Covid-19 Social Distancing, Lifestyle and Health Outcomes Among Persons Living with HIV (PLWH): A Web-based Survey.

We investigated changes in lifestyle, depressive symptoms, self-perception of health, and body weight changes of persons living with HIV (PLWH) during the COVID-19 social distancing (SD). In a Web-based cross-sectional survey, participants (n = 406) were questioned about lifestyle and health status before and during SD. Most responders were men, 50 + years old, high education level; 49.8% had their income reduced during SD. About 9% were diagnosed with COVID-19, of whom 13.5% required hospitalization. During SD: - most participants did not change their food intake, although 25% replaced healthy foods with unhealthy ones; -more than half mentioned poor sleep quality; -about 50% increased their sedentary behavior. Depressive symptoms (reported by 70.9%) were associated with sedentary behavior, poor sleep quality, and reduced income. About one-third had a negative perception of their health status, which was inversely associated with practicing physical exercises and positively associated with sedentarism and poor sleep quality. More than half increased their body weight, which was associated with a lower intake of vegetables. The older age reduced the odds of the three outcomes. Carefully monitoring PLWH regarding SD will enable early interventions toward health.

RESUMEN: En este trabajo investigamos los cambios en el estilo de vida, síntomas depresivos, autopercepción de salud y cambios en el peso corporal de las personas que viven con el VIH (PVCV) durante el distanciamiento social (DS) de COVID-19. En una encuesta transversal en línea, se preguntó a los participantes (n = 406) sobre el estilo de vida y el estado de salud antes y durante el DS. La mayoría de los encuestados eran hombres, mayores de 50 años, con alto nivel educativo. El 49,8% tuvo una disminución en sus ingresos durante el DS. El 9,1% fue diagnosticados con COVID-19, de los cuales 13,5% requirió hospitalización. Durante el DS: - la mayoría de los participantes no cambió su ingesta de alimentos, aunque el 25% reemplazó los alimentos saludables por los no saludables; más de la mitad mencionó mala calidad del sueño; cerca del 50% aumentó su comportamiento sedentario. Los síntomas depresivos (referidos por el 70,9%), fueron incrementados por el sedentarismo, la mala calidad del sueño y reducción de la renta. Cerca de un tercio tenía una percepción negativa de su estado de salud, que se redujo con la práctica de ejercicio físico y aumentó con el sedentarismo y la mala calidad del sueño. Más de la mitad aumentó su peso corporal, lo que se asoció con una menor ingesta de vegetales. Una edad más avanzada redujo las probabilidades de los tres desenlaces. El monitoreo cuidadoso de las PVCV con respecto al DS permitirá intervenciones tempranas para la salud.

HIV self-test: a tool to expand test uptake among men who have sex with men who have never been tested for HIV in São Paulo, Brazil.

OBJECTIVES: HIV self-testing is an effective tool to improve diagnostic coverage in key populations, enabling linkage to care and access to antiretroviral therapy. Its implementation requires better understanding of patients' perspectives on this novel strategy. The aim of the study was to investigate the perception of men who have sex with men (MSM) regarding the HIV oral fluid self-test (HIVST) in São Paulo, Brazil, and to analyse the sociodemographic characteristics and testing strategy preferences of individuals registered to undertake HIVST. METHODS: Preceding the implementation of HIVST use as public policy in 2019, we recruited MSM living in São Paulo to undertake HIVST using a digital platform, and investigated their sociodemographic profiles, testing experiences and testing preferences. Results were compared according to reported lifetime HIV testing. RESULTS: A total of 6477 MSM (median age 28 years) were recruited to the study from April 9th to December 31st, 2018. Seventy-eight per cent reported previous HIV testing. The opening hours of health facilities (53%), concern about disclosing intimate personal information to health care providers (34%) and fear of stigma (21%) were reported as the main barriers to testing. Older age, higher education, illicit drug use and self-identifying as gay were associated with prior HIV testing (P < 0.001). Most participants (67%) were unaware that HIVST was available before enrolling in the study. Preference for HIVST over other testing technologies was higher among those never tested (71%) than among participants with previous HIV testing (61%; P < 0.001). CONCLUSIONS: HIVST was found to be an effective tool to improve testing uptake among MSM, particularly those who had never been tested before. Characterization of the most likely users of HIVST among MSM will help to inform implementation and scaling up of this novel testing method in the Brazilian public health system.

