Auditing smear microscopy results according to time to detection using the BACTEC™ MGIT™ TB system.

Smear microscopy is a rapid method for the identification of the most infectious patients with mycobacterial infection. Suboptimal smear microscopy may significantly compromise or delay patient isolation and contact tracing. A stringent method for auditing mycobacterial smear results is thus needed. This article proposes an auditing tool based on time to detection (TTD) of culture-positive samples using the automated BACTEC™ MGIT™ 960 TB system. In our study, sputum samples subjected to liquefaction and concentration before staining with a TTD of ≤ 13 days using the BACTEC system should be positive on smear microscopy.

Cord formation in BACTEC(TM) medium aids rapid identification of Mycobacterium tuberculosis complex.

Mycobacterium tuberculosis complex (MTC) organisms form serpentine cords in fluid culture medium. Reporting of a presumptive identification of MTC based on cording allows rapid identification of patients with tuberculosis. A total of 612 positive mycobacterial cultures from 316 patients over 3 years (2008-2010) were evaluated for the presence of cord formation. Cording was identified in 426 (69.6%) specimens, while the reference laboratory confirmed M. tuberculosis in 424 specimens (69.3%). Sensitivity of the test in our laboratory was 99.1% (95%CI 97.4-99.7) and specificity was 96.8% (95%CI 92.8-98.7). Presumptive identification of M. tuberculosis by the presence of cording formation is both sensitive and specific.

Blood culture fluorescence rates predict severity and mortality of invasive pneumococcal pneumonia.

Invasive pneumococcal pneumonia is associated with high rates of mortality. Clinical assessment tools have poor sensitivity for predicting clinical outcomes. Molecular measurements of bacterial load correlate closely with clinical outcome but require specialist facilities and expertise. This study describes how routine blood culture testing can estimate bacterial load and predict clinical outcome for invasive pneumococcal pneumonia. Between December 2009 to March 2014, clinical and laboratory data were collected for 50 patients with Streptococcus pneumoniae bacteraemia secondary to community-acquired pneumonia. Fluorescence rates (FR) were calculated from growth curves generated by BACTEC blood culture analysers by dividing change in fluorescence units (FU), measured at the first point of detectable fluorescence and at the point of automated BACTEC positivity, by time in hours. The mean age of the patients was 70.6 years (49.6-86.3). Forty patients survived invasive pneumococcal disease and ten patients died. These two groups did not significantly differ by demographic or clinical characteristics. The mean FR for the non-survival group (3.62 × 10(-3) FU/h) was significantly higher (p < 0.001) than that of the survival group (1.73 × 10(-3) FU/h). FR did not vary by serotype. We determined that an FR of 2.59 × 10(-3) FU/h might represent a useful threshold for predicting high mortality risk with a sensitivity of 91 % and a specificity of 97 %. Our FR calculation uses cheap and accessible routine blood culture techniques to predict mortality in a small retrospective cohort study. In patients admitted to hospital with pneumococcal bacteraemia and, potentially, other organisms, this single tool could guide early escalation of clinical care.

Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn's disease.

The possible role of infection with Mycobacterium paratuberculosis (MAP) for the etiopathogenesis of Crohn's disease (CD) has been a matter of long-term controversy. In addition to similarities with the pathology of ruminant paratuberculosis, DNA fingerprinting confirmed the organism isolated from gut tissue, but the specificity of the immune repertoire has not as yet been evaluated. We report here on a serological study of 29 patients with CD, 20 patients with ulcerative colitis and 18 healthy control subjects, using three antigens attributed with species-specificity and selective immunogenicity following MAP infection. Antibodies binding to the 38-kD band of MAP extract were demonstrable by the Western blot technique in 57% of CD patients. Antibody levels to the 24-kD (p24BCD) cathodic bands, determined by competition ELISA using a monospecific murine antiserum, and to the 18-kD protease-resistant purified bacterioferritin, detected by standard ELISA, were significantly elevated in 53% of CD patients. However, these three antibody specificities tested in individual CD patients did not show any correlation with each other. Thus, 18% of patients were positive for all three specificities, whilst 84% had antibodies to at least one of the specific antigens. Although the exact proportion of affected patients is yet to be defined, the serological results obtained support the view that MAP infection may play an etiological role in Crohn's disease.

Localisation of linear epitopes at the carboxy-terminal end of the mycobacterial 71 kDa heat shock protein.

Four distinct linear epitopes localized within species-specific sequences at the carboxy-terminal end of the 71 kDa heat shock protein of M. tuberculosis have been identified by scanning 94 overlapping peptides with 13 human sera. One epitope ("C") of entirely M. tuberculosis-specific core sequence (GEAGPG) has been found immunogenic in smear-negative tuberculosis, but not in non-tuberculous mycobacterial diseases. This peptide appears to be a valuable candidate for further serodiagnostic evaluation.

Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stress proteins.

