각종 임상검체의 결핵균 검출 양성률과 염색 방법에 따른 비교 관찰

조금장 인제대학교 1986 仁濟醫學 Vol.7 No.1

결핵균 도말을 의뢰한 4,258건 중 균 양성 302건을 대상으로 검체별 양성률과 성별 · 연령별 균 양성 환자의 분포 상황과 함께 이들을 대상으로 배양 및 형광 염색과 Z-N염색을 동시 실시하여 진단을 위해서는 어떤 방법의 적용이 더 바람직한가를 조사하여 다음과 같은 결과를 얻었다. Even in decreasing trends of the prevalence of tuberculosis, the prevalence of bacteriologically confirmed pulmonary tuberculosis is 0.46% (estimated value, M.H.S.A., 1985) and death rate is still 31.7%(Based on vital registration, 1984) in Korea. Therefore prevention of occurrence of new patient by early detection as early as possible is one of the most important task in eradicating tuberculosis. Since Hagemann (1973) first described the fluorescence microscopy for demonstrating acid fast bacilli, there is convincing evidence that fluorescense microscopy is more sensitive than Ziehl-Neelsen method in literatures. Se author made a study to evaluate the usefulness of the fluorescent technique as a routine screening method for clinical materials suspected to contain acid fast bacilli. In this study, two smears were prepared before culture, one stained with Auramine fluorochrome procedure, and the other with conventional Ziehi-Neelsen procedure, from 4,258 specimens from various sources. Each was examined by Leitz Dialux 20 fluorescence microscope and conventional light microscope respectively. The results are summarized as follows 1.Number of specimen sources requested for AFB smear are; sputum, 3,329 cases (78.2%), bronchial washings, 268 cases (6.3%), urine, 263 cases (6.25) etc. among total 4,258 cases. 2.AFB smear-Positive rate is 7.1% (302/4,258 cases) and the rates in different specimen sources are; pus 15.4%, aspirated materials 11.9%, sputum 7.8% etc. 3.AFB smear-positive ratio in sex is 1.75:1 (M:F)-statistically significant. 4.Age distribution of AFB smear-positive rates are; 20-29 age group, 10.7%, 40-49 age group, 9.2%, 10-19 age group, 7.8%, etc. 5.Fluorescence microscopy revealed slightly higher sensitivity then Ziehl-Neelsen microscopy but the result is statistically insignificant. On the basis of this study, author recommend that the fluorochrome procedure should be used as the routine screening method for the early detection of acid-fast bacilli in various clinical specimens and the follow up study of the efficacy of antituberculous chemotherapy. But in the cases of doubtful result in fluorochrome procedure, confirmation should be made after a Ziehl-Neelsen procedure.

마이코박테리아의 자동형광염색기 AT-2000F의 유용성 평가

김신영,양은경,김영진,이선민,이은엽,박영길,배길한,장철훈 대한임상미생물학회 2006 Annals of clinical microbiology Vol.9 No.2

Background: Sputum smear microscopy is rapid, economic, and useful to detect patients with transmittable tuberculosis, albeit laborious. We aimed to evaluate the usefulness of an automated acid-fast bacilli stainer, which had been developed for lowering the labor and maintaining or increasing the staining quality. Methods: One hundred sputum samples including some known positive smear specimens which were selected from clinical specimens requested for smear and culture for mycobacteria at Pusan National University Hospital, were used for evaluation. Auramine/rhodamine fluorescent acid-fast stainings were performed manually or by using the automated stainer, AT-2000F (Dagatron, Ilsan, Korea). Ziehl-Neelsen stain was also performed simultaneously. Results: Concordance rate between automated and manual fluorescent stains was 98.0% and that between automated fluorescent and manual Ziehl-Neelsen stains was 88.0%. In all discordant cases, the automated stains showed one-grade higher results compared to the respective manual fluorescent or Ziehl-Neelsen stains. With the automatic stainer, all staining procedures were processed automatically except for slide loading and unloading. The process time was reduced by a half, and the slide-to-slide or day-to-day variations of staining quality were reduced compared with the manual fluorescent stain. Conclusion: Acid-fast bacilli stain using automated stainer AF-2000F can reduce the processing time, labor, and variations of staining quality, and enhance or maintain the detection of positive smears.