Use and misuse of biomarkers and the role of D-dimer and C-reactive protein in the management of COVID-19: A post-hoc analysis of a prospective cohort study.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is associated with high mortality among hospitalized patients and incurs high costs. Severe acute respiratory syndrome coronavirus 2 infection can trigger both inflammatory and thrombotic processes, and these complications can lead to a poorer prognosis. This study aimed to evaluate the association and temporal trends of D-dimer and C-reactive protein (CRP) levels with the incidence of venous thromboembolism (VTE), hospital mortality, and costs among inpatients with COVID-19. METHODS: Data were extracted from electronic patient records and laboratory databases. Crude and adjusted associations for age, sex, number of comorbidities, Sequential Organ Failure Assessment score at admission, and D-dimer or CRP logistic regression models were used to evaluate associations. RESULTS: Between March and June 2020, COVID-19 was documented in 3,254 inpatients. The D-dimer level ≥4,000 ng/mL fibrinogen equivalent unit (FEU) mortality odds ratio (OR) was 4.48 (adjusted OR: 1.97). The CRP level ≥220 mg/dL OR for death was 7.73 (adjusted OR: 3.93). The D-dimer level ≥4,000 ng/mL FEU VTE OR was 3.96 (adjusted OR: 3.26). The CRP level ≥220 mg/dL OR for VTE was 2.71 (adjusted OR: 1.92). All these analyses were statistically significant (p<0.001). Stratified hospital costs demonstrated a dose-response pattern. Adjusted D-dimer and CRP levels were associated with higher mortality and doubled hospital costs. In the first week, elevated D-dimer levels predicted VTE occurrence and systemic inflammatory harm, while CRP was a hospital mortality predictor. CONCLUSION: D-dimer and CRP levels were associated with higher hospital mortality and a higher incidence of VTE. D-dimer was more strongly associated with VTE, although its discriminative ability was poor, while CRP was a stronger predictor of hospital mortality. Their use outside the usual indications should not be modified and should be discouraged.

Incidence and associated factors of type 2 diabetes mellitus onset in the Brazilian HIV/AIDS cohort study.

BACKGROUND: People living with HIV (PLH) under combined antiretroviral therapy (cART) are at risk of developing type 2 diabetes mellitus (T2DM). OBJECTIVE: We examined the incidence of T2DM, associated factors and mean time to outcome in PLH under cART. METHOD: Data for this multicenter cohort study were obtained from PLH aged over 18, who started cART in 13 Brazilian sites from 2003 to 2013. Factors associated with incident T2DM were evaluated by Cox multiple regression models. RESULTS: A total of 6724 patients (30,997.93 person-years) were followed from January 2003 to December 2016. A T2DM incidence rate of 17.3/1000 person-years (95%CI 15.8-18.8) was observed. Incidence of isolated hypertriglyceridemia and impaired fasting glucose (IFG) were 84.3 (95%CI 81.1-87.6) and 14.5/1000 person-years (95%CI 13.2-15.9), respectively. Mean time to T2DM onset was 10.5 years (95%CI 10.3-10.6). Variables associated with incident T2DM were age 40-50 [Hazard Ratio (HR) 1.7, 95%CI 1.4-2.1] and ≥ 50 years (HR 2.4, 95%CI 1.9-3.1); obesity (HR 2.1, 95%CI 1.6-2.8); abnormal triglyceride/HDL-cholesterol ratio (HR 1.8, 95%CI 1.51-2.2). IFG predicted T2DM (HR 2.6, 95%CI 1.7-2.5) and occurred on average 3.3 years before diabetes onset. Exposure to stavudine for ≥ 2 years was independently associated with incident T2DM [HR 1.6, 95%CI 1.0-2.2). CONCLUSION: Brazilian PLH under cART are at significant risk of developing T2DM and share risk factors for diabetes onset with the general population, such as older age, obesity, and having metabolic abnormalities at baseline. Moreover, stavudine use was independently associated with incident T2DM. Identifying PLH at a higher risk of T2DM can help caretakers trigger health promotion and establish specific targets for implementation of preventive measures.

Human T-cell leukemia virus type 1 infection among Japanese immigrants and their descendants living in Southeast Brazil: A call for preventive and control responses.

