Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis.

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1ß secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.IMPORTANCEAddressing drug-resistant tuberculosis with existing therapies is challenging and the treatment success rate is lower when compared to drug-susceptible infection. This study demonstrates that pairing beta-lactams with ethambutol (EMB) significantly improves their efficacy against Mycobacterium tuberculosis (Mtb). The presence of EMB enhances beta-lactam access through the cell wall, which may translate into a prolonged contact between the drug and its targets at a concentration that effectively kills the pathogen. Importantly, we showed that the effects of the EMB and meropenem (MEM)/clavulanate combination were maintained intracellularly. These results are of high significance considering that the time above the minimum inhibitory concentration is the main determinant of beta-lactam efficacy. Moreover, a correlation was established between incubation with higher MEM concentrations during macrophage infection and increased IL-1ß secretion. This finding unveils a previously overlooked aspect of carbapenem repurposing against tuberculosis, as certain Mtb strains suppress the secretion of this key pro-inflammatory cytokine to evade host surveillance.

Plasmodium falciparum Chloroquine-pfcrt Resistant Haplotypes in Brazilian Endemic Areas Four Decades after CQ Withdrawn.

(1) Background: Malaria is a public health problem worldwide. Despite global efforts to control it, antimalarial drug resistance remains a great challenge. In 2009, our team identified, for the first time in Brazil, chloroquine (CQ)-susceptible Plasmodium falciparum parasites in isolates from the Brazilian Amazon. The present study extends those observations to include survey samples from 2010 to 2018 from the Amazonas and Acre states for the purpose of tracking pfcrt molecular changes in P. falciparum parasites. (2) Objective: to investigate SNPs in the P. falciparum gene associated with chemoresistance to CQ (pfcrt). (3) Methods: Sixty-six P. falciparum samples from the Amazonas and Acre states were collected from 2010 to 2018 in patients diagnosed at the Reference Research Center for Treatment and Diagnosis of Malaria (CPD-Mal/Fiocruz), FMT-HVD and Acre Health Units. These samples were subjected to PCR and DNA Sanger sequencing to identify mutations in pfcrt (C72S, M74I, N75E, and K76T). (4) Results: Of the 66 P. falciparum samples genotyped for pfcrt, 94% carried CQ-resistant genotypes and only 4 showed a CQ pfcrt sensitive-wild type genotype, i.e., 1 from Barcelos and 3 from Manaus. (5) Conclusion: CQ-resistant P. falciparum populations are fixed, and thus, CQ cannot be reintroduced in malaria falciparum therapy.

Spatial Dispersal of Epstein-Barr Virus in South America Reveals an African American Variant in Brazilian Lymphomas.

Epstein−Barr virus (EBV) is a saliva-borne É£-herpesvirus associated with benign and malignant lymphoproliferation. EBV-mediated tumorigenic mechanisms are not fully understood and may be related to viral genetic variations. In this work, we characterize the genetic diversity of EBV from Brazil, assessing 82 samples derived from saliva from asymptomatic carriers (n = 45), biopsies of benign reactive hyperplasia (n = 4), and lymphomas (n = 33). Phylogenetic and phylogeographic analysis of the entire coding region of the LMP-1 was performed. Additionally, type 1/type 2 distinction by the EBNA3C gene and Zp variants were evaluated. Our results revealed a high diversity of EBV in Brazil, with the co-circulation of four main clades, described here as: Mediterranean (40.2%, n = 33), Raji/Argentine (39%, n = 32), B95-8 (6.1%, n = 5), and Asian II (1.2%, n = 1). The Raji/Argentine and Mediterranean clades were the most prevalent in South America (45% and 28%, respectively). The Raji/Argentine clade was associated with polymorphisms I124V/I152L, del30 bp, and ins15 bp (p < 0.0001, to all clades) and with a high haplotype diversity related to EBV type and Zp variants. We found that a Raji/Argentine subclade spread primarily from Brazil and later to other South American countries. Although no LMP1 variant has been directly associated with disease, the Raji/Argentine clade was predominantly clustered with lymphomas (61%) and the Mediterranean clade with non-malignant cases (59%) (p = 0.1). These data highlight the high genetic diversity of EBV circulating in Brazil, calling attention to a Raji-related variant with great recombination potential in Brazilian lymphomas.

