Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.     

2-Hour cytomegalovirus pp65 antigenemia assay for rapid quantitation of cytomegalovirus in blood samples

Of 109 blood samples tested for cytomegalovirus (CMV) antigenemia, 18 (16.5%) were positive. CMV Brite detected 13 and CMV Brite Turbo detected 16 of the 18 positives. There was no significant difference in the number of positive cells detected per sample. The seven discrepant samples contained a median of only one positive cell.     

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

A multiplex PCR-immunoassay for the rapid diagnosis of bacterial meningitis from cerebrospinal fluid (CSF) or peripheral blood was compared with conventional microbiological techniques used in the routine diagnostic laboratory. The multiplex PCR was designed to detect simultaneously a universal conserved sequence coding for bacterial 16S rRNA and the Neisseria meningitidis porA gene. The PCR-immunoassay had a detection limit of (3-5) x 10(2) cfu/ml (equivalent to 1-3 cfu/PCR) with spiked CSF or blood samples, compared with (3-5) x 10(3) cfu/ml for PCR followed by conventional agarose gel electrophoresis for detection of PCR products. Of 294 CSF samples from patients suspected on clinical grounds of suffering from meningitis, the PCR-immunoassay detected bacterial DNA in 29 CSF samples, 15 of which were also positive for N. meningitidis DNA. The 29 positive CSF samples comprised 25 samples that were also reported positive and four that were reported negative by conventional methodology; the latter four were all positive for N. meningitidis by the PCR-immunoassay. Of 173 peripheral blood samples examined, the PCR-immunoassay detected bacterial DNA in 18 samples, 14 of which were also positive for N. meningitidis DNA. In comparison, only 10 samples were reported positive for N. meningitidis by conventional methodology. All negative PCR-immunoassay results correlated with those obtained by conventional methodology for both CSF and blood samples. The sensitivity and speed of the PCR-immunoassay system indicated that it could be used as a routine diagnostic test for meningococcal meningitis, enabling a diagnosis to be made within 4 h of receipt of the specimen.     

Evidence against the practicality and cost-effectiveness of a gram-positive coccal selective plate for routine urine cultures.

A total of 899 urine cultures were evaluated to assess the need for and cost-effectiveness of using a gram-positive coccal selective plate in the initial plating of urine cultures. Of these cultures, 437 were examined retrospectively and 462 were examined prospectively. Urines were quantitatively plated to three media: sheep blood sugar, MacConkey agar, and phenyl ethyl alcohol agar. Of all urine samples in both studies, 52% yielded no growth on any of the three media. Of all 899 urine cultures, there were only 5 cultures (less than 1%) in which a significant count of a gram-positive organism was detected only on the phenyl ethyl alcohol agar plate and not recoverable on the sheep blood agar plate. In each of these five instances, the need for the use of the selective plate occurred when a Proteus mirabilis strain swarmed over an enterococcus. The inclusion of a selective gram-positive coccal medium for initial plating of urine cultures is unnecessary and not cost-effective. When Proteus swarms on sheep blood agar, a sweep should be made with an inoculating loop from the sheep blood agar and streaked to phenyl ethyl alcohol agar or a similar gram-positive coccal selective medium.     

Blood alcohol levels in persons who died from accidents and suicide

Although it is known that alcohol is associated with a high proportion of fatal accidents and suicides, little information is available in Ireland on blood alcohol concentrations (BACs) of those who died. This study was undertaken to identity the (BACs) in persons who died as a result of suicide or injury. The study was a retrospective review of coroners' records to identify BACs in three counties in Ireland. All cases where the person died as a result of injury or suicide in 2001 and 2002 were included. There were 129 deaths eligible for inclusion. Of these, 98 (76%) were male, 55 (42.6%) were road traffic accidents (RTAs), 31 (24.0%) suicides, 12 (9.3%) substance misuse, 11 (8.5%) house fires and 20 (15.5%) others. Of the 55 who died as a result of RTAs, 22 (40%) had positive BACs ranging from 16mg/100 ml to 325 mg/100 ml. Of the 31 who died as a result of suicide, 28 (90.3%) were male. BACs were available for 29 (93.5%). Of these, 16 (55.5%) had alcohol detected. Persons aged less than 30 years were more likely to have alcohol in their blood (p < 0.002). The mean BAC for persons aged less than 30 was 191.5 mg/100 ml compared to 84.0 mg/100 ml for those aged 30 and over. The mean BAC for adults who died in house fires was 225.2 mg/100 ml. The high BACs in those who died as a result of suicide or injury reflect the high level of alcohol consumption and binge drinking in Ireland.     

