <95% and at least 1 increased inflammatory biomarker received baricitinib plus dexamethasone or dexamethasone alone. The primary endpoint was 28-day mortality. Main secondary endpoint was 28-day disease progression or critical care unit admission or mortality. The trial was stopped before reaching planned sample size due to the decrease in the number of cases and a mortality rate substantially lower than expected. RESULTS: Of the 355 included participants, 97% were hospitalized at baseline. Overall, 28-day mortality was 3.1%. The 28-day mortality relative risk (RR) for participants treated with TDF/FTC was 1.76 (95% confidence interval [CI], .52-5.91; P = .379); it was 0.42 (95% CI, .11-1.59; P = .201) for those treated with baricitinib. The 28-day RR for the main secondary combined endpoint for participants treated with TDF/FTC was 0.95 (95% CI, .66-1.40; P = .774); it was 0.90 (95% CI, .61-1.33; P = .687) for those treated with baricitinib. CONCLUSIONS: Our results do not suggest a beneficial effect of TDF/FTC; nevertheless, they are compatible with the beneficial effect of baricitinib already established by other clinical trials. CLINICAL TRIALS REGISTRATION: EudraCT: 2020-001156-18.

Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples.

Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014-2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.

SARS-CoV-2 intralineage variation and temporal patterns of COVID-19 risk factors in three cities of southeastern Brazil: Age, sex, and race.

The Santo André Regional Center from Adolfo Lutz Institute evaluated 91 537 samples by reverse transcription-polymerase chain reaction (RT-PCR) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from March 2020 to April 2021. The age, sex, and race of patients from three cities in southeastern Brazil, namely São Bernardo do Campo (SBC), Diadema, and Mauá were assessed in association to the rate of positive results using generalized linear models. Circulating lineages were obtained from GISAID and intralineage genetic variation was investigated employing Lasergene software. A declining number of reported cases around October to November 2020 separate two epidemic waves in the three cities. Mauá differed by the highest positive RT-PCR scores in January and February. GISAID classification of 38 SARS-CoV-2 complete genomic sequences showed the circulation of lineages P.1, B.1.1.28, P.2, B.1.1.332; P.1, P.2, B.1.1.28, B.1.1.33; and P.1, P.2 in SBC, Diadema and Mauá, respectively. Intralineage variation revealed a significant amino-acid substitution in the ORF3a encoding protein (A33S) present in four out of six (67%) P.1 Mauá isolates. As ORF3a encodes a nonselective Ca2+ permeable cation channel, supposed to interfere in airway homeostasis, specific mutations could increase its pathogenic effect resulting in a higher number of symptomatic individuals explaining why the second wave was more intense in Mauá city.

COVID-19 in hospitalized HIV-positive and HIV-negative patients: A matched study.

OBJECTIVES: We compared the characteristics and clinical outcomes of hospitalized individuals with COVID-19 with [people with HIV (PWH)] and without (non-PWH) HIV co-infection in Spain during the first wave of the pandemic. METHODS: This was a retrospective matched cohort study. People with HIV were identified by reviewing clinical records and laboratory registries of 10 922 patients in active-follow-up within the Spanish HIV Research Network (CoRIS) up to 30 June 2020. Each hospitalized PWH was matched with five non-PWH of the same age and sex randomly selected from COVID-19@Spain, a multicentre cohort of 4035 patients hospitalized with confirmed COVID-19. The main outcome was all-cause in-hospital mortality. RESULTS: Forty-five PWH with PCR-confirmed COVID-19 were identified in CoRIS, 21 of whom were hospitalized. A total of 105 age/sex-matched controls were selected from the COVID-19@Spain cohort. The median age in both groups was 53 (Q1-Q3, 46-56) years, and 90.5% were men. In PWH, 19.1% were injecting drug users, 95.2% were on antiretroviral therapy, 94.4% had HIV-RNA < 50 copies/mL, and the median (Q1-Q3) CD4 count was 595 (349-798) cells/µL. No statistically significant differences were found between PWH and non-PWH in number of comorbidities, presenting signs and symptoms, laboratory parameters, radiology findings and severity scores on admission. Corticosteroids were administered to 33.3% and 27.4% of PWH and non-PWH, respectively (P = 0.580). Deaths during admission were documented in two (9.5%) PWH and 12 (11.4%) non-PWH (P = 0.800). CONCLUSIONS: Our findings suggest that well-controlled HIV infection does not modify the clinical presentation or worsen clinical outcomes of COVID-19 hospitalization.

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype.