Evaluation and Comparison of Molecular and Conventional Diagnostic Tests for Detecting Tuberculosis in Korea, 2013

박상희,김창기,정혜란,손현진,김성한,박미선 질병관리본부 2014 Osong Public Health and Research Persptectives Vol.5 No.-

Objectives: A fast and accurate diagnosis is necessary to control and eliminate tuberculosis (TB). In Korea, TB continues to be a serious public health problem. In this study, diagnostic tests on clinical samples from patients suspected to have TB were performed and the sensitivity and specificity of the various techniques were compared. The main objective of the study was to compare various diagnostic tests and evaluate their sensitivity and specificity for detecting tuberculosis. Methods: From January 2013 to December 2013, 170,240 clinical samples from patients suspected to have TB were tested with smear microscopy, acid-fast bacilli culture, and real-time polymerase chain reaction (PCR). The test results were compared and data were analyzed. Results: A total of 8216 cultures tested positive for TB (positive detection rate, 4.8%). The contamination rate in the culture was 0.6% and the isolation rate of nontuberculous mycobacteria was 1.0%. The sensitivity and specificity of smear microscopy were 56.8% and 99.6%, respectively. The concordance rate between the solid and liquid cultures was 92.8%. Mycobacterium isolates were not detected in 0.4% of the cases in the liquid culture, whereas no Mycobacterium isolates were detected in 6.8% of the cases in the solid culture. The sensitivity and specificity of real-time PCR for the solid culture were 97.2% and 72.4%, respectively, whereas the corresponding data for the liquid culture were 93.5% and 97.2%. Conclusion: The study results can be used to improve existing TB diagnosis procedure as well as for comparing the effectiveness of the assay tests used for detecting Mycobacterium tuberculosis isolates.

Clinical significance of smear positivity for acid-fast bacilli after ≥5 months of treatment in patients with drug-susceptible pulmonary tuberculosis

Kang, Hyung Koo,Jeong, Byeong-Ho,Lee, Hyun,Park, Hye Yun,Jeon, Kyeongman,Huh, Hee Jae,Ki, Chang-Seok,Lee, Nam Yong,Koh, Won-Jung Williams & Wilkins Co 2016 Medicine Vol.95 No.31

<P><B>Abstract</B></P><P>Patients with pulmonary tuberculosis (TB) with acid-fast bacilli (AFB)-positive sputum smear at 5 months or later during treatment are considered to be cases of treatment failure according to World Health Organization guidelines. This study evaluated the proportion, clinical characteristics, and significance of positive sputum smears after ≥5 months of standard treatment in patients with drug-susceptible pulmonary TB.</P><P>This was a retrospective cohort study of 1611 patients with culture-confirmed drug-susceptible pulmonary TB who received standard anti-TB treatment from January 2009 to February 2014. Forty-one patients (2.5%) who were smear-positive after ≥5 months of treatment and 123 age- and sex-matched control patients were evaluated.</P><P>Among the 41 smear-positive patients, culture of the sputum specimens yielded <I>Mycobacterium tuberculosis</I> (MTB) in 1 patient (2.4%), nontuberculous mycobacteria (NTM) in 7 (17.1%), and no growth in the remaining 33 patients (80.5%). Treatment was successfully completed in 40 patients (97.6%) with prolongation of the continuation phase regimens without change to second-line anti-TB treatment. In patients with smear positivity after ≥5 months of treatment compared with controls, cavitation on chest radiographs (53.7% vs. 25.2%, <I>P</I> = 0.001), bilateral involvement (51.2% vs. 30.1%, <I>P</I> = 0.01) and combined pleural effusion (26.8% vs. 10.6%, <I>P</I> = 0.01) were found more frequently at the time of treatment initiation, and paradoxical response occurred more commonly (19.5% vs. 3.3%, <I>P</I> = 0.002) during treatment.</P><P>Smear-positive sputum after ≥5 months of standard anti-TB treatment was mainly because of nonviable MTB bacilli or NTM in patients with drug-susceptible pulmonary TB. AFB smear alone should not be used to assess treatment failure and careful examination of microbiologic status, including culture and drug susceptibility testing, is needed before making changes to retreatment regimens or empirical second-line anti-TB regimens in these patients.</P>

