Biomedical subjects

N Yamane

Publications and source records attributed to N Yamane.

At least 19 recordsLinked to original sources

PubMed2006

Detection of legionella species in clinical samples: Comparison of polymerase chain reaction and urinary antigen detection kits.

BACKGROUND: Recently, two excellent methods have been used for the diagnosis of Legionnaires' disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR. MATERIALS AND METHODS: A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used. RESULTS: Positive results were shown in all patients' urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR. CONCLUSION: In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients' serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.

Adult↗

PubMed2005

Determination of in vitro synergy when three antimicrobial agents are combined against Mycobacterium tuberculosis.

We determined the in vitro antimycobacterial activity of rifampicin, isoniazid and a third agent in combination using a three-dimensional chequerboard in Middlebrook 7H9 broth microdilutions. Of 28 agents screened, ethambutol, streptomycin, clarithromycin, minocycline, ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin and sitafloxacin were potentially synergistic. A further three-dimensional chequerboard assay quantitatively looked for synergy against ten clinical isolates of Mycobacterium tuberculosis, including seven multidrug-resistant isolates. Sitafloxacin, gatifloxacin and clarithromycin showed significant synergy, with fractional inhibitory concentration indices ranging from 0.41 to 0.79, 0.39 to 0.90 and 0.48 to 0.95, respectively. It is concluded that three-dimensional chequerboard assay can quantitatively determine antimycobacterial synergy, and that fluoroquinolones and antibacterial agents such as clarithromycin are effective against multidrug-resistant isolates of M. tuberculosis when combined with rifampicin and isoniazid.

Anti-Bacterial Agents↗

PubMed2004

Human herpesvirus 8 (HHV8) sequence variations in HHV8 related tumours in Okinawa, a subtropical island in southern Japan.

BACKGROUND: Although rare in mainland Japan, classic Kaposi's sarcoma (KS) is frequently reported in Okinawa, a subtropical island in southern Japan. Human herpesvirus 8 (HHV8) has been identified in the tumours and geographical differences occur. AIM: To sequence HHV8 in classic and AIDS associated KS in Okinawa. MATERIALS/METHODS: Eight classic KS cases, one AIDS associated KS, five granuloma pyogenicum cases, two inflammatory pseudotumours, two Castleman's disease cases, one angiosarcoma, and one primary effusion lymphoma (PEL) were studied. As a control, HHV8 positive cultured PEL cells (TY-1) were used. The presence of HHV8 sequences was evaluated by PCR and in situ hybridisation. PCR products were sequenced. RESULTS: There were no histological differences among KS resulting from the different virus genotypes. HHV8 was detected in all cases of KS, in one PEL, and one granuloma pyogenicum. Eight classic KS cases and one granuloma pyogenicum were infected with HHV8 genotype II/C (K1 region) or subtype C (ORF26 region), which had a five amino acid deletion at K1 VR2 region. An AIDS associated KS and a PEL were infected with type I/A virus. CONCLUSION: In Okinawa, classic KS cases and one granuloma pyogenicum case were infected with HHV8 genotype II/C, also classified as subtype C. AIDS associated KS and PEL were infected with a different HHV8 (genotype I/A), similar to that found in the USA. In Okinawa, HHV8 infection is more than four times higher than in mainland Japan, resulting in many cases of KS because of HHV8 genotype II/C infection.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Inflammatory pseudotumor of lymph nodes: clinicopathologic and immunohistological study of 11 Japanese cases.

