A blind study of the polymerase chain reaction for the detection of Mycobacterium tuberculosis DNA. Azay Mycobacteria Study Group.

SETTING: Nine French laboratories routinely involved in mycobacterial work. OBJECTIVE: To assess the detection of Mycobacterium tuberculosis in experimental samples by polymerase chain reaction (PCR) using the insertion sequence IS6110 as a target for deoxyribonucleic acid (DNA) amplification. DESIGN: Nine laboratories participated in a blind study of the detection of M. tuberculosis by PCR in 20 coded samples containing either a definite number of M. tuberculosis complex (positive samples) or environmental mycobacteria (four samples) or no mycobacteria (five samples). RESULTS: Five laboratories reported false-positive PCR results, with an average rate of 7%. All laboratories except one reported positive PCR results for samples containing 10(5) cfu/ml or more. M. tuberculosis DNA was detected in two thirds of samples containing 10(4) and 10(3) cfu/ml, and in one third of the samples containing 10(2) cfu/ml. CONCLUSION: The results of the study suggest that PCR using IS6110 as a target for DNA amplication is neither very sensitive nor really specific for the detection of M. tuberculosis.

Diagnosis of tuberculosis by Amplicor Mycobacterium tuberculosis test: a multicenter study.

The Amplicor Mycobacterium tuberculosis test is a new PCR assay for the direct detection of Mycobacterium tuberculosis from clinical samples. A multicenter study that included six laboratories was done to evaluate the Amplicor test in comparison with direct microscopy and culture (solid or radiometric media), and the culture method was used as the "gold standard." A total of 2,073 specimens, i.e., 1,749 respiratory specimens and 324 other specimens, were tested. A total of 184 cultures yielded M. tuberculosis. Of these 184 cultures, 77 (42%) were smear negative and 23 (12.5%) concerned extrapulmonary specimens. The sensitivity of the Amplicor test for all of the specimens and for extrapulmonary, smear-positive, and smear-negative specimens was 86, 83, 94.5, and 74%, respectively. The sensitivity of direct microscopy in comparison with that of culture was 58%. A total of 95% of patients with culture-proven tuberculosis were diagnosed by the Amplicor test, whereas direct microscopy detected mycobacteria in only 72% of these patients. The Amplicor test exhibited a high degree of specificity (98%). The assay was very rapid and easy to perform.

[Use of the Bactec TB 460 method for the bacteriological diagnosis of tuberculosis. Results of a multicenter study]. / Utilisation de la méthode Bactec TB460 pour le diagnostic bactériologique de la tuberculose. Résultats d'une étude multicentrique.

Isolation of Mycobacteria on Loewenstein-Jensen medium lasts many weeks. The use of Radiometric method (Bactec TB 460) reduces the delays. Results of 79,064 cultures are reported from a multicentric study associating 16 laboratories. The average was 4.8% of positivity and 2.51% of contamination. The comparison of the results with conventional method previously obtained shows that radiometric method is more sensitive and contaminations are less numerous. Concerning hemocultures the Bactec method is very usefull. Among 11,277 tests performed 907 were positive (8.04%). Mycobacterium avium was identified in 89% of the cases. Identification test utilizes Biochemical and NAP tests, but also more and more Nucleic probes. The antibiotic sensitivity is performed in five days. The mean delay of analysis is about 25 days, lessening by half the conventional method delays. Nevertheless, Bactec method has the following inconveniences: syringe inoculation, use of radiolabelled products, expensive cost.

French multicenter study involving eight test sites for radiometric determination of activities of 10 antimicrobial agents against Mycobacterium avium complex.

