Acridine orange stain of Mycobacterium leprae / 나학회지

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the diagnosis of leprosy. However, the fluorescent stain performs better and allows the detection of more positive smears. The limitation for its widespread use has been the high cost for fluorescent microscopes. Novel light-emitting diodes (LED) are inexpensive solutions for fluorescent microscopes, and thus fluorescent stain may be a cost-effective step to improve the diagnosis of leprosy in resource-poor countries. And the comparison of auramine and acridine orange for staining of acid-fast bacteria was showed significantly more acid-fast rods after using acridine orange and the number of "false positive" results was somewhat higher on auramine staining. So acridine orange offers a good alternative to auramine which is considered carcinogenic. This study evaluated the comparison of the Ziehl-Neelson's AFB stain and the acridine orange stain in the skin smear based on PCR. As PCR results were taken as gold standard, results of the study revealed that the sensitivity of Ziehl-Neelson's AFB stain was 50% and that of acridine orange stain was 92.2%. This study confirmed that the fluorescence stain method is more sensitive than the Ziehl-Neelsen's staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.

Comparative analysis of Fluorescence stain and Ziehl-Neelsen's AFB stain for Mycobacterium leprae (pilot study) / 나학회지

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the mycobacteria. However, this method has several issues of false negative results, and hence a comparative experiment of the Ziehl-Neelson's AFB staining and the fluorescence staining method was done to remedy this problem. As the fluorescence staining method brightly highlights the AFB in a dark field, and also as it is observed with the lower power objective, it is a method that can better the observation and shorten the time of observation as well. The fluorescence staining method that was used in this experiment did a comparative analysis of the Auramine O-Rhodamine B and the Acridine Orange. The results showed that although the Auramine O-Rhodamine B allows easier observation of the AFB with a high fluorescence expression rate for the multibacillary leprosy sample, the darkness on the periphery makes it hard to observe anything else, while also making it hard to observe the cell changes and paucibacillary leprosy of the AFB. However, the Acridine Orange staining method highlights the cells in dark green and changes the color of the AFB from bright red to orange making it easier to observe bacilli. The results of the study show that the Acridine Orange method is superior to the Auramine O-Rhodamine B method in detecting acid fast bacilli in specimen.

Comparison between auramine-rhodamine [AR] and Ziehl-Neelsen [ZN] staining in cases of chronic granulomatous lymphadenopathy

Experimental study. This study was conducted at various hospitals of Quetta. This study includes 103 cases of chronic granulomatous lymphadenitis, with 101 cases of tuberculosis lymph nodes amongst a total of 200 cases of non-neoplastic lymphadenopathy.Their ages ranged from 2 to 79 years. Maximum number of cases were in 10-29 years age group. Females [69.31%] were affected more as compared to males. The commonest presenting symptom was fever. Cervical lymph nodes [83 cases] were the commonest site of involvement whereas 18 cases showed multiple site involvement. Fluorescent staining of histopatholigical sections from 103 chronic granulomatous lymphadenitis gave positive results in 76 out of 103 [73.78%] cases, however Ziehl-Neelsen staining was positive only in 29 out of 103 [28.15%] cases. The yield of mycobacteria on fluorescent staining was higly significant [p<0.00l] as compared to Ziehl-Neelsen staining thereby providing the superiority of fluorescent stain. In a total of 200 cases of non-neoplastic lymphadenopathy 101 cases showed granulomatous lesions, histologically consistant with tuberculosis. Other causes of lymphadnopathy were chronic non-specific lymphadenitis [n=87] viral lymphadenitis [n=8], fungal lymphadenitis [n=2] and acute bacterial lymphadenitis [n=2]. Fluorescent staining of histopathological sections from 103 chronic granulomatous lymphadenitis gave positive results in 76 out of 103 [73.78%] cases, however Ziehl-Neelsen staining was positive only in 29 out of 103 [28.15%] cases. In 101 cases, the finding were consistent with the diagnosis of tuberculous lymphadenopathy. In our study, significantly greater number of cases, 78 out of 101 [P<0.001] diagnosed as tuberculous lymphadenitis were in age groups 10-29 years. Female [69.31%] were more affected than males [30.69%]. The common presenting symptom was fever. Cervical lymph nodes were commonest site of biopsy. Haemoglobin estimation in 98 cases revealed anaemia in 65 out of 101 [66.32%] patients of tuberculous lymphadenopathy. Erythrocyte sedimentation rate was performed in 63 cases and was raised in 52 [82.53%] cases. In a total of 80 cases in whom X-ray chest was performed, 14 [15.5%] cases revealed foci of tuberculosis. Thus, in conclusion, this study has highlighted the superiorty of fluorescent stain over ZN stain

