Rabies virus diversification in aerial and terrestrial mammals.

Rabies is a fatal zoonotic infection of the central nervous system of mammals and has been known to humans for millennia. The etiological agent, is a neurotropic RNA virus in the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. There are currently accepted to be two cycles for rabies transmission: the urban cycle and the sylvatic cycle. The fact that both cycles originated from a common RABV or lyssavirus ancestor and the adaptive divergence that occurred since then as this ancestor virus adapted to a wide range of fitness landscapes represented by reservoir species in the orders Carnivora and Chiroptera led to the emergence of the diverse RABV lineages currently found in the sylvatic and urban cycles. Here we study full genome phylogenies and the time to the most recent common ancestor (TMRCA) of the RABVs in the sylvatic and urban cycles. Results show that there were differences between the nucleotide substitution rates per site per year for the same RABV genes maintained independently in the urban and sylvatic cycles. The results identify the most suitable gene for phylogenetic analysis, heterotachy among RABV genes and the TMRCA for the two cycles.

Asaia (Rhodospirillales: Acetobacteraceae) and Serratia (Enterobacterales: Yersiniaceae) associated with Nyssorhynchus braziliensis and Nyssorhynchus darlingi (Diptera: Culicidae)

Abstract Midgut transgenic bacteria can be used to express and deliver anti-parasite molecules in malaria vector mosquitoes to reduce transmission. Hence, it is necessary to know the symbiotic bacteria of the microbiota of the midgut to identify those that can be used to interfering in the vector competence of a target mosquito population. The bacterial communities associated with the abdomen of Nyssorhynchus braziliensis (Chagas) (Diptera: Culicidae) and Nyssorhynchus darlingi (Root) (Diptera: Culicidae) were identified using Illumina NGS sequencing of the V4 region of the 16S rRNA gene. Wild females were collected in rural and periurban communities in the Brazilian Amazon. Proteobacteria was the most abundant group identified in both species. Asaia (Rhodospirillales: Acetobacteraceae) and Serratia (Enterobacterales: Yersiniaceae) were detected in Ny. braziliensis for the first time and its presence was confirmed in Ny. darlingi.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Shifts in intestinal microbiota after duodenal exclusion favor glycemic control and weight loss: a randomized controlled trial.

BACKGROUND: In recent years, studies indicate gut microbiota as an important modulator in the pathophysiology of type 2 diabetes. Environmental and genetic factors interact to control the host's intestinal microbiota, triggering metabolic disorders such as obesity and insulin resistance. OBJECTIVES: The objective of this study was to identify the fecal microbiota in adult type 2 diabetes patients and to assess changes in composition after metabolic surgery. SETTING: University Hospital of the University of São Paulo. METHODS: Twenty-one patients were enrolled in a randomized controlled study divided into 2 arms. One group underwent duodenal-jejunal bypass surgery with minimal gastric resection, and fecal samples were collected before the operation and after 6 and 12 months. The other group received medical care (standard care group) and was followed for 12 months. Fecal samples were collected at baseline and after 6 and 12 months. Fecal microbiota was analyzed using high-throughput sequencing with V4 16 S rRNA primers. RESULTS: The fecal microbiota in duodenal-jejunal bypass surgery with minimal gastric resection group (Bacteroides, Akkermansia, and Dialister) exhibited increased abundance and diversity compared with that in the standard care group; however, the increase in A. muciniphila was only statistically significant in the surgical group, probably due to the study's small sample size. CONCLUSIONS: The data presented suggest that duodenal-jejunal bypass surgery with minimal gastric resection increases microbial richness and abundancy, mainly for those bacteria related to weight loss and metabolic control (Akkermansia), providing a better understanding of the role of microbiota in type 2 diabetes regulation and its changes after metabolic surgery.

High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

BACKGROUND: An improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM) in therapy-naïve HIV-1-infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS) of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs. MATERIALS AND METHODS: The MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS) or "detuned" enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2) and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses. RESULTS: No TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR) and reverse transcriptase (RT) regions of the HIV-1 pol gene was 44.5% (57/128), including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI) and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI). Of the 57 subjects, 43 (75.4%) harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7%) had detectable minority resistant variants to NRTIs, 6 (13.9%) to NNRTIs, and 16 (37.2%) to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis. CONCLUSION: Our findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these mutations. Sequencing proviral DNA provided additional information about TDRM, which may impact treatment decisions. The overall results emphasize the importance of continuous monitoring.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin.

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors.

BACKGROUND: Here, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70). MATERIALS AND METHODS: A total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. RESULTS: Of the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1). CONCLUSION: Based on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Investigation Into an Outbreak of Dengue-like Illness in Pernambuco, Brazil, Revealed a Cocirculation of Zika, Chikungunya, and Dengue Virus Type 1.

