Innate immunity induced by Plasmodium liver infection inhibits malaria reinfections.

Following transmission through a mosquito bite to the mammalian host, Plasmodium parasites first invade and replicate inside hepatocytes before infecting erythrocytes and causing malaria. The mechanisms limiting Plasmodium reinfections in humans living in regions of malaria endemicity have mainly been explored by studying the resistance induced by the blood stage of infection. However, epidemiologic studies have suggested that in high-transmission areas, preerythrocytic stages also activate host resistance to reinfection. This, along with the recent discovery that liver infections trigger a specific and effective type I interferon (IFN) response, prompted us to hypothesize that this pre-erythrocyte-stage-induced resistance is linked to liver innate immunity. Here, we combined experimental approaches and mathematical modeling to recapitulate field studies and understand the molecular basis behind such resistance. We present a newly established mouse reinfection model and demonstrate that rodent malaria liver-stage infection inhibits reinfection. This protection relies on the activation of innate immunity and involves the type I IFN response and the antimicrobial cytokine gamma IFN (IFN-γ). Importantly, mathematical simulations indicate that the predictions based on our experimental murine reinfection model fit available epidemiological data. Overall, our study revealed that liver-stage-induced innate immunity may contribute to the preerythrocytic resistance observed in humans in regions of malaria hyperendemicity.

Relative incidence of interstitial lung diseases in Brazil.

OBJECTIVE: To assess the relative frequency of incident cases of interstitial lung diseases (ILDs) in Brazil. METHODS: This was a retrospective survey of new cases of ILD in six referral centers between January of 2013 and January of 2020. The diagnosis of ILD followed the criteria suggested by international bodies or was made through multidisciplinary discussion (MDD). The condition was characterized as unclassifiable ILD when there was no specific final diagnosis following MDD or when there was disagreement between clinical, radiological, or histological data. RESULTS: The sample comprised 1,406 patients (mean age = 61 ± 14 years), and 764 (54%) were female. Of the 747 cases exposed to hypersensitivity pneumonitis (HP)-related antigens, 327 (44%) had a final diagnosis of HP. A family history of ILD was reported in 8% of cases. HRCT findings were indicative of fibrosis in 74% of cases, including honeycombing, in 21%. Relevant autoantibodies were detected in 33% of cases. Transbronchial biopsy was performed in 23% of patients, and surgical lung biopsy, in 17%. The final diagnoses were: connective tissue disease-associated ILD (in 27%), HP (in 23%), idiopathic pulmonary fibrosis (in 14%), unclassifiable ILD (in 10%), and sarcoidosis (in 6%). Diagnoses varied significantly among centers (c2 = 312.4; p < 0.001). CONCLUSIONS: Our findings show that connective tissue disease-associated ILD is the most common ILD in Brazil, followed by HP. These results highlight the need for close collaboration between pulmonologists and rheumatologists, the importance of detailed questioning of patients in regard with potential exposure to antigens, and the need for public health campaigns to stress the importance of avoiding such exposure.

Venous thromboembolism risk score during hospitalization in pregnancy: results of 10694 prospective evaluations in a clinical trial.

