Detection of Mycobacterium tuberculosis growth by direct microscopic observation has a lower cost, higher sensitivity and higher diagnostic value than liquid and solid media cultures.

BACKGROUND: Culture is needed to confirm tuberculosis but results are generally obtained after several weeks. OBJECTIVES: We compared a direct microscopic observation technique for detection of mycobacterial culture positivity (MODS) with the classic solid and MB/BacT cultures in terms of sensitivity, contamination rate, speed and cost on 488 samples. RESULTS: The sensitivity of the MODS technique--99,2% (162 positive samples) was higher than MB/BacT 78,4% (125 positive samples) and solid culture 69,6% (113 positive samples) P<0.005 for all comparisons. The median times to positivity were 21, 13.3 and 3 days on solid media, B/BacT and MODS respectively. CONCLUSIONS: The MODS technique is faster and more sensitive than both solid media and MB/BacT culture.

[Assessment of Tc-99m MIBI uptake mechanism in Mycobacterium tuberculosis and normal cells (fibroblasts and myocytes) cultures]. / Studiul mecanismului de captare a Tc-99m MIBI pe culturi de Mycobacterium tuberculosis si de celule normale (fibroblaste si miocite).

One of the factors which can explain the scintigraphic Tc-99m MIBI positives images in pulmonary tuberculosis could be the Mycobacterium tuberculosis radiotracer uptake; this can be investigated in vitro, on cell culture, in comparison with normal cell types known to have high (myocites) or low (fibroblasts) uptake. Mycobacterium tuberculosis cultures were realised on Löwenstein Jensen medium, by standard methodology. Myocites and fibroblasts cultures were realized from neonatal rat hearts. Monolayer cells culture were incubated with a same 1.85 kBq/?l Tc-99m MIBI concentration, at 37 degrees C for 15, 60 and 90 minutes. The kinetic was stopped by rapidly washing the cells, with a 4 degrees C physiological saline solution and than scrapped cells were counted for the uptaken radioactivity. The results show that radiotracer cellular uptake (reported at the protein concentration) in myocites was maximum at 60 minutes. Cellular uptake in fibroblasts was very low at all the intervals. In Mycobacterium tuberculosis a peak was observed after 15 minutes, the uptake being similar to that of 60 minutes incubated myocites, considered 100%. After 90 minutes the Mycobacterium tuberculosis uptake was smaller than the 15 minutes value (65.82%). In conclusion, Mycobacterium tuberculosis in vitro uptake results could explain the more positives scintigraphic images in BK positive patients obtain at 15 minutes, in comparison with the delayed images.

Gas chromatographic detection of Mycobacterium tuberculosis complex in pure cultures from respiratory specimens.

It is important to identify mycobacteria to the species level in order to establish their clinical significance and to take the appropriate therapeutical decision. Biochemical tests on primary cultures take time (3-6 weeks) until the report of results; that's why more rapid techniques are needed. We have used gas chromatography with flame-ionisation detection (GC-FID) as an alternative identification method for 53 mycobacterial strains isolated from respiratory specimens. We have extracted fatty acids from whole mycobacterial cells, then derivatized them into methylesters, detectable by GC-FID. All the strains were identified as M. tuberculosis complex (MTC), using the Microbial Identification System (MIS) software. The specificity of the identification by GC-FID of MTC is 100%. In conclusion, pulmonary mycobacteriosis are dominated by MTC; GC-FID is a rapid and accurate method for the identification of MTC.

[Empyema caused by Corynebacterium striatum]. / Empiem determinat de Corynebacterium Striatum.

We present the case of a patient, male aged 21 years, admitted in our hospital for an empyema. A strain of C. striatum has been isolated from the pus sampled through the drainage tube. The isolate was identified on Api Coryne gallery (bio Mérieux S.A., France) (identity 97.1%). The disk sensitivity test showed sensitivity to penicillin, cefotaxime, amoxicillin-clavulanate and resistance to rifamycin, kanamycin, pefloxacin, ofloxacin, chloramphenicol, erythromycin and lincomycin. The arguments involving the isolated strain as the aetiological agent were: 1. The bacterium was isolated in pure culture and in significant quantity and 2. The existence of a favourizing clinical condition for an infection caused by a normal resident of the skin.

[The postantibiotic effect of azithromycin on respiratory pathogens]. / Efectul postantibiotic al azitromicinei asupra patogenilor respiratori.