We report 11 Japanese cases of inflammatory pseudotumor (IPT) of the lymph node. There were 7 males and 4 females with ages ranging from 5 to 68 years (median; 48). Only 2 patients had systemic lymphadenopathy, and all others had involvement of only 1 lymph node group. Constitutional symptoms such as fever were present in 8 patients and laboratory abnormalities were detected in 5. All patients recovered and were alive and well after 2 to 180 months (median; 32 months). Histologically, the process mainly involved the connective tissue framework of the lymph node, secondarily spreading into the lymph node parenchyma and the perinodal tissue. It was characterized by a storiform growth pattern of myofibroblasts, marked vascularity with associated vascular lesions, and a polymorphous reactive cellular infiltrate in a collagen-rich stroma. An immunohistochemical study revealed numerous myofibroblasts, histiocytes, and vascular endothelial cells expressing vascular endothelial growth factor (VEGF) in 6 cases. It was suggested that VEGF may be involved, in part, in the induction of the angiogenesis of IPT. Moreover, the present study indicates that follicular dendritic cell sarcoma, nasal T/natural killer cell lymphoma, and anaplastic large cell lymphoma should be added to the differential diagnosis from IPT of the lymph node. Int J Surg Pathol 9(3):207-214, 2001

Adolescent↗

PubMed2001

[Aeromonas species infection with severe clinical manifestation in okinawa, Japan -association with gas gangrene-].

We experienced two patients having Aeromonas species infection with severe clinical manifestations. The one patient was a 15-year-old high school girl student, who had been healthy in her school life, was admitted to the hospital with a sudden onset of left thigh muscle pain and swelling. She subsequently went into septic shock and died one day after admission. Pathological examination on autopsy revealed massive gas formation, skin bullas and ulcers, and extensive severe soft tissue damage throughout the body. Also, all the specimens, including brain, liver, spleen, thigh muscle, and blood in cardiac cavity, were positive for A. veronii biovar sobria. The other patient was 35-year-old man, who suffered from multiple bone fractures during the work in the harbor. One day after admission, he became febrile and went into septic shock. With the presumptive diagnosis of sepsis and gas gangrene, amputation of left thigh was performed. The exudate and aspirate of the amputated portion were repeatedly positive for A. hydrophila. Through the surveillance in Okinawa, Kagoshima, Miyazaki, and Kumamoto Prefectures, a total of 426 isolates from blood cultures were collected in the period from August, 1999 to February, 2000. Of these, 14 isolates (3.3%) were the species of Aeromonas. Of 14 isolates of Aeromonas, 13 were reported from Okinawa and the remaining one was from Kumamoto. Most patients had underlying diseases, particularly liver diseases including liver cirrhosis. The mortality rate was extremely high at 62.5%, and the patients died in short terms after blood culture became positive. With these, Aeromonas species infection is unique to Okinawa, and positive blood culture for Aeromonas species potentially indicates a high-risk, particularly among the patients with underlying diseases.

Adolescent↗

PubMed2000

[Comparative evaluation of BACTEC MGIT 960 system with MB/BacT and egg-based media for recovery of mycobacteria].

Fully automated, nonradiometric mycobacteria culture systems, BACTEC MGIT 960 (Becton Dickinson Microbiology Systems, Sparks, Md, U.S.A.) and MB/BacT (Organon Teknika, Durham, NC, U.S.A.) were evaluated in comparison with three different eggbased media (3% Ogawa, Ogawa K, and Vite) for the ability to detect mycobacteria in clinical sputum specimens. Sputum specimens were processed by semi-alkaline protease-N-acetyl-L-cysteine-NaOH (SAP-NALC-NaOH) for the automated systems, and by cetylpyridium chloride-succinic acid-NaCl for the egg-based media. A total of 954 sputum specimens were processed, and the recovery of mycobacteria by the BACTEC MGIT 960 was performed in a commercial laboratory. Overall, the frequency of breakthrough contamination was <1% for the three egg-based media, ranging from 0. 42% to 0.63%. Whereas, the frequency of false positives due to breakthrough contamination was 1.89% for MB/BacT and 20.1% for BACTEC MGIT 960. A total of 237 isolates of Mycobacterium tuberculosis complex and 167 isolates of nontuberculous mycobacteria (NTM) were recovered. The highest recovery ratio was obtained by MB/BacT (95.8%), followed by the egg-based media, Vite (74.3%), Ogawa K (65.8%), and 3% Ogawa (58.9%). The recovery ratio by BACTEC MGIT 960 was the lowest, and estimated at 43.1%, mainly due to a high frequency of breakthrough contamination. However, even after omission of these false positives reported by BACTEC MGIT 960, the recovery ratio by this system was comparable to that of 3% Ogawa media. The time to detection of 50% of positive cultures of M. tuberculosis complex by BACTEC MGIT 960 and MB/BacT was 20 days and 17 days, respectively.>From these results, it may be concluded that MB/BacT is superior to BACTEC MGIT 960 and egg-based media for the recovery of mycobacteria from sputum specimens. Furthermore, based on the outcome of this study, we think that considerable improvements are necessary for the clinical application of BACTEC MGIT 960. These improvements should particularly be focused on reducing the false positive ratio caused by contamination, and culture media, which effectively support mycobacterial growth.

