Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan / Fixação de alelos de resistência à cloroquina pfcrt em isolados clínicos de Plasmodium falciparum coletados de agitações de agências tribais do Paquistão

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.

Prevalence of antifolate drug resistance markers in Plasmodium vivax in China / 医学前沿

The dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes of Plasmodium vivax, as antifolate resistance-associated genes were used for drug resistance surveillance. A total of 375 P. vivax isolates collected from different geographical locations in China in 2009-2019 were used to sequence Pvdhfr and Pvdhps. The majority of the isolates harbored a mutant type allele for Pvdhfr (94.5%) and Pvdhps (68.2%). The most predominant point mutations were S117T/N (77.7%) in Pvdhfr and A383G (66.8%) in Pvdhps. Amino acid changes were identified at nine residues in Pvdhfr. A quadruple-mutant haplotype at 57, 58, 61, and 117 was the most frequent (57.4%) among 16 distinct Pvdhfr haplotypes. Mutations in Pvdhps were detected at six codons, and the double-mutant A383G/A553G was the most prevalent (39.3%). Pvdhfr exhibited a higher mutation prevalence and greater diversity than Pvdhps in China. Most isolates from Yunnan carried multiple mutant haplotypes, while the majority of samples from temperate regions and Hainan Island harbored the wild type or single mutant type. This study indicated that the antifolate resistance levels of P. vivax parasites were different across China and molecular markers could be used to rapidly monitor drug resistance. Results provided evidence for updating national drug policy and treatment guidelines.

Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast / Abordagens fenotípica e genotípica para detecção de resistência anti-helmíntica em nematoides gastrintestinais de ovinos no nordeste do Brasil

Abstract The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.

Resumo O objetivo deste estudo foi caracterizar a resistência anti-helmíntica (RA) da população de nematoides gastrintestinais de ovinos, denominada Caucaia, do Nordeste brasileiro. Os testes fenotípicos foram: eclosão de ovos (TEO), desenvolvimento larvar (TDL) e redução da contagem de ovos nas fezes (TRCOF). A avaliação genotípica para benzimidazóis (BZs) foi por frequência de polimorfismos de nucleotídeo único (SNPs) F200Y, F167Y e E198A; e para levamisol (LEV), pela frequência alélica para resistência no gene Hco-acr-8. Os "primers" foram específicos para Haemonchus contortus. As concentrações efetivas 50% (CE50) para BZs (TEO) e para lactonas macrocíclicas (LMs) e LEV (TDL) foram 1,02 µg/mL, 1,81 ng/mL e 0,04 µg/mL, respectivamente. Os fatores de resistência para LMs e LEV foram 0,91 e 3,07, respectivamente. As eficácias para BZs, LMs, monepantel (MPTL) e LEV no TRCOF foram 52,4; 87,0; 94,5 e 99,6%, respectivamente. A qPCR para BZs demonstrou frequências de 0%, 26,24% e 69,08% para SNPs E198A, F200Y e F167Y, respectivamente. Para LEV foram encontrados 54,37% de alelos resistentes. Houve concordância entre TEO, TRCOF e qPCR para BZs, e entre TDL e qPCR para LEV. Baseada na maior sensibilidade da qPCR e avaliação fenotípica, a população Caucaia foi considerada resistente a BZs, LMs, LEV e suspeita para MPTL.

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum

Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.

Distribuição das espécies de crípticas de Candida em isolados de corrente sanguínea: identificação e perfil de sensibilidade antifúngica / Distribution of Candida cryptic species in bloodstream isolates: identification and antifungal sensitivity profile