In April 2015, an outbreak of dengue-like illness occurred in Tuparetama, a small city in the northeast region of Brazil; this outbreak was characterized by its fast expansion. An investigation was initiated to identify the viral etiologies and advise the health authorities on implementing control measures to contain the outbreak. This is the first report of this outbreak in the northeast, even though a few cases were documented earlier in a neighboring city.Plasma samples were obtained from 77 suspected dengue patients attending the main hospital in the city. Laboratory assays, such as real-time reverse transcription polymerase chain reaction, virus cDNA sequencing, and enzyme-linked immunosorbent assay, were employed to identify the infecting virus and molecular phylogenetic analysis was performed to define the circulating viral genotypes.RNA of Zika virus (ZIKV) and Dengue virus (DENV) or IgM antibodies (Abs) to DENV or chikungunya (CHIKV) were detected in 40 of the 77 plasma samples (51.9%). DENV was found in 9 patients (11.7%), ZIKV was found in 31 patients (40.2%), CHIKV in 1 patient (1.3%), and coinfection of DENV and ZIKV was detected in 2 patients (2.6%). The phylogenetic analysis of 2 available partial DENV and 14 ZIKV sequences revealed the identities of genotype 1 and the Asiatic lineage, respectively.Consistent with recent reports from the same region, our results showed that the ongoing outbreak is caused by ZIKV, DENV, and CHIKV. This emphasizes the need for a routine and differential diagnosis of arboviruses in patients with dengue-like illness. Coordinated efforts are necessary to contain the outbreak. Continued surveillance will be important to assess the effectiveness of current and future prevention strategies.

Frequent detection of CXCR4-using viruses among Brazilian blood donors with HIV-1 long-standing infection and unknown clinical stage: Analysis of massive parallel sequencing data.

The determination of viral tropism is critically important and highly recommended to guide therapy with the CCR5 antagonist, which does not inhibit the effect of X4-tropic viruses. Here, we report the prevalence of HIV-1×4 HIV strains in 84 proviral DNA massively parallel sequencing "MPS" data from well-defined non-recently infected first-time Brazilian blood donors. The MPS data covering the entire V3 region of the env gene was extracted from our recently generated HIV-1 genomes sequenced by a paired-end protocol (Illumina). Of the 84 MPS data samples, 63 (75%) were derived from donors with long-standing infection and 21 (25%) were lacking stage information. HIV-1 tropism was inferred using Geno2pheno (g2p) [454] algorithm (FPR=1%, 2.5%, and 3.75%). Among the 84 data samples for which tropism was defined by g2p2.5%, 13 (15.5%) participants had detectable CXCR4-using viruses in their MPS reads. Mixed infections with R5 and X4 were observed in 11.9% of the study subjects and minority X4 viruses were detected in 7 (8.3%) of participants. Nine of the 63 (14.3%) subjects with LS infection were predicted by g2p 2.5% to harbor proviral CXCR4-using viruses. Our findings of a high proportion of blood donors (15.5%) harboring CXCR4-using viruses in PBMCs may indicate that this phenomenon is common. These findings may have implications for clinical and therapeutic aspects and may benefit individuals who plan to receive CCR5 antagonists.

Mitochondrial genomes and comparative analyses of Culex camposi, Culex coronator, Culex usquatus and Culex usquatissimus (Diptera:Culicidae), members of the coronator group.

BACKGROUND: The Coronator Group currently encompasses six morphologically similar species (Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex usquatus Dyar, Culex usquatissimus Dyar, and Culex ousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to provide genetic information about these species. RESULTS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi varied from 15,573 base pairs in Cx. usquatus to 15,576 in Cx. coronator. They contained 37 genes (13 protein-encoding genes, 2 rRNA genes, and 22 tRNA genes) and the AT-rich control region. Comparative analyses of the 37 genes demonstrated the mitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plus NADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control region contained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and the Culex pipens complex are monophyletic taxa. CONCLUSIONS: The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi share the same gene composition and arrangement features that match to those reported for most Culicidae species. They are composed of the same 37 genes and the AT-rich control region, which contains poly-T stretches that may be involved in the functional role of the mitochondrial genome. Taken together, results of the dN/dS ratios, the sliding window analyses and the Bayesian phylogenetic analyses suggest that ATP6, ATP8 and NADH5 are promising genes to be employed in phylogenetic studies involving species of the Coronator Group, and probably other species groups of the subgenus Culex. Bayesian topology corroborated the morphological hypothesis of the Coronator Group as monophyletic lineage within the subgenus Culex.

