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CHAPTER I Introduction

January 31, 2019 0 Comment

CHAPTER I
Introduction:
M.tuberculosis is the most frequent member for the cause of TB among M. tuberculosis complex(MTC) members worldwide (Albert 2004). Direct and Concentrated Smear Microscopy is used for the diagnosis of Tuberculosis but the direct smear microscopy is economical, simplest and relatively easy to perform for the poor staffed (Suarezet al., 2002, van Cleeff et al., 2005, Eisenach et al.,1990) and low income laboratories. This method reports within hours of receipt of the sample and provides reliable epidemiological indicators needed for the assessment of the National Tuberculosis Control Program. The limitation is for detection at least 5×103 Bacilli per ml of sputum is required. Smear microscopy is useful for initial diagnosis of tuberculosis from respiratory specimens of Acid-Fast Bacilli (AFB).On the other hand, detection of Pulmonary Tuberculosis by concentrated smear microscopy needs well-equipped setting in the laboratories with proper staff. Centrifuge machine is necessary for concentrating the sample, but the machine is not available in remote laboratories. In AFB, staining method is best to characterize theM. tuberculosis, treated with acidic solution. Ziehl-Neelsen staining method is most commonly used which is stain the AFB with bright red on the blue background. Lowenstein-Jensen, Middle-Brook 7H11 are used as a solid media and BECTEC radiometric system as a liquid media can be useful technique for Mycobacterium identification within two weeks but this method is the slower detection for diagnosis TB.

Microscopy clearly has many advantages when it comes to speed and feasibility, and if sensitivity could be improved it has the potential to become an even more valuable tool for National TB Control Programs (NTPs) around the world. In the last decade many researchers have suggested that the performance of sputum smear microscopy can be significantly improved if sputum is liquefied with chemical reagents and then concentrated by centrifugation or sedimentation prior to acid-fast staining.Out of them, the technique using N-acetyl-L-cysteine (NALC) with 2% sodium hydroxide (NaOH) is considered to be the best. NALC acts as a strong mucus digester and the smear processed by it has less debris and a greater concentration of AFB.This method has been found to increase the sensitivity of microscopy substantially . However, it requires some level of staff training, increases time needed for diagnosis, and requires some level of biosafety arrangement to ensure the security of the lab personnel. Due to resource constraints, it is not applied in the majority of TB laboratories in developing countries.

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As microscopy is the mainstay of TB diagnosis in our country, in this study we wanted to compare the sensitivity obtained with smears for detection of AFB prepared directly from respiratory specimens (direct AFB smears) to that obtained with the parallel smears prepared from concentrate of the specimens (concentrated AFB smears) in our country context. This information may be of great help to the NTP in formulating effective TB control guidelines.

CHAPTER II
Literature Review
(Md. Raihan Chowdhury et al 2013)conducted a study out of 305 patients that submitted 915 specimens forAFB smear, culture and PCR investigation, most of the patients suffered with cough (93%), fever (66%) and smoking habit (86%) . No gender distributions were observed with 100% male patients having the age of 14 to 65. A total of 4 patients were under previously treatment or ongoing treatment condition where rest of the patients were either highly suspected or having TB. From the studied specimens, 74 were found as AFB positive and AFB negative, Scanty negative, 1+, 2+, 3+ were found at the number of 9, 19, 27 and 19 respectively. It was conferred that 92% specimens were identified as negative by direct smearmicroscopy. In the concentrated AFB smear microscopy 87 and 828 were found as positive and negative AFB respectively. Others specimens were determined as scanty positive, 1+, 2+, 3+ at the number of 13, 18, 26, and 30 respectively.