The radiometric BACTEC 460-TB methodology has filled an increased need in the screening of a wide range of antimicrobial agents against Mycobacterium avium (MAC) isolates on a patient-to-patient basis. In this context, a multicenter study involving eight test sites across France was performed to determine the MICs of 10 antimicrobial agents for MAC organisms. The aim of the investigation was to compare the in vitro activities of D-cycloserine, ethambutol, ethionamide, rifampin, amikacin, streptomycin, ciprofloxacin, sparfloxacin, clofazimine, and clarithromycin against MAC isolates. All of the test sites received the same clinical isolates of MAC, and the MICs were determined by a common protocol. The overall interlaboratory reproducibility of the MICs within +/- 1 dilution of the modal MICs varied from 79.70 to 100% (mean, 95.2% +/- 2.1%), whereas overall agreement of the MICs among the test sites varied from a mean of 91% +/- 4.1% to a mean of 98 +/- 1.3%. We confirmed that the proposed methodology is easy, accurate, and sufficiently reproducible to be used routinely in a clinical laboratory. Despite variations in the MICs of the same drug among strains, no link between the origin of MAC isolates (from human immunodeficiency virus-positive or -negative patients) and their drug susceptibilities was established. On the basis of the MICs that inhibited 50 and 90% of isolates tested for the drugs used, clarithromycin, clofazimine, ethambutol, and streptomycin were the most uniformly active against MAC; this was followed by amikacin, rifampin, and sparfloxacin. On the other hand, ciprofloxacin, D-cycloserine, and ethionamide showed only marginal in vitro activities.

Rapid identification of Mycobacterium species.

Identification of Mycobacterium species is currently a long and fastidious procedure. We have developed a rapid (5-h) standard method using the API-ZYM system and rapid nitratase, urease and catalase tests. Pigmentation and growth rate were noted (but were only necessary for complete identification of 6% of strains). The tests were assigned numerical values from which a profile number was derived. A total of 716 strains were studied: 434 belonging to the M. tuberculosis complex and 282 other mycobacteria including 21 from M. avium complex. All M. tuberculosis complex strains were differentiated from all other mycobacteria and M. bovis was clearly separated from M. tuberculosis. All M. avium complex strains were differentiated from other mycobacteria. Among mycobacteria other than tubercle bacilli, 97% of the strains studied were identified. The method has proven to be simple, rapid and standardizable. It is suggested that the use of a code list could permit identification of most mycobacteria.

[Bacteriological aspects of 64 cases of severe pneumonia seen in a respiratory intensive care unit]. / Aspects bactériologiques de 64 pneumopathies graves observées en réanimation respiratoire.

We report on the bacterial aspects of 64 cases of severe pneumonia in an intensive care over a two year period, excluding cases occurring during artificial ventilation. In all the cases, the grave respiratory and haemodynamic signs, the blood gas and radiological findings justified admission of these patients to an intensive care unit. Specimens for bacteriology, virology and parasitology enabled a precise microbiological diagnosis 43 times (63%); in 44% of these diagnosis the possibility of the protected specimens (trans-tracheal, protected brushing) were confirmed by another specimen (pleural or blood). The germs identified were: Streptococcus pneumoniae (13), Staphylococcus aureus (9), Haemophilus influenzae (6), other gram negatives (12), Mycoplasma pneumoniae (1), Koch's bacillus (1), Aspergillus fumigatus, Cytomegalovirus, Myxovirus, Pneumocystis carinii (5). Twenty eight patients died of whom eight had marrow failure following chemotherapy; amongst the non-leukopenic patients an analysis of the records allowed certain presumptions as to the causative organism according to the patients mode of referral and immune state. The prognosis of these pneumonias remains serious in spite of improved bacteriological diagnosis, above all in the elderly, poorly nourished or with marrow aplasia.

[Disseminated invasive aspergillosis associated with Mycoplasma pneumoniae infection]. / Aspergillose invasive disséminée associée à une infection à Mycoplasma pneumoniae.

Disseminated aspergillosis is rare in patients not suffering from haematological disorders and myelo-suppression. We report a case of pulmonary cardiac and renal aspergillosis diagnosed at autopsy in a patient with chronic respiratory failure dying in the intensive care unit during an acute exacerbation. The role of risk factors associated with invasive aspergillosis in patients not suffering from myelosuppression is discussed (steroid therapy, chronic respiratory disease, concomitant viral or bacterial infection). The discovery in this patient of a pulmonary infection associated with mycoplasma pneumonia (the micro-organisms were found in necropsy specimens of pulmonary tissue) does not seem to have been reported before in the literature.

