Proteome and transcript analysis of Vitis vinifera cell cultures subjected to Botrytis cinerea infection.

Gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine resulting in significant reductions in yield and fruit quality. In order to examine the molecular mechanisms that characterize the interaction between B. cinerea and the host plant, the grapevine cytoplasmic proteome was analyzed by two-dimensional polyacrylamide gel electrophoresis. The interaction between Vitis vinifera cv. Gamay cells and B. cinerea was characterized by the increase in spot abundance of 30 proteins, of which 21 were successfully identified. The majority of these proteins were related to defence and stress responses and to cell wall modifications. Some of the modulated proteins have been previously found to be affected by other pathogens when they infect V. vinifera but interestingly, the proteins related to cell wall modification that were influenced by B. cinerea have not been shown to be modulated by any other pathogen studied to date. Transcript analysis using the quantitative real time polymerase chain reaction additionally revealed the up-regulation of several acidic, probably extracellular, chitinases. The results indicate that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are likely to be important mechanisms in the defence of grapevine against B. cinerea. BIOLOGICAL SIGNIFICANCE: Although gray mold caused by Botrytis cinerea is one of the most important diseases of grapevine, little information is available about proteomic changes in this pathosystem. These results suggest that cell wall strengthening, accumulation of PR proteins and excretion of lytic enzymes are important molecular mechanisms in the defence of grapevine against B. cinerea. Surprisingly, the proteins related to cell wall modification that were modulated by B. cinerea have not been shown to be affected by any other pathogen studied to date.

Mycobacterium marinum epididymoorchitis: case report and literature review.

Mycobacterium marinum is the most frequent non-tuberculous Mycobacterium in humans. We report the first ever described case of epididymoorchitis resulting from hematogenous spread of M. marinum from hand oligoarthritis. This was initially mistaken for rheumatoid disease and methylprednisolone-induced immunosuppression led to hematogenous spread of infection to the testis and epididymis.

Factors associated with multidrug-resistant tuberculosis: comparison of patients born inside and outside of the Czech Republic.

Multidrug-resistant tuberculosis (MDR-TB) is defined as resistance of Mycobacterium tuberculosis complex (MTB) to at least isoniazid and rifampicin. The aim of this study was to evaluate and compare a cohort of 50 patients with MDR-TB according to birthplace, resistance type, clinical outcome, length of bacteriological positivity of sputum and length of hospitalization. Thirty-three of the patients were born in the Czech Republic (group A) and 17 were immigrants to the Czech Republic (group B). Patients in group B were significantly younger (mean [range] age 33 [19 - 56] years) than those in group A (mean [range] age 48 [33 - 80] years). Primary resistance was present in 16 (48%) cases in group A and in 13 (76%) cases in group B. There were 36 (72%) cured patients, five (10%) remained on treatment and nine (18%) died; no patients failed or transferred out of the study. The mean length of bacteriological positivity of sputum samples was 5.9 months and the mean length of hospitalization was 8.2 months. Resistance to capreomycin was an important predictor of poor outcome.

[Laboratory diagnosis of mycobacterial infections]. / Laboratorní diagnostika mykobakteriálních infekcí.

Each year, tuberculosis claims nearly 2 million lives, especially in developing countries. Laboratory diagnosis of mycobacteria remains to be based on conventional approaches such as staining, culture and proportional methods for testing drug susceptibility. In recent years, new molecular methods have been proposed for mycobacterial diagnosis. Whereas the conventional methods for identification and detecting drug resistance are lengthy and cumbersome, the new assays are rapid, highly sensitive and specific and may be used for detecting resistance to antituberculosis drugs. The review describes the possibilities of laboratory diagnosis including rapid detection of mycobacteria directly from clinical specimens. For detecting latent tuberculosis, the determination of interferon gamma is important.

QuantiFERON-TB Gold in the diagnosis of active tuberculosis.

QuantiFERON-TB Gold (QFT) is an indirect diagnostic test for latent tuberculosis (TB) infection and active TB. This study aimed to evaluate and compare QFT and the tuberculin skin test (TST) for the diagnosis of active TB disease, not bacteriologically verified at the time of therapy initiation. Seventy-three patients with suspected active TB were examined. Further diagnostic steps confirmed the diagnosis of active TB in 53 persons. QFT was positive in 43 persons (81%), negative in seven (13%), and indeterminate results were found in three (6%). The sensitivity of the test was 86% in those with valid results, significantly higher than that for the TST (62%), and the correlation between the two tests was not high (55%). The QFT test is a useful addition to existing methods for the diagnosis of active TB.

