Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Leprosy and pregnancy: detection coefficient and proposal for a new index

Introduction Our study presents a method to generate a novel detection coefficient for the association between leprosy and pregnancy (DCLP). Methods The DCLP was calculated for women from the State of Pará (2007-2009), Brazil. Data were ordered, divided into five equal parts (corresponding to the P20, P40, P60, and P80 percentiles), and classified as low, medium, high, very high, or hyperendemic. Results Using the new index, we established the DCLP parameters for low (<0.36), medium (0.36-0.69), high (0.70-1.09), very high (1.10-1.50), and hyperendemic (>1.50). Conclusions The new DCLP is more appropriate than the overall detection coefficient (DC), which does not take into account the particularities of the interaction between a disease and a specific physiological state.

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH - 95% (81.8-99.1), EMB - 94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.

Characterization of Shigella spp. by antimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho (Western Amazon region), Brazil

The incidence of Shigella spp. was assessed in 877 infants from the public hospital in Rondônia (Western Amazon region, Brazil) where Shigella represents the fourth cause of diarrhea. Twenty-five isolates were identified: 18 were Shigella flexneri, three Shigella sonnei, three Shigella boydii and one Shigella dysenteriae. With the exception of S. dysenteriae, all Shigella spp. isolated from children with diarrhea acquired multiple antibiotic resistances. PCR detection of ipa virulence genes and invasion assays of bloody diarrhea and fever (colitis) were compared among 25 patients testing positive for Shigella. The ipaH and ipaBCD genes were detected in almost all isolates and, unsurprisingly, all Shigella isolates associated with colitis were able to invade HeLa cells. This work alerts for multiple antibiotic resistant Shigella in the region and characterizes presence of ipa virulence genes and invasion phenotypesin dysenteric shigellosis.

Adenovirus, calicivirus and astrovirus detection in fecal samples of hospitalized children with acute gastroenteritis from Campo Grande, MS, Brazil

We analyzed fecal samples from hospitalized children up to three years of age with acute gastroenteritis at Campo Grande, Mato Grosso do Sul, Brazil, from May 2000-January 2004. Astrovirus and calicivirus were detected by Reverse Transcription-Polymerase Chain Reaction and adenovirus was detected using the Rotavirus and Adenovirus combined immunoenzyme assay. Astrovirus, adenovirus and calicivirus were detected at rates of 3.1%, 3.6% and 7.6%, respectively. These results re-emphasize the need for the establishment of regional vigilance systems to evaluate the impact of enteric viruses on viral gastroenteritis.

Evaluation of Lionex TB kits and mycobacterial antigens for IgG and IgA detection in cerebrospinal fluid from tuberculosis meningitis patients

To evaluate commercial Lionex TB together with four antigens of Mycobacterium tuberculosis (MPT-64, MT10.3, 16 kDa and 38 kDa) for IgG and IgA cerebrospinal fluid (CSF) detection in the diagnosis of tuberculosis meningitis (TBM) with CSF negative acid-fast bacilli staining, 19 cases of TBM, 64 cases of other infectious meningoencephalitis and 73 cases of other neurological disorders were tested by enzyme linked immunosorbent assay. IgA-MPT-64 and IgG Lionex showed the highest sensitivities, specificities, positive predictive value and negative predictive value (63.2%, 47.4%; 95%, 93.7%; 40%, 98% and 28.4%, 97.1%, respectively). However, while grey zone was 12.7% and 6%, respectively, lowering sensitivity but maintains high specificity (> 95%). High protein concentration in CSF was associated with antibody positivity CSF/HIV+ which did not influence the sensitivity of both tests. To our knowledge, this is the first description of IgA-MPT-64 and IgG Lionex antibodies in CSF-TBM and, although there is good specificity, adjustments are needed based on antigen composition to enhance sensitivity.

Coverage mobilization in the sowing line and its influence on temperature and water content and on maize emergence

Straw on sowing line modifies seed germination environment regarding temperature and water content. Given these considerations, the aim of this study was to evaluate different mechanisms for coverage mobilization on the sowing line and their effect on germination environment of maize seeds, mainly in relation to the dynamics of straw in the seedbed, water content and soil temperature. Treatments consisted on the combination of two mechanisms at front of furrow opener, composed of cutting disc and row cleaners, with three mechanisms behind the seed furrower for returning the soil, consisting of three covering mechanisms, commercial and prototype models. It was found that straw presence on the surface of sowing line contributed to germination of maize seeds, maintenance of temperature and soil water content. The cutting disc treatment, associated with prototype, introduced percentages of water content near the ones in bottom layer, and this soil water content was 29.7% with 93.75% of straw coverage and deeper seeding depth, granting better conditions for seed germination. However, the straw coverage removal on soil by the row cleaners and its low sowing depth caused water loss in the lines resulting in great reduction of the emergence speed index in maize seedlings.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

AbstractMycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Central nervous system virion detection in acute measles: histopathological, ultrastructural and pathogenetic aspects

Histopathological and ultrastructural studies of 23 patients who died with clinical diagnosis of measles were carried out. In 12 cases viral nucleocapsids were searched by electron microscopy and detected in 100% of the cases in the lungs and in 50% of the cases in the central nervous system. They were mostly intranuclear. Histopathological changes associated to neurological alterations and the detection of virion are discussed in relation to acute and delayed clinical manifestations.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAV IgM, 6 and 95% for anti-HBs, 13 and 100% for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV. The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.