Bacteriological Techniques↗

PubMed2000

[Application of DNA-DNA hybridization, DDH MYCOBACTERIA 'Kyokuto' to species-identification of mycobacteria grown in Middlebrook 7H9 broth].

A colorimetric DNA-DNA hybridization for the genetic identification of mycobacteria, DDH MYCOBACTERIA 'KYOKUTO' (Kyokuto Pharmaceuticals, Tokyo) was evaluated for the clinical isolates of mycobacteria grown in Middlebrook 7H9 broth of MB/BacT (Organon Teknika, Durham, NC, U.S.A.). When the MB/BacT gave the positive interpretation, 10 ml of Middlebrook 7H9 broth was collected from the bottle. After centrifugation at 3,000 rpm for 10 min, two drops of acetone were added to the pellet, then let it stand for one hour at the room temperature. The air-dried pellet was resuspended in a small volume of distilled water, and proceeded to the identification described. Of 136 clinical isolates of mycobacteria, comprising of 76 M. tuberculosis complex and 60 nontuberculous mycobacteria (NTM), ninety-five (70%) were correctly identified when compared to the reference identification. Thirty (22%) isolates resulted in the unidentified due to negative reaction throughout the test wells, and the remaining 11 (8%) were also unidentified due to low likelihoods. According to the package insert, the DDH MYCOBACTERIA may not be applicable to the isolates grown in Middlebrook broth. However, our test procedure using acetone prior to the extraction of bacterial DNA enables us to directly identify the isolates of mycobacteria grown in Middlebrook 7H9 broth of MB/BacT.

Acetone↗

PubMed2000

[Comparative evaluation of two different formulae of Middlebrook 7H9 broth in a fully automated mycobacteria culture system, MB/BacT; the effect of Tween 80].

Two different formulae of Middlebrook 7H9 broth, one containing Tween 80 [Tween (+) broth] and the other containing vancomycin but not Tween 80 [Tween (-) broth], were evaluated in parallel for a fully automated mycobacteria culture system, MB/BacT(Organon Teknika, Durham, NC, U.S.A.). A total of 586 clinical sputum specimens were digested and decontaminated by the semi-alkaline protease-N-acetyl-L-cysteine-NaOH (SAP-NALC-NaOH). Each part of sample treated was inoculated into the MB/BacT Process Bottle containing the respective Middlebrook 7H9 broth. Culture bottles were incubated in the MB/BacT at 37 degrees C for up to 56 days. Of 586 samples, 110 isolates of Mycobacterium tuberculosis complex and 77 of nontuberculous mycobacteria (NTM) were isolated. The occurrence of false alarm due to breakthrough contamination was 3.2 in Tween (+) broth and 2.9% in Tween (-) broth. Also, the positivities of mycobacteria by the respective culture media were comparable. However, Tween (-) broth could detect positive cultures for mycobacteria, particularly for M. tuberculosis complex at the earlier incubation cycle when compared to Tween (+) broth. The time to detect 50% positive cultures for M. tuberculosis complex was 20.5 days for Tween (-) broth and 34.3 days for Tween (+) broth, respectively. With the results, it was concluded that; Tween (+) broth produced homogeneous mycobacterial growth in culture media, and thus, it was easy to prepare the inoculum directly adjusted to McFarland turbidity to the susceptibility test. However, the present formula of Middlebrook 7H9 broth supplemented with Tween 80 was not enough suitable for the rapid detection of positive cultures and needs some revisions to improve.