Candidemias caracterizam um grave problema de saúde pública em todo mundo pela alta mortalidade dos casos, onde as espécies apresentam variação epidemiológica e na sensibilidade aos antifúngicos. Objetivou-se demonstrar a frequência de espécies de Candida, enfatizando as espécies crípticas e caracterizar o perfil de sensibilidade antifúngica de cepas em casos de candidemia, internados em hospitais do estado de São Paulo, onde Instituto Adolfo Lutz é o laboratório de referência. As cepas, únicas de cada paciente, foram recebidas de 22 hospitais públicos gerais, filantrópico, escola e especializado em infectologia. A identificação fenotípica para determinação dos complexos deu-se por análise morfológica e bioquímica, por métodos auxanográficos. Para discriminar espécies crípticas aplicaram-se técnicas moleculares das mais simples às complexas, sendo elas: PCR, PCR-RFLP, MALDI-TOF e sequenciamento. Os antifúngicos utilizados nos testes de sensibilidade foram: fluconazol, voriconazol, caspofungina, micafungina, anidulafungina e anfotericina B. Nos anos de 2017 e 2018, foram estudadas 144 cepas de candidemia com as seguintes espécies crípticas: C. parapsilosis sensu stricto (47/144; 32,6%), C. orthopsilosis (4/144; 2,7%), C. metapsilosis (2/144; 1,4%), C. albicans ssss (40/144; 27,8%), C. dubliniensis (2/144, 1,4%), C. glabrata (14/144; 9,7%), C. haemulonii (2/144; 1,4%), C. haemulonii var. vulnera (3/144; 2,1); C. duobushaemulonii (1/144; 0,7%) e C. guilliermondii (2/144; 1,4%). As demais espécies foram: C. tropicalis (21/144; 14,6%), C. krusei (4/144; 2,8%), C. pelliculosa (1/144; 0,7%) e C. kefyr (1/144; 0,7%). Para FCZ foram encontradas 3 cepas de C. parapsilosis (3/46; 6,5%; 0,12->64 µg/mL) e em uma de C. tropicalis (1/21; 4,76%; 64 µg/mL) resistentes; observou-se uma cepa non-wild type de C. guilliermondii (1/2; 50%; 64 µg/mL) e altos MICs para 2 cepas de C. haemulonii var. vulnera (2/3; 66,6%; 16-32 µg/mL) e para a única cepa de C. duobushaemulonii (64 µg/mL). Alta taxa de cepas non-wild type ao VCZ (6/14; 42,8%) foi encontrada em C. glabrata. Reafirma-se neste estudo que as espécies do complexo C. haemulonii, consideradas multirresistentes aos antifúngicos, despontam com maior frequência em nosso estado, se comparado aos dados da literatura. De acordo com os resultados obtidos, a identificação por métodos moleculares representou importante estratégia para demonstrar a variedade de espécies causais de candidemias e alertar para necessidade de terapias apropriadas. A determinação de espécies crípticas propensas à resistência pode ter impacto na sobrevida de pacientes por fornecer subsídios para terapia empírica com base no perfil epidemiológico da candidemia em cada hospital, região e país. (AU)

