Preparation of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> DNA from respiratory samples for real-time PCR by commercial kits

Bordetella pertussis is increasingly detected by real-time PCR, but most kits for extracting Bordetella-DNA from respiratory samples are not validated for this material. Respiratory clinical materials were spiked with Bordetella pertussis cells. Four ion-exchange chromatography methods from one manufacturer were used for DNA preparation. Two real-time PCRs detecting the IS481 of Bordetella pertussis and based either on a hybridisation probes format (LightCycler®) or on a TaqMan® format were used. All kits effectively prepared DNA for Bordetella pertussis real-time PCR. The procedures were linear over a broad range, and the lower level of sensitivity was similar. Sensitivities measured as CT values were different. Inter-assay CVs were between 6.8% and 17.3%. Two kits did not effectively remove inhibitory substances from the respiratory samples. Commercial kits are useful for preparing Bordetella pertussis DNA from respiratory samples, but even kits from one manufacturer show significant differences in effectiveness and removal of inhibitory substances.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Evaluation of the BactiCard <Emphasis Type="Italic">Neisseria</Emphasis> for Identification of Pathogenic <Emphasis Type="Italic">Neisseria</Emphasis> Species and <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), γ-glutamyl aminopeptidase (GLUT), and ?-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory. Electronic Publication     

Differences in the in vitro susceptibility of mono-eukaryotic cultures of <Emphasis Type="Italic">Histomonas meleagridis,Tetratrichomonas gallinarum</Emphasis> and <Emphasis Type="Italic">Blastocystis</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> to natural organic compounds

Currently, all pharmaceuticals for the treatment or prophylaxis of blackhead disease (histomonosis) caused by the flagellate Histomonas meleagridis are banned from the market. Consequently, great interest exists on the finding of alternative drugs for the abatement of histomonosis. In this study, carvacrol, Cassia oil, an essential oil (EO) mixture containing thyme and rosemary EO and a Quillaja saponaria saponin were examined using in vitro assays for antiprotozoal and antibacterial activity testing established against cloned xenic cultures of different isolates of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. Whereas similar minimal lethal concentrations (MLCs) of five Histomonas isolates were obtained for both carvacrol and the EO mixture as well as for the saponin, significantly different MLCs were observed for them with Cassia oil, ranging from 0.25 up to 0.50 μl/ml. Testing the Blastocystis isolates, different MLCs were obtained for all substances, whereas the Tetratrichomonas gallinarum isolates showed identical susceptibilities. The effects are independent of the bacteria, underlining the need of well-defined protozoan cultures for these investigations.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

<Emphasis Type="Italic">In vivo</Emphasis> activity of <Emphasis Type="Italic">Sapindus saponaria</Emphasis> against azole-susceptible and -resistant human vaginal <Emphasis Type="Italic">Candida</Emphasis> species

Background  

Study of in vivo antifungal activity of the hydroalcoholic extract (HE) and n-BuOH extract (BUTE) of Sapindus saponaria against azole-susceptible and -resistant human vaginal Candida spp.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Transmission of <Emphasis Type="Italic">Yersinia pestis</Emphasis> cultures with different plasmid content from <Emphasis Type="Italic">Xenopsylla cheopis</Emphasis> to <Emphasis Type="Italic">Calomys callosus</Emphasis>

Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.     

Antiviral biflavonoids from <Emphasis Type="Italic">Radix Wikstroemiae</Emphasis> (<Emphasis Type="Italic">Liaogewanggen</Emphasis>)

Background  

Radix Wikstroemiae is a common Chinese herbal medicine. The ethyl acetate fraction of the ethanolic extract of W. indica possesses potent in vitro antiviral activity against respiratory syncytial virus (RSV). This study aims to identify the antiviral components of the active fraction.     

<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Subcellular localization of an extracellular serine protease in<Emphasis Type="Italic"> Leishmania</Emphasis> (<Emphasis Type="Italic">Leishmania</Emphasis>)<Emphasis Type="Italic"> amazonensis</Emphasis>

Extracellular proteolytic activity was detected in a Leishmania (L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH₄)₂SO₄ precipitation followed by affinity chromatography on aprotinin–agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. (L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.     

<Emphasis Type="Italic">Blastocystis</Emphasis> sp. subtype 5: a possibly zoonotic genotype

Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. Previous studies show that Blastocystis sp. subtypes 1–4, 6, and 7 were composed of isolates from humans and animals, while Blastocystis sp. subtype 5 included only pig and cattle isolates. A more recent study on the basis of the SSU rDNA sequence has showed that a single Blastocystis isolate amplified directly from the faeces of a Thai human belongs to Blastocystis sp. subtype 5, but that study failed to cultivate this isolate. We report herein two human isolates from in vitro cultures belonging to Blastocystis sp. subtype 5 and one human isolate from in vitro culture containing two distinct genotypes of Blastocystis sp. subtypes 3 and 5 using PCR amplification with seven kinds of sequence-tagged site (STS) primers. Additionally, 16 Blastocystis isolates from pigs living in the same rural area with the three humans infected Blastocystis sp. subtype 5 were also genotyped by PCR with the STS primers, and all isolates from pigs and humans were compared by small-subunit ribosomal RNA (SSU rRNA) restriction-fragment-length polymorphism (RFLP) analyses using two restriction endonucleases (HinfI and RsaI). The results indicated that all of the isolates from pigs showed Blastocystis sp. subtype 5 and the RFLP patterns of all of the isolates from humans except for the mixed one were identical or quite similar to those of the 16 pig isolates with both HinfI and RsaI enzymes. These findings provide additional molecular-based evidence supporting the zoonotic potential of Blastocystis sp. subtype 5. This study also showed that Blastocystis sp. subtype 3 overgrew Blastocystis sp. subtype 5 in vitro.