Structural homology between microbial and human stress proteins has been postulated to be a basis for autoimmunization in chronic inflammatory diseases. Therefore, we estimated by ELISA titration the antibody levels to mycobacterial (M) and human (H) recombinant hsp70 and M-hsp65 heat-shock proteins in sera of patients with Crohn's disease (n = 29), ulcerative colitis (n = 20) and nontuberculous mycobacterial disease of the lungs (n = 20). Antibodies to H-hsp60, separated by two-dimensional gel electrophoresis, were tested in six sera of each group of patients. In Crohn's disease, antibody titres to the M-hsp65 antigen without detectable H-hsp60 binding were significantly elevated in 52% of the patients. In contrast titres to both M-hsp70 and H-hsp70 were demonstrable and correlated, but increased over control values only in four (14%) patients. The antibody pattern in ulcerative colitis was found to be quite different: anti-H-hsp60 binding was demonstrable in most patients, although anti-M-hsp65 titres were not elevated. Furthermore, 25% of patients had significantly elevated titres to M-hsp70, but not to H-hsp70. In non-tuberculous mycobacterial pulmonary disease, about 50% of patients had elevated titres to both hsp65 and hsp71 mycobacterial antigens but not to the corresponding human proteins; patients with Mycobacterium xenopi infection had the highest titres in this group. These results demonstrate the existence of distinct disease-associated patterns in the human antibody response to stress protein antigens. However, these data are not sufficient to imply sensitization with mycobacteria in patients with inflammatory bowel diseases, since certain epitopes of heat-shock proteins are shared by several bacterial genera.

Distinctive western blot antibody patterns induced by infection of mice with individual strains of the Mycobacterium avium complex.

Systemic infection of mice with organisms of the Mycobacterium avium complex (MAC) induced antibody responses, characteristic for each of the three tested individual strains. The influence of host genetic factors was reflected up to 3 months after infection by the finding of generally oligobanded and multibanded Western blot patterns in C57B1/6 and BALB/c mice, respectively. Nevertheless, more bands developed at 6 months in C57BL/6 mice. The response to three antigens of 18,000, 38,000 and 24,000 MW was analysed in greater detail. Antibodies to a protease-resistant 18,000 MW band produced only by BALB/c mice were either strain specific, following infection with M. avium, strain Maa-B2, or cross-reactive within MAC, following infection with M. avium strain Maa-A6 and M. paratuberculosis, strain Map-203. Another protease-resistant antigen of 38,000 MW was immunogenic only in Maa-B2 infected mice. This constituent was found to be related to the protease-sensitive antigen of corresponding molecular weight from M. tuberculosis. Two 24,000 MW proteins of M. paratuberculosis were separated by two-dimensional gel electrophoresis: antibodies to the anodic band were induced by Map-203 infection, whilst the cathodic band was revealed by heteroclitic antibodies from Maa-B2-infected mice. The latter antigen is apparently expressed during in vivo replication, but not during in vitro culture of Maa-B2 bacteria. We generally conclude, that the selective antibody patterns after live infection, could be attributed to differences in the release of native antigens within mycobacterial lesions. In view of a high degree of species specificity, some of the immunogenic constituents identified may also be useful for serodiagnostic application.

M. tuberculosis-complex specific T-cell stimulation and DTH reactions induced with a peptide from the 38-kDa protein.

An immunodominant T-cell-stimulatory epitope located near the carboxy terminus of the 38-kDa antigen from M. tuberculosis (38.G, residues 350-369) was found to be M. tuberculosis-complex specific. This was demonstrated by the presence of proliferative and delayed type hypersensitivity (DTH) responses in mice immunized with Mycobacterium tuberculosis and Mycobacterium bovis BCG, whereas mice immunized with M. avium or other non-tuberculous species of mycobacteria showed no such responses. Peptide 38.G stimulated the proliferation of peripheral blood lymphocytes from healthy purified protein derivative (PPD)-positive individuals but not from PPD-negative individuals. It also elicited DTH responses in M. tuberculosis sensitized mice and in PPD-positive healthy human volunteers. Peptide 38.G could therefore prove to be an important component in any new molecularly defined reagent used in the immunodiagnosis of tuberculous infection.

Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy.

Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes.

Schistosoma mansoni: evidence that 'non-permissiveness' in 129/Ola mice involves worm relocation and attrition in the lungs.

