Growth curves of Leishmania braziliensis braziliensis Promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera

Leishmania braziliensis braziliensis(MHOM/BR/75/M2903) was grown in Schneider's Drosophila medium. In one set of experiments promastigotes were already adapted to the medium by means of serial passages whereas in the second cells were grown in a biphasic medium and transfered to the liquid. Growth was more abundant for culture medium adapted cells; degenerate cells in small numbers as well as dead ones were present from day 5 for promastigotes adapted to liquid medium and from day 3 for newly adapted cells. Synthesis of surface antigens differed according to length of cell culture as assessed by the titer of five mucocutaneous leishmaniasis sera on subsequent days. Five days of culture for cells already adapted to the culture medium and 3 days for newly adapted ones were judged to be the best for the preparation of immunofluorescence antigens.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Endemic Pemphigus foliaceus ("Fogo selvagem"): a series from the Northeastern region of the State of São Paulo, Brazil, 1973-1998

Endemic Pemphigus Foliaceus (EPF) is a bullous autoimmune skin disease whose incidence used to be high in the State of São Paulo (SP), Brazil, during the forties, but has declined thereafter. OBJECTIVES: to report a series of EPF patients from the northeastern region of SP. METHODS: a retrospective study concerning demographic and epidemiological data of patients seen from 1973 to 1998 was conducted at the University Hospital, Faculty of Medicine of Ribeirão Preto, SP. RESULTS: bullous disease was diagnosed in 340 patients, 245 with EPF (72.1%), 9.4 cases per year, 60.4% females, and 70.2% white, 7 to 82 year-old (29.4% in their teens); 46.9% lived in the rural zone. Concerning profession, housewives predominated among women (67.6%) and agricultural workers among men (40.2%). The time of disease was less than 1 year in 62.0% of cases, followed by 1 and 5 years (27%), and more than 5 years for the remaining patients (11%). 36.7% of patients were referred by the Direção Regional de Saúde (DIR) XVIII of Ribeirão Preto, with the largest number of cases being from Ribeirão Preto and Batatais: 33.3% and 23.3%, respectively; 22% from DIR XIII (Franca); 13.5% from DIR VII (Araraquara); 2.9% from DIR IX (Barretos); 4.1% from other DIRs of SP, and 20.8% from other States (16.7% from Minas Gerais). Thirteen (5.3%) patients reported occurrence of the disease in some relative, and 4 (1.6%) in neighbors. CONCLUSIONS: the present data characterize the northeastern region of the state of São Paulo as a remaining endemic focus of EPF.

Glucose consume and growth of E. coli under electromagnetic field

E. coli was submitted to a 5G electromagnetic field generated by a alternate 60 Hz voltage source. The differences on growth and glucose consume in control and exposed groups were evaluated using the non-parametric Mann-Whitney U-test. There was a significant difference in glucose consume and growth in E. coli after 8 hours of exposition to electromagnetic field. It can be concluded that electromagnetic field had a positive effect in consume of glucose and growth of E. coli. The cause of these results can be explained by an increasing of glucose entrance through membrane due to the stimulated transport system via Facility Diffusion or cyclotron resonance. The growth can be caused by shortening of lag phase and excitement of log phase.

First record of Desmodus rotundus in urban area from the city of Olinda, Pernambuco, Northeastern Brazil: a case report

The objective of this report is to describe the first record of Desmodus rotundus in urban area from the city of Olinda, Pernambuco State, Northeastern Brazil, and to draw attention to the possible risk of rabies transmission in this place. After the complaint of a dog owner who observed three bats attacking his dog, images registering attacks of D. rotundus were captured with a video camera. From 09:00 p.m. on 13 February 2004 to 04:00 a.m. of the next day, a high frequency of haematophagic activity and the presence of several bites on the dog's body were observed. This finding represents a serious risk to public health. Thus, it is necessary to further study the bat fauna, with special attention to their feeding behaviour in this place, in order to better know their biology and to adopt pertinent control measures. This is, to our knowledge, the first record of D. rotundus in urban area of Olinda.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µ L and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

The use of the antifungal agent miconazole as an inhibitor of Blastocystis hominis growth in Entamoeba histolytica/E. dispar cultures

In regions with high prevalence, Blastocystis hominis is frequently found in association with Entamoeba histolytica/E. dispar in xenic cultures. Its exacerbated growth is often superimposed on the growth of amebas, thus impeding the continuation of the amebas in the culture, within a few generations. The present study reports on the excellent efficacy (100%) of the antifungal agent miconazole in eliminating B. hominis from cultures of E. histolytica/E. dispar, thereby maintaining the integrity of the trophozoites of the amebas. Nystatin presented low efficacy (33.3%).