Anti-Bacterial Agents↗

PubMed2000

[Rapid diagnosis for infectious diseases: the respective roles of physician and medical technologist].

The outcome of infectious disease greatly depends on the rapidity of making a definite diagnosis. For this, smooth and tight cooperation between physician and laboratory where medical technologists are working will become to the key. Rapid diagnosis for infectious diseases can contribute on the patient care, however the laboratory has a self-limitation. That is a seesaw phenomenon; rapidity vs accuracy, rapidity vs sensitivity, rapidity vs easygoing, rapidity vs carefulness, etc. From the laboratory aspect, medical technologists should function as the effective access-point in the evidence-based medicine (EBM), and should have a reasonable balance in judging seesaw phenomena, and should be keeping up their knowledge and technical skill.

Humans↗

PubMed1999

Clinical significance of spontaneous apoptosis in advanced gastric adenocarcinoma.

BACKGROUND: Spontaneous apoptosis has been detected in gastric carcinoma. However, the clinicopathological significance of this remains unclear. The objective of this study was to investigate the correlation between spontaneous apoptosis and the clinicopathologic and biologic characteristics of advanced gastric carcinoma. In addition, the prognostic significance of spontaneous apoptosis of tumors was evaluated. METHODS: The occurrence of apoptotic cell death (apoptotic index [AI]) in 97 patients with advanced gastric carcinoma was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling method. The Ki-67 labeling index (LI), expression of p53 and p21, DNA ploidy pattern, microvessel density (MVD), density of dendritic cells (DCs) in tumors, and degree of lymphocytic infiltration (LI) of the tumors were compared with the AI for each patient. In addition, the prognostic significance of AI was evaluated in these patients. RESULTS: The mean AI of the 97 tumors was 2.05% (range, 0-11.31%). Statistical analyses revealed significant correlations between the AIs and the Ki-67 LIs (P = 0.0004) and between the AIs and the density of DCs (P = 0.0007), as well as an inverse correlation between the AIs and the intratumoral MVD (P = 0.0064). In addition, the AI of 47 tumors with high grade LI (2.94+/-2.51%) was significantly higher than that of 50 tumors with low grade LI (1.22+/-0.93%) (P<0.0001). However, the authors failed to find a significant correlation between the AIs and expression of p53 and p21 and the DNA ploidy pattern. In 93 surviving patients, the 5-year survival rate of 45 patients who had tumors with high AIs (> or =1.47; 82%) was significantly better than that of 48 patients who had tumors with low AIs (<1.47; 60%) (P = 0.0264). CONCLUSIONS: The findings of the current study suggest that the tumors with low neovascularization and high local immunoreactivity may regulate their progression by apoptosis. Moreover, less extensive apoptosis in gastric adenocarcinoma may contribute to disease progression and could be correlated with a poorer prognosis.

Adenocarcinoma↗

PubMed1999

S-phase accumulation precedes apoptosis induced by preoperative treatment with 5-fluorouracil in human colorectal carcinoma cells.

