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Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning. [ABSTRACT FROM AUTHOR]

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption. [ABSTRACT FROM AUTHOR]

Background: The objective of this study is to evaluate the diagnostic accuracy of plasma‐based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell‐free DNA from patients with ameloblastoma. Methods: This is a prospective diagnostic accuracy study conducted based on the Standards for Reporting Diagnostic Accuracy recommendations. The index test was the plasma‐based liquid biopsy, whereas the reference standard was the conventional tissue biopsy. The target condition was the detection of BRAF V600E mutation. The study population consisted of individuals with ameloblastoma recruited from three tertiary hospitals from Brazil. A negative control group composed of three individuals with confirmed wild‐type BRAF lesions were included. The participants underwent plasma circulating cell‐free DNA and tumor tissue DNA isolation, and both were submitted to using competitive allele‐specific TaqMan™ real‐time polymerase chain reaction technology mutation detection assays. Sensitivity and specificity measures and positive and negative predictive values were calculated. Results: Twelve patients with conventional ameloblastoma were included. BRAF V600E mutation was detected in 11/12 (91.66%) ameloblastoma tissue samples. However, the mutation was not detected in any of the plasma‐based liquid biopsy circulating cell‐free DNA samples in both ameloblastomas and negative control group. The sensitivity and specificity of plasma‐based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell‐free DNA was 0.0 and 1.0, respectively. The agreement between index test and reference standard results was 26.66%. Conclusion: Plasma‐based liquid biopsy does not seem to be an accurate method for the detection of the BRAF V600E mutation in circulating circulating cell‐free DNA from patients with ameloblastoma, regardless of tumor size, anatomic location, recurrence status, and other clinicopathological features. [ABSTRACT FROM AUTHOR]

The emergence of SARS-CoV-2 variants can affect their detection via RT-qPCR. The Omicron variant has a greater affinity for the upper respiratory system and causes clinical conditions similar to bronchitis, as opposed to the pneumonitis-like conditions caused by other SARS-CoV-2 variants. This characteristic increases the viscosity of clinical samples collected for diagnosis. Coinciding with the arrival of the Omicron variant, we observed a failure in control gene expression in our laboratory. In this report, we have optimized a rapid nucleic acid extraction step to restore gene expression and detect the presence of the SARS-CoV-2 virus. We reevaluated 3000 samples, compared variant types occurring in different time periods, and confirmed that the presence of the Omicron variant was responsible for changes observed in the characteristics of these clinical samples. For samples with large amounts of mucus, such as those containing the Omicron variant, a modification to the nucleic acid extraction step was sufficient to restore the quality of RT-qPCR results. [ABSTRACT FROM AUTHOR]

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10−1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1–2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings. Author summary: Having standardized, comparative studies of Zika virus (ZIKV) tests are important for implementing optimal diagnostic testing and disease surveillance. Detection of ZIKV nucleic acid is ideal for confirming infection; however, knowing the sensitivity and specificity of a test is needed for selecting the right test in a given laboratory. We looked at the analytical performance of 15 diagnostic and blood screening ZIKV molecular tests, including tests that detect ZIKV only and tests that detect ZIKV plus other arboviruses. We found donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, mostly due to the higher sample input volumes. We also found no difference in analytical sensitivity between tests that only detected ZIKV compared to tests that detected ZIKV and related viruses. The ideal molecular test will depend on the testing context, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection. [ABSTRACT FROM AUTHOR]