Frequency of coreceptor tropism in PBMC samples from HIV-1 recently infected blood donors by massively parallel sequencing: the REDS II study.

BACKGROUND: The interaction of HIV-1 and target cells involves sequential binding of the viral gp120 Env protein to the CD4 receptor and a chemokine co-receptor (either CCR5 or CXCR4). CCR5 antagonists have proved to be an effective salvage therapy in patients with CCR5 using variants (R5) but not with variants capable of using CXCR4 (×4) phenotype. Thus, it is critically important to determine cellular tropism of a country's circulating HIV strains to guide a management decision to improve treatment outcome. In this study, we report the prevalence of R5 and ×4 HIV strains in 45 proviral DNA massively parallel sequencing "MPS" data from recently infected Brazilian blood donors. METHODS: The MPS data encompassing the tropism-related V3 loop region of the HIV-1 env gene was extracted from our recently published HIV-1 genomes sequenced by a paired-end protocol (Illumina). HIV-1 tropism was inferred using Geno2pheno[coreceptor] algorithm (3.5 % false-positive rate). V3 net charge and 11/25 rules were also used for coreceptor prediction. RESULTS: Among the 45 samples for which tropism were determined, 39 were exclusively R5 variants, 5 ×4 variants, and one dual-tropic or mixed (D/M) populations of R5 and ×4 viruses, corresponding to 86.7, 11.1 and 2.2 %, respectively. Thus, the proportion of all blood donors that harbor CXCR4-using virus was 13.3 % including individuals with D/M-tropic viruses. CONCLUSIONS: The presence of CCR5-tropic variants in more than 85 % of our cohort of antiretroviral-naïve blood donors with recent HIV-1 infection indicates a potential benefit of CCR5 antagonists as a therapeutic option in Brazil. Therefore, determination of viral co-receptor tropism is an important diagnostic prerequisite.

Enhanced detection of viral diversity using partial and near full-length genomes of human immunodeficiency virus Type 1 provirus deep sequencing data from recently infected donors at four blood centers in Brazil.

BACKGROUND: Here, we report application of high-throughput near full-length genome (NFLG) and partial human immunodeficiency virus Type 1 (HIV-1) proviral genome deep sequencing to characterize HIV in recently infected blood donors at four major blood centers in Brazil. STUDY DESIGN AND METHODS: From 2007 to 2011, a total of 341 HIV+ blood donors from four blood centers were recruited to participate in a case-control study to identify HIV risk factors and motivations to donate. Forty-seven (17 from São Paulo, eight from Minas Gerais, 11 from Pernambuco, and 11 from Rio de Janeiro) were classified as recently infected based on testing by less-sensitive enzyme immunoassays. Five overlapping amplicons spanning the HIV genome were polymerase chain reaction amplified from peripheral blood mononuclear cells. The amplicons were molecularly barcoded, pooled, and sequenced by a paired-end protocol (Illumina). RESULTS: Of the 47 recently infected donor samples studied, 39 (82.9%) NFLGs and six (12.7%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Subtype B was the only nonrecombinant virus identified in this study and accounted for 62.2% (28/45) of samples. The remaining 37.8% (17/45) of samples showed various patterns of subtype discordance in different regions of HIV-1 genomes, indicating two to four circulating recombinant subtypes derived from Clades B, F, and C. Fourteen samples (31.1%) from this study harbored drug resistance mutations, indicating higher rate of drug resistance among Brazilian blood donors. CONCLUSION: Our findings revealed a high proportion of HIV-1 recombinants among recently infected blood donors in Brazil, which has implications for future blood screening, diagnosis, therapy, and vaccine development.

Deep sequencing of HIV-1 near full-length proviral genomes identifies high rates of BF1 recombinants including two novel circulating recombinant forms (CRF) 70_BF1 and a disseminating 71_BF1 among blood donors in Pernambuco, Brazil.