OBJECTIVES: Hospitalization during pregnancy and childbirth increases the risk of Venous Thromboembolism Risk (VTE). This study applied a VTE risk score to all hospitalized pregnant women to ascertain its effectiveness in preventing maternal death from VTE until 3 months after discharge. METHODS: In this interventional study, patients were classified as low- or high-risk according to the VTE risk score (Clinics Hospital risk score). High-risk patients (score ≥ 3) were scheduled for pharmacological Thromboprophylaxis (TPX). Interaction analysis of the main risk factors was performed using Odds Ratio (OR) and Poisson regression with robust variance. RESULTS: The data of 10694 cases (7212 patients) were analyzed; 1626 (15.2%, 1000 patients) and 9068 (84.8%, 6212 patients) cases were classified as high-risk (score ≥ 3) and low-risk (score < 3), respectively. The main risk factors (Odds Ratio, 95% Confidence Interval) for VTE were age ≥ 35 and < 40 years (1.6, 1.4-1.8), parity ≥ 3 (3.5, 3.0-4.0), age ≥ 40 years (4.8, 4.1-5.6), multiple pregnancies (2.1, 1.7-2.5), BMI ≥ 40 kg/m2 (5.1, 4.3-6.0), severe infection (4.1, 3.3-5.1), and cancer (12.3, 8.8-17.2). There were 10 cases of VTE: 7/1636 (0.4%) and 3/9068 (0.03%) in the high- and low-risk groups, respectively. No patient died of VTE. The intervention reduced the VTE risk by 87%; the number needed to treat was 3. CONCLUSIONS: This VTE risk score was effective in preventing maternal deaths from VTE, with a low indication for TPX. Maternal age, multiparity, obesity, severe infections, multiple pregnancies, and cancer were the main risk factors for VTE.

Tele-Intensive Care Unit Program in Brazil: Implementation and Expansion.

In this scientific report, we aimed to describe the implementation and expansion of a Tele-Intensive Care Unit (Tele-ICU) program in Brazil, highlighting the pillars of success, improvements, and perspectives. Tele-ICU program emerged during the COVID-19 pandemic at the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), focusing on clinical case discussions and training of health practitioners in public hospitals of the state of São Paulo in Brazil, to support health care professionals for treating COVID-19 patients. The success of implementing this initiative endorsed the project expansion to other five hospitals from different macroregions of the country, leading to the Tele-ICU-Brazil. These projects assisted 40 hospitals, allowing more than 11,500 teleinterconsultations (exchange of medical information between health care professionals using a licensed online platform) and training more than 14,800 health care professionals, reducing mortality and length of hospitalized patients. A segment in telehealth for the obstetrics health care was implemented after detecting these were a susceptible group of patients to COVID-19 severity. As a perspective, this segment will be expanded to 27 hospitals in the country. The Tele-ICU projects reported here were the largest digital health ICU programs ever established in Brazilian National Health System until know. Their results were unprecedented and proved to be crucial for supporting health care professionals nationwide during the COVID-19 pandemic and guide future initiatives in digital health in Brazil's National Health System.

Risk factors for oxygen requirement in hospitalized pregnant and postpartum women with COVID-19.

OBJECTIVE: To identify risk factors for Oxygen (O2) needs in pregnant and postpartum women with COVID-19. METHODS: Prospective cohort involving pregnant women hospitalized with COVID-19 from April to October 2020. The oxygen need was analyzed regarding risk factors: demographic characteristics, clinical and laboratory parameters at hospital admission, and chest Computer Tomography (CT) findings. Poisson univariate analysis was used to estimate the Relative Risk (RR) and 95% Confidence Intervals. RESULTS: 145 patients, 80 who used and 65 who did not use O2, were included. Body mass index ≥ 30, smoking, and chronic hypertension increased the risk of O2 need by 1.86 (95% CI 1.10-3.21), 1.57 (95% CI 1.16‒2.12), and 1.46 (95% CI 1.09‒1.95), respectively. Patients who were hospitalized for COVID-19 and for obstetric reasons had 8.24 (95% CI 2.8‒24.29) and 3.44 (95% CI 1.05‒11.31) times more use of O2 than those admitted for childbirth and abortion. Respiratory rate ≥ 24 breaths/min and O2 saturation < 95% presented RR for O2 requirements of 2.55 (1.82‒3.56) and 1.68 (95% CI 1.27-2.20), respectively. Ground Glass (GG) < 50% and with GG ≥ 50%, the risk of O2 use were respectively 3.41-fold and 5.33-fold higher than in patients who haven't viral pneumonia on CT. The combination of C-reactive protein ≥ 21 mg/L, hemoglobin < 11.0 g/dL, and lymphopenia < 1500 mm3 on hospital admission increased the risk of O2 use by 4.97-times. CONCLUSIONS: In obstetric patients, clinical history, laboratory, clinical and radiological parameters at admission were identified as a risk for O2 need, selecting the population with the greatest chance of worsening.

