Role of the alveolar macrophage in host defense and immunity to Legionella micdadei pneumonia in the guinea pig

Guinea pigs develop a lethal pneumonia after intratracheal infection with Legionella micdadei, and the lung displays pathological changes similar to those observed in humans. To investigate the role of the resident alveolar macrophage in the pathogenesis of L. micdadei pneumonia, guinea pig alveolar macrophages obtained by bronchoalveolar lavage were cultured in vitro and infected with L. micdadei. In the absence of opsonins L. micdadei was phagocytized by, and multiplied within, alveolar macrophages with greater than a 100-fold increase in cell-associated colony forming units over 20 h. L. micdadei opsonized with complement or antibody multiplied within alveolar macrophages at the same rate as unopsonized bacteria. Guinea pigs which were treated with antimicrobials after infection with L. micdadei and recovered from the pneumonia were immune to challenge with an otherwise lethal inoculum of L. micdadei. However, the growth curve of both unopsonized and opsonized L. micdadei in the alveolar macrophages from immune animals was essentially identical to that in macrophages from susceptible animals. Thus, the resident alveolar macrophage is not capable of limiting the growth of Legionella. Rather, the alveolar macrophages appear to be the primary site of Legionella multiplication within the lung. Although alveolar macrophages may participate in other aspects of pulmonary immunity to the legionellae, these data indicate that the alveolar macrophage alone does not act as an effector cell in cell-mediated immunity to Legionella.     

Evaluation of the MS-2 automated antimicrobial susceptibility testing system: Report of a European collaborative study

The MS-2 is an instrument for rapid automated testing of antimicrobial susceptibility. Its performance was evaluated by comparison with disc diffusion and MIC tests in a collaborative study in four European laboratories. Tests on independently isolated organisms showed the MS-2 to be in essential agreement with conventional methods in 94.8% of tests. A further series of 170 welldefined pathogens for which a reference antibiogram was available were tested by MS-2 and the manual methods in all of the participating laboratories. MS-2 results were in full accord in 90 % of tests and in essential agreement in 94 %. MS-2 results compared at least as well with the reference values as did either of the manual methods. Initial problems of false susceptibility results with erythromycin and penicillin were resolved by (1) the addition of small amounts of erythromycin which acted as an inducer and (2) by the use of a low content penicillin disc. MS-2 was found to be reliable and needed no attention following loading of the test cuvette cartridges. A print-out of the results was available 2–5 h after inception of the test.     

Evaluation of an enzyme immunoassay for detection of herpes simplex viras antigen in genital lesions

To evaluate a non-marketed research prototype of a solid-phase enzyme immunoassay for detection of herpes simplex virus in genital lesions, 154 clinical specimens were collected from 127 men and 27 women with symptoms suggestive of herpes simplex virus infection (erythema, vesicles, ulcers and crustae). The samples were tested using the assay and cultures on four monolayers of human embryonic lung fibroblasts and Vero cells. When the culture was used as reference method, sensitivity was 76.9% and specificity 100% (prevalence 42.4%). Comparison of results by patient group showed that sensitivity was highest in material from patients with vesicles and ulcers. The highest sensitivity was obtained in specimens which developed a cytopathological effect within 48 h and in specimens with three or four positive cell cultures. These findings suggest that the assay is more successful in specimens with high virus titres. The enzyme immunoassay was found to be a rapid, moderately sensitive, highly specific test for detection of herpes simplex virus from genital lesions, but the usefulness of the assay is limited and culture methods should be preferred.     

Suppression of human lymphocyte responses to specific and non-specific stimuli in human onchocerciasis.

Characterization of in vitro lymphocyte responsiveness was performed on selected groups of onchocerciasis patients from Sudan and Sierra Leone. These patients manifested a very broad range of clinical signs and showed widely divergent parasite infection intensities. Lymphocyte proliferative responses to soluble Onchocerca volvulus antigen (sAg) were poor in infected persons; mitogen and PPD responses were maintained in the normal range in one group of patients from southwestern Sudan, but were profoundly depressed in a group from N.E. Sudan. Proliferative responses and interferon-gamma (INF-gamma) secretion were very significantly depressed in the presence of live microfilariae of O. volvulus or secretions/excretions (S/E) from microfilariae (mf) or from female, but not male, adult parasites. Lymphocyte responses were maintained near normal when exogenous IL-2 was added to these cultures. The results indicate that O. volvulus infection and its clinical consequences are not consistently associated with systemic deficits in immune responsiveness. However, suppression of lymphocyte reactivity by mf and S/E in vitro suggests that direct parasite intervention in host cell responses could be taking place in vivo, perhaps at the local microenvironment level; mediated by effects on cytokine production.     