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilSEc404 and pilSC1096) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilSEc404 occurring more often (30.7%) than pilSC1096 (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilSEc404+ strains and in 65.1% of pilSC1096+ strains, but only pilSEc404+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.

Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli.

Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

Recurrent community-acquired pneumococcal meningitis in adults with and without identified predisposing factors.

Bacterial meningitis is still a significant public health concern, with high morbidity and mortality rates. Despite this, it is still a rare event that requires the bacterial invasion of the meninges. However, some predisposing factors can trigger recurrent episodes of meningitis. This study is aimed at determining the clinical characteristics and the molecular epidemiology of episodes of recurrent community-acquired meningitis with and without predisposing factors. For this purpose, we performed a retrospective study of our laboratory database during the period of 2010 to 2020. Additionally, using molecular tools developed in our previous works, the epidemiology of the pathogens causing these episodes was analyzed using cerebrospinal fluid samples, especially in the absence of isolated strains. We observed a total of 1,779 meningitis cases and 230 were caused by Streptococcus pneumoniae. Of those, 16 were recurrent meningitis episodes (16/1,779; 0.9%) from seven patients. Pneumococcus was the main agent responsible in these recurrent episodes and only two episodes were caused by Haemophilus influenzae. The mean age of these patients was 20 years old and three had predisposing factors which could have led to contracting meningitis. The samples presented different pneumococcal serotypes. Most of them were non-vaccine-covered serotypes and antibiotic susceptible strains. Therefore, it was demonstrated how the practical employment of molecular tools, developed for research, when applied in the routine of diagnosis, can provide important information for epidemiological surveillance. Furthermore, it was shown how pneumococcus was the leading cause of recurrent community-acquired meningitis without predisposing factors, suggesting that pneumococcal vaccination may be necessary, even in those groups of individuals considered to be less susceptible.

Enteroaggregative Escherichia coli (EAEC) isolates obtained from non-diarrheic children carry virulence factor-encoding genes from Extraintestinal Pathogenic E. Coli (ExPEC).

Enteroaggregative E. coli (EAEC) is one of the most frequent pathogens isolated from diarrheal patients as well as from healthy individuals in Brazil and has recently also been implicated as an extraintestinal pathogenic E. coli (ExPEC) associated with bloodstream and urinary tract infections. In this study, 37 EAEC isolates, obtained from fecal samples of non-diarrheic children, were molecularly and phenotypically characterized to access the pathogenic features of these isolates. The EAEC isolates were assigned into the phylogroups A (54.1%), D (29.7%), B1 (13.5%) and B2 (2.7%); and harbored genes responsible for encoding the major pilin subunit of the aggregative adherence fimbriae (AAFs) or aggregate-forming pili (AFP) adhesins as follows: aggA (24.3%), agg3A (5.4%), agg4A (27.0%), agg5A (32.4%) and afpA (10.8%). The most frequent O:H serotypes were O15:H2 (8.1%), O38:H25 (5.4%) and O86:H2 (5.4%). Twenty-one isolates (56.8%) produce the aggregative adherence (AA) pattern on HeLa cells, and biofilm formation was more efficient among EAEC isolates harboring the aggA and agg5A genes. PFGE analysis showed that 31 (83.8%) of the isolates were classified into 10 distinct clusters, which reinforces the high diversity found among the isolates studied. Of note, 40.5% (15/37) of the EAEC isolates have a genetic profile compatible with E. coli isolates with intrinsic potential to cause extraintestinal infections in healthy individuals, and therefore, classified as EAEC/ExPEC hybrids. In conclusion, we showed the presence of EAEC/ExPEC hybrids in the intestinal microbiota of non-diarrheic children, possibly representing the source of some endogenous extraintestinal infections.

Symptomatic remission and recovery in major psychosis: Is there a role for BDNF? A secondary analysis of the LABSP cohort data.

