REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria.

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.

Kinetics of neutrophil extracellular traps and cytokines in oral mucositis and Candida infection.

OBJECTIVE: This study investigated the concentrations of neutrophil extracellular traps (NET) and salivary cytokines (IL-1ß, IL-6, IL-8/CXCL8, TNF, and TGF-ß1) in patients undergoing chemotherapy and their associations with oral mucositis (OM) and Candida infection. MATERIALS AND METHODS: This prospective longitudinal study performed at a Brazilian service included 60 adults diagnosed with hematolymphoid diseases. Saliva samples were collected on days D0, D3, D10, and D15. Cytokines were analyzed by ELISA and NET formation by identification of the myeloperoxidase-DNA complex. Oral Candida spp. was cultured. RESULTS: OM occurred in 43.3% of patients and oral candidiasis in 20%. However, 66% of individuals had positive cultures for C. albicans. Higher concentrations of IL-6, IL-8/CXCL8, and TNF and lower concentrations of TGF-ß1 were observed in patients with OM. C. albicans infection contributed to the increase in IL-8/CXCL8, TGF-ß1, and TNF. Individuals with OM or with oral candidiasis had significant reductions in NET formation. In contrast, individuals with C. albicans and with concomitant C. albicans and OM exhibited higher NET formation. CONCLUSION: The kinetics of cytokine levels and NET formation in chemotherapy-induced OM appears to be altered by Candida infection, even in the absence of clinical signs of oral candidiasis.

Diet of the critically endangered blackchin guitarfish Glaucostegus cemiculus revealed using DNA metabarcoding.

We present the first assessment of the diet of the blackchin guitarfish Glaucostegus cemiculus (Geoffroy Saint-Hilaire, 1817) for West Africa using DNA metabarcoding on stomach contents of individuals captured in the Bijagós Archipelago, Guinea-Bissau. The diet was dominated by crustaceans, particularly caramote prawn Penaeus kerathurus (frequency of occurrence [FO] = 74%, numerical frequency [NF] = 54%) and fiddler crab Afruca tangeri (FO = 74%, NF = 12%). Bony fishes were present in 30% of the stomachs. We highlight the importance of conservation action for intertidal habitats and their associated benthic invertebrates for the survival of the critically endangered blackchin guitarfish.

Remediation through the coordinated use of local rice husk residues for the selective adsorption of iron and nickel in real landfill leachate.

Herein, we demonstrate the prospects of tackling several environmental problems by transforming a local rice husk residue into an effective adsorbent, which was then applied for the treatment of real landfill leachate (LL). The study focused on establishing (i) the effect of simple washing on morphological aspects, (ii) evaluating target adsorption capacity for total iron (Fe) and nickel (Ni), (iii) determining regeneration and reuse potential of the adsorbent and (iv) complying to the requirements of worldwide legislations for reuse of treated LL wastewater. The adsorbent was prepared by employing a simple yet effective purification process that can be performed in situ. The LL was collected post-membrane treatment, and the characterizations revealed high concentrations of Fe, Ni, and organic matter content. The simple washing affected the crystallinity, resulting in structural alterations of the adsorbents, also increasing the porosity and specific surface. The adsorption process for Ni occurred naturally at pH 6, but adjusting the pH to 3 significantly improved removal efficiency and adsorption capacity for total Fe. The kinetics were accurately described by the pseudo-second-order model, while the Langmuir model provided a better fit for the isotherms. The adsorbent was stable for 5 reuses, and the metals adsorbed were recovered through basic leaching. The removal capacities achieved underscore the remarkable effectiveness of the process, ensuring the treated LL wastewater meets rigorous global environmental legislations for safe use in irrigation. Thus, by employing the compelling methods herein optimized it is possible to refer to the of solving three environmental problems at once.

No Association Between Interleukin 6 and Inducible Nitric Oxide Synthase Polymorphisms and Dengue Infection: A Case-Control Study.

BACKGROUND: Dengue is a life-threatening disease. The factors that lead to severe cases are not completely understood. The host immune system is involved in the response to infections and plays an important role in dengue infection. IL-6 and iNOS are components of the immune system and genetic polymorphisms in these genes may be involved in dengue virus infection. The study aimed to investigate the association of genetic polymorphisms in the IL6 and iNOS genes and dengue. METHODS: We performed a case-control study using 60 dengue-infected individuals and 119 healthy controls. Polymorphisms in the IL6 (T15A) and iNOS (-1173CT) genes were amplified by Real-Time PCR. Statistical analyses were performed using BioEstat 5.0. RESULTS: We identified that the frequency of T/A genotype of IL6 was higher in dengue fever patients and C/T genotype of iNOS was higher in dengue hemorrhagic fever patients, however, no association was found between these polymorphisms and dengue. CONCLUSION: Polymorphisms in iNOS and IL6 were not associated with dengue infection.

