Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

Detection of non-enterotoxigenic and enterotoxigenic Bacteroides fragilis in stool samples from children in São Paulo, Brazil

Non-enterotoxigenic bacteria of the Bacteroides fragilis group and enterotoxigenic B. fragilis were identified from children with and without aqueous acute diarrhea. In this study, 170 stool samples from 96 children with and 74 without diarrhea were analyzed. Enterotoxin production and the toxin gene detection were detected by cytotoxicity assay on HT-29/C1 cells and by PCR, respectively. B. fragilis species was prevalent in both groups and enterotoxigenic B. fragilis strains were isolated from two children with diarrhea. More studies are important to evaluate the role of each bacteria of the B. fragilis group, including enterotoxigenic strains play in the diarrheal processes in children.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

Immunogenicity of three recombinant hepatitis B vaccines administered to students in three doses containing half the antigen amount routinely used for adult vaccination

We evaluated the immunogenicity of three recombinant hepatitis B vaccines, one Brazilian (Butang, Instituto Butantan) and two Korean vaccines (Euvax-B, LG Chemical Ltd. and Hepavax-Gene, Greencross Vaccine Corp.), administered intramuscularly to students aged 17 to 19 years in three 10-µg doses (corresponding to half the amount of antigen routinely used for adult vaccination) at intervals of one month between the first and second dose, and of four months between the second and third dose. A total of 316 students non-reactive for any serological marker of hepatitis B virus infection were vaccinated: 77 (24.4%) with the Butang vaccine, 71 (22.5%) with Euvax-B, 85 (26.9%) with Hepavax-Gene and, for comparison, 83 (26.2%) with Engerix-B (GlaxoSmithKline), whose efficacy in young adults at the dose used here has been confirmed in previous studies. Similar seroconversion rates (anti-HBs > 10 mIU/mL about one month after application of the third dose) were obtained for the Butang, Euvax-B, Hepavax-Gene and Engerix-B vaccines (96.2%, 98.6%, 96.5% and 97.6%, respectively). The frequency of good responders (anti-HBs > 100 mIU/mL) was also similar among students receiving the four vaccines (85.8%, 91.6%, 89.4% and 89.2%, respectively). The geometric mean titers (GMT) of anti-HBs about one month after the third dose obtained with these vaccines were 727.78 ± 6.46 mIU/mL, 2009.09 ± 7.16 mIU/mL, 1729.82 ± 8.85 mIU/mL and 2070.14 ± 11.69 mIU/mL, respectively. The GMT of anti-HBs induced by the Euvax-B and Engerix-B vaccines were higher than those obtained with the Butang vaccine (p < 0.05); this difference was not significant when comparing the other vaccines two-by-two. No spontaneous adverse effects attributable to the application of any dose of the four vaccines were reported.

Transformation of a Cutaneous Follicle Center Lymphoma to a Diffuse Large B-Cell Lymphoma - An Unusual Presentation

Primary cutaneous follicle center lymphoma (PCFCL) is characterized by a proliferation of follicle center cells in the skin. A definitive diagnosis is frequently delayed because of difficulties in interpretation of the histopathologic findings. It has an excellent prognosis with a 5-year survival over 95% and its risk of transformation has not been established. We describe a case report of man with a gastric diffuse large B-cell lymphoma (DLBCL) referred to our clinic because of nodules in the back that had gradually developed over a period of 10 years. A biopsy performed 3 years before was interpreted as reactive follicular hyperplasia. A new skin biopsy revealed a diffuse large B-cell lymphoma and immunoglobulin heavy chain gene rearrangements from the initial skin biopsy (PCBCL) and the DLBCL gastric biopsy were studied by polymerase chain reaction and an identical clonal rearrangement was detected which was highly suggestive of a transformation lymphoma.

Comparative study of the fecundity and fertility of Biomphalaria glabrata (Say, 1818) and Biomphalaria straminea (Dunker, 1848) in a laboratory through self-fertilization and cross-fertilization

The aim of this study was to compare the fecundity and fertility of B. glabrata and B. straminea by cross- and self-fertilization. To attain this objective, laboratory-raised strains of B. glabrata and B. straminea were used. The former originated from natural breeding grounds in the municipality Paulista, state of Pernambuco, Brazil. The latter originated from irrigation ditches in the municipality of Petrolândia, in the same state. Snail populations of B. glabrata and B. straminea were maintained for 240 days in laboratory. Their fecundity was evaluated by noting the number of egg-masses, eggs and eggs per mass. Their fertility was evaluated by the number of viable eggs and the hatching rate. B. straminea was markedly more fecund than B. glabrata through cross- and self-fertilization, namely: greater egg-mass; higher egg production and more eggs per mass. Regarding fertility, there seemed to be no preferential period for occlusion to occur or a trend in the rhythm of producing viable eggs.

Candidemia in a Brazilian tertiary care hospital: species distribution and antifungal susceptibility patterns

Recent studies have shown differences in the epidemiology of invasive infections caused by Candida species worldwide. In the period comprising August 2002 to August 2003, we performed a study in Santa Casa Complexo Hospitalar, Brazil, to determine Candida species distribution associated with candidemia and their antifungal susceptibility profiles to amphotericin B, fluconazole and itraconazole. Antifungal susceptibility was tested according to the broth microdilution method described in the NCCLS (M27A-2 method). Only one sample from each patient was analyzed (the first isolate). Most of the episodes had been caused by species other than C. albicans (51.6%), including C. parapsilosis (25.8%), C. tropicalis (13.3%), C. glabrata (3.3%), C. krusei (1.7%), and others (7.5%). Dose-dependent susceptibility to itraconazole was observed in 14.2% of strains, and dose-dependent susceptibility to fluconazole was found in 1.6%. Antifungal resistance was not found, probably related to low use of fluconazole. Further epidemiological surveillance is needed.