Métodos convencionais e moleculares para o diagnóstico da tuberculose pulmonar: um estudo comparativo / Conventional and molecular techniques in the diagnosis of pulmonary tuberculosis: a comparative study

OBJETIVO: Comparar quatro métodos laboratoriais no diagnóstico de tuberculose pulmonar. MÉTODOS: Foram realizadas pesquisa direta pelas colorações de Ziehl-Neelsen e auramina, cultura para micobactérias em meio Lõwenstein-Jensen (LJ) e polymerase chain reaction (PCR, reação em cadeia da polimerase) para Mycobacterium tuberculosis em 160 amostras de secreção respiratória de pacientes com suspeita de tuberculose pulmonar. As cepas isoladas foram identificadas por método radiométrico utilizando-se p-nitro-alfa-acetilamino-beta-hidroxipropiofenona (NAP) e métodos clássicos. A sensibilidade dos métodos foi comparada com o padrão ouro para o diagnóstico da tuberculose pulmonar, definido por critérios clínicos, radiológicos e microbiológicos. RESULTADOS: Dos 160 pacientes, 142 foram diagnosticados com tuberculose pulmonar de acordo com o padrão ouro. As técnicas de Ziehl-Neelsen e auramina, cultura em meio LJ e PCR apresentaram sensibilidade de 54,2 por cento, 58,4 por cento, 67,6 por cento e 77,5 por cento, respectivamente, quando comparados ao critério diagnóstico adotado. A especificidade dos quatro métodos foi de 100 por cento. A concordância na identificação da micobactéria entre PCR e o método radiométrico utilizando NAP foi alta (96,8 por cento). A sensibilidade da PCR foi de 50,8 por cento nas amostras com baciloscopia negativa e de 98,8 por cento naquelas com baciloscopia positiva. Nas amostras com resultados negativos na baciloscopia e cultura, a sensibilidade da PCR foi menor que nas com resultados positivos (25,6 por cento e 99,0 por cento, respectivamente). CONCLUSÕES: A PCR é método promissor no diagnóstico da tuberculose pulmonar, mesmo em amostras paucibacilares. Além disso, apresenta a vantagem da identificação simultânea e rapidez do resultado.

OBJECTIVE: To compare four laboratory methods in the diagnosis of pulmonary tuberculosis. METHODS: Respiratory secretion specimens were collected from 160 patients suspected of having pulmonary tuberculosis. Direct testing for Mycobacterium tuberculosis was carried out using Ziehl-Neelsen and auramine staining. In addition, culture in Lõwenstein-Jensen (LJ) medium and polymerase chain reaction (PCR) were used. The strains isolated were identified by means of a radiometric method using p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) and classical methods. The sensitivity of the methods was compared to the gold standard for the diagnosis of pulmonary tuberculosis, based on clinical, radiological and microbiological criteria. RESULTS: Of the 160 patients, 142 were diagnosed with pulmonary tuberculosis according to the gold standard. The sensitivity of Ziehl-Neelsen staining, auramine staining, culture in LJ medium and PCR was 54.2 percent, 58.4 percent, 67.6 percent and 77.5 percent, respectively, when compared with the diagnostic criterion adopted. All four methods presented 100 percent specificity. In the identification of mycobacteria, there was high (96.8 percent) concordance between PCR and the radiometric method using NAP. The sensitivity of PCR was 50.8 percent in samples with negative sputum smear microscopy results and 98.8 percent in those with positive results. The sensitivity of PCR was lower in specimens with negative results in sputum smear microscopy and culture than in those with positive results (25.6 percent and 99.0 percent, respectively). CONCLUSIONS: We found PCR to be a promising method for the diagnosis of pulmonary tuberculosis, even in paucibacillary specimens. Simultaneous identification and faster results are additional advantages of this method.