결핵균 자동염색기의 개발 및 평가

김수찬,강승일,김승철,황정호,김성녕,김영,송선대,조상래,김덕원,Kim, S. C.,Kang, S. I.,Kim, S. C.,Hwang, J. H.,Kim, S. Y.,Kim, Y.,Song, S. D.,Cho, S. N.,Kim, D. W. 대한의용생체공학회 2002 의공학회지 Vol.23 No.3

결핵을 진단하는 방법 중에서 신속하고 비교적 비용이 적게드는 방법은 객담을 통한 결핵균 도말 검사이다. 결핵균 도말 검사는 슬라이드에 도말한 환자의 객담을 가온 과정을 통해 고착시키고. acid-fast 염색방법을 통해 염색시킨 후 현미경으로 결핵균을 관찰하는 것이다. Acid-fast 염색방법은 크게 hot staining과 cold staining 방법 두 가지가 있으며, 우리나라에서는 염색 결과가 선명한 hot staining 방법인 Ziehl-Neelsen 방법을 주로 이용한다. 그러나, 기존의 결핵균 자동염색기는 가온 기능이 없어 환자의 객담을 슬라이드에 검사자가 고착을 시켜야 하고. 선명도도 낮은 문제점을 가지고 있다. 본 연구에서는 검사자의 인력 절감과 검사자 개인의 염색 능력에 따른 염색 정도의 변화를 줄이기 위해 가온이 가능한 결핵균 자동염색기를 개발하였다 개발된 염색기는 객담의 고착에서 염색 그리고 건조가지 전 과정이 자동으로 이루어진다. 염색 시간은 5개의 슬라이드를 고품질로 염색할 경우 21분이 소요되었다. 성능 평가를 위해 총 91개 객담을 대상으로 자동과 수동염색을 시행하여 일치율을 비교해 본 결과 75%로 통계적으로 유의한 차이를 보이지 않았다 (P>0.05). The detection of tubercle bacilli (TB) from sputum smear is one of the fast and inexpensive methods for diagnosis of tuberculosis. For this method. sputum smears are usually flexed by heating and stained by acid-fast staining method, and then examined under an optical microscope. Two Procedures are commonly used fur TB staining. One is hot staining and the other is cold staining method. The Ziehl-Neelsen method which is a hot staining method is widely used in Korea because its stained color is more vivid However, the conventional automated stainer has to fix the sputum smear on a slide manually and the stain is not so vivid because it has not heating function. In an effort to save labor and minimize variations in manual staining Procedure. we developed an automated stainer with heating function. The entire staining process is fully automated. from fixation to final washing and drying. With the automated methods, five slides can be flexed and stained in 21 minutes at consistent high quality We compared the concordance rate between the two methods for 91 sputum samples to validate the stain quality of the developed automated stainer. As the results, the concordant rate between the two methods was 95% and there was no significant difference (p>0.05)

A Case of Primary Lid Tuberculosis after Upper Lid Blepharoplasty

Jae Wook Yang,Yoon Duck Kim 대한안과학회 2004 Korean Journal of Ophthalmology Vol.18 No.2

Primary lid tuberculosis after lid surgery is a very rare condition and is likely caused by the introduction of bacilli through epithelial injury. Secondary infection, due to direct hematogenous spread or contiguous spread from adjacent structures are more common presentations of lid tuberculosis. The authors experienced a case of primary lid tuberculosis occurring in a 19 year old female after blepharoplasty for making a eyelid crease. Her upper lid skin showed a reddish and non-tender mass lesion measured 3x1 cm, which was diagnosed as the tuberculosis through typical histopathological findings(caseous necrosis), acid-fast bacilli stain and PCR, and treated with anti-tuberculosis medications.

