MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

RESOLUTION OF CUTANEOUS LEISHMANIASIS AFTER ACUTE ECZEMA DUE TO INTRALESIONAL MEGLUMINE ANTIMONIATE

We report a case of a 42 year-old female, who came to a leishmaniasis reference center in Rio de Janeiro, Brazil, presenting a cutaneous leishmaniasis lesion in the right forearm. Treatment with low-dose intramuscular meglumine antimoniate (MA) (5 mg Sb5+/kg/day) was initiated, with improvement after 28 days, although with the development of generalized eczema. After 87 days, the lesion worsened. Patient refused treatment with amphotericin B. MA was then infiltrated in the lesion, in two sessions, resulting in local eczema, with bullae formation; however, twenty days after, both the ulcer and eczema receded. Intralesional administration of MA should be used carefully when previous cutaneous hypersensitivity is detected.

Infliximab in Recalcitrant Generalized Pustular Arthropatic Psoriasis

Generalized pustular psoriasis is an unstable inflammatory type of psoriasis, with widespread areas of erythema and sterile pustules, associated with fever and systemic symptoms. Infliximab is a monoclonal antibody with anti-TNFalpha activity, approved for use in psoriasis. We describe a male patient with a long history of stable arthropathic psoriasis, hospitalized with a generalized pustular psoriasis and acute exacerbation of articular complaints. The disease was resistant to multiple therapies (acitretin, methotrexate and corticosteroids), so the patient was started on infliximab, with a very rapid response of both cutaneous and articular symptoms. He had complete clearing of lesions at week 12, and marked improvement of the articular symptoms. No recurrence occurred at 8 months of follow-up with infliximab every 8 weeks. Infliximab had an extremely rapid therapeutic action response on a recalcitrant generalized pustular psoriasis. The articular response was also excellent, with significant improvement of quality of life.

The impact of questions rotation in response accuracy

Dissertação apresentada como requisito parcial para obtenção do grau de Mestre em Estatística e Gestão de Informação

Cortisol and Anxiety Response to a Relaxing Intervention on Pregnant Women Awaiting Amniocentesis

Background: Stress and anxiety during pregnancy have been associated with premature and low birth weight babies, presumably through fetus over exposion to glucocorticoids. Antenatal stress also seems to have long-term effects upon infant development and adult health. However, medication for stress may carry risks to the expectant mother, therefore the efficacy of nonpharmacological interventions should be investigated. Methods: Pregnant women (n = 154) awaiting amniocentesis, were randomly assigned in the morning and the afternoon to three groups for 30 min: (1) listening to relaxing music, (2) sitting and reading magazines, and (3) sitting in the waiting-room. Before and after that period, they completed the Spielberger’s State and Trait anxiety inventory and provided blood samples for cortisol. The groups were then compared regarding change in cortisol levels and anxiety. Results: Maternal cortisol and state anxiety were correlated (r = 0.25, p = 0.04) in the afternoon, but not in the morning. The larger decreases in cortisol occurred in the music group ( 61.8 nmol/L, ANOVA: p = 0.01), followed by magazine, being differences among groups more pronounced in the morning. Women in the music group also exhibited the greater decreases in state anxiety ( p < 0.001). Younger mothers with less gestational age were on average the most anxious, and also the ones with greater decreases in cortisol and anxiety levels after relaxation. Conclusion: A relaxing intervention as short as 30 min, especially listening to music, decreases plasma cortisol and self-reported state anxiety score. Pregnant women might benefit from the routine practice of relaxation in the imminence of clinical stressful events.

The Role of the Humoral Immune Response in the Molecular Evolution of the Envelope C2, V3 and C3 Regions in Chronically HIV-2 Infected Patients

BACKGROUND: This study was designed to investigate, for the first time, the short-term molecular evolution of the HIV-2 C2, V3 and C3 envelope regions and its association with the immune response. Clonal sequences of the env C2V3C3 region were obtained from a cohort of eighteen HIV-2 chronically infected patients followed prospectively during 2-4 years. Genetic diversity, divergence, positive selection and glycosylation in the C2V3C3 region were analysed as a function of the number of CD4+ T cells and the anti-C2V3C3 IgG and IgA antibody reactivity RESULTS: The mean intra-host nucleotide diversity was 2.1% (SD, 1.1%), increasing along the course of infection in most patients. Diversity at the amino acid level was significantly lower for the V3 region and higher for the C2 region. The average divergence rate was 0.014 substitutions/site/year, which is similar to that reported in chronic HIV-1 infection. The number and position of positively selected sites was highly variable, except for codons 267 and 270 in C2 that were under strong and persistent positive selection in most patients. N-glycosylation sites located in C2 and V3 were conserved in all patients along the course of infection. Intra-host variation of C2V3C3-specific IgG response over time was inversely associated with the variation in nucleotide and amino acid diversity of the C2V3C3 region. Variation of the C2V3C3-specific IgA response was inversely associated with variation in the number of N-glycosylation sites. CONCLUSION: The evolutionary dynamics of HIV-2 envelope during chronic aviremic infection is similar to HIV-1 implying that the virus should be actively replicating in cellular compartments. Convergent evolution of N-glycosylation in C2 and V3, and the limited diversification of V3, indicates that there are important functional constraints to the potential diversity of the HIV-2 envelope. C2V3C3-specific IgG antibodies are effective at reducing viral population size limiting the number of virus escape mutants. The C3 region seems to be a target for IgA antibodies and increasing N-linked glycosylation may prevent HIV-2 envelope recognition by these antibodies. Our results provide new insights into the biology of HIV-2 and its relation with the human host and may have important implications for vaccine design.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

Introduction:American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo.Methods:Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus.Results:Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample.Conclusion:These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci.