Human T-cell leukemia virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in southwestern Japan. HTLV-1 infection has been associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) besides other diseases. This cross-sectional study aimed to investigate the prevalence, risk factors and molecular characterization of HTLV-1, among the world's largest population of Japanese immigrants and their descendants outside of Japan, in São Paulo, Southeast Brazil, as well as to analyze the phylogenetic relationship among isolates of HTLV-1. From July to December 2017, 2,139 individuals from five Japanese associations were interviewed and submitted to blood collection. All serum samples were first tested for the presence of anti-HTLV-1/2 antibodies by ELISA and then peripheral blood from individuals with positive serological results were analyzed for the presence of HTLV-1 5'LTR proviral DNA. Partial sequencing of the 5'LTR region of HTLV-1 proviral DNA was performed by Sanger. The prevalence of HTLV-1 infection was 5.1% (CI 95%: 4.2-6.0). In the multiple logistic regression model, HTLV-1 infection was associated with age ≥ 45 years, female sex, being first and second-generation Japanese immigrants, and having sexual partners with history of blood transfusion. The phylogenetic analysis revealed that all HTLV-1 were classified as Cosmopolitan (1a) subtype. Of them, 47.8% were classified as Transcontinental (A) subgroup and 52.2% as belonging to the Japanese (B) subgroup. Although most HTLV-1-infected patients were asymptomatic (97.3%), blurred vision was associated with HTLV-1 infection. The high prevalence of HTLV-1 infection found in this studied population and especially the intra- and interfamily HTLV-1 transmission presents an urgent call for preventive and control responses of this infection in Brazil.

High-risk human papillomavirus test in anal smears: can it optimize the screening for anal cancer?

OBJECTIVE: The current method for screening anal cancer is anal cytology, which has low sensitivity. Since high-risk human papillomavirus (HR-HPV) is associated with almost 90% of cases of anal cancer, the objective of this study is to evaluate whether testing for HR-HPV can optimize the screening. DESIGN: Prospective study with patients enrolled in a screening program for anal dysplasia. Considering high-resolution anoscopy (HRA)-guided biopsy as the gold standard for diagnosis of high-grade squamous intraepithelial lesions, the diagnostic performance of anal cytology, HR-HPV testing, and the combination of both was calculated. SETTINGS: A single center for anal dysplasia. PARTICIPANTS: A total of 364 patients (72% males, 82% HIV-positive). INTERVENTION: Patients underwent anal cytology, HR-HPV test, and HRA-guided biopsy of the anal canal. MAIN OUTCOME MEASURES: Ability of cytology and HR-HPV test (individually and combined) to detect high-grade squamous intraepithelial lesions, and analysis of the cost of each diagnostic algorithm. RESULTS: Cytology alone was the cheapest approach, but had the lowest sensitivity [59%, 95% confidence interval (CI) 46-71%], despite of highest specificity (73%, 95% CI 68-78%). Cotesting had the highest sensitivity (85%, 95% CI 74-93%) and lowest specificity (43%, 95% CI 38-49%), and did not seem to be cost-effective. However, HR-HPV testing can be used to triage patients with normal and atypical squamous cells of undetermined significance cytology for HRA, resulting in an algorithm with high sensitivity (80%, 95% CI 68-89%), and specificity (71%, 95% CI 65-76%), allied to a good cost-effectiveness. CONCLUSION: HR-HPV testing is helpful to optimize the screening in cases of normal and atypical squamous cells of undetermined significance cytology.

Use and misuse of biomarkers and the role of D-dimer and C-reactive protein in the management of COVID-19: A post-hoc analysis of a prospective cohort study

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is associated with high mortality among hospitalized patients and incurs high costs. Severe acute respiratory syndrome coronavirus 2 infection can trigger both inflammatory and thrombotic processes, and these complications can lead to a poorer prognosis. This study aimed to evaluate the association and temporal trends of D-dimer and C-reactive protein (CRP) levels with the incidence of venous thromboembolism (VTE), hospital mortality, and costs among inpatients with COVID-19. METHODS: Data were extracted from electronic patient records and laboratory databases. Crude and adjusted associations for age, sex, number of comorbidities, Sequential Organ Failure Assessment score at admission, and D-dimer or CRP logistic regression models were used to evaluate associations. RESULTS: Between March and June 2020, COVID-19 was documented in 3,254 inpatients. The D-dimer level ≥4,000 ng/mL fibrinogen equivalent unit (FEU) mortality odds ratio (OR) was 4.48 (adjusted OR: 1.97). The CRP level ≥220 mg/dL OR for death was 7.73 (adjusted OR: 3.93). The D-dimer level ≥4,000 ng/mL FEU VTE OR was 3.96 (adjusted OR: 3.26). The CRP level ≥220 mg/dL OR for VTE was 2.71 (adjusted OR: 1.92). All these analyses were statistically significant (p<0.001). Stratified hospital costs demonstrated a dose-response pattern. Adjusted D-dimer and CRP levels were associated with higher mortality and doubled hospital costs. In the first week, elevated D-dimer levels predicted VTE occurrence and systemic inflammatory harm, while CRP was a hospital mortality predictor. CONCLUSION: D-dimer and CRP levels were associated with higher hospital mortality and a higher incidence of VTE. D-dimer was more strongly associated with VTE, although its discriminative ability was poor, while CRP was a stronger predictor of hospital mortality. Their use outside the usual indications should not be modified and should be discouraged.

