A Comparative Study on Ziehl-Neelsen Staining ( Light Microscopy ) , Auramine O Staining ( iLED Fluorescent Microscopy ) and Culture on LJ Media of Sputum Samples for the Diagnosis of Pulmonary Tuberculosis

Background: Tuberculosis remains as a major public health threat in Nepal and one of the leading causes of death from communicable diseases among adults. The majority of tuberculosis is pulmonary tuberculosis infecting lungs. Sputum examination is important for this as it is the major way of transmission of disease. The diagnostic procedures rely on simple and inexpensive methods, mostly of microscopy and culture examination. Therefore, the evaluation of these diagnostic approaches has great importance. We have evaluated and compared the efficacy of Ziehl-Neelsen staining, Auramine O staining and culture of sputum samples for the diagnosis of pulmonary tuberculosis. Methods: Total 299 sputum samples (170 samples from 78 Group I suspected cases with no treatment; 42 samples from 22 Group II DOTS follow-up cases; and 87 samples from 87 Group III MDR follow-up cases) were subjected to direct smear preparation, each for ZN and AO staining for 1000x light microscopy and 400x fluorescent microscopy examination respectively. Remaining samples were further processed with NALC-NaOH method for culture on modified Lowenstein-Jensen Media for detection of Mycobacterium tuberculosis. Positive smears were graded according to IUATLD/WHO guideline. Result: Out of total 299 sputum samples of all types of cases, 19.06%, 29.1% and 24.41% were found pulmonary tuberculosis positive by ZN, AO and culture respectively. The case detection rates for suspected patients with no treatment were 20%, 25.88% & 28.24%; for DOTS follow-up patients were 30.95%, 57.14% & 19.05%; and for MDR follow-up patients were 11.49%, 21.84% & 19.54%, for ZN, AO and culture respectively. The difference in their case detection rates was statistically significant (p < 0.01). No AO negative result with ZN positive samples was found. More number of paucibacillary cases was detected by AO method than ZN. 25 samples were contaminated during the culture process. Removing contaminated cultural samples and taking culture as gold standard, the sensitivity and specificity of direct microscopic examination were found to be 60.03% and 98.51% for ZN method; and 83.56% and 94.53% for fluorescent AO method respectively. The percentage of false negative by AO staining was only 16.44%, which was in sharp contrast to that of ZN (39.73%). Conclusion: This comparative study proves that AO staining (Fluorescent microscopy) is superior to ZN staining (Light microscopy) in several aspects such as efficacy, sensitivity, and false negativity. Thus the AO staining, aided with culture, can prove to be important tool for the effective and reliable diagnosis and screening of pulmonary tuberculosis.

Copyright © 2016 International Journal of Medicine & Biomedical sciences.All right Reserved.

Tuberculosis is a particular infectious disease caused by
Mycobacterium tuberculosis [1].The disease primarily affects the lungs and cause pulmonary tuberculosis diagnosed with smear positive [4].Over 80,000 people in Nepal have the disease.Nearly 20,000 people develop infectious sputum positive TB every year and about 45,000 new cases of all forms of TB occur in the same period.It is estimated that about 5,000-7,000 people die from TB, every year [5].Therefore, proper and efficient early screening and diagnosis of cases, along with good treatment strategies are needed for successful TB control.
The highly accurate diagnosis of pulmonary tuberculosis in developing countries relied mostly on light microscopy with ZN staining in most of the laboratory settings, and in very few settings with Fluorescent microscopy and culture method of sputum sample [5][6].Though culture is regarded as gold standard highly sensitive method for diagnosis allowing susceptibility testing of the isolates, poor availability of culturing facilities, the long time span (6-9 weeks) required and chances of contamination make it difficult to follow [3].Studies have shown that the rapid diagnosis with smear microscopy is highly specific in areas of high TB prevalence.The speed, feasibility and cost effectiveness of microscopy aided with high sensitivity make it valuable tool for TB diagnosis, but for this technique the yield requirement is between 5,000 to 10,000 organisms per ml sample [7][8].The use of fluorescent microscopy enhances the visualization by providing contrast coloration to the bacilli present in the sample [9].There are also other methods like ELISA, Mycodot, X-ray, which are not confirmatory tests, and similarly, the advanced techniques like BACTEC, gene probe, PCR, gas liquid chromatography are costly and only available in very few laboratories and lack in specificity [10][11].
Therefore, this study only focuses on the comparison of the commonly used diagnostic methods in developing countries such as microscopy, and culture, which is very useful to give idea about the effectiveness of diagnosis.