Candidemia is a serious public health problem worldwide due to the high mortality of the cases. The species present epidemiological diversity and different profiles of sensitivity to antifungals. The aim is to show the frequency of Candida species, emphasizing the cryptic species and to characterize the antifungal sensitivity profile of strains in cases of candidemia, admitted to hospitals in the state of São Paulo, where Adolfo Lutz Institute is the reference laboratory. The strains, unique to each patient, were received from 22 general public hospitals, philanthropic, sshool, and specialized in infectious diseases. The phenotypic identification to determine the complex was done by morphological and biochemical analysis, using auxanographic methods. To discriminate cryptic species, molecular techniques from the simplest to the most complex were applied, namely: PCR, PCR-RFLP, MALDI-TOF, and Sequencing. The antifungals used in the susceptibility tests were: fluconazole, voriconazole, caspofungin, micafungin, anidulafungin and amphotericin B. In the years 2017 and 2018, 144 strains of candidemia were studied with the following cryptic species: C. parapsilosis sensu stricto ss (47/144; 32.6%), C. orthopsilosis (4/144; 2.7%), C. metapsilosis (2/144; 1.4%), C. albicans ssss (40/144; 27.8%), C. dubliniensis (2/144, 1.4%), C. glabrata (14/144; 9 , 7%), C. haemulonii (2/144; 1.4%), C. haemulonii var. vulnera (3/144; 2.1); C. duobushaemulonii (1/144; 0.7%) and C. guilliermondii (2/144; 1.4%). The other species were: C. tropicalis (21/144; 14.6%), C. krusei (4/144; 2.8%), C. pelliculosa (1/144; 0.7%) and C. kefyr (1/144; 0.7%). Resistance to FCZ was found in 3 strains of C. parapsilosis (3/46; 6.5%; 0.12-> 64 µg / mL) and 1 of C. tropicalis (1/21; 4.76%; 64 µg / mL) and non-wild type for a strain of C. guilliermondii (1/2; 50%; 64 µg / mL) and high MICs for 2 C. haemulonii var. vulnera (2/3; 66.6%; 16-32 µg / mL) and in the single strain of C. duobushaemulonii (64 µg / mL). A high rate of non-wild type to VCZ (6/14; 42.8%) was found for C. glabrata. It is reaffirmed in this study that the species of the C. haemulonii complex, considered multiresistant to antifungals, appear more frequently in our state when compared to the literature data. According to the results, the identification by molecular methods becomes an important tool for the construction of surveillance strategies in hospitals. The determination of cryptic species prone to resistance may have an impact on patient survival by providing subsidies for empirical therapy based on the epidemiological profile of candidemia in each hospital, region, and country. (AU)

Association of CYP2C19*2 polymorphism with clopidogrel resistance among patients with high cardiovascular risk in Northeastern Mexico / Asociación del polimorfismo CYP2C19*2 con resistencia a clopidogrel en pacientes con alto riesgo cardiovascular en el noreste de México

abstract Objective: Oral antiplatelet drugs are a key to modern pharmacotherapy in cardiovascular atherothrombotic diseases. Clopidogrel (CLO) constitutes the main preventive treatment of atherothrombosis. However, a considerable inter-individual variation in CLO response has been documented, resulting in suboptimal therapy and an increased risk of recurrent adverse effects in some patients. The enzyme CYP2C19 has been reported to be the CYP isoform that activates CLO to its active metabolite. Several single nucleotide polymorphisms in the CYP2C19 gene have been identified as strong predictors of CLO-impaired pharmacological response. At least 16 variants have been associated with changes in CYP2C19 activity. Materials and Methods: The following research was composed of a total of 102 subjects with high cardiovascular risk in the northeast of Mexico, with a maintenance dose of 75 mg of CLO per day. The platelet reactivity was measured with VerifyNow P2Y12 assay, while the presence of CYP2C19*2 was identified by real-time polymerase chain reaction. Results: Patients were categorized by CYP2C19 metabolizer status based on *2 genotypes using the common consensus star allele nomenclature as normal metabolizer (G/G), intermediate metabolizer (G/A), and poor metabolizer (A/A), respectively. The phenotype frequency for CYP2C19*2 was 74.5% (G/G), 21.6% (G/A), and 3.9% (A/A). The subjects with the A allele presented ≥235 P2Y12 reaction unit levels, classifying them how poor metabolizer. The prevalence of reduced CLO effectiveness was associated with the presence of CYP2C19*2 polymorphism among Mexican patients. Conclusion: The presence of the CYP2C19*2 allele is related to resistance to the antiplatelet effect of CLO (p = 0.003).