We assessed the post-antibiotic effect (PAE) of azithromycin against 3 strains of Streptococcus pneumoniae 2 strains of Haemophilus influenzae and 2 strains of Moraxella catarrhalis. The strains were exposed for 2 hours to a concentration of 0.5 mg/l. A stationary phase inoculum of 1 x 10(6)-5 x 10(6) CFU/ml in IsoSensitest Broth with 5% lysed horse blood and 20 mg/l NAD was used and shaken for the duration of the experiment. Antibiotic was neutralised by dilution 1:1000 into pre-warmed medium. [table: see text] In conclusion, even at such low concentration as achieved in serum, azithromycin has a PAE against the respiratory pathogens studied. In our opinion this could allow the use of azithromycin, in the usual regimen even in bacteremic respiratory infections.

[The postantibiotic effect of azithromycin on respiratory pathogens]. / Efectul postantibiotic al azithromycinei asupra patogenilor respiratori.

Azithromycin has in vitro activity which includes important respiratory pathogens and is successful in treatment of respiratory tract infections. We assessed postantibiotic effect (PAE) of azithromycin against 3 stains of Streptococcus pneumoniae, 2 strains of Haemophilus influenzae and 2 strains of Moraxella catarrhalis. The strains were exposed for 2 hours to an azithromycin concentration of 0.5 mg/L (maximum serum concentration achieved by azithromycin after the usual dosing regimen). A stationary phase inoculum of 1 x 10(6)-5 x 10(6) UFC/ml in IsoSensitest Broth with 5% lysed horse blood and 20 mg/L NAD was used and shaken for the duration of the experiment. Antibiotic was neutralised by dilution 1:1000 into pre-warmed medium. Viable counts were determined before and after antibiotic exposure and then hourly for 7 hours by Miles and Misra method. The experiment was performed in triplicate. Even at such low concentration as that achieved in serum, azithromycin exhibits a PAE of 119 min for pneumococci, 130 min for haemophili and 155 min for moraxellae, fact which could allow the use of usual oral regimen in bacteraemic respiratory infection, as well.

[HIV infection in a seronegative child diagnosed by isolation of the virus. The significant immunological and epidemiological signs]. / Infectie cu HIV la un copil seronegativ diagnosticata prin izolarea virusului. Semnificatii imunologice si epidemiologice.

HIV-1, subtype F was isolated from a seronegative child aged 2.5 yr. ELISA tests (Behring HIV-1 + 2, Abbott HIV-1 + 2, Wellcozyme HIV-1 Recombinant, Clonatec HIV-1 + 2, Genelavia Mixt), and also rapid tests (Abbott Pack, Serodia) were all negative, although some of them presented borderline reactivities. Western Blot (Cambridge Biotech) revealed an undetermined profile (traces of anti-gp160 plus anti-p24). A new WB test (Sanofi Dg. Pasteur) performed at a higher serum concentration (1/25) revealed a complete antibody profile, despite the very low intensity of bands. A new serum sample prelevated 6 month later was completely negative on all tests used. Both samples, were negatives in WB for HIV2 and HTLVs. A heparinised blood sample was used for the co-cultivation of PBMC and was proven to be positive in the 14th day of culture. The isolated DNA from end-culture cells was subjected to PCR amplifications for Heteroduplex Mobility Assay direct subtyping (primers ES7 and ES8) and for the investigation of genotypic sensitivity to AZT (primers A/NE1). Lymphocyte populations phenotyping revealed leukocytosis (> 15,000/mL) with a predominance of the CD8+ subset CD4/CD8 ratio was < 1. Plasmatic HIV-1 load (measured by bDNA--Chiron) did not reached detectable levels of HIV-1 RNA. p24 Ag assay (EIA-Coulter) revealed a detectable p24 antigenemia only in the first serum sample and only after acid dissociation. So, this patient may present an integrated HIV-1 infection until now "silent".

[The role of C-reactive protein (CRP) determination in the early diagnosis of infections with opportunistic microorganisms in HIV-infected children]. / Rolul dozarii proteinei C-reactive (CRP) în depistarea precoce a suprainfectiilor cu microorganisme conditionat patogene la copii HIV-infectati.