Can torque teno virus be a predictor of SARS-CoV-2 disease progression in cancer patients?

INTRODUCTION: Cancer patients with SARS-CoV-2 infection can experience a broad range of clinical manifestations and outcomes. Previous studies have demonstrated an association between torque teno virus (TTV) load and deficiencies of the immune system. The impact of SARS-CoV-2 and TTV viral loads in cancer patients is unknown. METHODS: In this retrospective study, 157 cancer patients and 191 noncancer controls were analysed for SARS-CoV-2 RNA and TTV DNA presence. RESULTS: SARS-CoV-2 RNA was detected in 66.2% of cancer patients and in 68.6% of noncancer control subjects. In SARS-CoV-2-positive patients, TTV was detectable in 79.8% of cancer patients, while in controls, TTV was detected in 71.7% of subjects. No statistically significant correlation was found between TTV and SARS-CoV-2 loads in cancer patients. However, the 100-day survival rate in cancer patients who died from COVID-19 was significantly lower in the TTV-positive group than in the TTV-negative group (P = 0.0475). In the cancer TTV-positive group, those who died also had a higher load of TTV than those who did not die (P = 0.0097). CONCLUSIONS: Our findings indicated that the presence of TTV in nasopharyngeal swabs from cancer patients was related to a higher number of deaths from COVID-19 and to a higher TTV DNA load.

Molecular aspects of Chikungunya virus infections in cancer patients.

BACKGROUND: Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016. OBJECTIVE: The aim of this work was to evaluate CHIKV viral load in serum, plasma and urine in cancer patients to determine the best sample for diagnosis, as well as perform molecular characterisation and phylogenetic analysis of circulating strains. METHODS: Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced. FINDINGS: We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients. MAIN CONCLUSION: The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution.

Molecular aspects of Chikungunya virus infections in cancer patients

BACKGROUND Chikungunya virus (CHIKV) is an arbovirus that can cause chronic and debilitating manifestations. The first autochthonous case in Rio de Janeiro state was diagnosed in 2015, and an outbreak was declared in 2016. OBJECTIVE The aim of this work was to evaluate CHIKV viral load in serum, plasma and urine in cancer patients to determine the best sample for diagnosis, as well as perform molecular characterisation and phylogenetic analysis of circulating strains. METHODS Paired serum, plasma and urine collected from 31 cancer patients were tested by real-time quantitative polymerase chain reaction (qPCR) and a segment of the CHIKV E1 gene was sequenced. FINDINGS We detected 11 CHIKV+ oncological patients. Paired samples analyses of nine patients showed a different pattern of detection. Also, a higher viral load in plasma (6.84 log10) and serum (6.07 log10) vs urine (3.76 log10) was found. Phylogenetic analysis and molecular characterisation revealed East/Central/Southern Africa (ECSA) genotype circulation and three amino acids substitutions (E1-K211T, E1-M269V, E1-T288I) in positive patients. MAIN CONCLUSION The results indicate the bioequivalence of serum and plasma for CHIKV diagnosis, with urine being an important complement. ECSA genotype was circulating among patients in the period of the 2016 outbreak with K211T, M269V and T288I substitution.

Parvovirus B19 in the Context of Hematopoietic Stem Cell Transplantation: Evaluating Cell Donors and Recipients.