Methacholine Dose-Response Slopes from Maximal Bronchial Challenge Tests in Asthmatic Children: Methodological Aspects

To determine whether the slope of a maximal bronchial challenge test (in which FEV1 falls by over 50%) could be extrapolated from a standard bronchial challenge test (in which FEV1 falls up to 20%), 14 asthmatic children performed a single maximal bronchial challenge test with methacholin(dose range: 0.097–30.08 umol) by the dosimeter method. Maximal dose-response curves were included according to the following criteria: (1) at least one more dose beyond a FEV1 ù 20%; and (2) a MFEV1 ù 50%. PD20 FEV1 was calculated, and the slopes of the early part of the dose-response curve (standard dose-response slopes) and of the entire curve (maximal dose-response slopes) were calculated by two methods: the two-point slope (DRR) and the least squares method (LSS) in % FEV1 × umol−1. Maximal dose-response slopes were compared with the corresponding standard dose-response slopes by a paired Student’s t test after logarithmic transformation of the data; the goodness of fit of the LSS was also determined. Maximal dose-response slopes were significantly different (p < 0.0001) from those calculated on the early part of the curve: DRR20% (91.2 ± 2.7 FEV1% z umol−1)was 2.88 times higher than DRR50% (31.6 ± 3.4 DFEV1% z umol−1), and the LSS20% (89.1 ± 2.8% FEV1 z umol−1) was 3.10 times higher than LSS 50% (28.8 ± 1.5%FEV1 z umol−1). The goodness of fit of LSS 50% was significant in all cases, whereas LSS 20% failed to be significant in one. These results suggest that maximal dose-response slopes cannot be predicted from the data of standard bronchial challenge tests.

Evolution of the Human Immunodeficiency Virus Type 2 Envelope in the First Years of Infection is Associated with the Dynamics of the Neutralizing Antibody Response

Background: Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. Results: CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Conclusion: Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Quantification of the TMS-EEG response in epilepsy

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

MOLECULAR CHARACTERIZATION OF AMERICAN CUTANEOUS LEISHMANIASIS IN THE TRI‑BORDER AREA OF ASSIS BRASIL, ACRE STATE, BRAZIL

SUMMARY In this study, Leishmaniaspecies were identified by Polymerase Chain Reaction (PCR). The epidemiology of patients suspected of having American Cutaneous Leishmaniasis in the municipality of Assis Brasil, Acre State, located in the Brazil/Peru/Bolivia triborder was also investigated. By PCR, the DNA of Leishmaniawas detected in 100% of the cases (37 samples) and a PCR-Restriction Fragment Length Polymorphism (RFLP) of the hsp 70gene identified the species in 32 samples: Leishmania (Viannia) braziliensis (65.6%) , L. (V.) shawi (28.1%) , L. (V.) guyanensis (3.1%) and mixed infection L. (V.) guyanensis and L. (Leishmania) amazonensis (3.1%)This is the first report of L. (V.) shawiand L. (L.) amazonensis in Acre. The two predominant species were found in patients living in urban and rural areas. Most cases were found in males living in rural areas for at least three years and involved in rural work. This suggests, in most cases, a possible transmission of the disease from a rural/forest source, although some patients had not engaged in activities associated with permanence in forestall areas, which indicate a possible sandflies adaptation to the periurban setting.

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

FIRST REPORT OF CUTANEOUS LEISHMANIASIS CAUSED BY Leishmania (Leishmania) infantum chagasi IN AN URBAN AREA OF RIO DE JANEIRO, BRAZIL

SUMMARY American tegumentary leishmaniasis (ATL) is an infectious disease caused by protozoa of the genus Leishmania, and transmitted by sandflies. In the state of Rio de Janeiro, almost all of the cases of American tegumentary leishmaniasis (ATL) are caused by Leishmania (Viannia) braziliensis, while cases of visceral leishmaniasis (VL) are caused by Leishmania (Leishmania) infantum chagasi. The resurgence of autochthonous VL cases in Rio de Janeiro is related to the geographic expansion of the vector Lutzomyia longipalpis and its ability to adapt to urban areas. We report the first case of leishmaniasis with exclusively cutaneous manifestations caused by L. (L.) infantum chagasi in an urban area of Rio de Janeiro. An eighty-one-year-old woman presented three pleomorphic skin lesions that were not associated with systemic symptoms or visceromegalies. Multilocus enzyme electrophoresis identified L. (L.) infantum chagasi, but direct smear and PCR of bone narrow were negative for Leishmania sp. (suggesting exclusively cutaneous involvement). We discuss the different dermatological presentations of viscerotropic leishmaniasis of the New and Old World, and the clinical and epidemiological importance of the case. Etiologic diagnosis of ATL based upon exclusive clinical criteria may lead to incorrect conclusions. We should be aware of the constant changes in epidemiological patterns related to leishmaniases.

HEPATOCELLULAR CARCINOMA IN A NON-CIRRHOTIC PATIENT WITH SUSTAINED VIROLOGICAL RESPONSE AFTER HEPATITIS C TREATMENT

Chronic infection by hepatitis C virus (HCV) is one of the main risk factors for the development of liver cirrhosis and hepatocellular carcinoma. However, the emergence of hepatocellular carcinoma (HCC) in non-cirrhotic HCV patients, especially after sustained virological response (SVR) is an unusual event. Recently, it has been suggested that HCV genotype 3 may have a particular oncogenic mechanism, but the factors involved in these cases as well as the profile of these patients are still not fully understood. Thus, we present the case of a non-cirrhotic fifty-year-old male with HCV infection, genotype 3a, who developed HCC two years after treatment with pegylated-interferon and ribavirin, with SVR, in Brazil.