Non-tuberculous mycobacteria: patterns of isolation. A multi-country retrospective survey.

OBJECTIVE: To collect data on non-tuberculous mycobacteria (NTM) isolated from clinical laboratories in different countries to establish: 1) whether the isolation of NTM was increasing, 2) which species were increasing, and 3) whether there was any pattern of geographical distribution. DESIGN: In 1996, the Working Group of the Bacteriology and Immunology Section of the International Union Against Tuberculosis and Lung Disease contacted 50 laboratories in different countries for the necessary information. RESULTS: The number of patients reported with NTM was 36099 from 14 countries. Mycobacterium avium complex, M. gordonae, M. xenopi, M. kansasii and M. fortuitum were the five species most frequently isolated. There was a significant upward trend for M. avium complex and M. xenopi. Pigmented mycobacteria predominated in Belgium, the Czech Republic and the Mediterranean coast of Spain. Non-chromogenic mycobacteria were found to be predominant in the area of the Atlantic coast of Brazil and in Turkey, the United Kingdom, Finland and Denmark. CONCLUSIONS: There was an increase in the number of NTM isolated from clinical samples of patients. Isolation of the most frequent species is constantly changing in most of the geographical areas, and newer species are emerging due to better diagnostic techniques to detect and identify NTM.

[Resistance of Mycobacterium tuberculosis to antituberculotic drugs in Czech Republic in 2003]. / Rezistence kmenu Mycobacterium tuberculosis na antituberkulotika v Ceské republice v roce 2003.

BACKGROUND: The results of Mycobacterium tuberculosis susceptibility testing in 2003 were analysed in 568 (84%) pulmonary TB definite cases in the Czech Republic. METHODS AND RESULTS: The resistance on antituberculotic drugs was detected in 4.9% of TB bacilli. Most frequently, resistance on one drug--isoniazid (1.8% of pulmonary TB definite cases) has been observed. Multidrug resistant TB was found in two cases in 2003. CONCLUSIONS: The resistance of Mycobacteria was found significantly more often in foreign-born persons with pulmonary TB (25% of strains of TB bacilli were resistant on one or more AT drugs). Resistance on AT drugs was more frequent also in previously treated TB cases, especially if the relapse occurred after 3,5 years after finishing previous treatment.

[Multiresistant tuberculosis in the Czech Republic (1999-2001)]. / Multirezistentní tuberkulóza v Ceské republice (1999-2001).

BACKGROUND: Multidrug-resistant (MDR) tuberculosis, defined as a disease caused by Mycobacterium tuberculosis strains, which are resistant to more antituberculous drugs (at least to isoniazid and rifampicin), is a problem frequently discussed in the Czech Republic. Cases of specific disease refractive to causal antituberculous therapy are associated with the risk of the spread of the causative agent among the population. METHODS AND RESULTS: The National Reference Laboratory for Mycobacteria collected 2813 Mycobacterium tuberculosis strains isolated by Czech mycobacteriological laboratories in 1999 to 2001. All strains were tested for susceptibility to basic antituberculous drugs and then the MDR strains were further tested for susceptibility/resistance to other antituberculous and antibacterial drugs. The MDR strains were studied by DNA analysis (DNA fingerprinting restriction analysis, RFLP-Restriction Fragment Length Polymorphism) as well. Thirty-nine patients who had MDR tuberculosis were excretors of 56 Mycobacterium tuberculosis strains. In average, MDR tuberculosis accounted for 1.96% (1.7-2.4) of all cases of bacillary tuberculosis. The most frequent type of the multidrug resistance was that resistant to four basic antituberculous drugs (isoniazid, rifampicin, ethambutol and streptomycin). It was confirmed in 48.2% multidrug resistant strains. CONCLUSIONS: Isepamicin, clofazimin, capreomycin and amikacin are considered to be the most promising antituberculosis drugs. Based on RFLP profiles, 61.5% of strains were placed into 8 clusters while the other strains remained unclustered. No significant differences in geographical distribution and population structure were found between the excretors of clustered strains and those of unclustered strains. Preliminary comparison with restriction profiles of the MDR Mycobacterium tuberculosis strains in the international database suggests the uniqueness of Czech strains showing the profiles not found elsewhere to date.

The first occurrence of a multi-drug resistant tuberculosis epidemic in the Czech Republic caused by genetically closely related Mycobacterium tuberculosis strains.