Impact of SARS-CoV-2 on pregnancy and neonatal outcomes: An open prospective study of pregnant women in Brazil.

OBJECTIVES: To determine the incidence and risk of adverse obstetric and neonatal outcomes according to SARS-CoV-2 infection severity in pregnant women. METHOD: Open prospective study of pregnant women tested for SARS-CoV-2 by serological and molecular assays during pregnancy or delivery in two hospitals in Sao Paulo, Brazil from April 12, 2020, to February 28, 2021. Five groups were considered for analysis: C0, negative COVID-19 results and no COVID-19 symptoms; C1, positive COVID-19 results, and no symptoms; C2, positive COVID-19 results with mild symptoms; C3, positive COVID-19 results with moderate symptoms; and C4, positive COVID-19 results with severe symptoms. The association between obstetric and neonatal outcomes and COVID-19 severity was determined using multivariate analysis. RESULTS: 734 eligible pregnant women were enrolled as follows: C0 (n = 357), C1 (n = 127), C2 (n = 174), C3 (n = 37), and C4 (n = 39). The following pregnancy and neonatal outcomes were associated with severe COVID-19: oligohydramnios (adjusted Odds Ratio [aOR] = 6.18; 95% CI 1.87‒20.39), fetal distress (aOR = 4.01; 95% Confidence Interval [CI] 1.84‒8.75), preterm birth (aOR = 5.51; 95% CI 1.47‒20.61), longer hospital stay (aOR = 1.66; 95% CI 1.36‒2.02), and admission to the neonatal intensive care unit (aOR = 19.36; 95% CI, 5.86‒63.99). All maternal (n = 6, 15.4%, p < 0.001) and neonatal (n = 5, 12.5%, p < 0.001) deaths and most fetal deaths (n = 4, 9.8%, p < 0.001) occurred in C4 group. Moderate COVID-19 was associated with oligohydramnios (aOR = 6.23; 95% CI 1.93‒20.13) and preterm birth (aOR = 3.60; 95% CI 1.45‒9.27). Mild COVID-19 was associated with oligohydramnios (aOR = 3.77; 95% CI 1.56‒9.07). CONCLUSION: Adverse pregnancy and neonatal outcomes were associated with maternal symptomatic COVID-19 status, and risk increased with disease severity.

Disinfection of 3D-printed protective face shield during COVID-19 pandemic.

This study assessed the disinfection using 70% ethanol; H2O2-quaternary ammonium salt mixture; 0.1% sodium hypochlorite and autoclaving of four 3D-printed face shields with different designs, visor materials; and visor thickness (0.5-0.75 mm). We also investigated their clinical suitability by applying a questionnaire to health workers (HW) who used them. Each type of disinfection was done 40 times on each type of mask without physical damage. In contrast, autoclaving led to appreciable damage.

Accumulation of Plasmodium berghei-infected red blood cells in the brain is crucial for the development of cerebral malaria in mice.

Cerebral malaria is the most severe complication of human infection with Plasmodium falciparum. It was shown that Plasmodium berghei ANKA-induced cerebral malaria was prevented in 100% of mice depleted of CD8+ T cells 1 day prior to the development of neurological signs. However, the importance of parasites in the brains of these mice was never clearly investigated. Moreover, the relevance of this model to human cerebral malaria has been questioned many times, especially concerning the relative importance of leukocytes versus parasitized erythrocytes sequestered in the brain. Here, we show that mice protected from cerebral malaria by CD8+ T-cell depletion have significantly fewer parasites in the brain. Treatment of infected mice with an antimalarial drug 15 to 20 h prior to the estimated time of death also protected mice from cerebral malaria without altering the number of CD8+ T cells in the brain. These mice subsequently developed cerebral malaria with parasitized red blood cells in the brain. Our results clearly demonstrated that sequestration of CD8+ T cells in the brain is not sufficient for the development of cerebral malaria in C57BL/6 mice but that the concomitant presence of parasitized red blood cells is crucial for the onset of pathology. Importantly, these results also demonstrated that the experimental cerebral malaria model shares many features with human pathology and might be a relevant model to study its pathogenesis.