Sample Collection and Transportation
Individual were advised to rinse mouth with water, cough forcibly, and collect sputum in mouth and spit carefully and aseptically in to a sterile, wide mouth, unbreakable container.The lid was tightened properly and the container was transported to the lab immediately.Three sputum specimens were collected i.e. one spot, one early morning and one another spot when patient returns next day for suspected out-patients visiting GENETUP.For in patients of GENETUP, two early morning samples were collected.
Similarly, one early morning sputum sample was collected for MDR patients and maximum two samples were collected for follow-up patients visiting GENETUP.The samples were transported in the tray for processing within the day of collection.Early morning sputum samples, collected at patient's home, were transported by patients or their relatives to the laboratory.Samples from remote areas were transported through shipping in multilayered plastic packs.The minimum amount of sputum sample was 2 ml, the optimum being 5 ml.At the time of sample collection, data was collected with written consent from individuals taking part in this study.

Sample processing and smear preparation
The processing of sample was carried out in a biosafety cabinet (BSL-2) and subjected to two direct smear preparations from each sample with the help of sterile wooden stick in clean, dry, thin (1mm) glass slides free from scratches; as scratches may retain flurochrome and appear as fluorescent artifacts.The smear was air dried and fixed by flaming.The remaining sample was further processed with NALC-NaOH method for culture preparation on modified Lowenstein-Jensen media.

Staining and microscopic examination
From each sample, Ziehl-Neelsen staining and Auramine O staining was done on two prepared smear slides from each sample.Both slides were blinded by the reader and were read by two independent readers.For the ZN staining, solutions were prepared and staining was done as per the technique described in standard operative procedure [5].Acid Fast Bacilli (AFB) was demonstrated by oil immersion lens using 1000x magnification in light microscopy.The AFB appeared as red rods against blue background (Fig. 1).
For the Fluorescent AO staining, solutions were prepared and staining was done as per the technique recommended by WHO [1,7].The fluorescence microscope used is the iLED Primostar Zeiss with an attached illuminator manufactured by Karl Zeiss and is fitted in a dark room.

Smear reporting
Evaluation of smear was done by standardized procedure of scanning by grid pattern, proposed by National Tuberculosis Institute, with following reporting standards criteria proposed by IUATLD/WHO [8].In this study, 2+, and 3+ were classified as multibacillary and scanty, and 1+ as paucibacillary.2).

Quality control
Mycobacterium fortuitum was inoculated in modified LJ media in two tubes and incubated for 48 hours.Normally it should show growth after 48 hours.If there is no growth after 48 hours, the media should not be used for culture.
The sterility of media was also checked after incubation of 48 hours and any contaminated tubes must be removed from the media box.The study protocol was approved by the Ethical Review Committee of the institution.