Incidence and associated factors of type 2 diabetes mellitus onset in the Brazilian HIV/AIDS cohort study

ABSTRACT Background: People living with HIV (PLH) under combined antiretroviral therapy (cART) are at risk of developing type 2 diabetes mellitus (T2DM). Objective: We examined the incidence of T2DM, associated factors and mean time to outcome in PLH under cART. Method: Data for this multicenter cohort study were obtained from PLH aged over 18, who started cART in 13 Brazilian sites from 2003 to 2013. Factors associated with incident T2DM were evaluated by Cox multiple regression models. Results: A total of 6724 patients (30,997.93 person-years) were followed from January 2003 to December 2016. A T2DM incidence rate of 17.3/1000 person-years (95%CI 15.8-18.8) was observed. Incidence of isolated hypertriglyceridemia and impaired fasting glucose (IFG) were 84.3 (95%CI 81.1-87.6) and 14.5/1000 person-years (95%CI 13.2-15.9), respectively. Mean time to T2DM onset was 10.5 years (95%CI 10.3-10.6). Variables associated with incident T2DM were age 40-50 [Hazard Ratio (HR) 1.7, 95%CI 1.4-2.1] and ≥ 50 years (HR 2.4, 95%CI 1.9-3.1); obesity (HR 2.1, 95%CI 1.6-2.8); abnormal triglyceride/HDL-cholesterol ratio (HR 1.8, 95%CI 1.51-2.2). IFG predicted T2DM (HR 2.6, 95%CI 1.7-2.5) and occurred on average 3.3 years before diabetes onset. Exposure to stavudine for ≥ 2 years was independently associated with incident T2DM [HR 1.6, 95%CI 1.0-2.2). Conclusion: Brazilian PLH under cART are at significant risk of developing T2DM and share risk factors for diabetes onset with the general population, such as older age, obesity, and having metabolic abnormalities at baseline. Moreover, stavudine use was independently associated with incident T2DM. Identifying PLH at a higher risk of T2DM can help caretakers trigger health promotion and establish specific targets for implementation of preventive measures.

Setting up hospital care provision to patients with COVID-19: lessons learnt at a 2400-bed academic tertiary center in São Paulo, Brazil

ABSTRACT As of August 30, 2020, Brazil ranked second among countries with the highest number of COVID-19 cases, with the city of São Paulo as the national epidemic epicenter. Local public healthcare institutions were challenged to respond to a fast-growing hospital demand, reengineering care provision to optimize clinical outcomes and minimize intra-hospital coronavirus infection. In this paper we describe how the largest public hospital complex in Latin America faced this unprecedented burden, managing severe COVID-19 cases while sustaining specialized care to patients with other conditions. In our strategic plan a 900-bed hospital was exclusively designated for COVID-19 care and continuity of care to those not infected with coronavirus ensured in other inpatient facilities. After 152 days, 4241 patients with severe COVID-19 were hospitalized, 70% of whom have already been discharged, whereas the remaining Institutes of the complex successfully maintained high complexity inpatient and urgent/emergency care to non-COVID-19 patients.

Setting up hospital care provision to patients with COVID-19: lessons learnt at a 2400-bed academic tertiary center in São Paulo, Brazil.

As of August 30, 2020, Brazil ranked second among countries with the highest number of COVID-19 cases, with the city of São Paulo as the national epidemic epicenter. Local public healthcare institutions were challenged to respond to a fast-growing hospital demand, reengineering care provision to optimize clinical outcomes and minimize intra-hospital coronavirus infection. In this paper we describe how the largest public hospital complex in Latin America faced this unprecedented burden, managing severe COVID-19 cases while sustaining specialized care to patients with other conditions. In our strategic plan a 900-bed hospital was exclusively designated for COVID-19 care and continuity of care to those not infected with coronavirus ensured in other inpatient facilities. After 152 days, 4241 patients with severe COVID-19 were hospitalized, 70% of whom have already been discharged, whereas the remaining Institutes of the complex successfully maintained high complexity inpatient and urgent/emergency care to non-COVID-19 patients.

Epidemiology of gonorrhoea: a global perspective.