Remission, relapse prevention, and clinical recovery are crucial areas of interest in schizophrenia (SCZ) research. Although SCZ is a chronic disorder with poor overall outcomes, years of research demonstrated that recovery is possible. There are considerable data linking brain-derived neurotrophic factor (BDNF) to SCZ, however, evidence on the role of BDNF in remission in SCZ is scarce. This secondary analysis of the Longitudinal Assessment of BDNF in Sardinian patients (LABSP) data aimed to investigate the relationship between serum BDNF levels and symptomatic remission, simultaneous clinical and functional remission, and recovery in patients with SCZ. A total of 105 patients with SCZ or schizoaffective disorder were recruited for a longitudinal assessment of BDNF levels over 24 months. Longitudinal data were analyzed using mixed-effects linear regression models. The study found significant associations between use of long acting injectables (χ2 = 7.075, df = 1, p = 0.008), baseline serum BDNF levels (U = 701, z = -2.543, p = 0.011), and "childhood" (U = 475, z = -2.124, p = 0.034) and "general" (U = 55, z = -2.014, p = 0.044) subscales of the Premorbid Adjustment Scale (PAS) with patients maintaining remission and recovery. The diagnosis of SCZ was significantly associated with lower BDNF levels for patients with simultaneous clinical and functional remission (Z = 2.035, p = 0.0419) and recovery (Z = 2.009, p = 0.0445) compared to those without. There were no significant associations between remission in the entire sample and longitudinal serum BDNF levels or genetic variants within the BDNF gene. These findings provide further insight into the complex relationship between BDNF and SCZ.

Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid.

Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24-79.86; ermB: 80.88-82.56; mef: 74.85-76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S. pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.

Tuberculosis/COVID-19 co-infection detected in a single sputum sample using a rapid molecular test.

Tuberculosis (TB) and COVID-19 affect the lungs and are transmitted mainly by aerosols or particles of saliva from infected persons. Clinical similarities between diseases can affect correct diagnosis. Individuals belonging to the population deprived of liberty (PDL) are at increased risk of contagion due to precarious sanitary conditions and overcrowded environments. A variety of specimens may be suitable for the diagnosis of COVID-19, using molecular diagnostic techniques; however, there is little data on the analysis of sputum samples with the Xpert Xpress SARS-CoV-2® for the diagnosis of COVID-19, especially in this population group. The present study reports a case of TB and COVID-19 co-infection detected in sputum from an individual belonging to the PDL. For the detection, it used the GeneXpert platform (Cepheid, USA). Mycobacterium tuberculosis complex (MTC) was detected using the Xpert MTB/RIF Ultra® cartridge and SARS-CoV-2 was detected using the Xpert Xpress SARS-CoV-2® cartridge. The genes IS6110 and IS1081 were detected within 80 min indicating the presence of MTC, with no mutations related to resistance to rifampicin. The SARS-CoV-2 E and N2 genes were detected within 45 min. The result was confirmed by RT-qPCR with detection of E, N, and RdRP/S genes in the sputum and nasopharyngeal (NP) specimens. Rapid diagnoses that allow the identification and differentiation of such diseases are important for adequate epidemiological surveillance, isolation of infected individuals, and interruption of the transmission chain. Using the GeneXpert platform, specimens can be tested as soon as they are received, without the need for prior preparation. The US Food and Drug Administration has issued emergency authorization for the use of the Cepheid Xpert Xpress SARS-CoV-2 for the rapid detection of SARS-CoV-2 using specimens from a NP or nasal wash/aspirate. The case presented here gains an innovation with the use of the sputum to COVID-19 diagnosis.

Viability assessment of Mycobacterium tuberculosis complex in OMNIgene • SPUTUM reagent using the BACTEC MGIT 960 System and Xpert MTB/RIF assay.

The World Health Organization advocates that sputum specimens submitted to tuberculosis (TB) diagnostic should be processed within 48 h after collection and be stored under cooling. We aimed to assess the performance of OMNIgene • SPUTUM reagent in maintaining viable specimens of Mycobacterium tuberculosis complex (MTBC) during transportation of sputum samples without refrigeration, in comparison to the standard protocol of the National TB Control Program. Sputum samples obtained in southeastern Brazil (June 2017 to July 2018) from 100 sequential patients with positive acid-fast bacillus smear microscopy were divided into two portions. Portion 1 continued to be cooled (standard protocol, STA), but portion 2 was added to OMNIgene • SPUTUM reagent (alternative protocol, OMS) until concomitant further processing. Both portions of all samples were cultured using MGIT and tested by Xpert MTB/RIF assay. Growth of MTBC in the first 42 days was detected in 96% of the cultures under the STA and 88% under the OMS. Intervals between processing and detecting MTBC growth in the two portions significantly differed (p = 0.0001). Portions under the two protocols showed similar results in the MTBC detection by Xpert assay and culture contamination by non-MTBC. The OMNIgene reagent liquefies and decontaminates sputum leading to a decrease in processing time. Although there was a small delay in mycobacterial growth, the OMNIgene reagent can be useful in specimens transported from collection sites over a long distance to centralized testing centers, maintaining viable MTBC for at least 8 days at room temperature.