DNA fingerprinting based on the detection of the insertion sequence IS6110 in Pvull restriction fragments was applied to M. tuberculosis isolates originating in the first microepidemic of multidrug resistant tuberculosis recorded in the Czech Republic. Their disseminators were 21 individuals living in--or roaming between three distant areas. The age of 17 males ranged from 36 to 64 years (average 45 years) and of 4 females aged from 38 to 52 years. The index person was most probably a former male prisoner, aged 49 years, who disseminated multidrug resistant M. tuberculosis over a period of 28 months. In ten of the patients the following risk factors for tuberculosis were found: imprisonment, homelessness, immigration and previous stay in asylum--or in a psychiatric ward. In six cases, M. kansasii infection preceded tuberculosis. Four out of the 21 patients died. The RFLP analysis separated the patients into two distinct groups: group A comprising 14 members of which M. tuberculosis strains were isolated with six IS6110 copies, whereas the isolates of seven individuals of the group B, the RFLP profile displayed highly similar RFLP patterns compared to the isolates of group A, but with two additional IS6110 copies. In one patient, both A and B patterns were found: the first one in a M. tuberculosis strain isolated in 1993 and the second one in the isolate isolated two years later. Both the appearance of pattern B among the isolates of a part of patients and the switch from A to B pattern in one of patients can be plausibly explained by the unstability of DNA genotypes caused by transposition of IS6110 elements.

Present state of tuberculosis in the Czech Republic and in central European and Baltic countries.

The Central Europe forms a buffer zone between the countries of the European West reporting tuberculosis notification rates lower than 20 per 100,000, the cut-off set between low and high incidence areas, and the Eastern European countries including the republics of the former USSR, Russia and the Baltic States. The Czech Republic holds an intermediate place between these two territories with the total notification rate of tuberculosis cases 18.8, 9.7 bacteriologically verified and 5.7 positive in direct smear per 100,000 in 1996. Data on drug resistance obtained from the WHO/IUATLD Global Project on Anti-Tuberculosis Drug Resistance Surveillance were available from the Czech Republic, the only Central European country participating in the Project. The prevalence of resistant cases was here low: 2% primary and 13% acquired, and MDR cases were recorded in 1% of untreated and in 6% of repeatedly treated patients. The first microepidemic of MDR cases comprising 21 individuals was characterized by DNA fingerprinting. This outbreak pointed out the MDR tuberculosis as a new, extremely serious phenomenon in the epidemiology of tuberculosis. Corresponding data from Estonia and Latvia showed incomparably higher values in the drug resistance pattern: from 28 to 34% primary and 46 to 74% acquired resistance. MDR strains were reported in 9 to 14% of untreated and in 19 to 54% of repeatedly treated patients.

A multidrug-resistant tuberculosis microepidemic caused by genetically closely related Mycobacterium tuberculosis strains.

IS6110 DNA fingerprinting was used to characterize an outbreak of multidrug-resistant tuberculosis in 21 individuals (17 males and 4 females) living in or roaming among four distantly separated areas in the Czech Republic. The restriction fragment length polymorphism (RFLP) analysis separated the collected Mycobacterium tuberculosis strains into group A, including 14 patients with six IS6110 copies, and group B, with 7 patients displaying highly similar RFLP patterns but with two additional IS6110 bands. A switch from pattern A to pattern B was observed in one patient, and the subsequent detection of subclone B in seven more individuals has been explained by the instability of DNA genotypes caused by transposition of IS6110 elements.

[A micro-epidemic of tuberculosis in the South Moravia Region]. / Mikroepidemie tuberkulózy v Jihomoravském kraji.

In the South Moravian region which is the area with the lowest prevalence of bacillary tuberculosis in the Czech Republic (6.6 per 100,000 population in 1996) in 1993-1996 a total of six local and family microepidemics of tuberculosis were detected. For their identification the RFLP fingerprinting method was used based on evidence of repeated sequence of IS6110 in the chromosomal DNA of the examined strains of Mycobacterium tuberculosis predigested with restrictive enzyme PvuII. The profiles of fingerprints of each microepidemic were included by means of a computer programme into the hierarchy of the fingerprint dendrogram of 184 strains of M. tuberculosis which made it possible to identify possible identical profiles of strains from patients from remote places in the Czech Republic. In three family microepidemics involving always two members no identical fingerprint profiles of other Czech strains of M. tuberculosis were revealed. To the fourth cluster formed by six members of one family an identical RFLP profile of a female patient living in a nearby locality was added. In another microepidemic recorded in three brothers identical fingerprints were found another four patients from the South Moravian region and in one from the Central Bohemian region. The last cluster of two brothers was surprisingly enlarged by six identical RFLP profiles of patients from the West Bohemian region and one from Prague. These findings suggest that in areas with a low prevalence tuberculosis persists more frequently than in areas with a high prevalence as familial or local microepidemics.