BACKGROUND: Parvovirus B19 (B19V) is a common human pathogen, member of the family Parvoviridae. Typically, B19V has been found to infect erythroid progenitors and cause hematological disorders, such as anemia and aplastic crisis. However, the persistence of genomic deoxyribonucleic acid (DNA) has been demonstrated in tonsils, liver, skin, brain, synovial, and testicular tissues as well as bone marrow, for both symptomatic and asymptomatic subjects. Although the molecular and cellular mechanisms of persistence remain undefined, it raises questions about potential virus transmissibility and its effects in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. METHODS: With this aim, we retrospectively screened allogeneic stem cell donors from 173 patients admitted for allo-HSCT from January 2008 to May 2013 using a seminested polymerase chain reaction approach. RESULTS: We found 8 positive donor samples, yielding a 4.6% of parvovirus prevalence (95% confidence interval, 2.36-8.85). Pre- and post-HSCT samples (n = 51) from the 8 recipients of the positive donors were also investigated, and 1 case exhibited B19V DNA in the post-HSCT follow-up (D + 60). Direct DNA sequencing was performed to determine the genotype of isolates and classification, performed by phylogenetic reconstruction, showed a predominance of genotype 1a, whereas the rare genotype 3b was detected in 2 additional patients. By molecular cloning, different B19V 1a substrains polymorphisms were evidenced in the single case in which donor and its recipient were B19V+. CONCLUSIONS: Our results suggest that HSCT allografts are not a main source for B19V transmission, pointing to potential events of reinfection or endogenous viral reactivation.

Low-grade sulfadoxine-pyrimethamine resistance in Plasmodium falciparum parasites from Lubango, Angola.

BACKGROUND: Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively. METHODS: Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing. RESULTS: In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted. CONCLUSION: pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control.

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.

Molecular markers of antifolate resistance in Plasmodium falciparum isolates from Luanda, Angola.

BACKGROUND: Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. METHODS: Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. RESULTS: The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). CONCLUSION: This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.

Tipagem de marcadores moleculares e de genes potencialmente associados à resistência a antimaláricos em isolados de Plasmodium falciparum e P. vivaz do Brasil / Typing of molecular markers and genes potentially associated with resistance to antimalarial drugs in isolates of Plasmodium falciparum and P. Brazil's lively

A malária, doença infecciosa causada por parasitas do gênero Plasmodium, é um conhecido flagelo das populações humanas desde a antiguidade. Na falta de uma vacina, a política de controle baseia-se principalmente no diagnostico rápido e no tratamento dos casos. Devido ao impacto da quimiorresistência parasitária no controle, faz-se necessário o constante monitoramento da eficácia de drogas. Portanto, para contextualizar o Brasil no panorama mundial, nosso objetivo se constituiu em realizar um estudo descritivo sobre a ocorrência de mutações (SNPs) nos genes considerados marcadores moleculares associados à resistência como pfcrt, pfmdr1, pfdhfr, pfdhps e pvdhfr, assim como em genes potencialmente associados à quimiorresistência (pfatpase6 e pvmdr1) em isolados de P. falciparum e P. vivax coletados nas cidades Amazônicas de Porto Velho, Paragominas e Manaus. Para tal, utilizamos PCRs seguidas do sequenciamento de DNA. Ao investigarmos a ocorrência de mutações nos genes pfcrt, pfdhfr e pfdhps em amostras de P. falciparum provenientes de Porto Velho e Paragominas foi possível detectar 1 isolado apresentando um haplótipo ou perfil de sensibilidade CVMNK no gene pfcrt e ACNSVI no gene pfdhfr e, além disso, pudemos observar uma redução no numero de mutações no gene pfdhps, ao confrontarmos os nossos resultados com os de um estudo retrospectivo. Em relação ao P. vivax, a análise do gene pvdmr1 num conjunto de amostras provenientes de Paragominas revelou o predomínio de mutações no códon 976 do gene pvmdr1 (preliminarmente associado com a resistência à cloroquina) e a presença de haplótipos duplo-mutante FRTNI e triplo-mutante FRTNL para o gene pvdhfr. dando continuidade à caracterização de P. falciparum no Brasil, foi estabelecido um registro de base da ocorrência de mutações nos genes pfmdr1 e pfatpase6, em amostras procedentes de Porto Velho, Paragominas e Manaus. Tal análise permitiu a identificação de um haplótipo prevalente NEF/CDVY no pfmdr1, assim como identificação de mutantes em um único códon ou duplo-mutantes (630 e/ou 402) no caso do gene pfatpase6. também não foi encontrada a mutação 769N, associada com a diminuição da susceptibilidade ao arteméter. Concluímos que: (a) existem no Brasil popluações parasitárias de P. falcipoarum portando haplótipos sensíveis para o gene pfcrt e o pfdhfr; (b) a mutação no códon 976 no gene pfmdr1 pode não ser válida para o monitoramento da resistência à cloroquina em isolados brasileiros de P. vivax; (c) as populações de P. vivax avaliadas apresentaram mutações no gene pvdhfr, apesar de nunca ter sido instituído o tratamento com sulfadoxina-pirimetamina para malária vivax e; (d) as amostras de P. falciparum avaliadas não portavam todos os alelos do gene pfmdr1 associados a um potencial desenvolvimento de resistência à combinação arteméter-lumefantrina.