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels. In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL-2 or IL-12 family cytokines was detected. Bacteria-induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated. Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria. Although pathogenic S. pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Impairment of macrophage function by inhibitors of ornithine decarboxylase activity.

The effects of irreversible inhibition of ornithine decarboxylase on the capacity of murine macrophages to take up a protozoan organism (Trypanosoma cruzi) or inert particles were investigated. Incubation of macrophage cultures with four different ornithine decarboxylase inhibitors, namely, DL-alpha-difluoromethylornithine (DFMO, 0.5 to 20 mM), delta-methyl-acetylenic putrescine (1 to 5 mM), monofluoromethyldehydroornithine ethyl ester (1 to 5 mM), and monofluoromethyldehydroornithine methyl ester (1 to 5 mM), before the addition of the parasites significantly reduced the percentage of macrophages with parasites, indicating that some of the host cells were no longer capable of binding or ingesting the parasite. The average number of trypanosomes per 100 macrophages was also diminished, denoting a lesser phagocytic capacity as a consequence of the treatments. These effects were reversible within 2 h after removal of excess DFMO. No alteration in parasite-macrophage interaction was seen when the trypanosomes were treated with DFMO. That the effects of DFMO on the macrophages probably resulted from a reduction in polyamine levels caused by inhibition of ornithine decarboxylase was indicated by the fact that these effects were not seen when the macrophages were incubated with DFMO in the presence of putrescine, the product of ornithine decarboxylation by ornithine decarboxylase. DFMO treatment of macrophages also inhibited the capacity of these cells to ingest killed parasites or latex beads and thus appeared to generally affect phagocytosis. An effect of DFMO on the susceptibility of macrophages to penetration by the parasites seemed less likely because no significant alteration in cell-parasite association occurred when myoblasts--which, not being phagocytic, can be infected only by membrane penetration--were treated with DFMO. Taken together, these results emphasize a role of ornithine decarboxylase activity and polyamine biosynthesis in macrophage function.     

Vulvovaginal candidiasis among symptomatic women of childbearing age attended at a Medical Analysis Laboratory in Franceville,Gabon

BackgroundVulvovaginal candidiasis (VVC) is one of the most common lower genital tract infections in women; this unpleasant and extremely embarrassing pathology is one of the main reasons for gynaecological consultation. In Gabon, the prevalence of VVC remains poorly described even though VVC is known to be the leading gynaecological condition in several countries. This retrospective cross-sectional study sought to assess the prevalence of VVC among symptomatic women in southeastern Gabon.MethodsClinical samples were collected from patients suspected to have VVC during a 2-year period (from January 2016 to December 2017). Gram staining of vaginal smears provided indications of vaginal flora and confirmed the presence of yeast. Sabouraud-chloramphenicol and chromID Candida media were used to isolate yeast, and species identification was performed using morphological tests and the Vitek 2 Compact automated system.ResultsFor the 873 patients included in this study, the prevalence of VVC was 28.52%. Eleven Candida species were identified, with greater representation of Candida albicans (82.73%) than of Non C. albicans candida (NCAC) (17.27%), which were distributed as follows: Candida famata (4.02%), Candida spp. (3.61%), Candida rugosa (3.21%), Candida lipolytica (1.61%), Candida parapsilosis (1.61%), Candida glabrata (1.21%), Candida tropicalis (0.80%), Candida krusei (0.40%), Candida dubliniensis (0.40%), and Candida sphaerica (0.40%).ConclusionThis study offers the first estimation of VVC among Gabonese women in childbearing age with the symptoms. It showed that VVC is very common in Gabon. C. albicans as the most commonly represented species.