Cytological diagnosis of pulmonary nocardiosis in an immunocompromised patient.

We report a case of pulmonary nocardiosis in an immunosuppressed patient having vasculitis who presented with fever, cough and chest pain. A suspicion of nocardiosis was made on auramine O staining of material procured by CT guided fine needle aspiration cytology right lung. Modified Ziehl-Neelsen staining was useful in confirming the diagnosis. The patient showed remarkable recovery after treatment with co-trimoxazole. Quick identification of this uncommon pathogen in the cytological material using special stains helped in timely diagnosis and successful treatment of the patient.

Auramine phenol staining of smears for screening acid fast bacilli in clinical specimens.

Tuberculosis is a major threat to the public health allover world. Out of the total tuberculosis cases reported globally, more than half are reported from African continent and India. Two to three fold rises in tuberculosis cases has been reported in the last two decades. Early diagnosis and treatment is one of the effective tools to control the rapid spread of disease. The aim of this study was to find out the value of auramine phenol (AP) staining technique in diagnosis of the suspected tuberculosis cases. A total of 2000 samples which included sputum (746), gastric aspirates (380), urine (336), endometrial biopsy (150), pleural fulids (146), Synovial fluids (67), ascitic fluids (35), cerebrospinal fluids (43), bone marrow (18), lymph node biopsy (11), pericardial aspirates (6), skin biopsy (4), peritoneal fluids (2), and stool (1) were included in the study. Sample were subjected for decontamination procedure by using standard Petroffs method. The deposit smears were stained by auramine phenol (AP stain) and Ziehl-Neelsenstaining (ZN stain) and specimens were cultured for Mycobacterium tuberculosis. Of the total positive isolates 69.23% were having pulmonary tuberculosis and 30.76 had extrapulmonary tuberculosis Genitourinary tuberculosis was the most common diagnosis among the extrapulmonary tuberculosis followed by chronic synovitis, bursitis, meningitis, septic arthritis and pericardial effusion. Out of 130 positive samples 70 by culture, 66 smears were positive by auramine phenol stain and 62 were positive by ZN stain. A total of 27 samples were tested positive only by AP staining technique, which included (12) pulmonary and (15) extrpulmonary samples. The endometrial biopsy and pericardial fluid samples showed positive for acid fast bacilli by AP stain only, whereas ZN stain and culture technique failed to demonstrate any bacilli in the same sample. Auramine stain showed high sensitivity (47.14%) and specificity (96.58%). Result of the present study showed that the auramine stain is a better method for screening samples from the suspected cases of tuberculosis sample especially pulmonary and extrapulmonary cases where bacilli count is usually low.

Comparison of multiplex PCR and acid fast and auramine -rhodamine staining for detection of Mycobacterium tuberculosis and non tuberculosis mycobacteria in paraffin- embedded pleural and bronchial tissues with granulomatous inflammation and caseous necrosis

The aim of this study was to compare the sensitivity and specificity of Acid fast and Auramine-Rhodamine staining and Multiplex PCR for the detection of Mycobacterium tuberculosis complex and non tuberculosis Mycobacteria on formalin fixed paraffin embedded tissues [FFPE]. Forty cases of FFPE pleural and bronchial tissue with chronic granulomatous inflammation and caseous necrosis and 10 cases with bronchogenic carcinoma as controls were investigated. We designed a Multiplex PCR DNA amplification method with two targets: 123bp DNA fragment from IS6110, which is present only in mycobacterium tuberculosis complex and 162bp DNA encoding Ag 85complex which is present in all of mycobacteria. The FFPE also stained by Acid fast and Rhodamine-Auramine staining method. In 26 samples [65%] 123 bp and 162 bp DNA fragments were detected together [12 in bronchial samples and 14 in pleural samples].The 162 bp fragment wasn't detected alone. The sensitivity of PCR was 65% and the specificity was 100%. Eleven cases were positive for Acid fast staining. There was 27.5% sensitivity and 100% specificity. Thirteen cases were positive for Auramine-Rhodamine staining [A-R-S]; there was 32.5% sensitivity and 100% specificity. All of the 10 controls were negative for 123 bp, 162 bp DNA fragments, for Acid fast and Auramine-Rhodamine staining. Multiplex PCR is a sensitive, specific and rapid method for detection of M tuberculosis in FFPE tissues