BACKGROUND: The findings of frequent circulation of HIV-1 subclade F1 viruses and the scarcity of BF1 recombinant viruses based on pol subgenomic fragment sequencing among blood donors in Pernambuco (PE), Northeast of Brazil, were reported recently. Here, we aimed to determine whether the classification of these strains (n = 26) extends to the whole genome sequences. METHODS: Five overlapping amplicons spanning the HIV near full-length genomes (NFLGs) were PCR amplified from peripheral blood mononuclear cells (PBMCs) of 26 blood donors. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol. The prevalence of viral variants containing drug resistant mutations (DRMs) was compared between plasma and PBMCs. RESULTS: Of the 26 samples studied, 20 NFLGs and 4 partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Two distinct BF1 recombinant profiles designated CRF70_BF1 and CRF71_BF1, with 4 samples in profile I and 11 in profile II were detected and thus constitute two novel recombinant forms circulating in PE. Evidence of dual infections was detected in four patients co-infected with distinct HIV-1 subtypes. According to our estimate, the new CRF71_BF1 accounts for 10% of the HIV-1 circulating strains among blood donors in PE. Discordant data between the plasma and PBMCs-virus were found in 15 of 24 donors. Six of these strains displayed major DRMs only in PBMCs and four of which had detectable DRMs changes at prevalence between 1-20% of the sequenced population. CONCLUSIONS: The high percentage of the new RF71_BF1 and other BF1 recombinants found among blood donors in Pernambuco, coupled with high rates of transmitted DRMs and dual infections confirm the need for effective surveillance to monitor the prevalence and distribution of HIV variants in a variety of settings in Brazil.

Identification of a Novel HIV-1 Circulating Recombinant Form (CRF72_BF1) in Deep Sequencing Data from Blood Donors in Southeastern Brazil.

We report the identification of a novel HIV-1 circulating recombinant form (CRF72_BF1) in deep sequencing data from peripheral blood mononuclear cells (PBMC) of five blood donors in southeastern Brazil. Detection of this circulating recombinant form (CRF) confirms the need for effective surveillance to monitor the prevalence and distribution of HIV variants in a variety of settings in Brazil.

Genetic Characterization of HIV-1 Subtype D Near-Full-Length Proviral Genomes by Illumina Massively Parallel Sequencing Technology.

This study describes the near-full-length genome deep sequencing of two HIV-1 subtype D strains identified in blood donors in Rio de Janeiro, Brazil, in what seems to have been a small restricted subtype D epidemic in the country.

Short communication: HIV type 1 subtype BF leads to faster CD4+ T cell loss compared to subtype B.

Although it has been suggested that biological differences among HIV-1 subtypes exist, their possible influence on disease progression has not been fully revealed. In particular, the increasing emergence of recombinants stresses the need to characterize disease presentation in persons infected by these diverse HIV-1 forms. We explored this issue among 83 Brazilian subjects infected with either HIV-1 subtype B or recombinant subtype BF, all followed since incident infection in a cohort study. Viral subtypes were assigned by full length sequencing of HIV-1 genomes. We observed that the baseline measures for CD4(+) T cells and viral load did not differ between the groups. However, longitudinal analysis revealed that subtype BF was clearly associated with a faster CD4(+) T cell decline compared to infection with subtype B, in spite of a similar plasma HIV-1 load. While subtype B-infected subjects presented a loss of 3.6 CD4(+) T cells/µl per month, subtype BF-infected individuals showed a monthly decay of 6.3 CD4(+) T cells/µl (p<0.01). The time to reach 350 CD4(+) T cells/µl and the time to start antiretroviral treatment were also shorter in subtype BF-infected persons. The elucidation of an accelerated CD4(+) T cell loss associated with subtype BF suggests that this HIV-1 genetic form could be more pathogenic than subtype B.

Dasatinib overrides imatinib resistance mediated by the F359I residue mutation in two patients with chronic myeloid leukemia.

Despite the beneficial effects of imatinib mesylate, some patients may either not respond or respond suboptimally. Here, we report two chronic myelogenous leukemia patients; one had a suboptimal response according to European LeukemiaNet criteria (a major molecular response was not achieved after 18 months of standard-dose imatinib therapy) and the other had failure with a standard dose of imatinib. At the time of the suboptimal response in patient 1 and the failure in patient 2, we were able to detect the F359I mutation in the BCR-ABL tyrosine kinase domain using DNA sequencing in both patients. Therefore, it was decided to change the therapeutic regimen to dasatinib at a dose of 100 mg once daily in both patients. This change resulted in the achievement of complete cytogenetic remission in patient 1 after 4 months and a major molecular response within 2 and 3 months in both patients. Detection of the F359I mutation in our two cases likely explains the suboptimal response to imatinib in case 1 and the failure in case 2. This implies that in such cases dasatinib should be considered to effectively suppress the mutated clones.

Correlation between LTR point mutations and proviral load levels among human T cell lymphotropic virus type 1 (HTLV-1) asymptomatic carriers.

BACKGROUND: In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of Tax-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1-infected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden. METHODS: A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR) and a PvL for Tax DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared. RESULTS: Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both p < 0.05), but not between subjects with a low or intermediate PvL (p > 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p < 0.03). No significant difference was found between the groups in relation to age, sex or viral subtypes (p > 0. 05). CONCLUSIONS: The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission.

BACKGROUND: The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. METHODS: The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. RESULTS: Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. CONCLUSIONS: Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.