129/Ola mice resemble WEHI 129J mice in that around 70% of the individuals in any given population resist a primary infection with Schistosoma mansoni. Squashed-organ autoradiographic tracking of [75Se]selenomethionine-labelled parasites has shown that the kinetics of worm migration in 129/Ola mice follows the expected pattern, and that all rodents harbour essentially similar numbers of worms on day 14 post-infection. Combined lung and liver worm recovery techniques have revealed, however, that segregation of mice into 'permissive' and 'non-permissive' individuals can first be detected on day 20. 'Non-permissive' mice are characterized by the absence of schistosome eggs at all times in the liver parenchyma and, in consequence, lack the attendant manifestations of pathology; they do, however, harbour a few stunted worms in the liver and significant numbers of adult schistosomes in the pulmonary vasculature. Histological analysis of sectioned lung tissue from such animals indicates that some lung-located schistosomes feed, pair and lay eggs. Nevertheless, eosinophil-enriched inflammatory reactions develop around such worms and the parasites themselves exhibit various manifestations of trauma, ranging from minor vacuolation to gut herniation and extrusion. The phenomenon of 'non-permissiveness' thus involves retardation of worm development in the liver and, in consequence, relocation of the parasites to the lungs, where they become subject to host effector responses.

Schistosoma mansoni: evidence that vascular abnormalities correlate with the 'non-permissive' trait in 129/Ola mice.

The pulmonary and portal vasculature of naïve mice of the 129/Ola and CBA/Ca strains has been studied by means of the vasculature casting technique. This involve injection of pigmented vinylite resin into the arterial and venous systems, followed by digestion of the tissues with KOH. The peripheral vessels of the arterial and portal systems of CBA/Ca mice were numerous and highly branched. In contrast, casts prepared from 70-80% of naïve 129/Ola mice showed dramatic reductions in the number and extent of the peripheral vessels. In addition, such vessels appeared severely truncated. The remaining 20-30% of naïve 129/Ola mice yielded lung and liver casts that were indistinguishable from the CBA/Ca casts. Casts prepared from 129/Ola mice infected 6 weeks previously with Schistosoma mansoni cercariae showed the same segregation; faecal smears, together with observations of presence or absence of gross pathology in such mice confirmed that the vascular changes correlated with the 'non-permissive trait'. We propose that such alterations facilitate the reportedly abnormal migration of schistosomes from the liver to the lungs in 'non-permissive' 129/Ola mice.

Effect of praziquantel on the migration and survival of developmental stages of Schistosoma mansoni in mice.

Compressed organ autoradiography has been used to determine whether the anthelmintic drug praziquantel (Pzq) modifies the migration of isotopically labelled Schistosoma mansoni during the first 16 days of infection in CBA/Ca mice. The mice were treated with 500 mg kg-1 body weight of the drug on day 1 or day 6. Treatment caused a marked delay in parasite migration from the skin when the drug was administered intradermally at the site of infection on day 1; migration from the lungs was also delayed after such treatment. Pzq injected either intradermally on day 1 or intramuscularly on day 6 effectively reduced the number of parasites that finally arrived in the lungs and the livers by 41 and 47%, respectively. Intramuscular administration of the drug on day 1 had a negligible effect. Worm recoveries assessed on day 38 by perfusion of the hepatic portal system were greatly reduced when Pzq was administered on day 14. The worms proved less susceptible when the drug was administered on day 21 and were completely resistant following drug delivery on day 28. The influence of drug preparation and route of delivery on parasite migration and survival are discussed.

Schistosoma mansoni: influence of immunization and challenge schedules on the sites and mechanisms of resistance in CBA/Ca mice vaccinated with highly irradiated cercariae.

These studies address current controversies over the site(s) of challenge attrition in the murine irradiated vaccine model of immunity to Schistosoma mansoni. Two possibilities have been investigated. Firstly, that the site of death of the radiation-attenuated schistosomes used to vaccinate the mice may vary in different laboratories and secondly, that the skin sites selected for presentation of the immunizing and challenge parasites may influence the final site at which immunity is effected (i.e. ear/abdomen vs tail/tail). The migration of radiolabelled cercariae exposed to 0, 20 or 50 krad of gamma irradiation from the NIMR 60Cobalt source was examined in CBA/Ca mice by squashed organ autoradiography. Unirradiated parasites all migrated from the skin to the lungs, and 65% moved on to the liver. Migration of parasites attenuated by exposure to 20 krad of gamma irradiation was delayed, but 76% finally reached the lungs; only 1% was recruited to the liver. The majority of 50 krad attenuated parasites died in the skin, with only 4% accomplishing migration to the pulmonary vasculature. The major site of death (and by implication of antigenic stimulation) of the 20 krad attenuated NIMR strain S. mansoni used routinely for vaccination purposes in our laboratory, is thus the lungs, a finding that does not explain the fact that immunity is mediated primarily in the skin in our model system. Site elimination experiments and squashed organ autoradiography showed conclusively that, irrespective of the skin sites chosen for presentation of the immunizing and challenge population of worms, NIMR challenge parasites are killed predominantly in the skin of vaccinated CBA/Ca mice. Moreover, qualitative and quantitative histological examination of the challenged tail skin of vaccinated mice revealed that inflammatory reactions comprising mononuclear cells and eosinophils develop in this site and function to trap and eliminate challenge larvae, despite a reported reduction in antigen presenting cells in this region.