Development of antigen detection assay for diagnosis of tuberculosis using sputum samples

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 10(4) bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 10(4) M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 microgram of LAM/ml. The detection limit in sputum pretreated with N-acetyl-L-cysteine and proteinase K was 10(4) M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis.     

Problems encountered in using Cefinase disks to predict in vitro penicillin susceptibility of coagulase-negative staphylococci

The value of Cefinase (BBL Microbiology Systems, Cockeysville, M.D.) disks in predicting penicillin responses of coagulase-negative staphylococci was studied with the use of clinical isolates. The beta-lactamase activity of each isolate was determined both before and after enzyme induction for 18-24 hours with a 5-micrograms methicillin disk. Penicillin responses were determined in microdilution minimum inhibitory concentration (MIC) panels after 18 hours of incubation at 35 degrees C. Of 485 isolates tested, 338 (70%) were beta-lactamase positive, 168 (35%) before and 170 (35%) after induction. Only two (0.6%) of these isolates had a penicillin MIC of less than or equal to 0.12 microgram/mL (less than or equal to 0.12 mg/L). Of the 147 Cefinase-negative isolates, penicillin MICs of 0.12 microgram/mL or less (less than or equal to 0.12 mg/L) and 0.25 microgram/mL or more (greater than or equal to 0.25 mg/L) were obtained from 110 (75%) and 37 (25%), respectively. Although Staphylococcus saprophyticus accounted for only 30 (6%) of the total isolates, 54% of the organisms that were beta-lactamase negative with penicillin MICs of greater than or equal to 0.25 microgram/mL (greater than or equal to 0.25 mg/L) belonged to that species. A positive Cefinase test accurately predicted elevated penicillin MICs, but a negative test, especially with S. saprophyticus, was of less value.     

Simultaneous supercritical fluid extraction and chemical derivatization for the gas chromatographic-isotope dilution mass spectrometric determination of amphetamine and methamphetamine in urine

An in-situ supercritical fluid extraction (SFE) and chemical derivatization (ChD) procedure followed by gas chromatography-isotope dilution mass spectrometry (GC-MS) for the determination of amphetamines in urine is described and evaluated. While using celite as the SFE wet-support, the one-pot sample pretreatment procedure also employs ammonium water to alkalize the urine matrix that contains protonated amphetamine (AP) and methamphetamine (MA). The mean recoveries achieved by simultaneous SFE-ChD, i.e., 95% (RSD=3.8%) for AP and 89% (RSD=4.0%) for MA, are significantly better than the corresponding overall recoveries obtained upon stepwise SFE-ChD, suggesting the unreacted trifluoroacetic anhydride (TFA) in the former procedure has strengthened the extracting power of CO, fluid as has been evidenced by a control test. As to GC-MS analysis, the optimal qualitative ions and quantitative ions of the respective analytes were determined via a rigorous evaluation process. Thus, the regression calibration curves for AP and MA in urine are linear within 100 approximately 50,000 ng/ml, with correlation coefficients typically exceeding 0.999. The limits of detection determined by two methods for AP and MA vary from 19 to 50 ng/ml, and limits of quantitation from 21 to 100 ng/ml. Precisions calculated for the triplicate analyses of AP and MA in a 500-ng/ml spiked control, two real-case samples and two quasi real-case samples, respectively, using regression calibration are typically below 10%. The method is simple and reliable. It may serve as an alternative to the existing confirmatory protocol for forensic urine drug testing.     

Gentamicin-blood agar for isolation of Streptococcus pneumoniae from respiratory secretions.

Previous studies have suggested that the yield of Streptococcus pneumoniae from respiratory secretions can be increased by using a 5% sheep blood agar plate supplemented with 5 microgram of gentamicin (GBA) per ml. We report our experience with 245 lower respiratory specimens in which this method was compared with 5% sheep blood agar (SBA) alone. Of 35 specimens with growth of S. pneumoniae on either plate, 21 were detected exclusively on SBA, whereas only 3 were detected on GBA alone (P less than 0.01). By subculturing representative alpha-hemolytic colonies from the final 169 specimens, the yield of S. pneumoniae was increased by 27% compared with the number of identifications that could be made directly from the primary culture. Minimal inhibitory concentrations of gentamicin for the last 25 isolates were greater than or equal to 8 microgram/ml. Our results do not substantiate the previous observations that S. pneumoniae from respiratory secretions gives an increased yield in cultures on GBA.     

Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium.