Relative incidence of interstitial lung diseases in Brazil / Incidência relativa de doenças pulmonares intersticiais no Brasil

ABSTRACT Objective: To assess the relative frequency of incident cases of interstitial lung diseases (ILDs) in Brazil. Methods: This was a retrospective survey of new cases of ILD in six referral centers between January of 2013 and January of 2020. The diagnosis of ILD followed the criteria suggested by international bodies or was made through multidisciplinary discussion (MDD). The condition was characterized as unclassifiable ILD when there was no specific final diagnosis following MDD or when there was disagreement between clinical, radiological, or histological data. Results: The sample comprised 1,406 patients (mean age = 61 ± 14 years), and 764 (54%) were female. Of the 747 cases exposed to hypersensitivity pneumonitis (HP)-related antigens, 327 (44%) had a final diagnosis of HP. A family history of ILD was reported in 8% of cases. HRCT findings were indicative of fibrosis in 74% of cases, including honeycombing, in 21%. Relevant autoantibodies were detected in 33% of cases. Transbronchial biopsy was performed in 23% of patients, and surgical lung biopsy, in 17%. The final diagnoses were: connective tissue disease-associated ILD (in 27%), HP (in 23%), idiopathic pulmonary fibrosis (in 14%), unclassifiable ILD (in 10%), and sarcoidosis (in 6%). Diagnoses varied significantly among centers (c2 = 312.4; p < 0.001). Conclusions: Our findings show that connective tissue disease-associated ILD is the most common ILD in Brazil, followed by HP. These results highlight the need for close collaboration between pulmonologists and rheumatologists, the importance of detailed questioning of patients in regard with potential exposure to antigens, and the need for public health campaigns to stress the importance of avoiding such exposure.

RESUMO Objetivo: Avaliar a frequência relativa de casos incidentes de doenças pulmonares intersticiais (DPI) no Brasil. Métodos: Levantamento retrospectivo de casos novos de DPI em seis centros de referência entre janeiro de 2013 e janeiro de 2020. O diagnóstico de DPI seguiu os critérios sugeridos por órgãos internacionais ou foi feito por meio de discussão multidisciplinar (DMD). A condição foi caracterizada como DPI não classificável quando não houve um diagnóstico final específico após a DMD ou houve discordância entre dados clínicos, radiológicos ou histológicos. Resultados: A amostra foi composta por 1.406 pacientes (média de idade = 61 ± 14 anos), sendo 764 (54%) do sexo feminino. Dos 747 casos expostos a antígenos para pneumonite de hipersensibilidade (PH), 327 (44%) tiveram diagnóstico final de PH. Houve relato de história familiar de DPI em 8% dos casos. Os achados de TCAR foram indicativos de fibrose em 74% dos casos, incluindo faveolamento, em 21%. Autoanticorpos relevantes foram detectados em 33% dos casos. Biópsia transbrônquica foi realizada em 23% dos pacientes, e biópsia pulmonar cirúrgica, em 17%. Os diagnósticos finais foram: DPI associada à doença do tecido conjuntivo (em 27%), PH (em 23%), fibrose pulmonar idiopática (em 14%), DPI não classificável (em 10%) e sarcoidose (em 6%). Os diagnósticos variaram significativamente entre os centros (c2 = 312,4; p < 0,001). Conclusões: Nossos achados mostram que DPI associada à doença do tecido conjuntivo é a DPI mais comum no Brasil, seguida pela PH. Esses resultados destacam a necessidade de uma estreita colaboração entre pneumologistas e reumatologistas, a importância de fazer perguntas detalhadas aos pacientes a respeito da potencial exposição a antígenos e a necessidade de campanhas de saúde pública destinadas a enfatizar a importância de evitar essa exposição.