BACKGROUND: In this study, the authors examined the correlations among enhancement of apoptosis, changes in cell cycle distribution, and expression of Ki-67 in human colorectal carcinoma cells during different durations of preoperative treatment with 5-fluorouracil (5-FU). METHODS: Forty-one patients with advanced colorectal carcinoma were divided into 4 groups, which received intravenous 5-FU at 500 mg/body/day preoperatively for 3 days (n = 11), 5 days (n = 13), 7 days (n = 9), or 10 days (n = 8). Patients were further divided into two subgroups according to the DNA ploidy pattern of their tumors, i.e., diploid or aneuploid. Apoptotic cells were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The expression of Ki-67 was examined by immunohistochemical staining. Flow cytometry was used to analyze changes in cell cycle distribution. RESULTS: Apoptosis of cancer cells was mostly increased in the 7-day group (apoptotic index [Al]: 18.3+/-1.7%] compared with that of the 3-day, 5-day (AI: 11.3+/-2.3% and 13.2+/-2.8%, respectively) (P < 0.01), or 10-day (Al: 16.2+/-2.1%) (P < 0.05) groups. The expression of Ki-67 was reduced with increasing prolongation of 5-FU administration, from 55.2+/-2.1% in the 3-day group to 38.1+/-2.7% in the 10-day group. The authors also assessed the S-phase fraction (SPF) of cancer cells to evaluate changes in cell cycle distribution caused by 5-FU. All tumor samples from patients treated by 5-FU showed S-phase accumulation. The ratio of SPF (after 5-FU/before 5-FU) was higher in the 5-day group (2.35+/-0.78) than in the 3-day (1.71+/-0.48), 7-day (1.68+/-0.55), or 10-day (1.20+/-0.20) groups (P < 0.05). DNA ploidy pattern of the tumor had no influence on the enhancement of apoptosis, the increased ratio of SPF, or the decrease in proliferative activity of colorectal carcinoma cells induced by 5-FU. CONCLUSIONS: S-phase accumulation preceded apoptotic cell death when intravenous 5-FU was administered continuously to human colorectal carcinoma cells.

Apoptosis↗

PubMed1999

Soluble CD44 variant 6 as a prognostic indicator in patients with colorectal cancer.

The expression of CD44v6 is well known as a useful marker of tumor progression and prognosis in colorectal cancer. In this study, we evaluated the serum levels of soluble CD44 splice variants containing exon v6 (sCD44v6) and examined the histological expression of CD44v6 in patients with colorectal cancer. Serum samples were obtained from 44 primary colorectal cancer patients before surgery. We used enzyme-linked immunosorbent assay to determine the serum levels of sCD44v6. The expression of CD44v6 was examined by immunohistochemical staining of the primary tumors obtained from the same patients. Both the serum concentration of sCD44v6 and the expression of CD44v6 were significantly associated with lymph node metastasis (p < 0.05). Furthermore, the serum level of sCD44v6 was higher in those patients with CD44v6-positive tumor tissues (154.4 +/- 34.8 ng/ml) than in those with CD44v6-negative ones (130.7 +/- 32.3 ng/ml; p < 0.05). The 5-year survival rate was significantly lower in patients with high serum levels of sCD44v6 (52.4%) than in those with low levels of sCD44v6 (78.0%; p < 0.05), and it was also significantly lower in patients with CD44v6-positive cancer (42.1%) than in those with CD44v6-negative cancer (84%; p < 0. 01). We concluded that preoperative elevation in the serum levels of sCD44v6 might be a prognostic indicator for patients with colorectal cancer.

Adenocarcinoma↗

PubMed1999

[So-called benign metastasizing leiomyoma of the lung presenting with bone metastases].

The patient, a 42-year-old woman, was admitted to our hospital because of abnormal shadows on chest X-ray films obtained during a routine medical check-up. Her medical history included a uterine myomectomy at the age of 21, and thereafter, periodic lumbago and back pain for which she had not sought any medical treatment. Chest computed tomography detected a number of pulmonary nodules in both whole lung fields, and magnetic resonance imaging revealed many spherical metastatic lesions in the thoracic and lumbar vertebrae. Although we initially suspected lung cancer, no primary lesion was found. A thoracoscopic lung biopsy revealed leiomyomatous tumors that were histologically similar to the uterine myoma removed 21 years previously. The final diagnosis was so-called benign metastasizing leiomyoma (BML). Because the removed tumor contained a high concentration of progesterone receptors (240 fmol/mg), a gonadotropin-releasing hormone analogue was administered, and proved effective in relieving the patient's periodic lumbago and back pain. The findings in this case suggested that the so-called BML was in fact a metastasis of a low-grade uterine leiomyosarcoma.

Adult↗

PubMed1999

[Laboratory-based evaluation of DainaScreen TPAb to detect specific antibodies against Treponema pallidum].