A selective medium was developed and used successfully to isolate Legionella pneumophila and Legionella-like organisms from environmental specimens previously positive by animal inoculation methods. This medium consists of charcoal-yeast extract agar to which have been added cephalothin (4 micrograms/ml), colistin (16 micrograms/ml), vancomycin (0.5 microgram/ml), and cycloheximide (80 micrograms/ml). Pretreating of the environmental water samples with an acid buffer (pH 2.2), followed by plating on the selective medium, improved the rate of recovery of both Legionella and Legionella-like organisms relative to that with direct plating on selective media.     

Rapid Semiautomated Screening and Processing of Urine Specimens

A rapid urine culture procedure was evaluated in which positive urines were detected by using light-scatter photometry (Autobac). Specimens were analyzed at 3, 5, and 6 h. Specimens detected as positive at 3 h were then further evaluated by a direct 3-h susceptibility procedure (Autobac) and by a 4-h identification procedure (Micro-ID). Of 949 specimens, 175 had >10⁵ colony-forming units per ml by colony count. Of these latter specimens, 75.4% had been detected by 3 h, and 95.4% were detected by 6 h. Of specimens positive by Autobac at 3 h, 96% (95.7%) had >10⁵ colony-forming units per ml. If pure by Gram stain, those positive specimens were inoculated to direct susceptibility and identification systems. When direct Autobac susceptibilities were compared with the standard Autobac method done from the plate the following day, discrepancy rates were 1.3% very major, 2.1% major, and 7.4% total. The direct identifications were 94% (94.2%) correct when using the Micro-ID manual and a collection of octal patterns unique to this system, in which urine/broth culture inoculum was employed instead of the usual organism colony suspension. Those urine specimens negative after screening at 3 h were evaluated at 5 and 6 h, and an additional 126 specimens were detected as positive. These were then processed by routine plate inoculation, due to the limitations of the work day. By 6 h, 95.4% of specimens with >10⁵ colony-forming units per ml were detected. The 4.6% false-negative results consisted of patients on antibiotics, or slowly growing bacteria suspected of being distal urethral contaminants. Thus, 83.5% of the urine cultures received by 9:00 a.m. (10.6% 3-h positives and 72.9% negative at 6 h) could be evaluated and reported within one 8-h work day.     

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin

A procedure was devised for routine examination of feces for Clostridium difficile with selective enrichment broth culture containing increased levels of carbohydrates and antibiotics to detect cytotoxin and volatile acids in broths inoculated with fecal samples. C. difficile was detected and identified with a rapidity comparable to that of conventional culture on selective cycloserine-cefoxitin fructose agar. Detection rates for C. difficile in inoculated broths (111/401 or 27%) were significantly higher than for culture on cycloserine-cefoxitin fructose agar (47/401 or 11%, P greater than 0.001). All fecal samples containing C. difficile and cytotoxin were correctly identified by the procedure. Isocaproic acid peak heights greater than 2 mm in selective enrichment broths inoculated with fecal samples indicated that C. difficile was present in the fecal sample examined. Of the positive specimens examined, 58% (64/111) produced peak heights greater than 10 mm. Peak heights less than 2 mm were not associated with C. difficile in the fecal sample. The investigated procedure provided a reliable alternative to the routine processing of feces for detecting C. difficile and associated cytotoxin in feces. Inoculated broths with isocaproic acid peak heights greater than 2 mm, after 24 to 48 h of incubation, and in which cytotoxin was detected, were subcultured to blood agar to obtain isolates of the organism as required. Broths which showed isocaproic acid peak heights less than 2 mm, and in which cytotoxin was not detected, were discarded as negative for C. difficile. The procedure was deemed potentially useful for epidemiological surveys of C. difficile.     

Correlation if circulating immune complexes and disease status in patients with leukaemia.

The occurrence of soluble immune complexes (IC) was investigated in 177 serum samples from 92 patients with various leukaemias using the Raji cell immunoassay. In general, patients with myeloproliferative diseases had a higher incidence and higher quantities of IC than did patients with lymphoproliferative disorders. Elevated levels of IC were found in the sera of patients as follows: 17% with chronic lymphocytic leukaemia (mean value of 13.1 microgram/ml), 67% with acute lymphocytic leukaemia (54.1 microgram/ml), 65% with chronic myelocytic leukaemia (86.7 microgram/ml), 70% with acute myelocytic leukaemia (202.5 microgram/ml) and 56% with acute myelomonocytic leukaemia (41.9 microgram/ml). Patients in terminal blastic crisis of chronic myelocytic leukaemia had the highest levels, with a mean level of 1,364.1 microgram/ml. Serial samples were obtained, as available, from individual patients during the course of the disease in an attempt to relate severity with the incidence and quantity of IC. No significant correlation could be made between the occurrence or levels of IC and the presence of absence of systemic symptoms. Similarly, no correlations could be made between levels of IC and haematological parameters, infection, or therapy. However, the data does indicate a positive relationship between the levels of IC and the progressive state of the leukaemia, especially, the myelocytic leukaemias.     