Pedagogical practices developed with children through hospital classes: An integrative literature review.

OBJECTIVE: To analyze the evidence available in the literature on the pedagogical practices developed with children through hospital classes. METHOD: An integrative review was conducted on July 20, 2022, in Scopus, MEDLINE/PubMed, CINAHL, LILACS, Web of Science, ERIC, Educ@, and Scielo using the following descriptors in English, Portuguese, and Spanish, extracted from DECS/MeSH, CINAHL, Brased/INEP, and ERIC Thesaurus: "Child, Hospitalized", "Education, Special", "Education Department, Hospital", "Hospital Classroom", "Hospital Class", "Child Rearing", "Educational Practices", "Early Childhood Education", "Education", "Hospital Pedagogy", and "Hospital Special Class". No time restriction was applied. The EndNot Web reference manager and the Rayyan software were used to select studies, and later, the methodological rigor and level of evidence were assessed. RESULTS: The 22 articles described pedagogical practices, including ludic activities, individualized work, working with regular school content, stimulation activities, pedagogical and dialogic listening, learning based on the exchange of knowledge, video games, computational robotics, and theatrical performance. CONCLUSION: Although difficulties were identified in implementing pedagogical practices in the hospital, they were shown to allow educational continuity and clinical improvement of hospitalized children. IMPLICATIONS FOR PRACTICE: Studies on the educational process within the hospital setting can contribute to the development of public policies and the guarantee of the right to education for hospitalized children. DESCRIPTORS: Special education; Hospital education department; Hospitalized child; Child rearing; Teaching.

Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.

In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.

MIBG cardiac imaging compared to ejection fraction in evaluation of cardiotoxicity: a systematic review.

BACKGROUND: Advances in diagnosis and treatment of cancer has improved survival but resulted in increased cardiotoxic effects. The decrease in left ventricular ejection fraction (EF), one of the pillars of diagnosis of cardiotoxicity, seems to be a late process in the evolution of the disease, so 123I-metaiodobenzylguanidine (MIBG) cardiac imaging has been proposed to detect early cardiac impairment. The aim of this systematic review was to evaluate the performance of MIBG cardiac scan in this scenario. METHODS AND RESULTS: A systematic search was conducted in five international databases comparing MIBG parameters with EF for evaluation of cardiotoxicity. Twelve studies were included and separated in three groups. First, studies evaluating patients with established cardiotoxicity, in which EF was reduced and MIBG parameters were abnormal. Second, studies analyzing patients during or after treatment compared to controls, with MIBG parameters significantly different between groups in most studies, even when EF remained normal. Finally, studies analyzing anthracycline (ATC) dose-related changes, with alteration in MIBG parameters occurring even when EF was preserved. CONCLUSION: Although studies had high methodological variability, cardiac sympathetic innervation imaging seems to be a promising tool for assessing early cardiotoxicity. Further studies are needed to analyze its diagnostic value in this scenario.

Leishmanicidal activity of Morita-Baylis-Hillman adducts.

Leishmaniasis is a neglected disease that affects millions of people, mostly in developing countries. Although this disease has a high impact on public health, there are few drug options to treat the different leishmaniasis forms. Additionally, these current therapies have various adverse effects, including gastrointestinal disturbances, headache, pancreatitis, and hepatotoxicity. Thus, it is essential to develop new drug prototypes to treat leishmaniasis. Accordingly, the present study aimed to evaluate the leishmanicidal activity of Morita-Baylis-Hillman adducts and their O-acetylates, carboxylic acid derivatives, and acid and ester derivatives of 2-methyl-phenylpropanoids against Leishmania chagasi. Initially, we evaluated the cytotoxicity of 16 derivatives (1-16G) against J774A.1 macrophages. Eight derivatives (2G, 4G, 5G, 7G, 9G, 10G, 13G, and 15G) showed no cytotoxicity at up to the maximum concentration tested (100 µM). When evaluated for antileishmanial effect against promastigote forms, 1G, 6G, 8G, 10G, 11G, 13G, 14G, 15G, and 16G displayed significant toxicity compared to the control (0.1% DMSO). Additionally, the compounds 1G, 5G, 7G, 9G, 11G, 13G, 14G, and 16G reduced macrophage infection by amastigotes. Thus, we conclude that these derivatives have antileishmanial effects, particularly 1G, which showed activity against promastigotes and amastigotes, and low toxicity against macrophages.