The global impact of coronavirus disease 2019 (COVID-19) is unprecedented, and many control and prevention measures have been implemented to test for and trace COVID-19. However, invisible-spreaders, who are associated with nucleic acid detection and asymptomatic infections, have received insufficient attention in the current COVID-19 control efforts. In this paper, we analyze the time series infection data for Italy, Germany, Brazil, India and Sweden since the first wave outbreak to address the following issues through a series of experiments. We conclude that: 1) As of June 1, 2020, the proportion of invisible-spreaders is close to 0.4% in Sweden, 0.8% in early Italy and Germany, and 0.4% in the middle and late stages. However, in Brazil and India, the proportion still shows a gradual upward trend; 2) During the spread of this pandemic, even a slight increase in the proportion of invisible-spreaders could have large implications for the health of the community; and 3) On resuming work, the pandemic intervention measures will be relaxed, and invisible-spreaders will cause a new round of outbreaks. [ABSTRACT FROM AUTHOR]

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods. [ABSTRACT FROM AUTHOR]

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research. Image 1 • We standardized a specific LAMP reaction for Leishmania amazonensis. • LAMP reaction amplifies a sample with a parasite load of only 28 parasites in 50 ng of DNA. • LAMP results could be visualized by PAGE and/or dye SYBR® Safe. • Our work could contribute with identification of Leishmania species. [ABSTRACT FROM AUTHOR]

Multidrug-resistant Salmonella enterica serovar Heidelberg isolates are frequently recovered in the Netherlands from poultry meat imported from South America. Our aim was to retrospectively assess the characteristics of the antimicrobial determinants, gene content and the clonal relatedness of 122 unique S. Heidelberg isolates from chicken meat from Brazil (n = 119) and Argentina (n = 3) that were imported between 2010 and 2015. These isolates were subjected to antimicrobial susceptibility testing, PCR and Illumina HiSeq2500 whole genome sequencing. Draft genomes were assembled to assess the gene content, and the phylogenetic relationships between isolates were determined using single nucleotide polymorphisms. Ciprofloxacin-resistance was identified in 98.4% of the isolates and 83.7% isolates showed resistance to the extended-spectrum cephalosporins cefotaxime and ceftazidime (83.6% and 82.8% respectively). Of the latter, 97.1% exhibited an AmpC phenotype and contained blaCMY-2, whereas the remaining three isolates contained an extended spectrum beta-lactamase. Of the 99 extended-spectrum cephalosporins-resistant isolates harboring CMY-2 plasmids, 56.6% contained the incompatibility group I1 replicon. Phylogenetic cluster analysis showed that all isolates from Brazil clustered together, with 49% occurring in clusters larger than 5 isolates that revealed intra-cluster similarities based on geographical location and/or resistance profiles. The remaining isolates were classified in smaller clusters or as singletons, highlighting the large diversity of S. Heidelberg in the poultry chain in Brazil that was revealed by this study. Considering the potential public health risk associated with multidrug-resistant S. Heidelberg in imported poultry, collaborative whole genome sequencing-based surveillance is needed to monitor the spread, pathogenic properties and epidemiological distribution of these isolates. [ABSTRACT FROM AUTHOR]

In the border region between Brazil and French Guiana, American cutaneous leishmaniasis is a worrisome public health issue, and entomological studies are required there to better identify classical and putative emerging transmission patterns. The present study aimed to detect and characterize Leishmania DNA in the phlebotomine population of Oiapoque (Amapá State, Brazil). Phlebotomines were captured in anthropized and wild environments in the outskirts of Oiapoque municipality, using CDC light traps installed in vertical (ground/canopy level) and horizontal (peridomicile/extradomicile/forest-edge/forest) strata. Captured specimens were identified according to their morphology. Females were processed for Leishmania DNA detection and characterization using a multiplex polymerase chain reaction targeting kinetoplast DNA (kDNA) and the phlebotomine cacophony gene. The kDNA positive samples were characterized by cloning and sequencing the Leishmania 234 bp-hsp70 gene. Among the 3957 phlebotomine specimens captured, 26 pooled female samples were positive for Leishmania (Viannia) spp. DNA. Sequencing analysis allowed species-specific identification of L. (V.) braziliensis DNA in Trichophoromyia ininii, Bichromomyia flaviscutellata, Nyssomyia umbratilis, and Evandromyia infraspinosa, and L. (V.) guyanensis DNA in Ny. umbratilis. A pooled sample of Ny. umbratilis was positive for both L. (V.) braziliensis and L. (V.) guyanensis DNA. The present study provided additional information regarding ACL ecology in Oiapoque, highlighting the presence of L. (V.) braziliensis DNA in different phlebotomine species. The epidemiological implications of these findings and the determinant incrimination of L. (V.) braziliensis as proven vectors in that region must be clarified. In this regard, studies on Leishmania spp. infection and suggestive anthropophilic behavior of associated phlebotomines need to be prioritized in entomological surveillance. [ABSTRACT FROM AUTHOR]

