Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Relevant prevalence of Mycoplasma hominis and Ureaplasma urealyticum serogroups in HIV-1 infected men without urethritis symptoms

M. hominis and U. urealyticum are the better-known mycoplasma species pathogenic to the human genitourinary tract, causing mainly urethritis, bacterial vaginosis and pregnancy complications. In HIV-infected patients, the prevalence and role of these species is still not well known. The aim of this work was to determinate the prevalence of these species in this group of male patients (HIV group), in comparison to a group of men with clinical symptoms of urethritis (STD group). M. hominis was isolated from 7.5% patients (8/106) and U. urealyticum from 18.9% patients (20/106) from the HIV group, being among these 62.5% and 85% in significant concentrations, respectively. In the STD group these rates were 0.9% (1/110) for M. hominis and 13.6% (15/110) for U. urealyticum, being 100% and 93.3% in significant concentrations, respectively. We could demonstrate infection rates by these mycoplasma species in the HIV group as high as the one found in the STD one, what may indicate the occurrence of opportunistic infections in our population. This fact is discussed here because in immunosuppressed patients, specially M. hominis has been reported causing severe infections, even systemically.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Liver histology in co-infection of hepatitis C virus (HCV) and Hepatitis G virus (HGV)

As little is known about liver histology in the co-infection of hepatitis C virus (HCV) and hepatitis G virus (HGV), HGV RNA was investigated in 46 blood donors with hepatitis C, 22 of them with liver biopsy: co-infection HCV / HGV (n = 6) and HCV isolated infection (n = 16). Besides staging and grading of inflammation at portal, peri-portal and lobular areas (Brazilian Consensus), the fibrosis progression index was also calculated. All patients had no symptoms or signs of liver disease and prevalence of HGV / HCV co-infection was 15.2%. Most patients had mild liver disease and fibrosis progression index, calculated only in patients with known duration of infection, was 0.110 for co-infection and 0.130 for isolated HCV infection, characterizing these patients as "slow fibrosers". No statistical differences could be found between the groups, although a lesser degree of inflammation was always present in co-infection. In conclusion co-infection HCV / HGV does not induce a more aggressive liver disease, supporting the hypothesis that HGV is not pathogenic.

A preliminary study of the prevalence of intestinal parasites in immunocompromised patients with and without gastrointestinal manifestations

The objective of the present study was to determine the prevalence of the intestinal parasites most commonly found in immunocompromised patients. A group of 111 individuals with acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), human immunodeficiency virus (HIV) and other immunocompromised conditions (principally haematological disorders) was selected. A battery of tests was performed on each individual to identify the presence of parasites (three stool specimens with saline solution and Lugol both directly and by concentration, culture and special staining). No significant differences were found among the frequencies of the different parasites with the several types of immunocompromised conditions. The overall frequencies of potentially pathogenic and opportunistic parasites were 32.4% (36/111) and 9% (10/111) respectively, the most frequently encountered among the latter being Cryptosporidium sp., Microsporidia spp. and Strongyloides stercoralis.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Staphylococcus aureus ampicillin-resistant from the odontological clinic environment

The aim of this research was to evaluate the prevalence of Sthaphylococcus spp. and S. aureus in the odontological clinic environment (air), their production of beta-lactamase and antibacterial susceptibility to the major antibiotics utilized in medical particle. During 12 months of samples collect were isolated 9775 CFU by MSA medium suggesting a high amount of Staphylococcus spp. in the clinic environment which can appear through aerosols. A total of 3149 colonies (32.2%) were suggestive of pathogenic staphylococci. Gram coloration, catalase test, colony-mallow growing on chromogenic medium, and coagulase test confirmed the identity of 44 (0.45%) S. aureus isolates. Of these, 35 isolates (79.5%) showed production of beta-lactamase by CefinaseTM discs and resistance to ampicillin, erythromycin (7 isolates) and tetracycline (1 isolate) suggesting the existence of multiresistant isolates. The evaluation of the oxacillin MIC by Etest® assays showed susceptibility patterns suggesting the inexistence of the mecA gene in chromosomal DNA. These results point out to the need of a larger knowledge on the contamination means and propagation of this microorganism into the odontological clinic.

