Preanalytical conditions can interfere with M. tuberculosis detection by PCR in respiratory samples

OBJECTIVES: Tuberculosis is one of the most prevalent infections in humans. Although culture is the reference for diagnosis, its sensitivity is compromised, especially in paucibacillary samples. Because polymerase chain reaction (PCR) amplifies mycobacterial DNA, it is more sensitive than culture for the diagnosis of Mycobacterium tuberculosis (Mtb). However, its performance can be affected by intrinsic sample inhibitors and by the extraction/detection techniques used. METHODS: We evaluated the influence of preanalytical conditions on Mtb detection in samples of sputum (SPU), bronchoalveolar lavage (BAL), and pleural fluid (PF) using combinations of extraction/detection methods. Respiratory samples were prepared to contain different concentrations of red blood cells and nucleated cells to which increasing amounts of Mtb colonies were inoculated and submitted to PCR. RESULTS: Up to 102 CFU/ml of Mtb were detected in the SPU in all methods, except for the Roche extraction/detection method, regardless of the preanalytical sample condition. In BAL samples, medium and high concentrations of cells and high concentrations of red blood cells contributed to a lower Mtb detection, regardless of the extraction method used. In PF, red blood cells were the variable that most interfered with Mtb detection, with better recovery (102 CFU/ml) observed with the Qiagen/Nanogen combination. CONCLUSION: The choice of Mtb extraction and detection method is of fundamental importance for PCR analytical sensitivity, especially when paucibacillary samples and/or samples containing potential PCR inhibitors are analyzed.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2 percent) and specificities (100 percent for rMSP5 and 93.8 percent for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15 percent (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1 percent by rMSP5 ELISA and 79.7 percent by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Malaysian mosquitocidal soil bacterium (Bacillus thuringiensis) strains with selective hemolytic and lectin activity against human and rat erythrocytes.

The objective of this study is to determine the role of carbohydrates on the toxic effect of parasporal inclusion proteins isolated from Malaysian mosquitocidal Bacillus thuringiensis (Bt) strains on erythrocytes (human and rat). Dose response analyses on the effect of these parasporal inclusions on human and rat erythrocytes suggest that toxin action is selective depending on bacterial strains and source of erythrocytes. Results from this study suggest Bt toxin is a lectin which recognizes specific plasma membrane glycoconjugate receptor(s) with a terminal residue of either D-mannose (Man), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc) or even a combination of these monosaccharides.

Some virulence characteristics of uropathogenic Escherichia coli in different patient groups.

BACKGROUND & OBJECTIVE: Uropathogenic Escherichia coli have virulence properties, that are absent in non pathogenic E. coli. The distribution of these markers can vary according to patient populations. Hence, a study was undertaken to describe the presence of virulence factors like Pfimbriae, type 1 fimbriae and haemolysin in E.coli causing urinary infections in three groups of patients. Antibiogram was also recorded to determine differences, if any, between the groups. METHODS: E. coli isolated from three groups of subjects, in counts of >10(5) CFU/ml and in pure growth were tested for mannose resistant haemagglutination (MRHA) to indicate P fimbriae and mannose sensitive haemagglutination (MSHA) to indicate type 1 fimbriae. Haemolysin production and antimicrobial susceptibility patterns were also recorded. RESULTS: Significantly more isolates from antenatal and postnatal women possessed P fimbriae compared to groups with urologic abnormalities (P=0.05). Haemolysin production was also significantly higher (P<0.001) in this group. Greater proportions of isolates from pregnant women were susceptible to commonly used antimicrobials. However, resistance to third generation cephalosporins was present even in these isolates from community infections. INTERPRETATION & CONCLUSION: In patients with urological abnormality, E. coli with lower virulence can cause infections. Isolates from these patients exhibited greater drug resistance. In pregnant women and in community acquired infections, simple antimicrobial drugs like nitrofurantoin might still be useful. However, urgent and stringent policies for antimicrobial use and infection control in hospitals are required in India.

Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars.

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.

Heterogenous adhesion properties of pseudomonas aeruginosa strains to different athrocytes species

The property of Pseudomonas aeruginosa strains and isolates to hemagglutinate erythrocytes of human ABO-blood groups, rat, rabbit, ginea pig, mouse, bovine and ovine were investigated. The findings showed a heterogeneous behaviour among strains and isolates to hemagglutinate different erythrocyte species. The basic results showed that adhesion could be mediated both through mannose containing entity and non-mannose dependent entity. Evidence for the implication of both ligand and receptor operating both on bacterium and target erythrocyte is presented. There was no correlation between the presence of terminal mannose, as evidenced by FITC-con-A binding among the strains and isolates and the mannose sensitive adherence [hemagglutination]. The results are discussed based on the importance of such heterogeneous adherence mechanisms operating in Pseudomonas aeruginosa and the new trends of adhesion importance

Fragilidad osmótica de los eritrocitos de carnero en relación con su uso en el laboratorio clínico / Osmotic fragility of sheep erythrocytes in relation with its use at clinic laboratory

Al notar que los eritrocitos de carnero del Bioterio del Hospital San Juan de Dios, San José, Costa Rica, hemolizaban espontáneamente, fueron comparados, por medio de una serie de ensayos de fragilidad osmótica, con los de la Universidad Nacional de Costa Rica, que no mostraban tal característica. Se encontraron diferencias significativas (P<0.05), las cuales se pueden relacionar con condiciones de higiene y nutrición en el mantenimiento de los carneros de laboratorio.

Infección "in vitro" de los hematies humanos por bartonella / Human hemocytes infection \"in vitro\" by bartonella

La Enfermedad de Carrión es producida por Bartonella bacilliformis a través de la picadura de insecto del género Lutzomyia. La bacteria después de un periodo de incubación en el RES, parasita a los hematies. Conociendo estos aspectos dinámicos de la Bartonella, nos llevó a realizar la réplica de la infección In-Vitro. Para lo cual utilizamos dos cepas de Bartonella (21 y 051), el medio Agar de Fases y hematíes de los diferentes grupos sanguíneos humanossanos. Cultivamos el complejo Hematíes-Bartonellas, utilizando 15 frascos de Kolle con Medio Agar de Fases para cada una de las cepas de Bartonella. Resultados: A partir del sexto día de incubación, se observó el parasitismo de los hematíes por la bacteria, siendo más acentuado a las dos semanas de incubación. Conclusión: el parasitismo hemático en la infección in-vitro, es parecido a la infección natural de las personas en zonas endémicas de Bartonelosis

Actividad hemolítica de Klebsiella pneumoniae sobre glóbulos rojos de conejo / Hemolytic activity of Klebsiella pneumoniae on rabbit erythrocytes

Se investigó la actividad hemolítica de Klebsiella pneumoniae, empleando cepas de referencia y cepas aisladas de infecciones. Todas las bacterias estudiadas resultaron hemolíticas en forma selectiva sobre eritrocitos de conejo. El frío intensificó el efecto lítico sobre los glóbulos rojos. Se estimó la capacidad hemolítica de las cepas mediante el diámetro de los halos producidos alrededor de orificios inoculados con 5 micron**l de suspensiones bacterianas. Cuando se realizó la detección de hemolisis en medios líquidos, sólo fue posible evidenciar lisis franca al tratar los cultivos con ciertos agentes reductores; de los cuales el más efectivo resultó el 2 mercaptoetanol. A la misma concentración los demás agentes químicos utilizados dieron menor porcentaje de eritrocitos lisados. La actividad hemolítica apareció en los cultivos durante la fase exponencial del crecimiento bacteriano y al cabo de 24 horas disminuyó al 33% del valor máximo alcanzado. Tanto la hemolisina cruda como la purificada reqirieron SH-activación y fueron inhibidas por suero