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) has been described in Native American populations prior to the arrival of European explorers, and in Brazilian populations dating from the Colonial Period. There are no studies demonstrating TB infection in native Brazilians, and the history and epidemiological scenario of TB in Brazil is still unknown. The aim of this study was to verify the presence of TB infection among the native Tenetehara-Guajajara population from Maranhão State, Brazil, 210 ± 40 years ago. A Tenetehara-Guajajara skeleton collection was submitted to paleopathological analysis, and rib bone samples (n = 17) were used for paleogenetic analysis based on Mycobacterium tuberculosis complex (MTC) targets. Porotic hyperostosis and cribra orbitalia were found in 10 and 13 individuals, respectively. Maternal ancestry analysis revealed Native American mtDNA haplogroups A and C1 in three individuals. Three samples showed osteological evidence suggestive of TB. katG and mtp40 sequences were detected in three individuals, indicating probable TB infection by two MTC lineages. Tuberculosis infection in the Tenetehara-Guajajara population since the 18th century points to a panorama of the disease resulting, most probably, from European contact. However, the important contribution of African slaves in the population of Maranhão State, could be also considered as a source of the disease. This study provides new data on TB during the Brazilian Colonial Period. This is the first report integrating paleopathological and paleogenetic data for the study of TB in Brazil. [ABSTRACT FROM AUTHOR]

Background: The history of the development and implementation of the Brazilian nucleic acid testing (NAT) platform to detect and discriminate among human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in blood donors is described here. The results for the sensitivity, reproducibility, and NAT yield of the platform since program implementation are provided.Study Design and Methods: The Brazilian NAT HIV, HCV, and HBV kit was developed and evaluated with regard to analytical sensitivity, specificity, intralot and interlot reproducibility, interfering substances, and genotype and diagnostic sensitivity. Additionally, a sample of identified NAT-yield cases was characterized with regard to viral load.Results: The 95% limits of detection for HIV, HCV, and HBV were 68.02, 102.35, and 9.08 IU/mL, respectively. All replicates were detected with reproducibility assays between the acceptable values. A total of 13,610,536 blood donors was screened from 2010 to 2016, and 63 HIV-yield cases and 28 HCV-yield cases were detected. Among 5,795,424 blood donors screened for HBV from 2014 to 2016, 42 yield cases were found.Conclusion: The Brazilian NAT HIV, HCV, and HBV kit is an automated NAT system suitable for routine blood donor screening in a completely traceable process. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements set by the health ministry for blood donor screening. A significant number of transmission cases were prevented by the implementation of this important program. [ABSTRACT FROM AUTHOR]

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes of the genus Meloidogyne, known as root-knot nematodes (RKN), have an important economic impact on golf course turfgrasses. The most prevalent RKN species associated with grasses are M. chitwoodi, M. graminicola, M. graminis, M. incognita, M. marylandi, M. microtyla, M. minor, M. naasi and M. sasseri. In 2010, slight thickening of the roots and RKN females with unusual features were observed in turfgrass roots on golf courses in Araras, São Paulo state, Brazil. This population (MgARA) was maintained in the lab and studied including morphological, morphometrical, biochemical and molecular markers. Morphology and morphometry were variable and not useful for identification, although perineal pattern morphology showed highly similarity with M. graminis description. Concerning to biochemical characterisation, the esterase phenotype Mg1, characterised by a very slow and fainter band, was detected in some protein homogenates. Regarding to molecular analysis, D2-D3 region of 28S rDNA gene and cytochrome oxidase subunit II region from mitochondrial DNA were amplified by PCR and sequenced. Phylogenetic analysis revealed that the Brazilian isolate, found associated with turfgrass, grouped with M. graminis isolates (98–99% bootstrap; variation of 8–11 and 0–24 bp, respectively), close to M. marylandi, supporting its identification as M. graminis. This is the first report of M. graminis on golf courses in Brazil. [ABSTRACT FROM AUTHOR]