A newly developed immunochromatography assay, DainaScreen TPAb (Dainabot, Tokyo), to detect antibodies specific to Treponema pallidum was evaluated. When we tested serum and plasma samples of Syphilis Mixed Titer Performance Panel PSS201 (Boston Biomedica, Inc. , Bridgewater, MA, U.S.A.), all the test results obtained by DainaScreen TPAb were comparable to those determined by fluorescent treponemal antibody absorption test (FTA-ABS). Both within-run and day-to-day variation tests were highly precise, and no discrepant interpretation was obtained by the different medical technicians performed. Also, the testings of whole blood and plasma for individual samples gave same interpretations. The minimum detectable antibody titer was equal to that of Mediace TPLA (Sekisui Chemicals, Osaka) determined by Behring Nephelometer Analyzer (Dade Behring, Marburg, Germany). All the test results by DainaScreen TPAb for clinical serum samples were comparable to those by Mediace TPLA. With these results, we can conclude that DainaScreen TPAb is a rapid, practical and easy-to-perform alternative to detect antibodies specific to Treponema pallidum, in particular as being a point-of-care testing.

Antibodies, Bacterial↗

PubMed1999

[Mycobacteria--methods to meet the CDC guidelines].

In response to the guidelines from the Centers for Disease Control and Prevention, Atlanta, U.S.A. (1994), a considerable effort has been focused on the development and application of rapid laboratory testings for Mycobacterium tuberculosis. Concerning the first goal, "acid-fast bacillus smear results should be reported within 24 hr of specimen collection", most laboratories latently have an ability to perform unless the specimens will be processed in a lump. However, the second goal, "isolation and identification of M. tuberculosis should be within 10 to 14 days", is almost impossible for most Japanese laboratories where the radiometric Bactec 460 system is not applicable. In recent years, several nonradiometric, automated or semiautomated mycobacterial culture systems are developed and applied. To date, the evaluated results indicate that the newly developed mycobacterial culture systems with Middlebrook 7H9 broth can report positive with means of 17 to 20 day incubation, 3 to 6 days behind the CDC guidelines. Finally, regarding the third goal, "susceptibility test results should be within a total of 15 to 30 days", our developed microdilution test method can report quantitative minimum inhibitory concentrations (MICs) for M. tuberculosis after 7 day incubation. Overall, our procedures are very close to the CDC guidelines, except for isolation and identification of M. tuberculosis. The remaining key to fulfil the guidelines is a rapid detection, that is, culture media which can support rapid mycobacterial growth and advanced technology to detect attenuated growth of M. tuberculosis.

Bacteriological Techniques↗

PubMed1999

[Multicenter evaluation of broth microdilution test, BrothMIC MTB, to determine minimum inhibitory concentrations (MICs) of antimicrobial agents for Mycobacterium tuberculosis--evaluation of interlaboratory precision and interpretive compatibility with agar proportion method].

A newly developed microdilution antimycobacterial susceptibility test, BrothMIC MTB (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) to determine minimum inhibitory concentrations (MICs) was evaluated at multisites. The test method utilizes air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight antimycobacterial agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, sparfloxacin and ciprofloxacin. The test plates were reconstituted by inoculation of 0.2 ml of cell suspensions (6 x 10(5) cells/ml) and were incubated at 36 degrees C in 5% to 10% CO2. The growth endpoints were visually read after 7-day and 10-day incubations. The reproducibility was evaluated with the four reference strains of Mycobacterium tuberculosis, and were compared with the agar proportion method described in the National Committee for Clinical Laboratory Standards (NCCLS) M24-T. Of the 1,022 testings of the reference strains, 1,020 (99.8%) of the MICs read after 7-day incubation fell within 3 log2 dilutions. The growth endpoints read after 7-day and 10-day incubations gave equal MIC ranges for the respective agents. The results obtained by the BrothMIC MTB for 93 clinical isolates of M. tuberculosis compared well with those determined by the NCCLS method with 98% to 99% agreements, except for ethambutol. According to the comparative analysis with the agar proportion method, the interpretive MIC breakpoints to discriminate between the isolates susceptible and resistant against the respective agents were proposed. In conclusion, this newly developed microdilution test for M. tuberculosis is a practical, rapid, quantitative, nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.