(Mohammad Khaja Mafij Uddin et al 2013)was conducted a study Out of 915 specimens 74 (8.1%) were found to be AFB positive and 841 (91.9%) were found AFB negative by direct AFB smear. Among the positive specimens, 9 (1%) were scanty positive, 19 (2%) were 1+, 27 (3%) were 2+ and 19 (2%) were 3+. In contrast, 87 (9.5%) were found to be AFB positive and 828 (90.5%) were AFB negative when smear prepared from the concentrated specimens. Among the positive specimens 13 (2%), 18 (2%), 26 (3%), 30 (3%) were scanty positive, 1+, 2+ and 3+ respectively .Grading of AFB between the slides prepared from the same sample using direct and concentrated method was compared. Of the 915 specimens, 73 (8%) specimens were positive both on direct and concentrated methods, one sample was positive on direct microscopy but was negative on concentrated method. An extra 14 (1.5%) samples were positive on concentrated method, which were negative on direct smear. More than 90% of the samples were found to be negative on both methods. Among 841 negative specimens on direct microscopy, 11 (1.3%) and 3 (0.4%) were found to be positive on concentrated methods. More than 66% of scanty, 52.6% of 1+ and 37.1% of 2+ AFB on direct microscopy were converted into 1+, 2+ and 3+ respectively on concentrated smear microscopy. No difference was found in case of high positive (3+) on both methods.Among the 915 specimens, 105 (11.5%) were found to be positive on culture.Considering 105 culture positive cases, 74 (70.5%) and 87 (82.9%) of them were AFB positive in direct and concentrated smear, respectively . About 17.1% culture positive cases were found to be negative on both direct and concentrated AFB microscopy.The sensitivity of direct and concentrated smear microscopy was different when using positive culture result as the gold standard. Applying the McNemar ?2 test, the difference between sensitivities (71% versus 83%; p?=?0.002) obtained by the two methods was found to be significant. These results showed that concentrated technique increases the sensitivity of microscopy up to 12% when performed with the same specimens. Among the 810 patients with negative cultures, the specificity (100%) was similar for both techniques.

In (a study done by Ellena M. Peterson et al 1999) total of 2,693 respiratory specimens were included in the study. Of these 67% were evaluated at the UCIMC laboratory, which serves inpatients in a tertiary-care hospital and several local outpatients clinics, and the remaining 33% were from MDL and came from clinics on Pacific islands. ? Eighteen specimens were positive for AFB with one of the smears but were culture negative. Although these specimens were from patients who had provided other specimens that were positive by culture during the evaluation period, data for these specimens were excluded from the data analysis for the smear comparison. Of the culture-positive specimens, AFB smears made from the concentrated specimen detected 56% (224 of 398) of the positive specimens. This was in contrast to the direct smear, which detected only 34% (136 of 398) of the positive specimens (P < 0.05). Of the 146 specimens culture positive for Mycobacterium spp. that were not M. tuberculosis, 19% (28 of 146) were positive when the concentrated smear was used and 8% (12 of 146) were positive when the direct smear was used. When only specimens that were culture positive for M. tuberculosis are considered, there was a significant difference in the abilities of the concentrated and directsmears to detect positive specimens (P < 0.05). Here, 78% (196 of 252) were positive when the concentrated smear was used, while 49% (124 of 252) were positive when the direct smear was used.Because of differences in patient populations and positivity rates, the smear results from the two settings were analyzed separately. At UCIMC, in parallel with the overall results presented above, there was a significant difference (P < 0.05) in the abilities of the concentrated and direct smears to detect all culture-positive specimens as well as those positive for M. tuberculosis. In contrast, at MDL, while the concentrated smear was able to detect more culture-positive specimens than the direct smear, the difference was not significant. In comparing the quantities of AFB in the two smears, the majority of smears from both settings were scored as having more AFB on the smear from the concentrated specimen?.At UCIMC multiple specimens for culture were available from several of the patients infected with M. tuberculosis, with the average number of specimens that were either smear or culture positive being 5.2; the range was from 1 to 20 specimens. Therefore, the results were analyzed separately for the first three specimens received from a patient since for most patients these would be the specimens that would aid in the initial diagnosis of the infection and that would be obtained either before or shortly after the institution of antimicrobial therapy. There were 28 patients who were culture positive for M. tuberculosis and from whom three or more specimens were received. Among these patients, the concentrated smear of the first specimen identified 20 patients who were infected with M. tuberculosis with an additional 3 patients and 1 patient found to be positive by use of concentrated smears of the second and third samples. The parallel direct smear for these patients was positive for 15 of the initial specimens, with the second direct smear identifying five more infected patients and the third one detecting one additional infected patient. No significant difference in the number of patients found to be positive with the first three samples was found between the concentrated and direct smears .Three or more specimens were received from 15 patients at MDL. The smear of the concentrated specimen identified 11, 12, and 14 infected patients from the cumulative results for the first, second, and third specimens. With the direct smear the cumulative results for the three specimens identified 9, 12, and 13 infected patients, respectively. As with the data from UCIMC, the difference between the initial direct and concentrated smears for the detection of patient’s culture positive for M. tuberculosis was not significant (P ; 0.05).By combining the results from the two settings for the initial three specimens, the concentrated smear identified more of the 42 infected patients for each of the three specimens submitted; however, the difference was not significant. The difference between the two smears was greatest with the first specimen; however, this difference was diminished with inclusion of a second and a third specimen. When comparing the abilities of the concentrated and direct smears to identify culture-positive patients, there was a significant difference in the ability of the initial three specimens to identify patients culture positive for M. tuberculosis in comparison to that of the first specimen alone (P ; 0.05).