Diagnosis of Intestinal Tuberculosis by Polymerase Chain Reaction

Oh, Hwa Eun,Cho, Hyun Deuk,Choi, Jong Sang 고려대학교 의과대학 1998 고려대 의대 잡지 Vol.35 No.3

장결핵이 의심되는 42예의 생검 혹은 절제된 조직에서 PCR과 AFB 염색, 그리고 Rhodamine-Auramine 형광 염색(RAF-AFB)을 시행했다 환자는 만성 설사와 복통을 호소하였고 복통이 가장 흔한 증상이었다(76%). 병리학적 소견상, 10예에서 만성 건락성 육아종성염증으로 진단되었고. 32예는 만성 비건락성 육아종성 염증으로 진단되었다. Ziehl-Neelsen(ZN) AFB 염색에서 25예 중 11예에서 결핵균이 관찰되었으며(44%) Rhodamine-Auramine 형광 염색에서는 25예 중 13예에서 결핵균이 관찰되었다(52%). 25예 중 9예에서 Ziehl-Neelsen(ZN) AFB염색과 RAF-AFB 염색 모두에서 결핵균이 관찰되었으며. 6예는 두 AFB 염색중 어느 하나에서 결핵균을 관찰할 수 있었으므로 15예에서 결핵균을 관찰할 수 있었다(60%). PCR에서 전체 42예 중 8예에서 결핵균의 DNA를 확인할 수 있었다(19%). 비록 PCR이 장결핵의 진단에 있어서 빠르고, 민감한 방법이기는 하나, 우리의 실험결과에 의하면 상대적으로 덜 민감한 방법이었다. 그러므로, 결핵균을 검출하는데 있어서 AFB 염색과 PCR을 병행하는 것이 진단의 정확도를 높일 수 있으리라 생각한다. With 42 endoscopically biopsied or resected tissues, we performed a polymerase chain reaction(PCR), acid fast bacilli(AFB) stain assay and Rhodamine-Auramine fluorescent acid-fast (RAF-AFB) staining. With clinical impressions of intestinal tuberculosis, specimens were obtained from patients with chronic diarrhea or abdominal pain. Abdominal pain was the most common symptom (76%). On pathologic findings, ten of forty-two cases were diagnosed as chronic caseating granulomatous inflammation and thirty-two cases were diagnosed as chronic noncaseating granulomatous inflammation. Ziehl-Neelsen(ZN) stain from formalin-fixed, paraffin-embedded tissue revealed AFB in eleven of twenty-five cases(44%). Rhodamine-Auramine fluorescent acid-fast staining revealed AFB in thirteen of 25 cases(52%). Nine of 25 cases showed AFB on both ZN and RAF-AFB stainings and six of 25 cases showed AFB one each of ZN and RAF-AFB stainings, the resulting fifteen of 25 cases(60%) showed AFB with special stains. M. tuberculosis DNA was identified in 8 of 42 cases(l9%) by PCR, two cases of the positive eight cases and six cases without AFB stainings revealed positive findings on Tb-PCR. Although PCR is known a s a rapid, sensitive, and specific method for the diagnosis of intestinal tuberculosis, our study showed relatively low sensitivity and specificity, which might be caused by low numbers of organisms in the specimen and/or inadequate specimen. Therefore, for the detection of M. tuberculosis, diagnostic accuracy will be increased when both AFB stain and PCR are determined.