Can thrombophilia worsen maternal and perinatal outcomes in cases of severe preeclampsia?

OBJECTIVE: To evaluate whether thrombophilia worsens maternal and foetal outcomes among patients with severe preeclampsia (PE). METHOD: From October 2009 to October 2014, an observational retrospective cohort study was performed on pregnant women with severe PE diagnosed before 34 weeks of gestation and their newborns hospitalized at the Clinics Hospital, FMUSP. Patients who had no heart disease, nephropathies, pre-gestational diabetes, gestational trophoblastic disease, foetal malformation, or twin pregnancy and who underwent thrombophilia screening during the postnatal period were included. New pregnancies of the same patient; cases of foetal morphological, genetic, or chromosomal abnormalities after birth; and women who used heparin or acetylsalicylic acid during pregnancy were excluded. Factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C, protein S, homocysteine, lupus anticoagulant, and anticardiolipin IgG and IgM antibodies were analysed. The groups with and without thrombophilia were compared regarding their maternal clinical and laboratory parameters and perinatal outcomes. RESULTS: Of the 127 patients selected, 30 (23.6%) had thrombophilia (hereditary or acquired). We found more white patients in thrombophilia group (p = .036). Analysis of maternal parameters showed a tendency of thrombophilic women to have more thrombocytopenia (p = .056) and showed worsening of composite laboratory abnormalities (aspartate aminotransferase ≥ 70 mg/dL, alanine aminotransferase ≥ 70 mg/dL, platelets < 100,000/mm3, serum creatinine ≥ 1.1 mg/dL; p = .017). There were no differences in foetal perinatal outcomes. CONCLUSION: The presence of thrombophilia leads to worsening of maternal laboratory parameters among patients with severe forms of PE but without worsening perinatal outcomes.

Venous thromboembolism risk score during hospitalization in pregnancy: results of 10694 prospective evaluations in a clinical trial

Abstract Objectives Hospitalization during pregnancy and childbirth increases the risk of Venous Thromboembolism Risk (VTE). This study applied a VTE risk score to all hospitalized pregnant women to ascertain its effectiveness in preventing maternal death from VTE until 3 months after discharge. Methods In this interventional study, patients were classified as low- or high-risk according to the VTE risk score (Clinics Hospital risk score). High-risk patients (score ≥ 3) were scheduled for pharmacological Thromboprophylaxis (TPX). Interaction analysis of the main risk factors was performed using Odds Ratio (OR) and Poisson regression with robust variance. Results The data of 10694 cases (7212 patients) were analyzed; 1626 (15.2%, 1000 patients) and 9068 (84.8%, 6212 patients) cases were classified as high-risk (score ≥ 3) and low-risk (score < 3), respectively. The main risk factors (Odds Ratio, 95% Confidence Interval) for VTE were age ≥ 35 and < 40 years (1.6, 1.4-1.8), parity ≥ 3 (3.5, 3.0-4.0), age ≥ 40 years (4.8, 4.1-5.6), multiple pregnancies (2.1, 1.7-2.5), BMI ≥ 40 kg/m2 (5.1, 4.3-6.0), severe infection (4.1, 3.3-5.1), and cancer (12.3, 8.8-17.2). There were 10 cases of VTE: 7/1636 (0.4%) and 3/9068 (0.03%) in the high- and low-risk groups, respectively. No patient died of VTE. The intervention reduced the VTE risk by 87%; the number needed to treat was 3. Conclusions This VTE risk score was effective in preventing maternal deaths from VTE, with a low indication for TPX. Maternal age, multiparity, obesity, severe infections, multiple pregnancies, and cancer were the main risk factors for VTE.