The levels of C-reactive protein (CRP) in sera of 71 HIV-seropositive children and of 71 apparently healthy children were determined by Mancini method. The results demonstrate that HIV-infection per se doesn't increase the concentration of CRP in serum. After this we wanted to determine the relationship between CRP and evolution of HIV-infection. For this we used a set of 8 children in different stages of HIV-infection. For each child we had at least 2 sera, used for diagnosis and CRP assay. Three children (one with AIDS [correction of SIDA] and 2 in intermediate stage) had elevated levels of CRP. The reason for these elevations were an acute salmonellosis, a febrile episode of unknown origin and for the last child, once a staphylococcal infection of the skin and once an acute bronchiolitis clinically but not microbiologically documented. In conclusion, HIV-infection per se doesn't induce increased levels of the CRP, in any stage; this protein could be used as a marker of bacterial, parasitic and cytomegalovirus infections.

[Pyogenic infections with different locations caused by Streptococcus anginosus alone or in association with anaerobic bacteria]. / Infectii piogene cu localizari diferite cauzate de Streptoccus anginosus, singur sau în asociatie cu bacterii anaerobe.

Isolation and identification of S. anginosus from pathologic products are possible to be realised in informed clinical laboratory, allowing so a better knowledge of these infections' incidences and on adequate treatment. The authors' report 11 cases of pyogenic infection caused by S. anginosus. Five out of these 11 cases evaluated as mixed infections, S. anginosus being associated with anaerobic bacteria. 10 children hospitalised in surgery Department of Children Hospital had infections with different localisations; necrotizing fasciitis, preknee cap abscess, generalised peritonitis, abscess postappendectomy, pleurisy and acute mediastinitis, knee arthritis, acute osteomyelitis of mandible and an infection of the fracture's focus in upper 1/3 of the thigh bone. For all these patients the favoring factor was represented by a traumatic or surgical lesion of the skin or diverse mucosa; oral, oesophageal, intestinal, allowing the access of the normal flora of these covers to normally sterile sites. The eleventh case was an adult with a lung abscess and pleurisy, as a complication of an aspiration pneumonia. The treatment of S. anginosus infections consisted especially in penicillin or ampicillin, associated with metronidazol when anaerobic bacteria were present.

[The determination of the prevalence of antibodies to the hepatitis C virus (anti-HCV) and of the serum levels of C-reactive protein in chronic dialysis patients]. / Determinarea prevalentei anticorpilor fata de virusul hepatitei C (anti-VHC) si a nivelelor serice ale proteinei C reactive la pacienti dializati cronic.

UNLABELLED: The chronic dialysis patients are subject at risk for the hepatitis C virus (HCV) infection. Among these subjects, the hemodialysis-induced and increased synthesis of IL6 which suggest an acute phase response. 58 chronic dialysis patients were tested for presence of anti-HCV antibodies; 45 (77.58%) presented this serological marker. None of the 15 nursing subjects tested presented anti-HCV. C reactive protein (CRP) levels were measured in 41 patients. Elevated levels were detected in 5 (12.19%) patients. Such a high prevalence of anti-HCV reveals the hemodialysis to be a risk factor for the HCV infection. CRP levels measured by Mancini method lack the sensitivity for a more accurate interpretation. CONCLUSION: 1. the high anti-HCV prevalence among hemodialysis patients emphasize the opportunity of blood testing also for serological markers in ante-transfusional screening. 2. the immunoturbidimetry method for CRP testing may be a better assay in order to establish significant correlations.

[The frequency of Streptococcus anginosus in the pharyngeal exudate from children and its differentiation from other beta-hemolytic streptococci]. / Frecventa Streptococcus anginosus în exsudatul faringian la copii si diferentierea de alti streptococi beta-hemolitici.

UNLABELLED: S. anginosus is a commensal of the oro-pharyngeal mucous membrane without any signification for the local pathology. Ignoring the existence of beta-haemolytic colonies of this species, the risk to report the presence of some beta-haemolytic streptococci that actually belong to the normal flora exists. The antigenic identifications of beta-haemolytic streptococci maintain the confusion either, S. anginosus being able to react with specific sera anti group G, C or A. In our study, two identification criteria out of those available demonstrated a high value: the small or very small colonies' size, especially in secondary cultures and the production of acetoin (Voges-Proskauer test). S. anginosus was isolated with a quite great frequency in pharyngeal exudate from children: 41 strains out of 90 strains of beta-haemolytic non-group A streptococci were S. anginosus. Antigenically they belonged, in numerical order to groups C, G, F or they were ungroupable. IN CONCLUSION: The microbiologist has to identify, but not to report the presence of S. anginosus in pharyngeal exudate, it being a normal component of the oro-pharyngeal flora. Doing so, a better evaluation of the clinical signification of the other beta haemolytic streptococci's non group A will be possible.