Comparison of the Rodac imprint method to selective enrichment broth for recovery of vancomycin-resistant enterococci and drug-resistant Enterobacteriaceae from environmental surfaces

We compared the Rodac imprint technique to selective enrichment broth for detecting vancomycin-resistant enterococci (VRE) and multidrug-resistant Enterobacteriaceae (MDRE) on surfaces. Rodac plates contained tryptic soy agar with 5% sheep blood, vancomycin (6 microg/ml), ceftazidime (2 microg/ml), amphotericin B (2 microg/ml), and clindamycin (1 microg/ml). Two types of broth were used: brain heart infusion (BHI) and BHI plus vancomycin (6 microg/ml) and ceftazidime (2 microg/ml) (BHIVC). Of the 46 surfaces cultured for VRE, 12 (26%) were positive. Of the 12 VRE-positive surfaces, 11 (92%) grew from Rodac, 8 (67%) grew from BHIVC, and 7 (58%) grew from BHI. A larger study is needed for MDRE, as only 4 of 43 surfaces were MDRE positive. The Rodac imprint technique successfully recovered VRE from environmental surfaces.     

Semisolid blood-free selective-motility medium for the isolation of campylobacters from stool specimens.

Isolation of Campylobacter jejuni and C. coli from stool specimens is done by growing campylobacter colonies on solid selective media with or without blood. However, recognition of these colonies can be difficult. Therefore, we decided to evaluate an isolation procedure based on the swarming of campylobacters through a semisolid medium. We developed a semisolid blood-free selective motility (SSM) medium which is composed of Mueller-Hinton broth with 0.4% agar and supplemented with cefoperazone (30 micrograms/ml) and trimethoprim (50 micrograms/ml). The SSM medium was compared with our previously described Butzler Medium Virion (Goossens et al., J. Clin. Microbiol. 24:840-843, 1986) and blood-free medium (Bolton and Coates, J. Appl. Bacteriol. 54:115-125, 1983) with cefoperazone (32 micrograms/ml) (Bolton et al., J. Clin. Pathol. 37:956-957, 1986). Of 1,890 routine stool specimens tested, 100 were found to be positive for campylobacters: 95 were recovered with the SSM medium, 94 with the Virion medium, and 90 with the blood-free medium. The SSM medium performed equally well whether it was incubated in the special incubator or the candle jar. Only 4.4 and 7.3% of the plates grew contaminating fecal flora when incubated in the special incubator and the candle jar, respectively. Clearly the SSM medium is easy, quick, cheap, sensitive, and more selective than any other medium which has been developed so far and does not require the addition of blood. We believe that this medium has a future in the routine microbiology laboratory in developed as well as in developing countries.     

Detection of campylobacter species: a comparison of culture and polymerase chain reaction based methods

AIMS: To investigate the optimal method for the detection of campylobacters from stool samples by comparing selective culture with membrane filtration and the polymerase chain reaction (PCR). METHODS: Three hundred and forty three stool samples were investigated by each of the three methods mentioned above. Selective culture was performed with charcoal cefoperazone desoxycholate agar plates. Membrane filtration was performed using cellulose triacetate membranes with 0.45 micro m pores placed on blood agar plates. Enteropathogenic campylobacters were detected using a PCR identification algorithm, consisting of screening PCRs and species identification using a PCR enzyme linked immunosorbent assay (PCR-ELISA), both based on the 16S rRNA gene. RESULTS: Of the 343 samples tested, 23 were positive by one or more method. Of these, 17 were positive by selective culture, 12 by membrane filtration, and 20 by the PCR identification algorithm. A total of 18 of 23 positives were identified as C jejuni and/or C coli by the PCR identification algorithm, compared with 14 identified to the genus level by selective culture, and 10 by membrane filtration. Among the remaining five positive samples, one C hyointestinalis was detected only by the PCR identification algorithm; one C upsaliensis was detected only by the PCR identification algorithm; one Campylobacter sp was detected by membrane filtration and selective culture and later identified as C concisus; one Campylobacter sp was detected by membrane filtration alone and later identified as Arcobacter sp; and one Campylobacter sp detected only by selective culture was lost to study and therefore not speciated. There was no significant difference between detection by selective culture and the other two methods. However, detection by PCR was significantly better than by membrane filtration (0.05 > p > 0.02). CONCLUSION: The PCR identification algorithm can detect and identify Campylobacter spp to the species level and the result is obtained on the same day. However, PCR is expensive, labour intensive, and does not provide an isolate for further identification or typing. Selective culture is as good as the PCR identification algorithm for the detection of the two most common species, C jejuni and C coli, and it is cheap and practical. However, it does miss the less common species, results take 48 hours, and identification is only to the genus level. Membrane filtration showed a low sensitivity compared with the other methods and is not appropriate for the diagnostic laboratory, although it was the only method to detect the Arcobacter sp. The optimum method for the detection of campylobacters from stool samples in the diagnostic laboratory remains selective culture.     