Although understanding the local epidemiology of gonorrhoea is critical for local efforts, understanding the multinational epidemiology may support development of national and international prevention and control policies and strategies. In this paper, current epidemiology of gonorrhoea is reviewed through an international lens and with a focus on selected populations. The World Health Organization (WHO) estimates that ~87 million new gonococcal infections occurred among people aged 15-49 years in 2016. Gonorrhoea rates are rising in many countries. Gay, bisexual and other men who have sex with men, racial or ethnic minorities, Indigenous populations and sex workers appear to bear disproportionate burdens of gonorrhoea. International travel can facilitate spread of gonorrhoea, including resistant strains, across international borders. Critical gaps in epidemiological knowledge are highlighted, including data on gonorrhoea among transgender persons and the burden of extragenital gonorrhoea. Even as further data are gathered, action - informed by currently available data - is needed now to confront this growing international threat.

Comprehensive Antiretroviral Restriction Factor Profiling Reveals the Evolutionary Imprint of the ex Vivo and in Vivo IFN-ß Response in HTLV-1-Associated Neuroinflammation.

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/ß) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-ß therapy decreases tax mRNA and lymphoproliferation. We hypothesize this "IFN paradox" in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4+ T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4+ T cells. This revealed two major clusters ("antiviral/protective" vs. "proviral/deleterious"), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-ß. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up-regulated by IFN-ß, but not IFN-α, in HAM/TSP. This was paralleled by a significant decrease in lymphoproliferation by IFN-ß, but not IFN-α treatment. Finally, using published ex vivo whole blood transcriptomic data of independent cohorts, we validated the significant positive correlation between TRIM5, TRIM22, and BST2 in HTLV-1-infected individuals and HAM/TSP patients, which was independent of the HAM/TSP disease signature. In conclusion, our results provide ex vivo mechanistic evidence for the observed immunovirological effect of in vivo IFN-ß treatment in HAM/TSP, reconcile an apparent IFN paradox in HTLV-1 research and identify biomarkers/targets for a precision medicine approach.

MAIT cells are reduced in frequency and functionally impaired in human T lymphotropic virus type 1 infection: Potential clinical implications.

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develop HAM/TSP. The cellular immune response has been implicated in the development of inflammatory alterations in these patients; however the pathogenic mechanisms for disease progression remain unclear. Furthermore, HTLV-1-infected individuals have an increase incidence of Mycobacterium tuberculosis (Mtb) infection, suggesting that immunological defect are associated with HTLV-1 infection. Evidence suggests an important role for Mucosal-associated invariant T (MAIT) cells in the early control of Mtb infection. Chronic viral infections like HIV and HCV have been associated with decreased frequency and functionality of MAIT cells. We hypothesized that HTLV-1 infection is associated with similar perturbations in MAIT cells. We investigated MAIT cell frequency, phenotype, and function by flow cytometry in a cohort of 10 asymptomatic and 10 HAM/TSP HTLV-1 infected patients. We found that MAIT cells from HTLV-1-infected subjects were reduced and showed high co-expression of the activation markers CD38 and HLA-DR but normal levels of CCR6 and CD127. MAIT cells had a lower expression of the transcription factor PLZF in HAM/TSP patients. Unlike Tax-specific CD8+T cells, which are hyperfunctional, MAIT cells from HTLV-1-infected subjects had a poor IFNγ response following antigen stimulation. MAIT cell perturbations in HTLV-1 infection were not associated with HTLV-1 proviral load and MAIT cells were not infected by HTLV-1 in vivo. Rather, MAIT cells loss was associated with immune activation. Overall, our results do not support a role for MAIT cells in HAM/TSP pathogenesis but reduced numbers of MAIT cells, together with their poor functionality, could contribute to the increased susceptibility of HTLV-1-infected individuals to other infectious agents.

Brainstem Auditory Evoked Potential in HIV-Positive Adults.

BACKGROUND: To characterize the findings of brainstem auditory evoked potential in HIV-positive individuals exposed and not exposed to antiretroviral treatment. MATERIAL AND METHODS: This research was a cross-sectional, observational, and descriptive study. Forty-five HIV-positive individuals (18 not exposed and 27 exposed to the antiretroviral treatment - research groups I and II, respectively - and 30 control group individuals) were assessed through brainstem auditory evoked potential. RESULTS: There were no significant between-group differences regarding wave latencies. A higher percentage of altered brainstem auditory evoked potential was observed in the HIV-positive groups when compared to the control group. The most common alteration was in the low brainstem. CONCLUSIONS: HIV-positive individuals have a higher percentage of altered brainstem auditory evoked potential that suggests central auditory pathway impairment when compared to HIV-negative individuals. There was no significant difference between individuals exposed and not exposed to antiretroviral treatment.

Analysis of polymorphisms in Plasmodium falciparum genes related to drug resistance: a survey over four decades under different treatment policies in Brazil.

BACKGROUND: Anti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile. METHODS: Samples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2. RESULTS: All 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2. CONCLUSIONS: The 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.