[The value of C-reactive protein for the differentiation of bacterial meningitis from viral meningitis]. / Valoarea proteinei C-reactive pentru diferentierea meningitelor bacteriene de meningitele virale.

In order to differentiate bacterial meningitis versus viral meningitis, we have comparatively tested the efficacy of the following tests: C-reactive protein (CRP), erythrocytes sedimentation rate (ESR), fever, level of glucose in cerebro-spinal fluid (CSF), glucose in CSF/glycemia ratio, number of white blood cells in peripheric blood, percentage of neutrophils in peripheric blood, level of proteins in CSF and number of nucleated cells in CSF for a group of 49 patients, both children and adults with central nervous system infection (37 patients with bacterial meningitis and 12 with viral meningitis) hospitalised between May 1993 and July 1994 in Clinical Hospital for Infectious Diseases in Iasi. The mean value of CRP in bacterial meningitis patients was 8.78 mg%, contrasting with the mean value of CRP = 1.92 mg% recorded in patients with viral meningitis. Ten out of 37 bacterial meningitis patients presented a CRP concentration < 1.85 mg%. All these 10 patients have already had an antibiotic treatment at the moment of the assay. One out of 12 cases of viral meningitis had a value of CRP = 3.3 mg%, all the remainder cases having values under 1.85 mg%. We recorded highly significant differences between the two patient groups for CRP (p < 0.001), ESR (p < 0.01), protein concentration in CSF (p < 0.001) and number of nucleated cells in CSF (p < 0.001). Differences recorded for fever, concentration of glucose in CSF, glucose in CSF/glycemia ratio, number of leucocytes in peripheric blood and percentage of neutrophils in peripheric blood, were not significant (p > 0.5). Data were analysed also by box-plot method which facilitates the visual appraisal of the differences recorded between the two aetiological groups. In conclusion, assays of CRP and ESR may be used as differentiation tests for bacterial meningitis versus viral meningitis, when assay is done before the antibiotic treatment, being sufficient sensitive, and easy to perform.

[The isolation and subtyping of the human immunodeficiency virus (HIV) from children in Moldova. The evidence for subtype F]. / Izolarea si subtiparea virusului imunodeficientei umane (HIV) la copii din Moldova. Evidentierea subtipului F.

HIV epidemic related to nosocomial transmission in Romanian nursed children represents up to now more than 50% of paediatric AIDS cases in Europe. Although the sources of this epidemic are obscure, HIV-1 subtype F, a minor form in Brasil and Africa, realised a founder effect in this risk group, thus becoming the major form in Romanian epidemic. This study presents the results of the virus isolation from HIV-infected children in Northeast Romania. Heteroduplex mobility assay (HMA) was performed in order to establish HIV-1 viral subtype for 6 isolates. All tested HIV-1 strains were proved to belong to F subtype. So, our study confirm that HIV-1 subtype F is responsible for the epidemic in the risk group of Romanian nosocomially infected children.

Detection of beta-lactamase production and enzyme profiles in Enterobacteriaceae isolated in Moldova.

In our study, out of 449 Enterobacteriaceae strains isolated between 1985 and 1990, 16 strains (3 Proteus, 6 nontyphoidal Salmonella, 7 Escherichia coli) were resistant both to Ampicillin- Sulbactam and Amoxycillin-Clavulanic acid associations. The activity profiles of the beta-lactamases produced by these resistant strains are described. Sarcina lutea ATCC 9341 was used as test strain. The effect of the enzymatic filtrate against beta-lactam antibiotics: Ampicillin, Cloxacillin, Cefadroxil, Cefuroxime, Cefotaxime, Ceftriaxone, was followed up. The enzyme types were established according to the ability of inactivating the tested antibiotics. Penicillins and cephalosporins were inactivated by these enzymes, except for Carbenicillin and Oxacillin. These beta-lactamases were resistant to Sulbactam and Clavulanic acid. In the studied Salmonella strains they are plasmidic codified, demonstrating that they belong to a new beta-lactamase class.