Resumen Objetivo: Los antiplaquetarios orales son clave en la farmacoterapia moderna de las enfermedades aterotrombóticas cardiovasculares. Clopidogrel (CLO) constituye el principal tratamiento preventivo de aterotrombosis (AT). Sin embargo, se ha documentado una considerable variación interindividual en la respuesta a CLO, lo que da como resultado una terapia subóptima y mayor riesgo de efectos adversos en algunos pacientes. La enzima CYP2C19 es la isoforma CYP que activa CLO a su metabolito activo. Se han identificado varios polimorfismos de un solo nucleótido en el gen CYP2C19 como fuertes predictores de respuesta farmacológica alterada a CLO. Al menos 16 variantes se han asociado con cambios en la actividad de CYP2C19. Método: Se reclutaron un total de 102 sujetos con alto riesgo cardiovascular del noreste de México, con dosis de mantenimiento de 75 mg de CLO/día. La reactividad plaquetaria se midió con el ensayo Verify Now P2Y12, la presencia de CYP2C19*2 se identificó mediante polymerase chain reaction en tiempo real. Resultado: Los pacientes fueron clasificados por el estado metabolizador CYP2C19*2 utilizando nomenclatura consenso, como metabolizador normal (G/G), metabolizador intermedio (G/A) y metabolizador pobre (A/A), respectivamente. La frecuencia del fenotipo para CYP2C19*2 fue 74.5% (G/G), 21.6% (G/A) y 3.9% (A/A). Los sujetos con alelo A presentaron ≥235 niveles P2Y12 reaction unit, clasificándolos como metabolizadores deficientes. La prevalencia de eficacia reducida a CLO se asoció con la presencia del polimorfismo CYP2C19*2 en pacientes mexicanos. Conclusiones: La presencia del alelo CYP2C19*2 se relaciona con resistencia al efecto antiagregante plaquetario del CLO (p = 0.003).

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Plasmodium falciparum malarial parasites from Brazil lack artemisinin resistance-associated mutations in the kelch13 gene

Abstract INTRODUCTION Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.

Falla o resistencia en la anestesia espinal para cesárea / Failure or resistance in spinal anesthesia for cesarean section

Introducción: La anestesia regional es la más empleada en la cesárea obstétrica. En particular, el uso de la anestesia intratecal tiene sus ventajas. Aunque la tasa de falla es baja, la aparición de este evento genera dificultades que merecen atención. Objetivo: Describir la conducta anestésica en una paciente obstétrica en la que falla la anestesia regional intratecal. Caso clínico: Paciente femenina, de 20 años a la que se le administró anestesia intratecal por el especialista. No hubo errores en la punción lumbar, esta se realizó con trócar 25 punta Whitacre. No se constató bloqueo sensitivo, motor ni simpático, por lo que se realizó anestesia general endotraqueal, la cual transcurrió sin dificultades. En el posoperatorio inmediato se observa hiperlaxitud articular lo que llevó a sospechar el diagnóstico. Este fue positivo conjuntamente con el servicio de Neurología, se determinó Síndrome de Ehlers danlos tipo III. Conclusiones: El índice de falla es muy bajo en anestesia suaracnoidea pero si se presenta un paciente de este tipo, debe descartarse por completo. Existen pocos casos documentados de resistencia a la anestesia local; pero si así fuera, debe estudiarse exhaustivamente para buscar estrategias que permitan un acto anestésico óptimo(AU)

Introduction: Regional anesthesia is the most used in obstetric caesarean section. In particular, the use of intrathecal anesthesia has its advantages. Although the failure rate is low, the onset of this event generates difficulties that deserve attention. Objective: To describe the anesthetic management in an obstetric patient with failure of regional intrathecal anesthesia. Clinical case: Female patient, aged 20 years, who was administered intrathecal anesthesia by the specialist. There were no errors in the lumbar puncture, this was done with a trocar 25 of Whitacre tip. No sensory, motor or sympathetic block was observed, so general endotracheal anesthesia was performed, which went on smoothly. In the immediate postoperative period, joint hypermobility was observed, leading to suspicion of the diagnosis. This was positive in conjunction with the Neurology service, Ehlers-Danlos syndrome type 3 was determined. Conclusions: The failure rate is very low for subarachnoid anesthesia. However, for a patient of this type, it should be completely ruled out. There are few documented cases of resistance to local anesthesia. If it were the case, it should be studied exhaustively to look for strategies that allow an optimal anesthetic management(AU)