(In a study conducted by Pranali Pingle et al, 2014)the cases used in this study covered a wide age group ranging from infants to adults and included both males and females. The total 365 samples studied consisted of sputum (n = 336 (112 × 3 samples each)), gastric aspirate (n = 15 (5 × 3 samples each)), endotracheal tube washing (n = 2) and bronchial lavage (n = 12). In the group of 112 three day pooled sputum samples 16 were positive by direct ZN staining and the positivity increased to 32 after bleach concentration. In the second group of single first morningsamples the positivity increased from 13 to 47 when the direct and bleach processed morning samples were compared.All the 5 gastric aspirate samples were negative by direct ZN staining, but after bleach concentration 2 were positive for AFB. The 2 endotracheal tube washing samples were negative by direct staining, but after bleach concentration both were positive for AFB. Of the 12 bronchial lavage samples only one was positive by direct staining, but after bleach concentration 5 were positive for AFB.

The positivity of AFB was also compared between the smears for direct staining and those processed by bleach concentration. All the 11 direct smear positive sputum samples which were in this study showed increased positivity with varying degrees depending on their initial positivity. Four samples showed scanty bacilli whose positivity rose to 1+ to 2+ after bleach concentration. Four were 1+ positive which became 2+ to 3+ after bleach concentration and three samples which were 2+ positive became 3+ after bleach concentration. Fourteen pooled sample smears which were negative by direct ZN staining gave a positivity ranging from scanty (n = 7)
Pooled and morning samples were treated by ZN staining and bleach concentration followed by ZN staining method. Out of total sample of 112, the number of positive samples by both methods has been shown, other samples were reported negative.

1+ (n = 4) and 2+ (n = 3) after bleach concentration. Twenty-seven first morning sample smears which were negative by direct ZN staining gave a positivity ranging from scanty (n = 18), 1+ (n = 5), 2+ (n = 4) after bleach concentration.

Tavares e Castro et al 2015, conducted a study in which a total of 694 patients were enrolled in this study (65% men, mean age 48.5 ± 18.6 years, 97% Portuguese), most of them exhibiting TB-related complaints. Pulmonary TB was diagnosed in 41% of the patients; 54% had non-specific radiological changes and 34% had pulmonary cavitation. The cumulative sensitivity rates of each of the three smears were 24.6%, 27.7% and 28.8% for concentrated samples and 19.3%, 20.4% and 22.5% for direct samples. The cumulative sensitivities of sputum culture were 33.3%, 37.9% and 41.8% for solid medium, and 43.9%, 51.6% and 55.4% for liquid medium. Pondering all forms of microbiological analysis, the cumulative sensitivities of each sample were 51.2%, 59.6% and 63.2%. There was an incremental yield of 8.4% for the second specimen and 3.5% for the third specimen. All sensitivity rates were higher among patients with pulmonary cavitation.

OBJECTIVE:
The objective of my study was to compare direct smear microscopy with concentrated smear microscopy in endobronchial washing samples for the detection of AFB.

CHAPTER III
Materials and Methods
Study Duration:
Six months after the approval of research proposal.

Study Design:
Cross sectional study.

Sample Size:
A total 150 sample were included in this study.

Sampling Technique:
Consecutive sampling method was used for this study.

Setting:
The study was carried out at the department of microbiology Armed Forces Institute of Pathology Rawalpindi.