Analysis of the Virulence Profile and Phenotypic Features of Typical and Atypical Enteroaggregative Escherichia coli (EAEC) Isolated From Diarrheal Patients in Brazil.

Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea in children and adults worldwide. Here we report a characterization of 220 EAEC isolates, 88.2% (194/220) of which were typical and 11.8% (26/220) were atypical, obtained from diarrheal patients during seven years (2010-2016) of epidemiological surveillance in Brazil. The majority of the isolates were assigned to phylogroups A (44.1%, 97/220) or B1 (21.4%, 47/220). The aggregative adherence (AA) pattern was detected in 92.7% (204/220) of the isolates, with six of them exhibiting AA concomitantly with a chain-like adherence pattern; and agg5A and agg4A were the most common adhesin-encoding genes, which were equally detected in 14.5% (32/220) of the isolates. Each of 12 virulence factor-encoding genes (agg4A, agg5A, pic, aap, aaiA, aaiC, aaiG, orf3, aar, air, capU, and shf) were statistically associated with typical EAEC (P < 0.05). The genes encoding the newly described aggregate-forming pili (AFP) searched (afpB, afpD, afpP, and afpA2), and/or its regulator (afpR), were exclusively detected in atypical EAEC (57.7%, 15/26), and showed a significant association with this subgroup of EAEC (P < 0.001). In conclusion, we presented an extensive characterization of the EAEC circulating in the Brazilian settings and identified the afp genes as putative markers for increasing the efficiency of atypical EAEC diagnosis.

Virulence Profiling and Molecular Typing of Shiga Toxin-Producing E. coli (STEC) from Human Sources in Brazil.

Since no recent data characterizing Shiga toxin-producing E. coli (STEC) from human infections in Brazil are available, the present study aimed to investigate serotypes, stx genotypes, and accessory virulence genes, and also to perform pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) of 43 STEC strains recovered from 2007 to 2017. Twenty-one distinct serotypes were found, with serotype O111:H8 being the most common. However, serotypes less frequently reported in human diseases were also found and included a hybrid STEC/ETEC O100:H25 clone. The majority of the strains carried stx1a as the sole stx genotype and were positive for the eae gene. Regarding the occurrence of 28 additional virulence genes associated with plasmids and pathogenicity islands, a diversity of profiles was found especially among the eae-harboring strains, which had combinations of markers composed of up to 12 distinct genes. Although PFGE analysis demonstrated genetic diversity between serotypes such as O157:H7, O111:H8, O26:H11, O118:H16, and O123:H2, high genetic relatedness was found for strains of serotypes O24:H4 and O145:H34. MLST allowed the identification of 17 distinct sequence types (STs) with ST 16 and 21 being the most common ones. Thirty-five percent of the strains studied were not typeable by the currently used MLST approach, suggesting new STs. Although STEC O111:H8 remains the leading serotype in Brazil, a diversity of other serotypes, some carrying virulence genes and belonging to STs incriminated as causing severe disease, were found in this study. Further studies are needed to determine whether they have any epidemiological relevance.

Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli

Although it has been hypothesized that the acquisition of plasmids—especially those bearing virulence factors and antimicrobial resistance genes—increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from − 4.7 to 5.2% per generation. This suggests that there are few biological barriers—or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model—against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

Epidemiology and genetic screening of diarrheagenic Escherichia coli among symptomatic and asymptomatic children.

PURPOSE: Enteropathogens are frequently associated with diarrheal disease. Knowledge of their etiology and epidemiology is essential for the prevention and control of the sickness. This study describes the microbiological and epidemiological features of diarrheal disease in 197 symptomatic and 223 asymptomatic under-five-year-old children from southeastern Brazil, between January 2015 and September 2016. METHODS: Isolation of Escherichia coli, Salmonella, Shigella and Campylobacter was realized by culture. E. coli strains were screened by multiplex PCR, PFGE and O:H serotyping. Antimicrobial susceptibility testing was also performed. RESULTS: Most of the 127 enteropathogens isolated were diarrheagenic E. coli (96.1 %), with predominance of several serotypes of enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC). Age, sex, rotavirus vaccination, recent use of antibiotics and previous contact with pets, were factors that revealed no significant effects on the probability of infection by the predominant pathogens. Even so, higher incomes could be related to a lesser chance of testing positive for EPEC. Evidence of possible EAEC clonal spread was detected, as well as genetic similarity among strains from both symptomatic and asymptomatic children. Resistance to antimicrobial agents was more pronounced among EAEC than EPEC. CONCLUSION: The occurrence of genetically similar diarrheagenic E. coli in both groups of children, likewise resistant to these agents, underscores the importance of establishing strategies for the prevention of outbreaks, especially among low-income households.

Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples

Streptococcus pneumoniae causes invasive diseases of significant public health concern, such as meningitis. The culture of cerebrospinal fluid (CSF) samples, the standard technique for meningitis diagnoses, is not always positive. Consequently, meaningful information about the etiological agent is lost, which can compromise effective epidemiological surveillance and the improvement of immunization policies. This study aims to standardize a method to genotype pneumococcus in the CSF samples which could mitigate the absence of isolated strains, and also evaluate the prediction of this assay. We applied eight multiplex PCR (mPCR) assays to CSF samples paired with the Quellung reaction applied to the isolated strains. We also compared different master mix kits in the mPCR. Moreover, a retrospective study was conducted with CSF samples considered pneumococcus positive due to the presence of the lytA gene. Results showed that genotyping by the mPCR correlated 100% with the Quellung reaction, and genotyping was dependent on the master mix applied. In the retrospective study (2014–2020), 73.4% were successfully genotyped. The analyses of the receiver operating characteristic curve showed that the cycle threshold (Ct value) around 30 for the lytA gene had a 75% positive chance of successful genotyping, whereas with a Ct value > 35, the chance was 12.5%. Finally, we observed that genotype 19A was prevalent in the period (12%), information unknown until now due to the lack of isolated strains. Therefore, the mPCR of CSF samples can efficiently predict S. pneumoniae serotypes, especially in the absence of isolated strains, which can be a great tool for pneumococcal serotype surveillance.

Distribution of virulence profiles related to new toxins and putative adhesins in Shiga toxin-producing Escherichia coli isolated from diverse sources in Brazil.

The distribution of virulence markers related to cytolethal distending toxin-V (CDT-V), subtilase cytotoxin (SubAB), the enterohemorrhagic Escherichia coli factor for adherence (Efa1), the adhesin similar to IrgA (Iha), the long polar fimbriae (LpfO113), the autoagglutinating adhesin (Saa), and the protein required for full expression of adherence of O157:H7 Sakai strain (ToxB) was investigated in 121 Shiga toxin-producing E. coli (STEC) strains isolated in Brazil. STEC strains were isolated from human infections (n=49), cattle (n=68) and ground meat samples (n=4). Overall, the lpfA(O113), iha, efa1, saa, and toxB sequences were observed in 89.2%, 87.6%, 47.1%, 43%, and 13.2% of the strains, respectively. The genes efa1 (96.6%) and toxB (27%) were only identified among eae-positive strains, while saa (83.8%), cdt-V (12.9%), and subAB (48.4%) just occurred in eae-negative STEC strains. STEC strains harboring cdt-V and subAB were for the first time described in the South American subcontinent. In addition, the simultaneous presence of cdt-V and subAB has not been previously reported, nor the presence of subAB in STEC O77, O79, O105, O174, and O178 serogroups. A diversity of virulence profiles was observed among the STEC strains studied. The most prevalent profile observed among eae-positive STEC strains mainly isolated from humans was eae efa1 iha lpfA(O113), whereas iha lpfA(O113) saa ehxA subAB prevailed among eae-negative STEC strains, mostly isolated from cattle and foods.

Presacral venous bleeding during mobilization in rectal cancer.

AIM: To analyze the anatomy of sacral venous plexus flow, the causes of injuries and the methods for controlling presacral hemorrhage during surgery for rectal cancer. METHODS: A review of the databases MEDLINE® and Embase™ was conducted, and relevant scientific articles published between January 1960 and June 2016 were examined. The anatomy of the sacrum and its venous plexus, as well as the factors that influence bleeding, the causes of this complication, and its surgical management were defined. RESULTS: This is a review of 58 published articles on presacral venous plexus injury during the mobilization of the rectum and on techniques used to treat presacral venous bleeding. Due to the lack of cases published in the literature, there is no consensus on which is the best technique to use if there is presacral bleeding during mobilization in surgery for rectal cancer. This review may provide a tool to help surgeons make decisions regarding how to resolve this serious complication. CONCLUSION: A series of alternative treatments are described; however, a conventional systematic review in which optimal treatment is identified could not be performed because few cases were analyzed in most publications.

Multiplex real-time PCR using SYBR Green: unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24­79.86; ermB: 80.88­82.56; mef: 74.85­76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S.pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains. (AU)