Acute and subacute (28 days) oral toxicity studies of tucum almond oil (Bactris Setosa Mart.) in mice.

Oils extracted from almonds are often used with particular interest due to their prospective health effects and benefits. Tucum is a Pantanal fruit widely consumed by local population and no in vivo toxicity studies regarding its safety are available in the literature to date. This study investigated the acute and subacute toxicity of tucum almond oil (TAO) in mice by evaluating its safety profile. For the acute (2000 mg/kg) and subacute (250, 500 and 1000 mg/kg) toxicity studies, TAO was administered orally to mice according to 425 and 407 Organization for Economic Cooperation and Development Guidelines, respectively. Food intake, body, and organ weight of animals were recorded. Signs of toxicity were assessed, and hematological, biochemical and histopathological analyses were performed. In the acute toxicity study, no mortality or behavioral changes were observed in mice treated with 2000 mg/kg, indicating that LD50 is higher than this dose. In the subacute toxicity test, the doses evaluated did not produce relevant changes in hematological, biochemical or histopathological parameters in the exposed animals. The data obtained suggest that TAO did not induce toxicity after exposure to a single or repeated doses and LD50 value may be considered to be more than 2000 mg/kg body weight.

IL-10 (-819C/T), TNFA (-30G/A) and ENOS (-786T/C) Polymorphisms Modulating the Outcome Related to Mental Disorders in Crack Addicted Users.

Background: Cocaine/crack use affects immune system molecules and development of mental disorders has been identified. Objective: To investigate the relationship of polymorphisms in the TNFA (-308G/A), IL-10 (-819C/T) and ENOS (-786T/C) genes with mental disorders in cocaine and crack users. Methods: A case-control study was carried out, which included 107 cocaine and crack users and 115 controls who never used healthy cocaine and crack. The SNPs in the TNFA (-308G/A), IL-10 (-819C/T) and ENOS (-786T/C) genes were genotyped by real time PCR. Results: As for the individuals included in this study, the average age of 31.4 years (± 8.59). We identified that the G/A genotype to TNFA (-308) (OR = 0.24; p = 0.03) and the A allele (OR = 0.30; p = 0.03) were associated with reduced risk for dysthymic disorder. The T allele of the IL-10 (-819) polymorphism was associated with decreased risk of developing panic disorder (OR = 0.44; p = 0.01), while the C allele was correlated with an increased risk for alcohol dependence (OR = 1.97; p = 0.04), alcohol abuse (OR = 1.81; p = 0.04) and psychotic syndrome (OR = 2.23; p = 0.01). C/C genotype was correlated with increased chances of developing current psychotic syndrome (OR = 4.23; p = 0.01). Conclusion: Our results suggest that genetic polymorphisms promote susceptibility or promote protection for clinical phenotypes of psychiatric comorbidities in cocaine and crack users and be considered as good prognostic markers.

Assessing Stress Response in Lizards from Agroecosystems with Different Management Practices.

Despite the importance of reptiles in agroecosystems, little is known about the effects of agricultural intensification and pesticide use on these animals. We compared antioxidant and haematological biomarkers in the wild Italian wall lizards Podarcis siculus from three olive groves representing a gradient of management intensity. Lizards from the conventional grove showed induced antioxidant defences relative to those from the organic field. However, this induction did not avoid the occurrence of oxidative stress in males from intensively managed olive groves, who showed TBARS levels 58%-133% higher than males from the other sites. Haematological responses also suggested increased stress in females from the intensively managed olive groves, with a heterophil-to-lymphocyte ratio 5.3 to 14.8-fold higher than in the other sites. The observed stress responses of lizards along the studied gradient of agricultural management suggest their potential usefulness as non-destructive biomarkers to environmental stressors associated with agricultural intensification.

Widespread contamination of SARS-CoV-2 on highly touched surfaces in Brazil during the second wave of the COVID-19 pandemic.