[Substituted benzo[b]thiophenes with anti-leukotriene activity]. / Substituované benzo[b]thiofeny s antileukotrienovou aktivitou.

The cyclizing reaction of cinnamic acid (Ia) with thionyl chloride was optimized and a series of 3-chloro-6-subst.benzo[b]thiophene-2-carbonyl chlorides Va-Vn was prepared. Chloride Va was transformed into a series of N-aryl-3-subst. (Cl, OCH3, OH) benzo[b]thiophene-2-carboxamides VII, IX, X. The drugs were subjected to an evaluation of selected antileucotriene activities in vitro and of the anti-inflammatory effect in vivo. In agreement with the assumed mechanism, inhibition of the ear inflammation is conditioned by the antileucotriene activity, whereas inhibition of the carrageen oedema is not due to this mechanism alone.

Molecular epidemiology of tuberculosis in Prague: analysis by restriction fragment length polymorphism.

OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.

[Serodiagnostic detection of IGA-specific antibodies in tuberculosis]. / Sérodiagnostický prukaz specifických IgA-protilátek pri tuberkulóze.

The objective of the submitted paper is evaluation of the possible use of detection of serum antibodies class IgA against the mycobacterial antigen Kp-90 ImCRA in the serodiagnostics of tuberculosis. Antibodies were detected by means of a commercially available IgA set Enzymimmunoassay Kreatech (Kreatech Diagnostics, Amsterdam, Netherlands). By means of the mentioned diagnostic kit antibodies class IgA were assessed in 26 patients with respiratory TB positive on microscopic examination and cultivation, 35 patients with tuberculosis of the lungs or extrapulmonary TB confirmed on cultivation and in 48 subjects with various clinical diagnoses, where TB was not confirmed clinically nor by bacteriological examination. In 64.3% of the patients with TB positive microscopical and cultivation optic density values (OD) higher than 1.2 were recorded, in 57% of these patients OD was higher than 1.5. The reactivity of patients in the control group (subjects with non-specific respiratory diseases) was strikingly low and all subjects with the exception of two had OD values lower than 1.2. The sensitivity of the test was 46.0%, the specificity 95.8%. The assembled results confirmed the reactivity of the majority of TB patients to mycobacterial antigen Kp-90 in the class of IgA serum antibodies and a low seroreactivity of controls with non-tuberculous affections of the airways where only two cases of high titres from a total of 48 examinations were recorded. The evaluated test--IgA Immunoassay Kreatech--thus extends the possibilities to detect the immunoreactivity of the patient when assessing the diagnosis and during the further follow up of the tuberculous finding.

Bacillary tuberculosis in the Czech Republic: developmental trends in the 1981-1993 period.

Results of an automated information system on bacillary tuberculosis and mycobacterioses (ISBT) operating in the Czech Republic since 1981 nation-wide have been employed in this study. This system collects and processes data reported by all mycobacteriology laboratories in the country (34 in 1993) on each person disseminating pathogenic and/or opportunistic mycobacteria, on pathological materials examined in these patients and on methods applied for detection and identification of isolated mycobacteria Results of the 1981-1993 period were analyzed in this study. The annual incidence of bacillary tuberculosis cases identified by culture fell down from 2655 (25.8 per 100,000 popul.) in 1981 to 1139 (11.0 per 100,000 popul.) in 1993, i.e. by 57.4% in total, and by 4.8% in average annually. The decrease of annual mean values differed between two periods, from 1981 to 1985 and from 1986 to 1993, being 8.7% in the first and 3.7% in the successive period. The incidence of cases detected by direct microscopy of sputum showed a decline from 615 to 410 cases (5.97 to 3.97 per 100,000 popul.) in the 1981 to 1993 period, i.e. 2.8% annually. The analysis of the development and of the present state of the bacillary tuberculosis is instrumental in estimating the today's burden of the tuberculosis problem in the Czech Republic. Although distinct sings of worsened epidemiological parameters were not shown in this study, some disturbing findings can be considered as alerting: (a) a slowdown of the declining trend of bacillary tuberculosis cases detected by culture techniques seen in a few recent years, and (b) conserving potential tuberculosis pools in patients suffering from serious forms of the disease detectable by direct microscopy.