Sample Preparation:
Endobronchial samples were used for the analysis of both direct and concentrated smear microscopy that is also performed with the culture method and PCR based IS6110 analysis. Unprocessed specimens were used to prepare smear, which is kept on slides. Both direct ; concentrated smear is prepared by Ziehl-Neelsen staining method using carbol fuchsin solution. For staining, the slides were arranged on a staining rack over a sink. The smear were flooded with carbol fuchsin. The carbol fuchsin is kept for 10 mintutes. The slides were washed with distilled water. The washed slides were decolorized with 20% H2SO4 for 2 to 3 minutes again washed the slides with distilled water. After washing the slides, counter stained with methylene blue and kept for 2 to 3 minutes. After washing with distilled water, the slides were dried by electrical heater.

The samples were diluted with the 4% NaOH and 0.5% N-acetyl-L-Cysteine (NALC, Sigma), and decontaminated the samples. Samples were vortex for 2 minutes and incubated at 37ºC for 30 minutes. Incubated samples were be diluted and centrifuged at 4000 rpm for 15 minutes.Deposits were used for AFB microscopy, culture method and extraction of DNA for PCR analysis
Processing and Analysis:
Collected data analyzed by SPSS version 20.

Data was store and processed by using Microsoft Excel and word version 2007.

CHAPTER IV
Results
A total of 150specimens were used for comparison.68.7% were smear negative ,12.0% showed1+,6.7% showed 2+, 7.3% showed 3+,and 5.3% showed 4+ results as shown in fig:1
D-Smear
Result FrequencyPercent
Negative 103 68.7% 1+ 1812.0%
2+10 6.7% 3+11 7.3% 4+ 08 5.3% Total 150 100% 1
Fig:1 Mycobacterial slide index in direct microscopy

Total 150 patient were included in concentrated smear .68.7% shown negative ,6.7% shown 1+,6.0% shown 2+, 10.7% shown 3+,8.0% shown 4+ result are shown in fig:2
C-Smear
Result Frequency Percent Negative 103 68.7% 1+ 106.7%
2+ 9 6.0% 3+ 16 10.7% 4+ 12 8.0% Total 150 100%
Fig:2 Mycobacterial slide index in concentrated microscopy

CHAPTER V
Discussion
This study showed that the use of the concentrated method for preparing smears for AFB microscopy increases sensitivity without a loss of specificity in identifying positive TB cases, compared to the direct method.The specimen that was positive with the direct method but negative with the concentrated method was also found to be positive by culture. It was an unusual phenomenon and theoretically, it is difficult to explain such cases. It may haveoccurred due to improper sample concentration and smear preparation, smear preparation from a negative sample accidentally, faulty staining process, or inappropriate microscopic observation.47 specimens were found to be positive in both direct and concentrated smear microscopy.

A study conducted by Barez, et al showed that the sensitivity was almost similar in both methods as described 81.6% for direct method and 82.7% for the concentrated method. In another study, Cattamanchi et al failed to find a difference in sensitivity between direct and concentrated sputum smear microscopy, the calculated sensitivity of direct and concentrated smear microscopy was not significantly different (51% vs. 52%).
In a similar study conducted by Peterson et al in two different laboratory settings (a tertiary-care laboratory and several local outpatients clinics) found that in a tertiary-care hospital the direct smear was significantly less sensitive than the concentrated smear (28% and 51%, respectively) and in the samples from outpatients of the Pacific islands the direct smear was less sensitive than that made from the concentrated specimen (82 versus 93%, respectively).

Despite some evidence that the concentrated method can be superior to the direct method, it is not being performed in peripheral TB laboratories in low-income countries, because of the following concerns: feasibility of centrifugation in settings with irregular power supply; limited human and financial resources; inadequate training capacity; lack of proper biosafety arrangements; and potential biohazard posed by centrifugation. In most resource poor countries like Pakistan where TB is common, most of the microscopy centers are using direct smear microscopy with low sensitivity for the diagnosis of TB.

However, in the light of our study findings, it may be recommend that concentrated smear microscopy can be used in place of conventional direct microscopy where appropriate facility is available, to get better diagnostic accuracy and make sure greater success of the TB control programs.
Conclusion:
Our study concludes that the concentrated method was superior to the direct method.The results showed that concentrated technique increases the mycobacterial index sensitivity of microscopy. Therefore, the national programs in high TB burden countries like Pakistan may consider incorporating the technique into their guidelines at least in the district and higher-level laboratories to improve case finding strategy.

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