Detection of Cryptosporidium oocysts by auramine and Ziehl Neelsen staining methods

Cryptosporidium spp is a common intestinal pathogen of animals and humans. It may have an important economic impact on farms and cause potentially zoonotic infections. Fecal specimens were collected from 331 domestic animals (81 beef cattle, 50 sheep, 100 pigs and 100 dogs) and checked for the presence of Cryptosporidium oocysts by way of Ziehl Neelsen and auramine staining methods. An overall positivity rate of 7.5 percent (25/331) was found, with rates of 10 percent (10/100) among the dogs and 18.5 percent (15/81) among the beef cattle. The feces of sheep and pigs tested negative. In beef cattle, 15 and 12 positive samples were detected by the auramine and Ziehl Neelsen staining techniques, respectively, with no statistically significant difference between the two methods. In dogs, the same number of positive samples was found by both techniques.

Effects on Detection Rate and Turnaround Time by Changes in the Mycobacterial Culture and Identification Methods / 대한진단검사의학회지

BACKGROUND: We evaluated the effects of changes in laboratory practices on the detection rates and turnaround time in a clinical mycobacteriology laboratory. METHODS: A total of 27,339 specimens from 9,947 patients were tested during the period from September 2000 to March 2004, which could be divided into the following four periods according the culture and identification methods used: Period I, use of 2% Ogawa medium for culture and niacin for identification; period II, introduction of PCR for identification; period III, introduction of BacT/Alert system (bioMerieux, Durham, USA) for culturing sterile body fluids; period IV, use of Bact/Alert system for CSF and the second of repeated sputum specimens from the same patients. During these periods, two technicians (one for the first half and the other for the second half periods) did all mycobacterial tests except PCR. RESULTS: Mean detection rates were 8.0% by auramine stain, 7.9% by nested PCR, and 6.6% by culture. The detection rates by culture for sputum specimens varied 11.4%, 7.7%, 8.1% and 5.9% in each of the four periods; for body fluids, the detection rate of 4.3% during the period III was the highest. The proportion of stain results reported within 24 hours and the identification of culture isolates within 21 days changed from 80.9% to 72.4% and from 2.3% to 30.8%, respectively. With an introduction of PCR for identification in the period II the time required for the identification of cultures decreased dramatically from 26.5 days to 4.8 days. The TAT of a direct detection by nested PCR changed from 7.2 days to 5.1 days. CONCLUSIONS: New tests should be introduced in the clinical mycobacteriology laboratory. But the cost and workload of the tests should be taken into consideration to make the laboratory service more efficient.

Enhanced detection of Mycobacteria stained with rhodamine auramine at 37 degrees C.

Sputum smear microscopy is the most efficient and rapid technique for detection of acid-fast bacilli (AFB). Fluorochrome method of staining is preferred for Mycobacteria in the overburdened laboratories as the fluorescing bacilli are more readily detected than the fuchsin stained bacilli in shorter period of time. A total of 300 sputum samples obtained from suspected cases of Tuberculosis were collected and were subjected to staining by rhodamine auramine at 37 degrees C and also at room temperature (conventional method). The smears were then blindly evaluated. Fifty-eight samples were positive by both methods and 5 were positive at 37 degrees C only. Staining at 37 degrees C increased the smear positivity by 8.6% over conventional staining at room temperature. No smears were positive only with staining at room temperature alone. Out of 58 smears positive by both methods, 25 had equal number of AFB in both smears, 22 had more AFB in smear stained at 37 degrees C and 11 had greater number of AFB in smears stained at room temperature. Our study, therefore, indicates that rhodamine auramine staining at 37 degrees C is superior to conventional auramine method at room temperature for detecting AFB in sputum smears.