[Bactericidal effect of piperacillin alone and combined]. / Activité bactéricide de la pipéracilline seule et associée.

The bactericidal activity of piperacillin was evaluated by the kill rate kinetics method. Piperacillin compared with amoxicillin, amdinocillin, mezlocillin, cefotaxime, ceftazidime and latamoxef. Against E. coli, piperacillin and antibiotics electively bound to penicillin-binding protein (PBP) 3 had the same, mainly time-dependent, bactericidal activity. Antibiotics bound to PBP 1 and 2 mainly have a dose-dependent activity. The bactericidal activity of piperacillin against E. coli was enhanced when the drug was combined with amoxicillin and amdinocillin; in contrast, the kill rate remained unmodified when piperacillin was combined with latamoxef. When the piperacillin-amikacin combination was tested against Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli and Serratia marcescens, early synergism (5th hour), was weak, but after 24 hours piperacillin reduced the regrowth observed with the aminoglycoside, and synergism was much more pronounced irrespective of the species investigated.

[Contribution of rapid methods to the diagnosis of Haemophilus influenzae infections and the conduct of antibacterial treatment]. / Apport des méthodes rapides dans le diagnostic des infections à Haemophilus influenzae et la conduite du traitement antibactérien.

In the last decade medical microbiologists could make use of a great number of techniques that allow to shorten length of time of report from the arrival of specimens in laboratory. The direct microscopical examination has been considerably revalued: it is very important. Now it can be completed by use of reagents that allow the presumptive identification of various bacteria. For Hemophilus influenzae the efforts are devoted only to serovar b. In the absence of visible bacteria, the research of the presence of bacterial antigen is now possible. The semiautomatic apparatus allows also to watch over the cultures and to detect the beginning of development more accurately than macroscopically. Finally, for antibiotic sensitivity test, we can also use the automatic apparatus that shortens length of time of report to clinician.

[Clinical aspects of 27 cases of severe primary infectious lung diseases]. / Aspects cliniques de 27 cas de pneumopathies infectieuses graves primitives.

The authors report 27 cases of severe primary pulmonary infections which resulted in acute respiratory failure and which were treated in a Respiratory Medicine Intensive Care Unit. A bacteriological specimen (blood culture, transtracheal aspiration, fibroscopy, pleural tap) and/or a serological examination permitted the responsible agent to be identified in 20 of the 27 cases (74%). Apart from antibiotics and symptomatic treatments, 13 patients also received oxygen therapy and 14 other patients were intubated and ventilated. In the first group, 3 patients died (24%) and in the second group, the mortality was much higher, with 9 deaths out of 14 patients (64%). The authors present the clinical features, the frequency of the various micro-organisms isolated, the therapeutic modalities and various prognostic factors. Although repeated early specimens usually provide bacteriological diagnosis and therefore appropriate antibiotic therapy, the prognosis of these conditions, at the stage of acute respiratory failure remains serious and is probably related to risk factors linked with predisposition. The exact nature and the relative importance of these risk factors still needs to be determined.

[Distribution of Streptococcus pneumoniae serotypes in Nantes Hospital (1969-1978) (author's transl)]. / Répartitions des serovars de Streptococcus pneumoniae au Centre Hospitalier Régional de Nantes de 1969 a 1978.

Between 1969 and 1978 at the CHR of Nantes we isolated Streptococcus pneumoniae from 815 swabs taken from 591 patients and which provided 595 different strains. Serotyping of all the strains was carried out using the capsular reaction test. The prevalence of the different serovars varied according to the year and the type of swab. No resistant strains to benzylpenicillin or ampicillin were observed. There was significant resistance to sulphonamides and tetracycline : 24,3 and 29,6% respectively. The American vaccine containing the greatest number of serovars was effective for only 69,8% of the strains isolated in Nantes.