Resistencia plasmídica a colistin por el gen mcr-1 en Enterobacteriaceae en Paraguay / Plasmid-Mediated Colistin Resistance Gene mcr-1 in Enterobacteriaceae in Paraguay

La resistencia a las polimixinas mediada por plásmidos (gen mcr-1) representa una amenaza para la salud pública, puesto que colistina es utilizada en la práctica médica como una de las últimas alternativas para el tratamiento de gérmenes multiresistentes. Este estudio describe la circulaciónde cepas de Enterobacterias que portan este gen de resistencia, aisladas de pacientes hospitalizados, así como también de la comunidad. Los hallazgos de la Red de Vigilancia de la Resistencia a los Antimicrobianos-Paraguay fueron de casi el 5 % (4,7) en cepas remitidas con criterio de sospecha, siendo las especies involucradas Escherichiacoli, Klebsiella pneumoniae y Salmonella Schwarzengrund. Además, por métodos moleculares se confirmaron en todas ellas la portación de otros genes de resistencia (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) asociados al mcr-1. Palabras claves: Enterobacterias, resistencia, colistina, mcr-1.

Resistance to polymyxins mediated by plasmids (mcr-1 gene) represents a threat to public health, since colistin is used in medical practice, as one of the last alternatives, for the treatment of multi-resistant germs. This study describes the circulation of strains of Enterobacteria that carry this resistance gene, isolated from hospitalized patients, as well as from the community. The findings of the Red de Vigilancia de la Resistencia a los Antimicrobianos­Paraguay were almost 5% (4.7) in strains submitted with suspicion criteria; the species involved being Escherichia coli, Klebsiella pneumoniae and Salmonella Schwarzengrund. In addition, molecular methods confirmed in all of them the carrying of other resistance genes (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) associated with mcr-1. Key words: Enterobacteria, resistance, colistin, mcr-1.

Overexpression of eukaryotic initiation factor 5A (eIF5A) affects susceptibility to benznidazole in Trypanosoma cruzi populations

Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER) Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A (in which serine 2 was replaced by alanine). Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions (e.g., spermidine and trypanothione), both of which alter BZ's effects in the parasite.

A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: A study from Eastern India.

Background: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very effi cient opportunity for the diagnosis of drug resistance by in vitro method. Aim: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. Methods: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplifi ed by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI – BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. Results: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp – 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specifi c for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). Limitations: The major limitations of multipleprimer PCR amplifi cation refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. Conclusion: The study indicates the existence of rifampicin drug resistance in Eastern India.

Diagnóstico da infecção pelo HIV-1: proposta de um algoritmo para identificação de infecção aguda / Diagnosis of HIV-1 infection: proposal of an algorithm for the identification of acute infection

A epidemia de AIDS em nosso País apresenta relativa estabilidade, concentrada em populações de maior vulnerabilidade, porém informações são em geral relacionadas à fase avançada da doença. Nosso estudo procura contribuir com informações sobre casos incidentes de infeção pelo HIV-1, através de um algoritmo que favorece a identificação de casos em fase aguda da doença. Esse algoritmo, com critérios clínicos e laboratoriais, permitiu o recrutamento consecutivo de 99 pacientes em um universo de cerca de 300 casos novos incorporados ao SAE do Município de Santo André, no período de outubro de 2011 a novembro de 2014. Nesses pacientes recém diagnosticados foram analisadas características clínicas, epidemiológicas e aspectos moleculares do HIV-1. Alguns aspectos clínicos e laboratoriais foram avaliados em adicionais 154 casos incorporados nesta época, assim como algumas características do HIV-1 identificadas em teste de genotipagem no período entre 2001 e 2014, em pacientes do mesmo SAE, que permitiram contextualizar a coorte. Entre os 99 pacientes... O algoritmo proposto identificou 5 casos em 8 suspeitos de infecção aguda. A genotipagem pré-tratamento avaliou...Na coorte foi identificado ainda, por estudo clínico e epidemiológico, confirmado por associação filogenética, uma possível transmissão do HIV-1 por procedimento de manicure (Matsuda et al., 2014b).