Usefulness of Kato-Katz and trichrome staining as diagnostic methods for parasitic infections in clinical laboratories

To assess the effi cacy of the Kato-Katz technique and to re-evaluate other routine procedures conducted in the Microbiology Clinical Laboratory at Sultan Qaboos University Hospital [SQUH] and to throw light on the prevalence of intestinal parasitic infections among a small group of food handlers in Muscat. Faecal samples collected from food handlers were examined using fi ve parasitological techniques. Out of 100 faecal samples, 53 were positive for one or more of 11 intestinal parasites. The Kato- Katz and trichrome stain methods were found superior to the other techniques in detecting helminthic and protozoan infections, respectively. The auramine stain was useful only in detecting Cryptosporidium parvum oocysts. A combination of trichrome stain and Kato-Katz techniques for stool examination is suffi cient and recommended for busy laboratories; auramine stain should be applied only to samples with suspected cryptosporidal infections

The value of fluorescence microscopy of auramine stained sputum smears for the diagnosis of pulmonary tuberculosis.

Laboratory diagnosis of pulmonary tuberculosis rests on the bacteriological examination of sputum smears stained by the Ziehl-Neelsen (ZN) method for acid fast bacilli (AFB). In the present study, we have compared light microscopy of ZN stained smears with that of fluorescence microscopy of sputum smears stained by auramine-phenol flurochrome dye for detection of AFB in sputum specimens. Sputum specimens from a total of 2,600 clinically suspected and diagnosed cases of pulmonary tuberculosis were examined by both the methods. Sputum specimens from a total of 1,104 patients were found to be positive for AFB. These included sputa from 975 (37.5%) patients positive for AFB by both ZN and auramine staining methods and sputa from an additional 129 (4.96%) patients positive for AFB by auramine staining only. Thus auramine staining of sputum smears in comparison to that of ZN staining is a better method of sputum microscopy for demonstration of AFB in sputum specimens. Fluorescence microscopy is relatively more sensitive and has the added advantage of allowing a large number of sputum specimens to be examined in a given time, in laboratories equipped with a fluorescent microscope.

Comparison of Stain Methods with PCR and Culture for the Detection of Mycobacterium Tuberculosis in the Sputum / 대한임상병리학회지

BACKGROUND: Currently, many laboratories have selected several different methods for the detection of M. tuberculosis in the sputum. To select efficient method for clinical laboratories among the various methods, we compared the results of several methods. METHODS: Total 72 sputums were examined by the six combinations of stain methods. The samples were constructed as follows on the result of direct smear ZN stain; negatives (26), traces (3), 1+(9), 2+(12), 3+(12) and 4+(10). The true positives were determined after close evaluation of the clinical, radiological and other laboratory findings. RESULTS: The sensitivities and specificities of each methods were as follows; direct smear ZN stain were 83.6% and 100%, direct smear Auramine stain were 90.9% and 100%, centrifugation ZN stain were 94.6% and 100%, centrifugation Auramine stain were 98.2% and 94.1%, cytocentrifugation ZN stain were 96.4% and 100%, cytocentrifugation Auramine stain were 100% and 64.7%, nested PCR were 80% and 94.1% and culture were 67.3% and 100% respectively. CONCLUSIONS: Concentration method by centrifugation is suitable for routine laboratory if enough centrifugal force were engaged. Auramine stain is more suitable staining method than ZN stain in direct smear but not in concentrated smear because it has the potency of false positivity. The PCR assay is thought to be not only a fast, sensitive method but also a specific method for the direct detection of M. tuberculosis in the sputum. The culture method using Ogawa media is specific but not sensitive.

Non acid-fast fluorescent M. leprae (?) in skin smears from leprosy patients.

Smears from 74 known smear negative cases of leprosy were examined after staining with Auramine 'O'. 40.54% cases were positive for fluorescent bacilli. 60.52% of cases on treatment and 19.44% cases after RFT had fluorescent bacilli in the skin smears. Results suggest the possibility of a non acid-fast fluorescent positive variant of M. leprae.

Auramine staining in histopathology sections.

Auramine staining was done on 65 histopathological sections from different types of treated leprosy cases which were negative by Fite-Farraco stain. All the sections except one showed auramine positive organisms. The organisms were mostly coccoid except in BL/LL cases where beaded bacilli could be seen.