중합효소연쇄반응을 이용한 결핵균과 비정형 결핵균의 감별동정

이성덕,이혜영,김현철,김수영 대한임상미생물학회 2007 Annals of clinical microbiology Vol.10 No.2

Background: The purpose of this study was to evaluate the usefulness of direct PCR for a rapid detection of Mycobacterium tuberculosis (MTB), the differentiation of MTB from nontuberculous mycobacteria (NTM), and the identification of NTM species in acid-fast bacilli (AFB) smear-positive specimens. Methods: A total of 255 AFB smear-positive respiratory specimens were studied. For the differentiation of MTB from NTM, the bands of the 360 bp, which exists both in MTB and NTM, and the 190 bp, which exists only in MTB, were amplified using the onetube nested PCR targeting regions of the rpoB gene. The two-tube nested PCR targeting 16S-23S rRNA spacer gene was done with 34 specimens that were negative by one-tube nested PCR. The specimens that were tested positive for NTMs were subsequently subjected to PCR-restriction fragment length polymorphism (PCR-RFLP) analysis based on the rpoB gene for mycobacterial species identification. Results: Detection rate of MTB and NTM was 87% after the one-tube nested PCR. The detection range of MTB and NTM increased up to 93% after the two-tube nested PCR. The results of PCR-RFLP analysis identified those NTMs as M. avium and M. intracellulare. Conclusion: This result seems to suggest strongly that the PCR testing especially aiming for differentiation of MTB from NTM, and identificatin of mycobacterial species using AFB smear-positive specimens would be highly importnat in clinical settings for effective treatment of patients.

복강경 검사로 진단한 결핵성 복막염 1례

박민경,서원석,박재옥,김창휘,고은석,임철완 순천향의학연구소 2004 Journal of Soonchunhyang Medical Science Vol.10 No.2

Tuberculous peritonitis is discovered from sixty to ninty percent of pulmonary tuberculosis patients. Clinical symptoms are nonspecific such as mild fever, weakness and malaise but show tenderness in right lower abdomen and abdominal distension. Diagnosis is confirmed by laparoscopic and pathologic findings. We report a case of tuberculous peritonitis in an 11-year-old boy who was hospitalized because of abdominal distension with pain on right lower abdomen which had developed one day before he came to hospital. Chest X-ray showed pleural effusion on both sides. We did laparoscopic examination and observed diffuse nodular infiltration. In biopsy specimen we observed osseous granuloma and acid-fast bacilli in AFB stain.

Factors Associated with the Performance of Direct PCR Detection of Mycobacteria in Clinical Specimens: Retrospective Real-world Data

박창훈 대한진단검사의학회 2023 Laboratory Medicine Online Vol.13 No.3

Background: Tuberculosis caused by Mycobacterium tuberculosis (MTB) remains a major health problem worldwide. Nontuberculous mycobacteria (NTM) infection is the primary cause of pulmonary disease. Currently, for early diagnosis of mycobacterial infection, direct PCR detection in clinical specimens is used. This study aimed to identify factors affecting the direct PCR detection of mycobacteria from clinical specimens. Methods: Records of mycobacterial culture from October 2016 to July 2020 were retrospectively reviewed. Using culture as the reference method, the performance of direct PCR detection was calculated. Differences in analytical performances among mycobacteria species, specimen type, and acid-fast bacillus (AFB) staining were determined using chi-squared or Fisher’s exact test. Results: Of the 27,267 culture datasets, 1,586 datasets were selected. The sensitivity of direct PCR detection for NTM was 27.6% (95% confidence interval [CI], 22.3–33.5) in sputum and 47.8% (95% CI, 37.3–58.5) in bronchial washing fluid (P<0.001). The sensitivity of direct PCR detection showed higher sensitivity in smear-positive AFB than in smear-negative AFB for both MTB (93.8% vs. 51.2%; P<0.001) and NTM (68.3% vs. 26.1%; P<0.001). Conclusions: AFB staining results were related with the direct PCR detection of MTB and NTM, whereas the respiratory specimen type was related with the direct PCR detection of NTM.