The New World genus Philonome Chambers, 1874 is revised. This genus comprises twelve species, seven of which are described as new: two species, P. nigrescens sp. n. and P. wielgusi sp. n., from the United States; four species, P. albivittata sp. n., P. curvilineata sp. n., P. kawakitai sp. n., and P. lambdagrapha sp. n., from French Guiana; and one species, P. penerivifera sp. n., from Brazil. Lectotypes are designated for Philonome clemensella Chambers, 1874 and P. rivifera Meyrick, 1915. Partially on evidence of their head morphology and particularly from molecular evidence, the genus Philonome, previously associated with Bucculatricidae or Lyonetiidae, is reassigned to Tineidae. A possible systematic position of Philonome within Tineidae is discussed. Eurynome Chambers, 1875, is synonymized with Argyresthia Hübner, 1825 (Argyresthiidae). Photographs of adults and illustrations of genitalia, when available, are provided for all described species of Philonome and two species previously misplaced in Philonome, Argyresthia luteella (Chambers, 1875) and Elachista albella (Chambers, 1877). In addition, DNA barcodes were used for the delimitation of most species. [ABSTRACT FROM AUTHOR]

People deprived of liberty in prisons are at higher risk of infection by the human immunodeficiency virus (HIV) due to their increased exposure through intravenous drug use, unprotected sexual activity, tattooing in prison and blood exposure in fights and rebellions. Yet, the contribution of intramural HIV transmission to the epidemic is scarcely known, especially in low- and middle-income settings. In this study, we surveyed 1,667 inmates incarcerated at Presídio Central de Porto Alegre, located in southern Brazil, for HIV infection and molecular characterization. The HIV seroprevalence was 6.6% (110/1,667). Further analyses were carried out on 40 HIV-seropositive inmates to assess HIV transmission clusters and drug resistance within the facility with the use of molecular and phylogenetic techniques. The molecular epidemiology of HIV-1 subtypes observed was similar to the one reported for the general population in southern Brazil, with the predominance of HIV-1 subtypes C, B, CRF31_BC and unique BC recombinants. In particular, the high rate (24%) of URF_BC found here may reflect multiple exposures of the population investigated to HIV infection. We failed to find HIV-infected inmates sharing transmission clusters with each other. Importantly, the analysis of HIV-1 pol genomic fragments evidenced high rates of HIV primary and secondary (acquired) drug resistance and an alarming proportion of virologic failure among patients under treatment, unveiling suboptimal access to antiretroviral therapy (ARV), low ARV adherence and dissemination of drug resistant HIV strains in primary infections. Our results call for immediate actions of public authority to implement preventive measures, serological screening and, for HIV-seropositive subjects, clinical and treatment follow-up in order to control HIV infection and limit the spread of drug resistance strains in Brazilian prisons. [ABSTRACT FROM AUTHOR]