Onychomycosis by Scytalidium dimidiatum: report of two cases in Santa Catarina, Brazil

Onychomycoses constitute pathologies frequently seen in dermatological practice worldwide. Usually, they are caused by two groups of pathogenic fungi: dermatophytes and yeasts of the Candida genus. However, in a small fraction of the cases, the etiologic agents comprise nondermatophyte molds, belonging to several genera and species. The objective of this study was to present two cases of onychomycosis associated to the mold Scytalidium dimidiatum in patients residing in two cities of Santa Catarina State, Brazil. Aspects of fungal pathogenesis, as well as the epidemiological characteristics and laboratory diagnosis, are discussed.

Comparative analysis of two-dimensional electrophoresis maps (2-DE) of Helicobacter pylori from Brazilian patients with chronic gastritis and duodenal ulcer: a preliminary report

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Mass treatment for intestinal helminthisis control in an Amazonian endemic area in Brazil

The objective of the present study was to estimate the prevalence of soil-transmitted helminthiasis and evaluate the sanitary conditions and the role of a mass treatment campaign for control of these infections in Santa Isabel do Rio Negro. A cross-sectional survey was carried out in 2002, to obtain data related to the sanitary conditions of the population and fecal samples for parasitological examination in 308 individuals, followed by a mass treatment with albendazole or mebendazole with coverage of 83% of the city population in 2003. A new survey was carried out in 2004, involving 214 individuals, for comparison of the prevalences of intestinal parasitosis before and after the mass treatment. The prevalences of ascariasis, trichuriasis and hookworm infection were 48%; 27% and 21% respectively in 2002. There was a significant decrease for the frequency of infections by Ascaris lumbricoides (p < 0.05; OR / 95% CI = 0.44 / 0.30 - 0.65), Trichuris trichiura (p < 0.05; OR / 95% CI = 0.37 / 0.22 - 0.62), hookworm (p < 0.05; OR / 95% CI = 0.03 / 0.01 - 0.15) and helminth poliparasitism (p < 0.05; OR / 95% CI = 0.16 / 0.08 - 0.32). It was also noticed a decrease of prevalence of infection by Entamoeba histolytica / dispar (p < 0.05; OR / 95% CI = 0.30 / 0.19 - 0.49) and non-pathogenic amoebas. It was inferred that a mass treatment can contribute to the control of soil-transmitted helminthiasis as a practicable short-dated measure. However, governmental plans for public health, education and urban infrastructure are essential for the sustained reduction of prevalences of those infections.

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Occurrence of Proteus mirabilis associated with two species of venezuelan oysters

The fecal contamination of raw seafood by indicators and opportunistic pathogenic microorganisms represents a public health concern. The objective of this study was to investigate the presence of enteric bacteria colonizing oysters collected from a Venezuelan touristic area. Oyster samples were collected at the northwestern coast of Venezuela and local salinity, pH, temperature, and dissolved oxygen of seawater were recorded. Total and fecal coliforms were measured for the assessment of the microbiological quality of water and oysters, using the Multiple Tube Fermentation technique. Analyses were made using cultures and 16S rRNA gene sequencing. Diverse enrichment and selective culture methods were used to isolate enteric bacteria. We obtained pure cultures of Gram-negative straight rods with fimbriae from Isognomon alatus and Crassostrea rhizophorae. Our results show that P. mirabilis was predominant under our culture conditions. We confirmed the identity of the cultures by biochemical tests, 16S rRNA gene sequencing, and data analysis. Other enterobacteria such as Escherichia coli, Morganella morganii and Klebsiella pneumoniae were also isolated from seawater and oysters. The presence of pathogenic bacteria in oysters could have serious epidemiological implications and a potential human health risk associated with consumption of raw seafood.

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

Enteroparasitosis and their ethnographic relationship to food handlers in a tourist and economic center in Paraná, Southern Brazil

This study reports on the occurrence of enteroparasites based on data from an ethnographic study of food handlers in the city of Cascavel, Paraná, Brazil. Fecal material from 343 food handlers of both sexes, between 14 and 75 years of age, was analyzed using Lutz, modified Ritchie and Ziehl-Neelsen techniques. Ethnographic relationships were investigated by means of specific questionnaires. Positive fecal samples were found for 131 (38.2%) handlers. Endolimax nana (67.9%) was the predominant species, followed by Entamoeba coli (35.9%), Blastocystis hominis (28.2%), Entamoeba histolytica/dispar (10.1%) and Giardia duodenalis (8.4%). Protozoan infections were more common than helminth infections (p = 0.00). The positive results for some parasites were associated with the male sex, professional category, and the performance of other activities (p < 0.05). The high overall occurrence of enteroparasites found indicates improper hygiene and sanitation conditions. Effective educational measures should be implemented to prevent the transfer of pathogenic organisms to food via handling.