Application of the Gen-Probe amplified MTD test (Mycobacterium tuberculosis Direct Test) in the diagnostics of tuberculosis.

A commercially available set, the Gen-Probe amplified MTD test (Mycobacterium tuberculosis Direct Test; Gen-Probe Incorporated, 9080 Campus Point Drive, 92121 California, USA) has been applied for the detection of the M. tuberculosis complex in clinical material in parallel to direct microscopy and cultivation in liquid and solid growth media. The method is based on the amplification of a specific 16 S rRNA segment. Detection of the amplified segment is facilitated by single-strand probes tagged with acridinium ester. Hybridization results are interpreted in RLU (Relative Light Unit) values and readings exceeding 30,000 RLU are considered positive, lower values being negative. In all, there have examined 69 samples of clinical material (35 sputums, 29 bronchoalveolar washings or bronchial aspirations, and 5 cerebrospinal fluids). As positive amplification controls served freshly cultured strains of M. tuberculosis and M. bovis BCG; negative controls were M. gordonae, M. xenopi, M. kansasii, and M. terrae. Out of a total 69 samples examined, M. tuberculosis was detected in 28 (40.7%) cases by the Gen-Probe amplified MTD test, in 10 (14.5%) cases by direct microscopy, and in 26 (37.7%) cases by cultivation. In samples from 8 patients M. tuberculosis was detected by just Gen-Probe amplified MTD test the results of cultivation and direct microscopy being negative. Culture-positive samples yielded no Gen-Probe amplified MTD test negative results. The Gen-Probe amplified MTD test contributes significantly to the speeding up of diagnostics in tuberculosis because the results is obtainable with six hours. Under the conditions of this pilot study the results were termed as preliminary and collaborating physicians were advised to wait for cultivation results and their definite evaluation.

[Drug resistance in M. tuberculosis in Prague 1987-1992]. / Léková rezistence M. tuberculosis v Praze v letech 1987-1992.

In 1987-1992 in Prague drug resistance of M. tuberculosis to four standard antituberculotics (isoniazide, streptomycin, rifampicin, ethambutol) was recorded in 39 patients, i.e. in 1.5-5% of patients with bacillary tuberculosis. Initial resistance was found in 25, secondary resistance in 13 subjects, in one patient both types of resistance were observed. In both groups men and patients born before 1941 predominated. In patients with initial resistance findings rated as small or medium-sized predominated, while in secondary resistance half the findings were medium-sized or extensive; an extrapulmonary form was recorded in one female patient. In the group of patients with an initially resistant M. tuberculosis monoresistant strains predominated and the highest ratio was accounted for by strains resistant to isoniazide; in patients with secondary resistance strains with combined resistance to two or more drugs predominated, however in all instances a combination of isoniazide with other antituberculotics was involved. With regard to the changing epidemiological situation as regards tuberculosis (in particular the arrested decline of the incidence of bacillary tuberculosis and the increase of drug resistance of M. tuberculosis), the authors recommend further monitoring and epidemiological analysis of the incidence of strains with initial and secondary resistance, systematic external checks of laboratory technique used for testing the sensitivity to antituberculotics and its centralization as well as the introduction of analyses of restrictive fragments of the DNA genome (RFLP method) to monitor the spread of tuberculous mycobacteria in the population.

[External control of drug sensitivity determination in mycobacteriologic laboratories in the Czech Republic]. / Externí kontroly stanovení lékové citlivosti v mykobakteriologických laboratorích Ceské republiky.

In 1992 and 1993 an external control of sensitivity assessment of coded strains of M. tuberculosis to five basic antituberculotics was made: isoniazide, streptomycin, pyrazinamide, ethambutol and rifampicin. In 1992 from 11 participating laboratories an erroneous result was recorded in 9 (3 laboratories made two mistakes, 6 laboratories one mistake), two laboratories did not provide complete results. Unsatisfactory results were obtained during external controls in 1993. Of 15 participating laboratories four laboratories made one mistake, two laboratories two mistakes, four laboratories three mistakes. Five laboratories (i.e. one third) made more than three mistakes. Analysis of the results revealed a low reproductibility of results of drug sensitivity tests. To eliminate these shortcomings it will be necessary to make systematic external controls with subsequent solution of the shortcomings, restriction of the number of laboratories making these examinations to those who will systematically produce correct results. And it will be also necessary to test the sensitivity tests to antituberculotics in resistant strains in the National reference laboratory for mycobacteria.