Murine monoclonal antibody which can distinguish cystatins SA1 and SA2

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.     

The endothelin-1 receptor antagonists ameliorate histology and ultrastructural alterations in the pancreas and decrease trypsinogen activation in severe taurocholate pancreatitis in rats

The role of potent vasoconstrictor endothelin-1 (ET-1) in acute pancreatitis (AP) remains controversial. The aim was to compare the effect of nonselective ET RA/B (LU-302872) and selective ET RA (LU-302146) antagonists on pancreatic histology, ultrastructure and trypsinogen activation in severe taurocholate AP in rats. Male Wistar rats with AP were treated with an intraperitoneous injection of 1, 5 and 10 mg/kg of body weight of each antagonist at 0, 6, 12 and 18 h after taurocholate administration. After 24 h, the samples of pancreases were taken for histological and ultrastructural examinations and for assessment of trypsinogen activation. Both antagonists, at all investigated doses, decreased the damage to the acinar cells detected in the light microscope and ultrastructurally. Trypsinogen activation increased to 29.7 +/- 3.9% in the AP untreated in comparison to the control group [12.7 +/- 1.4% (P<0.001)]. This increase was attenuated to 13.8 +/- 2.2% in AP treated with a high dose of the nonselective antagonist and to 8.4 +/- 1.7% with low dose of selective antagonist. The obtained results indicate that ET-1 could participate in the damage to the pancreas during AP. Both antagonists of ET-1 receptors exerted a similar beneficial effect on the morphological changes of the pancreas in AP. One of the probable mechanism could be the attenuation of trypsinogen activation.     

Comparison of specimen collection and laboratory techniques for isolation of Haemophilus ducreyi.

Sixteen patients with clinical chancroid were studied prospectively; different culture media and sampling techniques from genital lesions were evaluated. Technique A was aspiration of a saline wash from the ulcer which was pooled and inoculated into rabbit blood, rabbit blood + vancomycin (5 microgram/ml), and semisolid chocolate agar + vancomycin (3 microgram/ml). Each primary culture medium was subcultured to chocolate agar with 1% IsoVitaleX (CA), CA with vancomycin (3 microgram/ml) plus polymyxin (7.5 microgram/ml; CA + vp). Technique B was the use of a cotton swab, plated directly on CA, CA + v, and CA + vp. Nine strains of Haemophilus ducreyi were obtained. Technique A yielded seven strains, whereas technique B yielded eight strains; with each technique, five strains were isolated only after use of selective antibiotic media. CA + v medium yielded the largest number of isolates. Direct inoculation by swab to CA + v from chancroidal ulcers is effective as an isolation technique for growth of H. ducreyi.     

New medium for isolation of Actinomyces viscosus and Actinomyces naeslundii from dental plaque.

Metronidazole (10 microgram/ml) and cadmium sulfate (20 microgram/ml) were added to a gelatin-based medium to select for microaerophilic Actinomyces species from dental plaque samples. The new medium (GMC), when incubated anaerobically, allowed 98% recovery of seven pure cultures of Actinomyces viscosus and 73% recovery of eight pure cultures of Actinomyces naeslundii, while suppressing 76% of the total count of other organisms in dental plaque samples. In 203 plaque samples, recoveries of A. viscosus and A. naeslundii on GMC and another selective medium for oral Actinomyces (CNAC-20) were compared. Recovery of A. viscosus was comparable on the two media. Recovery of A. naeslundii was significantly higher on GMC than CNAC-20 (P is less than 0.001), and GMC allowed a more characteristic cell morphology of both organisms. GMC medium appears to be useful for the isolation and presumptive identification of A. viscosus and A. naeslundii from dental plaque.