Although SARS-CoV-2 surface contamination has been investigated in health care settings, little is known about the SARS-CoV-2 surface contamination in public urban areas, particularly in tropical countries. Here, we investigated the presence of SARS-CoV-2 on high-touch surfaces in a large city in Brazil, one of the most affected countries by the COVID-19 pandemic in the world. A total of 400 surface samples were collected in February 2021 in the City of Recife, Northeastern Brazil. A total of 97 samples (24.2%) tested positive for SARS-CoV-2 by RT-qPCR using the CDC-USA protocol. All the collection sites, except one (18/19, 94.7%) had at least one environmental surface sample contaminated. SARS-CoV-2 positivity was higher in public transport terminals (47/84, 55.9%), followed by health care units (26/84, 30.9%), beach areas (4/21, 19.0%), public parks (14/105, 13.3%), supply centre (2/21, 9.5%), and public markets (4/85, 4.7%). Toilets, ATMs, handrails, playgrounds and outdoor gyms were identified as fomites with the highest rates of SARS-CoV-2 detection. Taken together, our data provide a real-world picture of SARS-CoV-2 dispersion in highly populated tropical areas and identify critical control points that need to be targeted to break SARS-CoV-2 transmission chains.

Presentation of fatal stroke due to SARS-CoV-2 and dengue virus coinfection.

Herein, we report a case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and dengue coinfection, presented as a fatal stroke in our hospital, in São José do Rio Preto, São Paulo State, a Brazilian city hyperendemic for dengue viruses and other arthropod-borne viruses (arboviruses) and currently facing a surge of SARS-CoV-2 cases. This case is the first described in the literature and contributes to the better understanding of clinical presentations of two important diseases in a tropical setting.

Chikungunya Virus Infection Outcome: A Systematic Review of Host Genetics.

Background: Chikungunya virus (CHIKV) is a global concern, inducing chikungunya fever and trigging an arthritogenic chronic phase beyond some severe forms. Outcomes of CHIKV infections in humans are dependent on genetic variations. Here, a systematic review was performed to show evidence of genetic variations on infection outcomes of patients. Methods: Searches were performed in Scopus, SciELO, MEDLINE/PubMed, Web of Science, OneFile (GALE), Periódicos CAPES and ScienceDirect Journals databases. The PICOS approach was used to assess the eligibility of records. A meta-analysis was also conducted to show an association between described alleles/genes and CHIKV infection outcome. Results: Reviews of genetic variants were conducted on genes: CD 209, OAS1, OAS2, OAS3, MIF, TLR-3, TLR-7, TLR-8, MYD-88, KIR, HLA-B; HLA-C; DRB1 and DQB1. Studies were performed on Gabon, Singapore, and India, including Indians, Malay, Gabonese and Chinese ethnicities and published between 2009-2017. The meta-analysis was performed with DRB1 *01; *03; *04; *07; *10; *11; *13; *14 and *15 and DQB1 *02; *03; *05 and *06 alleles with Indian population sample. Sampling power was >80% and a significant positive association between DRB1*14 and CHIKV infection was found (OR = 1.67, 95% CI = 1.04-2.67; p = .03). Conclusion: Majority of the studies were conducted in India. Meta-analysis suggests that DRB1*14 is related to the susceptibility of symptomatic CHIKV infection in Indian population. The literature about CHIKV infection and genetic variations is scarce. The precise role of genetic variation in CHIKV is not clear yet. Further studies are necessary to provide more concrete evidences.

Fructose Intake Impairs Cortical Antioxidant Defenses Allied to Hyperlocomotion in Middle-Aged C57BL/6 Female Mice.

Recent evidence suggests that young rodents submitted to high fructose (FRU) diet develop metabolic, and cognitive dysfunctions. However, it remains unclear whether these detrimental effects of FRU intake can also be observed in middle-aged mice. Nine months-old C57BL/6 female mice were fed with water (Control) or 10% FRU in drinking water during 12 weeks. After that, metabolic, and neurochemical alterations were evaluated, focusing on neurotransmitters, and antioxidant defenses. Behavioral parameters related to motor activity, memory, anxiety, and depression were also evaluated. Mice consuming FRU diet displayed increased water, and caloric intake, resulting in weight gain, which was partially compensated due to decreased food pellet intake. FRU fed animals displayed increased plasma glucose, and cholesterol levels, which was not observed in overnight-fasted animals. Superoxide dismutase (SOD), and catalase (CAT) activities were markedly decreased in the prefrontal cortex of animals receiving FRU diet, while glutathione peroxidase (GPx) slightly increased. Liver (lower GPx), striatum (higher SOD and lower CAT), and hippocampus (no changes) were less impacted. No changes were observed in glutathione reductase, and thioredoxin reductase activities, two ancillary enzymes for peroxide detoxification. FRU intake did not alter serotonin, dopamine, and norepinephrine levels in the hippocampus, prefrontal cortex, and striatum. No significant alterations were observed in working, and short-term spatial memory; and in anxiety- and depressive-like behaviors in animals treated with FRU. Increased locomotor activity was observed in FRU-fed middle-aged mice, as evaluated in the open field, elevated plus-maze, Y maze, and object location tasks. Overall, these results demonstrate that high FRU consumption can disturb antioxidant defenses, and increase locomotor activity in middle-aged mice, open the opportunity for further studies to address the underlying mechanisms related to these findings.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli.