Foamy viruses naturally infect a wide range of mammals, including Old World (OWP) and New World primates (NWP), which are collectively called simian foamy viruses (SFV). While NWP species in Central and South America are highly diverse, only SFV from captive marmoset, spider monkey, and squirrel monkey have been genetically characterized and the molecular epidemiology of SFV infection in NWPs remains unknown. We tested a large collection of genomic DNA (n  = 332) comprising 14 genera of NWP species for the presence of SFV polymerase (pol) sequences using generic PCR primers. Further molecular characterization of positive samples was carried out by LTR-gag and larger pol sequence analysis. We identified novel SFVs infecting nine NWP genera. Prevalence rates varied between 14–30% in different species for which at least 10 specimens were tested. High SFV genetic diversity among NWP up to 50% in LTR-gag and 40% in pol was revealed by intragenus and intrafamilial comparisons. Two different SFV strains infecting two captive yellow-breasted capuchins did not group in species-specific lineages but rather clustered with SFVs from marmoset and spider monkeys, indicating independent cross-species transmission events. We describe the first SFV epidemiology study of NWP, and the first evidence of SFV infection in wild NWPs. We also document a wide distribution of distinct SFVs in 14 NWP genera, including two novel co-speciating SFVs in capuchins and howler monkeys, suggestive of an ancient evolutionary history in NWPs for at least 28 million years. A high SFV genetic diversity was seen among NWP, yet these viruses seem able to jump between NWP species and even genera. Our results raise concerns for the risk of zoonotic transmission of NWP SFV to humans as these primates are regularly hunted for food or kept as pets in forest regions of South America. [ABSTRACT FROM AUTHOR]

This research had as objective to characterize genetically individuals of physic nut cultivated in experimental areas in Sergipe, Brazil by means of RAPD molecular markers. Leaves of 40 individuals were collected and DNA was isolated using CTAB 2% method. Were used 30 primers RAPD for DNA amplification, and this data was used to estimate the genetic similarity among the pairs of individuals, using Jaccard coefficient, and group them out for the UPGMA method. Also, the genetic structure and diversity of the populations were assessed using AMOVA. Of the 100 fragments generated, 29 of were polymorphic. A similarity average of 0.54 among the individuals was found and the amplitude similarities varied from 0.18 to 1.00. One of them (U5) was unit clusters and formed by the most divergent individuals. AMOVA indicated that there is more variation within (63%) the population. In conclusion, it was possible verify genetic variability in physic nut using RAPD markers at these experimental areas. [ABSTRACT FROM AUTHOR]

The article presents information on a study related to the genetic diversity of Brazilian isolates of feline immunodeficiency virus. It is stated that the authors isolated feline immunodeficiency virus (FIV) from three adult domestic cats, originating from two open shelters in Brazil. The nucleic acid sequences of seven clones were determined and then compared with those of previously described isolates.

The article focuses on a study which proposed a new molecular phylogeny for akodontine rodents from Brazil. Total deoxyribonucleic acid (DNA) was obtained from grounded tissues of 20 samples belonging to 8 species of the genus Akodon,while the other 28 sequences were obtained from GenBank. The proposed three phylogenetic trees were constructed by the methods of Neighbor-Joining Minimum Evolution and Maximum Parsimony. The similarities and differences of the topology of the taxa are discussed. It describes how the dating dispersion events are calculated.

The I50V protease inhibitor (PI) resistance mutation was found in 87.4% of protease gene fragments sequenced from 199 nucleic acid isolates extracted using an NASBA virus load assay, performed between 1997 and 2001 in Brazil. This mutation is an amprenavir-related mutation, and at that particular time this PI was seldom used in Brazil. This mutation was found both in patients with and without therapeutic success. Q calibrators showed the PI resistance mutation I50V when directly amplified and sequenced from the 423-bp PCR product targeting protease gene. The majority of the patients’ samples had a mixture of I50I and I50V; however, this artifact was nor seen when a 989-bp PCR product was used. These results show that RNA extracted using virus load kits need to be critically evaluated before being used in home-brew genotypic tests. [ABSTRACT FROM AUTHOR]

Background: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions. [ABSTRACT FROM AUTHOR]