The AIDS epidemic in our country shows a relative stability, concentrated in the most vulnerable populations, but the limited information is generally related to the advanced stage of the disease. Our study aims to contribute with information on incident cases of HIV-1 infection. In a universe of about 300 new cases incorporated into the Santo André AIDS Program from Oct/2011 to Nov/2014, 99 patients were recruited consecutively. The study evaluated patients admitted after a recent diagnostic through the usual demand for follow-up and from an active case finding, using clinical laboratory algorithm that aimed to identify patients with acute infection. Available clinical and laboratory data from 154 additional cases incorporated in the service during this period and molecular data from HIV-1 genotypic tests from patients of this service in the period of 2001-2012, allowed to contextualize the cohort findings. Among the 99 patients studied ...The proposed algorithm has identified 5/8 cases of suspected acute infection. Pretreatment genotyping, evaluated...In the cohort, we identified, based in epidemiological clinical and subsequent phylogeny a possible transmission of HIV-1 for manicure procedure (Matsuda et al., 2014b).

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Genetics of chloroquine-resistant malaria: a haplotypic view

The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.

Frequency of benzimidazole resistance in Haemonchus contortus populations isolated from buffalo, goat and sheep herds / Frequência da resistência ao benzimidazol em populações de Haemonchus contortus isoladas de rebanhos de bubalinos, caprinos e ovinos

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

A resistência aos anti-helmínticos é um problema crescente que ameaça a produção pecuária em todo o mundo. A compreensão da base genética da resistência ao benzimidazol permitiu, recentemente, o desenvolvimento de métodos diagnósticos moleculares promissores. Neste estudo, isolados de Haemonchus contortus obtidos a partir de rebanhos de caprinos, ovinos e bubalinos criados no Brasil foram avaliados quanto à presença do polimorfismo F200Y no gene da β-tubulina1, o qual confere resistência ao benzimidazol. A frequência alélica da mutação variou de 7% a 43%, indicando que a resistência ao benzimidazol pode ser encontrada em nematoides isolados a partir de todas as espécies de ruminantes pesquisadas. Embora tenha sido observada variação significativa das frequências de mutação F200Y entre rebanhos/espécies hospedeiros distintos, não foi encontrada variação significativa entre populações isoladas de animais dentro de um mesmo rebanho. Estes achados sugerem que a avaliação de amostras de alguns poucos animais tem o potencial de fornecer informações sobre o nível de resistência ao benzimidazol de todo o rebanho, possibilitando uma redução considerável dos custos de diagnóstico para o produtor. O diagnóstico molecular apresenta vantagens práticas, uma vez que pode guiar a escolha da base anti-helmíntica a ser utilizada antes da sua aplicação no rebanho, reduzindo, portanto, as perdas ocasionadas pela resistência aos fármacos anti-helmínticos.

Pharmacogenetics in schizophrenia: a review of clozapine studies

Objectives: Clozapine is quite effective to treat schizophrenia, but its use is complicated by several factors. Although many patients respond to antipsychotic therapy, about 50% of them exhibit inadequate response, and ineffective medication trials may entail weeks of unremitted illness, potential adverse drug reactions, and treatment nonadherence. This review of the literature sought to describe the main pharmacogenetic studies of clozapine and the genes that potentially influence response to treatment with this medication in schizophrenics. Methods: We searched the PubMed database for studies published in English in the last 20 years using keywords related to the topic. Results and Conclusions: Our search yielded 145 studies that met the search and selection criteria. Of these, 21 review articles were excluded. The 124 studies included for analysis showed controversial results. Therefore, efforts to identify key gene mechanisms that will be useful in predicting clozapine response and side effects have not been fully successful. Further studies with new analysis approaches and larger sample sizes are still required. .