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of ∼38 kDa. The native enzyme was a tetramer with a molecular mass of approximately 178 kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.

PCR primers designed for new world Leishmania: A systematic review.

Studies of the primers that were designed to detect New World Leishmania were systematically reviewed to report the characteristics of each target, detection limit, specificity of the primers designed and diagnostic sensibility. The papers identified in the databases PubMed and Web of Science involved 50 studies. Minicircle is the most applied target in molecular research for diagnosis, due to its high sensitivity in detecting Leishmania in different clinical samples, a characteristic that can be partially attributed to the higher number of copies of the minicircle per cell. The other molecular targets shown in this review were less sensitive to diagnostic use because of the lower number of copies of the target gene per cell, but more specific for identification of the subgenus and/or species. The choice of the best target is an important step towards the result of the research. The target allows the design of primers that are specific to the genus, subgenus or a particular species and also imparts sensitivity to the method for diagnosis. The findings of this systematic review provide the advantages and disadvantages of the main molecular targets and primers designed for New World Leishmania, offering information so that the researcher can choose the PCR system best suited to their research need. This is a timely and extremely thorough review of the primers designed for New World Leishmania.

The Impact of the Ionic Cross-Linking Mode on the Physical and In Vitro Dexamethasone Release Properties of Chitosan/Hydroxyapatite Beads.

In this study, the effect of the ionic cross-linking mode on the ability to control physical properties and in vitro release behavior of the dexamethasone (DEX) drug from chitosan (CS) and chitosan/hydroxyapatite (CS/HA) beads was investigated. CS solutions without and with HA and DEX were dripped into two coagulation solutions, prepared with a non-toxic ionic crosslinker (sodium tripolyphosphate, TPP) and distilled water, one at pH = 9.0 and other at pH = 6.0. Optical microscopy (OM) and scanning electron microscopy (SEM) results showed changes on the surface topology of the beads, with a reduction of roughness for beads prepared at pH = 6.0 and an increase for the one prepared at pH = 9.0. The diameter and sphericity of the beads prepared at pH = 6.0 proved more uniform and had a larger pore size with a good interconnectivity framework. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) suggested a higher crosslinking degree for beads prepared at pH = 6.0, corroborated by X-ray diffraction profiles (XRD) analysis that indicated a decrease in the crystalline structure for such beads. In in vitro drug release data, all beads presented a sustained release during the studied period (24 h). The drug release rate was affected by the pH of the coagulation solution used in the preparation of the beads. The in vitro kinetics of the release process was of the Peppas-Sahlin model, controlled by both diffusion and relaxation of polymer chains or swelling (anomalous transport mechanism). Our results suggest that DEX-loaded CS/HA beads, crosslinked in TPP coagulation solution at pH = 9.0, led to a decrease in the DEX release rate and prolonged the release period. Thus, this composition might have prospective as a functional material for bone and cartilage tissue engineering.

Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil.

BACKGROUND: Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. METHODS: Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RDRio. The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. RESULTS: Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6-9 isolates/RFLP cluster were more likely belong to the LAM family, having the RDRio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08-1.26; adjusted OR = 1.25, 95% CI 1.14-1.37; crude OR = 2.68, 95% IC 1.13-6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6-9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10-0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2-5 isolates/RFLP cluster than unique patterns compared to individuals < 20 years of age (adjusted OR = 0.49, 95% CI 0.28-0.85, OR = 0.43 95% CI 0.24-0.77and OR = 0. 49, 95% CI 0.26-0.91), respectively. The extrapulmonary disease was less likely to occur in those infected with strains in the 2-5 isolates/cluster group (adjustment OR = 0.45, 95% CI 0.24-0.85) than unique patterns. CONCLUSIONS: We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes belonging to the LAM family and RDRio genotypes. Such information demonstrates that some genotypes are more likely to cause recent transmission. Targeting interventions such as screening in specific areas and social risk groups, should be a priority for reducing transmission.