The glacial refugia paradigm has been broadly applied to patterns of species dynamics and population diversification. However, recent geological studies have demonstrated striking Pleistocene climate changes in currently semiarid northeastern Brazil at time intervals much more frequent than the climatic oscillations associated with glacial and interglacial periods. These geomorphic data documented recurrent pulses of wet regimes in the past 210,000 years that correlate with climate anomalies affecting multiple continents. While analyzing DNA sequences of two mitochondrial genes (cytochrome b and NADH-dehydrogenase subunit 2) and one nuclear marker (cellular-myelocytomatosis proto-oncogene) in the forest-associated frogs Proceratophrys boiei and Ischnocnema gr. ramagii, we found evidence of biological responses consistent with these pluvial maxima events. Sampled areas included old, naturally isolated forest enclaves within the semiarid Caatinga, as well as recent man-made fragments of humid coastal Atlantic forest. Results show that mtDNA lineages in enclave populations are monophyletic or nearly so, whereas nonenclave populations are polyphyletic and more diverse. The studied taxa show evidence of demographic expansions at times that match phases of pluvial maxima inferred from geological data. Divergence times between several populations fall within comparatively drier intervals suggested by geomorphology. Mitochondrial and nuclear data show local populations to be genetically structured, with some high levels of differentiation that suggest the need of further taxonomic work. [ABSTRACT FROM AUTHOR]

Aims: A one-year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples. Methods and Results: Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT-PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real-time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB. Conclusions: Real-time PCR was more sensitive than nested RT-PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage. Significance and Impact of the Study: These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection. [ABSTRACT FROM AUTHOR]

In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of São Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, H.J#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown. [ABSTRACT FROM AUTHOR]

In an attempt to resolve whether two putative carlaviruses, potato rough dwarf virus (PRDV) and potato virus P (PVP), reported infecting potato in Argentina and Brazil, respectively, are the same virus in the genus Carlavirus, they were characterized using nucleotide sequence analysis, serology and bioassay. For PRDV and PVP, sequences of 2016 and 1492 bp, respectively, were obtained. Similarity searches of coat-protein amino acid sequences and the presence of the nucleic-acid-binding protein exhibiting the characteristic motif C-X2-C-X11-C-X4-C, highly conserved in carlaviruses, showed PRDV and PVP to be members of the genus Carlavirus. Further analysis showed that they were the same virus species since the full coat-protein sequence, translated from the nucleotide sequence, exceeded the 84% minimum identity required for members of the same species within the genus Carlavirus, at 90·8% identity in an ungapped alignment of 304 amino acids. Moreover, the viruses could not be distinguished using polyclonal antibodies raised to PRDV and PVP in ELISA. They could, however, be distinguished using an anti-PVP monoclonal antibody, which did not react to PRDV, and by differences in the susceptibility of plant species and potato cultivars to infection and/or symptom expression following mechanical inoculation. Host plants Datura metel, Nicandra physalodes and Nicotiana edwardsonii were reliably infected systemically with PVP, but not with PRDV. Although all potato cultivars tested were infected, there were marked differences between cultivars in the symptoms produced. The cultivars Ditta and King Edward showed symptoms (including stunting and mottle) when infected with PRDV, but not when infected with PVP. Based on the results, it is proposed that PRDV is a strain of PVP within the genus Carlavirus. For detection of the viruses in postentry quarantine, it is recommended that PRDV or PVP polyclonal antibodies are used, together with inoculation to Nicotiana occidentalis N. megalosiphon-P1. For differentiation of PRDV and PVP, the PVP monoclonal antibody may be used together with inoculation to D. metel and N. physalodes (systemic infection with PVP but not PRDV, determined using ELISA). [ABSTRACT FROM AUTHOR]