HISTOPLASMIN SURVEY IN HIV-POSITIVE PATIENTS: RESULTS FROM AN ENDEMIC AREA IN NORTHEASTERN BRAZIL

SUMMARY Background. Disseminated histoplasmosis is common in AIDS patients with advanced immunosuppression in Ceará, Northeastern Brazil. The goal of this study was to determine the prevalence of Histoplasma infection in patients with HIV/AIDS living in Fortaleza, the capital of Ceará. Methods. Intradermal tests with histoplasmin (mycelial phase) were performed in 161 HIV patients with CD4 ≥ 350 cells/mm 3 . Evidence of recent illness was evaluated with immunodiffusion (ID) tests in 76 of these individuals. Results. A total of 11.8% of patients reacted to histoplasmin and 2.63% had ID test positive to Histoplasma. The presence of mango trees (Mangifera indica) in the patient neighborhood (OR = 2.870; 95% CI = 1.081-7.617; p = 0.040) and past activity involving soil (OR = 2.834; 95% CI = 1.045-7.687; p = 0.045) or visits to a farm (OR = 3.869; 95% CI = 1.189-12.591; p = 0.033) were significantly associated with Histoplasma infection. Conclusions. Patients with HIV living in Fortaleza have an expressive prevalence of infection with Histoplasma.

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.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL

There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species.

PRETERM BIRTH AND FETAL GROWTH RESTRICTION IN HIV-INFECTED BRAZILIAN PREGNANT WOMEN

Introduction: Maternal HIV infection and related co-morbidities may have two outstanding consequences to fetal health: mother-to-child transmission (MTCT) and adverse perinatal outcomes. After Brazilian success in reducing MTCT, the attention must now be diverted to the potentially increased risk for preterm birth (PTB) and intrauterine fetal growth restriction (IUGR). Objective: To determine the prevalence of PTB and IUGR in low income, antiretroviral users, publicly assisted, HIV-infected women and to verify its relation to the HIV infection stage. Patients and Methods: Out of 250 deliveries from HIV-infected mothers that delivered at a tertiary public university hospital in the city of Vitória, state of Espírito Santo, Southeastern Brazil, from November 2001 to May 2012, 74 single pregnancies were selected for study, with ultrasound validated gestational age (GA) and data on birth dimensions: fetal weight (FW), birth length (BL), head and abdominal circumferences (HC, AC). The data were extracted from clinical and pathological records, and the outcomes summarized as proportions of preterm birth (PTB, < 37 weeks), low birth weight (LBW, < 2500g) and small (SGA), adequate (AGA) and large (LGA) for GA, defined as having a value below, between or beyond the ±1.28 z/GA score, the usual clinical cut-off to demarcate the 10th and 90th percentiles. Results: PTB was observed in 17.5%, LBW in 20.2% and SGA FW, BL, HC and AC in 16.2%, 19.1%, 13.8%, and 17.4% respectively. The proportions in HIV-only and AIDS cases were: PTB: 5.9 versus 27.5%, LBW: 14.7% versus 25.0%, SGA BW: 17.6% versus 15.0%, BL: 6.0% versus 30.0%, HC: 9.0% versus 17.9%, and AC: 13.3% versus 21.2%; only SGA BL attained a significant difference. Out of 15 cases of LBW, eight (53.3%) were preterm only, four (26.7%) were SGA only, and three (20.0%) were both PTB and SGA cases. A concomitant presence of, at least, two SGA dimensions in the same fetus was frequent. Conclusions: The proportions of preterm birth and low birth weight were higher than the local and Brazilian prevalence and a trend was observed for higher proportions of SGA fetal dimensions than the expected population distribution in this small casuistry of newborn from the HIV-infected, low income, antiretroviral users, and publicly assisted pregnant women. A trend for higher prevalence of PTB, LBW and SGA fetal dimensions was also observed in infants born to mothers with AIDS compared to HIV-infected mothers without AIDS.