Brazilian Plasmodium falciparum isolates: investigation of candidate polymorphisms for artemisinin resistance before introduction of artemisinin-based combination therapy.

BACKGROUND: This study was performed to better understand the genetic diversity of known polymorphisms in pfatpase6 and pfmdr1 genes before the introduction of ACT in Brazil, in order to get a genotypic snapshot of Plasmodium falciparum parasites that may be used as baseline reference for future studies. METHODS: Parasites from P. falciparum samples collected in 2002, 2004 and 2006-2007 were genotyped using PCR and DNA sequencing at codons 86, 130, 184, 1034, 1042, 1109 and 1246 for pfmdr1 gene, and 243, 263, 402, 431, 623, 630, 639, 683, 716, 776, 769 and 771 for pfatpase6 gene. RESULTS: A pfmdr1 haplotype NEF/CDVY was found in 97% of the samples. In the case of pfatpase6, four haplotypes, wild-type (37%), 630 S (35%), 402 V (5%) and double-mutant 630 S + 402 V (23%), were detected. CONCLUSION: Although some polymorphism in pfmdr1 and pfatpase6 were verified, no reported haplotypes in both genes that may mediate altered response to ACT was detected before the introduction of this therapy in Brazil. Thus, the haplotypes herein described can be very useful as a baseline reference of P. falciparum populations without ACT drug pressure.

Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene.

BACKGROUND: Effective treatment remains a mainstay of malaria control, but it is unfortunately strongly compromised by drug resistance, particularly in Plasmodium falciparum, the most important human malaria parasite. Although P. falciparum chemoresistance is well recognized all over the world, limited data are available on the distribution and prevalence of pfcrt and pfmdr1 haplotypes that mediate resistance to commonly used drugs and that show distinct geographic differences. METHODS: Plasmodium falciparum-infected blood samples collected in 2007 at four municipalities of Luanda, Angola, were genotyped using PCR and direct DNA sequencing. Single nucleotide polymorphisms in the P. falciparum pfcrt and pfmdr1 genes were assessed and haplotype prevalences were determined. RESULTS AND DISCUSSION: The most prevalent pfcrt haplotype was StctVMNT (representing amino acids at codons 72-76). This result was unexpected, since the StctVMNT haplotype has previously been seen mainly in parasites from South America and India. The CVIET, CVMNT and CVINT drug-resistance haplotypes were also found, and one previously undescribed haplotype (CVMDT) was detected. Regarding pfmdr1, the most prevalent haplotype was YEYSNVD (representing amino acids at codons 86, 130, 184, 1034, 1042, 1109 and 1246). Wild haplotypes for pfcrt and pfmdr1 were uncommon; 3% of field isolates harbored wild type pfcrt (CVMNK), whereas 21% had wild type pfmdr1 (NEYSNVD). The observed predominance of the StctVMNT haplotype in Angola could be a result of frequent travel between Brazil and Angola citizens in the context of selective pressure of heavy CQ use. CONCLUSIONS: The high prevalence of the pfcrt SVMNT haplotype and the pfmdr1 86Y mutation confirm high-level chloroquine resistance and might suggest reduced efficacy of amodiaquine in Angola. Further studies must be encouraged to examine the in vitro sensitivity of pfcrt SVMNT parasites to artesunate and amodiaquine for better conclusive data.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates.

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7 percent) and double (14.3 percent) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2 percent) and triple (42.8 percent) mutant haplotypes. The implications of these findings are discussed.

Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area.

BACKGROUND: The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. METHODS: Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. RESULTS: Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. CONCLUSION: This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed.

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia.

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.