Since we have limited knowledge about the occurrence of Helicobacter in wild animals, we searched for Helicobacter species in the gastrointestinal tract of 75 rodents captured in forest remnants of Minas Gerais, Brazil. Fragments from the antrum and corpus of the stomach and from the colon were taken for PCR assays for Helicobacter detection. Although gastric mucosa was Helicobacter-positive in only one animal, the bacterium was detected in the colonic mucosa of 23 rodents (30.7%). Helicobacter detection was more frequent in the colon of terraced rice rat (56%) and house rat (30%) in contrast to punare and Spix’s yellow-toothed cavy, in which the presence of the bacterium was not detected. Helicobacter rodentium, H. marmotae, H. cinaedi, and other species closely related to the murine helicobacters were presumptively identified by DNA sequencing. Wild rodents may serve as a reservoir of these Helicobacter species in nature. [ABSTRACT FROM AUTHOR]

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS I -ITS4, for the amplification of ITS I and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups. [ABSTRACT FROM AUTHOR]

Summary. Isolates of the newly characterized, single-stranded DNA virus TTV, have been tentatively classified into four major phylogenetic groups and at least 28 genotypes. Four Japanese isolates, designated as YONBAN viruses, belong to the fourth group and to genotype 21. In this study, a genotype 21-specific PCR assay was standardized. With this assay, 48/184 (26%) serum samples and 76/167 (46%) saliva samples, collected from unselected ambulatory patients (aged 2 to 82) of a Brazilian public hospital, were positive. A total of 110 (66%) patients had TTV genotype 21 DNA in serum, saliva, or both fluids. Furthermore, 18/37 (49%) serum samples, collected from Indians belonging to three ethnic groups of the Western Brazilian Amazon, were also positive. Nucleotide sequences (253 bases at the 3′ end of the non-coding region of the genome) were determined, that derived from 25 individuals, i.e. 17 patients and eight Indians. Phylogenetic analysis showed that three isolates from Indians of a particular ethnic group formed a separate subgroup within genotype 21. Among non-Indians, a clustering of strains was observed according to their country of origin (Japan or Brazil), with all 17 sequences derived from Brazilian patients located in a unique subgroup. [ABSTRACT FROM AUTHOR]

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors. [ABSTRACT FROM AUTHOR]

Human leucocyte antigen (HLA)-B*4509 differs from the closest HLA-B*4502 by three nucleotides that lead to changes of Tyr113His, Asn114Asp and Phe116Ser. HLA-B*5212 differs from HLA-B*520101 by a single nucleotide substitution, leading to a change of Asn114Asp. [ABSTRACT FROM AUTHOR]

A viroid was detected in Chrysanthemum plants showing symptoms of stunting in a commercial field in Brazil. Analysis by return polyacrylamide gel electrophoresis (R-PAGE) of the nucleic acid preparations of leaves and flowers revealed the presence of a nucleic acid of low molecular weight with mobility within the range of viroids. The viroid-like band was completely eliminated by ribonuclease treatment or alkaline hydrolysis. The Chrysanthemum viroid was readily transmissible to Chrysanthemum, tomato and Gynura, which suggests that it may be an isolate of chrysanthemum stunt viroid. [ABSTRACT FROM AUTHOR]

Inspired by Helmut Sies we continue the development of suitable chemical generators of (1)O2 based on the thermodissociation of naphthalene endoperoxide derivatives. The present manuscript focuses on how the use of [(18)O]-labeled endoperoxides and hydroperoxides can be applied to study mechanistic aspects related to the generation of singlet molecular oxygen and its reactions in biological systems. The peroxidation reactions of the main cellular targets including unsaturated lipids, proteins and nucleic acids have received major attention during the last three decades. Emphasis is placed in this manuscript on the description of the synthesis and the main use of [(18)O]-labeled compounds, and especially of peroxides and (1)O2, for tracer elucidation of reaction mechanisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Discusses the abstract of the research paper 'Residual Risk for Transfusion-Transmitted HIV and Cost-Benefit for HIV Nucleic Acid Amplification Technology in Brazil,' by L. Amorim, C. Silvia, et al, to be presented at the American Association of Blood Banks' 55th Annual Meeting on October 26-29, 2002.