Serpentine bacteria influence metal translocation and bioconcentration of Brassica juncea and Ricinus communis grown in multi-metal polluted soils

The aim of this study was to assess the effects of inoculation of rhizosphere or endophytic bacteria (Psychrobacter sp. SRS8 and Pseudomonas sp. A3R3, respectively) isolated from a serpentine environment on the plant growth and the translocation and accumulation of Ni, Zn, and Fe by Brassica juncea and Ricinus communis on a multi-metal polluted serpentine soil (SS). Field collected SS was diluted to 0, 25, 50, and 75% with pristine soil in order to obtain a range of heavy metal concentrations and used in microcosm experiments. Regardless of inoculation with bacteria, the biomass of both plant species decreased with increase of the proportion of SS. Inoculation of plants with bacteria significantly increased the plant biomass and the heavy metal accumulation compared with non-inoculated control in the presence of different proportion of SS, which was attributed to the production of plant growth promoting and/or metal mobilizing metabolites by bacteria. However, SRS8 showed a maximum increase in the biomass of the test plants grown even in the treatment of 75% SS. In turn, A3R3 showed maximum effects on the accumulation of heavy metals in both plants. Regardless of inoculation of bacteria and proportion of SS, both plant species exhibited low values of bioconcentration factor (<1) for Ni and Fe. The inoculation of both bacterial strains significantly increased the translocation factor (TF) of Ni while decreasing the TF of Zn in both plant species. Besides this contrasting effect, the TFs of all metals were <1, indicating that all studied bacteria–plant combinations are suitable for phytostabilization. This study demonstrates that the bacterial isolates A3R3 and SRS8 improved the growth of B. juncea and R. communis in SS soils and have a great potential to be used as inoculants in phytostabilization scenarios of multi-metal contaminated soils.

The hyperaccumulator Sedum plumbizincicola harbors metal-resistant endophytic bacteria that improve its phytoextraction capacity in multi-metal contaminated soil

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains.

Environmental straits of Cryptococcus neoformans variety grubii in the city of Santos, SP, Brazil

This study involved a total of 116 samples, 79 taken from pigeon droppings and 37 of atmospheric air taken close to accumulations of excrement. Cryptococcus neoformans var. grubii was isolated from 11 (13.9%) of these samples. Other species of Cryptococcus were also isolated from these samples, such as C. albidus (12.6%) and C. laurentii (8.9%). C. neoformans was not isolated from the air samples, though C. albidus (5.4%) was. All the strains of C. neoformans were found to belong to the A serotype (C. neoformans var. grubii). In regard to the studies with the antifungal agents 5-fluorocytosine, fluconazole, itraconazole, amphotericin B and voriconazole, by means of the microdilution method (EUCAST), we point out that one sample demonstrated resistance to fluconazole, this being especially significant because this is an environmental strain.

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Pará, Brazil

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Pará showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Serotyping and evaluation of the virulence in mice of Streptococcus suis strains isolated from diseased pigs

A total of 110 strains of Streptococcus suis, isolated from diseased pigs in Brazil were serotyped and analyzed for virulence. Serotyping of the strains resulted in the following classification: 42 strains of serotype 2 (38.2%), 10 strains of serotype 14 (9.1%), seven strains of serotype 9 (6.4%), three strains each of serotype 7 and 11 (2.7%), two strains each of serotype 1 and 8 (1.8%) and one strain each of serotypes ½, 3, 5, 6 and 10 (0.9%). Cross reactions among serotypes 1, 14 and 7 were observed in 21 strains (19.1%). Only 41.9% of the strains were lethal for mice using the pathogenicity test.

Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in goiânia city, Goiás, Brasil

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 µg/mL for amphotericin B, 0.125-8 µg/mL for fluconazole, 0.03-0.5 µg/mL for itraconazole and 0.03-0.25 µg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 µg/mL, 0.25-2.0 µg/mL, 0.007-0.125 µg/mL and 0.03-0.25 µg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Rotavirus serotypes and electropherotypes identified among hospitalised children in São Luís, Maranhão, Brazil

During June 1997-June 1999 rotavirus infection was screened in infants aged up to 2 years and hospitalised with acute diarrhoea in São Luís, Northeastern Brazil. Altogether, 128 stool samples were collected from diarrhoeic patients and additional 122 faecal specimens from age- and- temporal matched inpatients without diarrhoea were obtained; rotavirus positivity rates for these groups were 32.0% (41/128) and 9.8% (12/122), respectively (p < 0.001). Both electropherotyping and serotyping could be performed in 42 (79.2%) of the 53 rotavirus-positive stool samples. Long and short electropherotypes were detected at similar rates - 38.1% and 40.5% of specimens, respectively. Overall, a G serotype could be assigned for 35 (83.3%) of specimens, the majority of them (66.7%) bearing G1-serotype specificity. Taking both electropherotypes and serotypes together, G1 rotavirus strains displaying long and short RNA patterns accounted for 30.9% and 19.0% of tested specimens, respectively; all G2 strains had short electropherotype. Rotavirus gastroenteritis was detected year-round and, in 1998, the incidence rates tended to be higher during the second semester than in the first semester: 45.2% and 26.1% (p = 0.13), respectively. Rotavirus infections peaked at the second semester of life with frequencies of 30.1% and 13.5% for diarrhoeic children and controls, respectively. While the six rotavirus strains bearing G2-type specificity were circulating throughout the whole study period, G1 serotypes (n = 27) emerged as from June 1998 onwards, 20 (74.1%) of which clustering in 1998. These data underscore the importance of rotaviruses in the aetiology of severe infantile gastroenteritis in Northeastern Brazil and sustain the concept that a future vaccine should confer protection against more than one serotype.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.