Antitubercular Agents↗

PubMed1999

[Evaluation of inoculum density prepared by prompt inoculation system and antimicrobial susceptibility test results by the automated MicroScan WalkAway system].

The Prompt Inoculation System adapted to the susceptibility testing by the automated microbiology system, MicroScan WalkAway (Dade MicroScan Inc., West Sacramento, CA, U.S.A.) was evaluated by determining colony forming units (cfu) per ml of the inocula and by the susceptibility test results obtained through repeated testing of the American Type Culture Collection (ATCC) reference strains described by the National Committee for Clinical Laboratory Standards (NCCLS). The colony forming units per ml of the inocula prepared by the Prompt ranged 2x10++(5) to 2x10(6)++ cfu/ml for the ATCC reference strains, the results indicating that the Prompt gave a higher inoculum density and was more reproducible when compared to the standard turbidity, McFarland adjustment. Also, most inocula prepared from the clinical isolates, comprising the strains of Enterobacteriaceae,no-entericbacilli,staphylococci,enterococci, and streptococci,contained 1x10(6) to 3x10(6) cfu/ml. Although the inocula prepared by the Prompt contained more viable bacterial cells, the outcome results for susceptibility testing by the MicroScan WalkAway were highly acceptable. Four ATCC reference strains were repeatedly tested. Of 540 MIC determinations, 489 (90. 6%) were within the acceptable MIC ranges described by the NCCLS M100-S9, whereas the inocula prepared by the photometric adjustment gave 87.4%. In conclusion, the Prompt inocula were found to give more precise susceptibility test results mostly equivalent to those obtained from inocula prepared by the conventional photometric procedures.

Bacteriological Techniques↗

PubMed1999

[Evaluation of a fully automated mycobacteria culture system, MB/BacT using a newly developed digestion-decontamination procedure, semi-alkaline protease-N-acetyl-L-cysteine-NaOH (SAP-NALC-NaOH) method].

A fully automated non-radiometric mycobacteria culture system, MB/BacT (Organon Teknika, Durham, NC, U.S.A.), was recently introduced in Japan and evaluated for its ability to detect mycobacteria in clinical sputum specimens. A previous study yielded nearly a 40% contamination ratio from sputa treated with the standard N-acetyl-L-cysteine (NALC)-NaOH method. This study employed a mucolytic agent (semi-alkaline protease; SAP) in which the sputa were processed twice for digestion followed by decontamination at twice the standard volume of NALC-NaOH. The concentrated sediments were resuspended in phosphate buffer (0.067 M, pH 6.8), and inoculated into the MB/BacT Process Bottles supplemented with antibiotics. The bottles were incubated at 37 degrees C and monitored for up to fifty-six days. Recovery of mycobacteria was compared in three different egg-based Ogawa media in addition to a non-selective Middlebrook 7H10 agar. A total of 1, 124 clinical sputum specimens have been evaluated. Of these, 464 were positive for growth of mycobacteria, of which 447 (96.3%) were positive by the MB/BacT. False-positive alarms due to break through contamination, mainly by Pseudomonas aeruginosa and Candida spp., were observed in twenty-one specimens (1.9%). The three Ogawa media could detect only 283 (60.5%) to 353 (75.4%) positives, and Middlebrook 7H10 agar only 424 (90.6%) positives. The time to detect positive cultures of Mycobacterium tuberculosis complex by the MB/BacT ranged from 2.2 days to 52.3 days, and 50% of positive cultures were detected within 16.7 days of incubation. It can be concluded that the combination of SAP-NALC-NaOH digestion-decontamination procedure and the MB/BacT is particularly useful for the isolation of mycobacteria and has a faster time to detect than conventional methods. MB/BacT is a suitable alternative method for the detection of mycobacteria in Japan, where the radiometric Bactec System is not available.

Acetylcysteine↗