Risk factors for oxygen requirement in hospitalized pregnant and postpartum women with COVID-19

Abstract Objective: To identify risk factors for Oxygen (O2) needs in pregnant and postpartum women with COVID-19. Methods: Prospective cohort involving pregnant women hospitalized with COVID-19 from April to October 2020. The oxygen need was analyzed regarding risk factors: demographic characteristics, clinical and laboratory parameters at hospital admission, and chest Computer Tomography (CT) findings. Poisson univariate analysis was used to estimate the Relative Risk (RR) and 95% Confidence Intervals. Results: 145 patients, 80 who used and 65 who did not use O2, were included. Body mass index ≥ 30, smoking, and chronic hypertension increased the risk of O2 need by 1.86 (95% CI 1.10-3.21), 1.57 (95% CI 1.16‒2.12), and 1.46 (95% CI 1.09‒1.95), respectively. Patients who were hospitalized for COVID-19 and for obstetric reasons had 8.24 (95% CI 2.8‒24.29) and 3.44 (95% CI 1.05‒11.31) times more use of O2 than those admitted for childbirth and abortion. Respiratory rate ≥ 24 breaths/min and O2 saturation < 95% presented RR for O2 requirements of 2.55 (1.82‒3.56) and 1.68 (95% CI 1.27-2.20), respectively. Ground Glass (GG) < 50% and with GG ≥ 50%, the risk of O2 use were respectively 3.41-fold and 5.33-fold higher than in patients who haven't viral pneumonia on CT. The combination of C-reactive protein ≥ 21 mg/L, hemoglobin < 11.0 g/dL, and lymphopenia < 1500 mm3 on hospital admission increased the risk of O2 use by 4.97-times. Conclusions: In obstetric patients, clinical history, laboratory, clinical and radiological parameters at admission were identified as a risk for O2 need, selecting the population with the greatest chance of worsening. HIGHLIGHTS In unvaccinated pregnant and postpartum women, any need for oxygen supply increases the risk of invasive ventilation. Obesity, smoking and chronic arterial hypertension proved to be risk factors for the use of oxygen in pregnant and postpartum women with COVID-19. The combination of C-reactive protein ≥ 21 mg/L, hemoglobin < 11.0 g/dL, and lymphopenia < 1500 mm on hospital admission and the presence of ground glass ≥ 50% in computer tomography increased the risk of O2 use by 4.97 and 5.33 times respectively in pregnant and postpartum women with COVID-19.

Impact of SARS-CoV-2 on pregnancy and neonatal outcomes: An open prospective study of pregnant women in Brazil

Abstract Objectives: To determine the incidence and risk of adverse obstetric and neonatal outcomes according to SARS-CoV-2 infection severity in pregnant women. Method: Open prospective study of pregnant women tested for SARS-CoV-2 by serological and molecular assays during pregnancy or delivery in two hospitals in Sao Paulo, Brazil from April 12, 2020, to February 28, 2021. Five groups were considered for analysis: C0, negative COVID-19 results and no COVID-19 symptoms; C1, positive COVID-19 results, and no symptoms; C2, positive COVID-19 results with mild symptoms; C3, positive COVID-19 results with moderate symptoms; and C4, positive COVID-19 results with severe symptoms. The association between obstetric and neonatal outcomes and COVID-19 severity was determined using multivariate analysis. Results: 734 eligible pregnant women were enrolled as follows: C0 (n = 357), C1 (n = 127), C2 (n = 174), C3 (n = 37), and C4 (n = 39). The following pregnancy and neonatal outcomes were associated with severe COVID-19: oligohydramnios (adjusted Odds Ratio [aOR] = 6.18; 95% CI 1.87‒20.39), fetal distress (aOR = 4.01; 95% Confidence Interval [CI] 1.84‒8.75), preterm birth (aOR = 5.51; 95% CI 1.47‒20.61), longer hospital stay (aOR = 1.66; 95% CI 1.36‒2.02), and admission to the neonatal intensive care unit (aOR = 19.36; 95% CI, 5.86‒63.99). All maternal (n = 6, 15.4%, p < 0.001) and neonatal (n = 5, 12.5%, p < 0.001) deaths and most fetal deaths (n = 4, 9.8%, p < 0.001) occurred in C4 group. Moderate COVID-19 was associated with oligohydramnios (aOR = 6.23; 95% CI 1.93‒20.13) and preterm birth (aOR = 3.60; 95% CI 1.45‒9.27). Mild COVID-19 was associated with oligohydramnios (aOR=3.77; 95% CI 1.56‒9.07). Conclusion: Adverse pregnancy and neonatal outcomes were associated with maternal symptomatic COVID-19 status, and risk increased with disease severity. HIGHLIGHTS COVID-19 increases the rates of adverse pregnancy and neonatal outcomes. Serious cases are associated with oligohydramnios, fetal distress, prematurity, neonatal ICU admission, maternal and neonatal deaths. The maternal clinical status dictates obstetric and neonatal outcomes.

Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.

Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/ß receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Avaliação da exposição ambiental ao cromo na população residente no entorno de curtumes em Rondônia / Assessment of environmental exposure to chromium in the resident population in the vicinity of tanneries in Rondônia

O cromo é um metal de grande importância por ser amplamente utilizado em atividades industriais, como por exemplo, curtumes e fabricação de aço inoxidável. O Cr (III) é essencial para os seres-humanos, pois participa do metabolismo da glicose, enquanto o Cr (VI) é classificado pela IARC como carcinogênico. Nos curtumes, o sal Cr2(SO4)3 é utilizado durante a etapa de curtimento e o metal, apesar de encontrar sob a forma de Cr(III), pode seroxidado a Cr(VI) durante algumas reações químicas. O objetivo deste trabalho foi avaliar a exposição ambiental ao cromo da população residente no entorno de curtumes em Candeias do Jamari, município de Rondônia. Um grupo de pessoas residentes próximo aos curtumes foi comparado com um grupo de moradores do município de Itapuã do Oeste, RO. A metodologia para a determinação de cromo em urina foi validada por espectrometria de absorção atômica eletrotérmica. O uso de temperaturas de pirólise e de atomização iguais a 1400°C e 2100°C, respectivamente, urina diluída 1+2 em HNO3 0,2% (v/v) e modificador químico Mg(NO3)2 0,10% (v/v) permitiram que a melhor sensibilidade fosse alcançada. O cromo se mostrou linear até 30 µg L-1 na urina e a razão de sensibilidades apontou para a existência de efeito de matriz, 1,04 ± 0,10, o que impossibilita a calibração aquosa. O limite de detecção (3σ) calculado foi de 0,07 ± 0,001 µg L-1, enquanto que a exatidão foi verificada pela análise de material de referência para urina. O método foi aplicado na determinação de cromo em 42 amostras de urina da população e 49 águas de consumo, do rio Candeias do Jamari e do reservatório de Samuel, locais de despejo dos efluentes dos curtumes e reservatório de água para consumo do grupo de comparação, respectivamente. A concentração de cromo encontrada foi de 0,24 ± 0,22 nos expostos e de 0,13 ± 0,07 µg L-1 no grupo de comparação. Na água do rio Candeias do Jamari, o teor de cromo ficou em 212,38 ± 666,29, e foi de 0,27 ± 0,22 µg L-1 no reservatório de Samuel. As águas para consumo de ambos os grupos ficaram dentro do limite de 50 µg L-1, estabelecido pela resolução CONAMA nº 357. Os níveis do metal nas amostras de urina dos dois grupos não se mostraram estatisticamente diferentes (p = 0,087; IC 95%) de acordo com o teste t de Student assim como a concentração de Cr-U e as variavéis "tipo de fornecimento de água" (p = 0,170; IC 95%) e "sexo" (p = 0,593; IC 95%). O resultado da correlação de Pearson, porém, indicou uma associação positiva (r=0,297 e p=0,033). O estudo mostra que concentrações mais elevadas de cromo na urina estão associadas ao tempo de residência e com o passar dos anos poderá haver diferença estatística entre as concentrações de cromo na urina do grupo expostos e não exposto.