Padronização de reações de polimerização em cadeia(PCRs) convencional e em tempo real gênero-específicas para a detecção dos parasitas da malária em indivíduos pauciparasitados / Standardization of reactions of PCRs conventional and real time genus specific for the detection of the parasites of the malaria in low-grade individuals

A malária até hoje permanece como uma doença de alta prevalência nas áreas tropicais e subtropicais.Em regiões de transmissão perene, muitos indivíduos após várias infecções podem permanecer assintomáticos com parasitemias submicroscópicas necessitando de técnicas mais sensíveis do que o exame microscópico para a detecção dos parasitas.Essa condição dificulta o diagnóstico individual...Dentro dessa ótica nos propusemos a desenvolver PCRs gênero específicas convencional e em tempo real para a detecção dos parasitas da malária em indivíduos pauciparasitados.Para a padronização e validação das PCRs foram utilizadas 84 amostras de sangue no caso da PCR convencional e 33 dessas amostras no caso da PCR em tempo real,as quais foram coletadas nas cidades de Paragominas(PA)e Porto Velho(RO)...Ambas PCRs apresentaram a mesma especificidade frente a amostras clínicas de HBV e HIV,tendo sido assegurada frente a controles negativos de áreas endêmicas,no caso da PCR convencional.O limite de detecção para ambas PCRs foi de 0,5 parasita/µL.Contrariamente...Considerando as amostras extraídas pela técnica de fenol-clorofórmio,as PCRs de gênero mostraram 100 por cento de concordância de resultados quando comparadas com as PCRs espécie-específicas e com o exame microscópico realizado em nosso laboratório.Entretanto,quando consideramos o exame microscópico realizado em Paragominas,os resultados desse exame concordaram 98,8 por cento(n=84)com a PCR convencional e 96,9 por cento(n=33)com a PCR em tempo real.Assim,embora o tempo de realização do fenol-clorofórmio seja um fator limitante para sua utilização em larga escala,além da sua toxicidade,ele se mostrou como o método mais eficiente para a extração de DNA, seguido pelas técnicas das colunas e de Kawasaki.A escolha entre a técnica de PCR em seu formato convencional e a em tempo real... certamente deverá ser norteada pela disponibilidade de recursos financeiros. Devido ao seu menor custo,a PCR convencional poderá ser utilizada em...

Glycosylation influencing on fusion activity of HA and HEF structures of influenza viruses

Desde os tempos mais remotos, os vírus influenza têm sido o agente causal de doenças respiratórias letais. O seu processo de replicação ocorre nas células epiteliais do trato respiratório, causando uma síndrome respiratória aguda, na qual as glicoproteínas hemaglutinina (HA) e hemaglutinina-esterase-fusão (HEF), ancoradas na superfície dos vírus influenza A e C, respectivamente, são responsáveis pelos processos de fusão durante o ciclo de replicação viral. Ao contrário dos processos de adsorção, sialidase e esterase, atividades inatas desempenhadas por estas mesmas estruturas, a fusão depende de uma glicosilação e clivagem protéica prévias, para que o vírus se torne infeccioso. A glicosilação das proteínas de superfície está associada principalmente com a antigenicidade e com a estabilidade das proteínas de fusão. O presente trabalho analisa a influência da de-glicosilação sobre a atividade de fusão, usando vírus influenza A e C como modelos de estudo. A de-glicosilação parcial provocou significativa redução da atividade fusogênica, induzindo uma redução de 51,0 por cento, 87,5 por cento, 95,5 por cento e 79,3 por cento em relação às amostras de vírus influenza A/Memphis/102/ 72, A/FM/1/47, C/Taylor/1233/47 e C/Paris/1/67, respectivamente, acarretando também eventuais aumentos desta atividade biológica para certas amostras: 10,15 por cento (pH 5,8), 59,47 por cento (pH 5,8), 32,55 por cento (pH 5,8) e 80,7 por cento (pH 5,4) com relação às amostras de vírus influenza A/Memphis/102/72, A/FM/1/ 47, C/Taylor/1233/47 e A/Paris/1/67, respectivamente. Os resultados permitem concluir que o nível de glicosilação mostra-se estreitamente relacionado com a estabilidade das proteínas, causando o processo deglicosilação uma influência significativa sobre a atividade fusogênica viral.