RESULTS
Table 3 shows that, out of total 299 sputum samples of     time, it is regarded as gold standard for diagnosis and useful for Drug Susceptibility Test (DST).ZN stain can detect bacilli when they are in the order of 10 5 /ml of the sputum, whereas a more sensitive AO stain can detect in the order of 10  Similarly, the culture method is prerequisite for standard diagnosis and testing of drug.
The study clearly indicated that the case detection rate (efficacy) of fluorescent microscopy (AO stain) is remarkably higher than that of ZN (light microscopy) ,with aided advantages of less eye strain, easy visualization, less time consuming and even detection of low number of bacteria (paucibacillary cases) in comparison to ZN method.As the screening was done under lower power magnification (400x) i.e. larger area per field than ZN method (1000x), less time is consumed for examination of same area by AO method.Taking culture as gold standard, there is high agreement between AO staining and culture than that of ZN staining and culture.The use of AO staining alone could be reliable microscopic method as there were no cases of ZN positive where AO was negative.But ZN staining alone missed most of the positive pulmonary cases with respect to AO and culture.
In case of Fluorescent microscopy, though the capital cost is higher for expensive instrumentation, the overall cost with large number of sample processing by limited manpower makes it no difference in cost.Thus, it has been recommended as very effective method of choice in high risk areas, where a large number of sputum samples are to be examined.
Similarly, culture examination is very efficient, most The prospective case-control study was conducted in the Nepal Anti-Tuberculosis Association (NATA), GENETUP lab, Kalimati, Kathmandu from July 2010 to October 2010, on sputum samples from patient visiting at the GENETUP lab with suspected pulmonary tuberculosis cases without treatment and follow-up cases after DOTS and MDR treatment.A total of 299 sputum samples (170 from 78 suspected cases, 42 from 22 follow-up cases with DOTS treatment and 87 from Follow-up cases with MDR) were collected aseptically.
The film is examined at 400x magnification.The tubercle bacilli are seen as yellow luminous slight curved rods in a green background (Fig1) which was further cross examined by another examiner.Artifacts are distinguished from bacilli by their hazy yellow or grey coloration lacking reddish tinge and were poorly delineated[3].

Fig. 1 :
Fig. 1: (A) ZN stained sputum showing red TB bacilli in blue background as 1000x.(B) AO fluorescence stained sputum showing yellow luminous TB bacilli in green background as 400x.
pulmonary tuberculosis, including general suspected cases and with follow-up cases having treatment, 19.06%, 29.1% and 24.41% were found pulmonary tuberculosis positive by ZN, AO and culture respectively.In respective category of cases, the case detection rates for general suspected patients without treatment were 20%, 25.88% & 28.24%; for follow up patients of DOTS treatment were 30.95%,57.14% & 19.05%; and for MDR follow-up patients were 11.49%, 21.84% & 19.54% for ZN, AO and culturerespectively.The rate of case detection is significantly higher with culture in case of general suspected cases without treatment followed by AO and ZN.But the followup cases showed decrease in case of detection rate by cultural methods and in these, AO staining proved to be more sensitive than the other two.The difference in their case detection rates was statistically significant (p < 0.01).

Figure 3 : 3 . 1 )Fig. 4 :
Figure 3: Evaluation of ZN and AO microscopy with respect to culture in (3.1)General suspected cases (3.2) Follow-up patients with DOTS treatment (3.3) Follow-up MDR patients (3.4)Total sputum samples of all cases of pulmonary tuberculosis In a study conducted by Kumar et.al., 85% of culture positive cases could be diagnosed by microscopy alone.In this study, the diagnosis by ZN light microscopy alone was achieved in 60.03% (44/73) of the culture positive cases which is remarkably increased with AO fluorescent microscopy alone, 83.56% (61/73) of the culture positive cases.All cases of ZN positive were found to be positive by AO fluorescent method.Hence, it is apparent that the number of cases missed by ZN stain is higher which can be detected by AO fluorescent method.CONCLUSION The most commonly and routinely used method of diagnosis of pulmonary tuberculosis is the sputum examination by microscopy and culture method.The cost effectiveness and easy to perform with limited resources made these methods most prominently approached in resource poor countries, where the detection by molecular techniques are very limited and not usually done.In microscopy, there are conventional light microscopy (ZN staining) and fluorescent microscopy (AO staining).These two methods differ with each other in several aspects such as efficacy, cost of processing and instrumentation.Copyright © 2016 International Journal of Medicine & Biomedical sciences.All right Reserved.
reliable and gold standard technique, which is the prerequisite for determining strength of bacteria to antibiotics and differentiating from other non-pathogenic mycobacterium by growth rate and biochemical test.But the requirement of longer time duration (6-8 weeks), high cost, well trained manpower and chances of contamination are making it difficult to implement, in developing countries.Thus culture along with fluorescent microscopy should be method of diagnosis of pulmonary tuberculosis.Hence, the correct diagnosis of pulmonary tuberculosis requires combination of AO (fluorescent microscopy), culture and biochemical analysis.

Table 4 : Comparison between ZN and Fluorescent AO microscopy in total cases.
Copyright © 2016 International Journal of Medicine & Biomedical sciences.All right Reserved.