Application of cetylpyridinium chloride and sodium chloride decontamination method for recovery of Mycobacterium tuberculosis from clinically suspected cases of pulmonary tuberculosis

Diagnostic Microbiology and Infectious Disease 77 (2013) 150–157

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Application of cetylpyridinium chloride and sodium chloride decontamination method for recovery of Mycobacterium tuberculosis from clinically suspected cases of pulmonary tuberculosis☆ Pottathil Shinu a,⁎, Varsha Singh a, Anroop Nair b, Priya Mehrishi a, Sonia Mehta a, Ekta Joshi a a b

Department of Microbiology, M.M.I.M.S.R., MM University, Mullana, Ambala 133207, India Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, KSA

a r t i c l e

i n f o

Article history: Received 11 April 2013 Received in revised form 12 June 2013 Accepted 14 June 2013 Available online 6 August 2013 Keywords: Pulmonary tuberculosis Direct microscopy Cetylpyridinium chloride N-acetyl L-Cystine Decontamination

a b s t r a c t The study was designed to compare the efficacy of cetylpyridinium chloride (CPC) and sodium chloride (NaCl) decontamination method with N-acetyl L-Cystine (NALC) and sodium hydroxide (NaOH) decontamination (the reference method) method for the recovery of Mycobacterium tuberculosis (MTB) from clinically suspected cases of pulmonary tuberculosis. To evaluate CPC-NaCl and NALC-NaOH decontamination methods, sputum specimens (n = 796) were studied (culturing on Löwenstein-Jensen medium), and the performances were compared. The CPC-NaCl decontamination method demonstrated a sensitivity, specificity, negative predictive value, and positive predictive value of 97.99%, 87.53%, 70.19%, and 99.32%, respectively, when compared to NALC-NaOH decontamination method. In summary, CPC-NaCl decontamination method effectively detected significantly higher number of MTB cases (n = 208) than NALC-NaOH decontamination method (n = 149) particularly in sputum with scanty bacilli and smear-negative cases, indicating the potential of CPC-NaCl decontamination method to preserve paucibacillary cases more efficient than NALCNaOH decontamination method. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Tuberculosis (TB) is one of the leading causes of morbidity and mortality worldwide, affecting one-third of world's population. Geographically, the incidence is much higher in Southeast Asia (India and China together account for nearly 40% of the global TB cases) (WHO, 2012). However, the prevalence and transmission of TB and other mycobacterial infections may be considerably reduced by detecting in the early stage of the disease. The global TB control programs use direct microscopy for rapid detection of acid-fast bacilli (AFB) directly from sputum (RNTCP, 2009). Unfortunately, the direct microscopy has low sensitivity when compared to culture (Bonnet et al., 2011; Shinu et al., 2013). Although culture technique is more sensitive (detect 10–100 bacilli/mL) (Biotec Laboratories. 2000), the decontamination procedure [N-Acetyl L Cystiene (NALC)– sodium hydroxide (NaOH)] used for the culture is toxic (2–4% NaOH) to Mycobacterium tuberculosis (MTB) and other non-tuberculous mycobacteria (NTM) as well (Ratnam et al., 1987). Further, the NALC solution is unstable (to be utilized within 24 h after preparation) and also requires additional training to perform, which restrict their extensive use worldwide (Ganoza et al., 2008; Kent and Kubica, 1985a). ☆ Conflict of interest: The authors declare that there is no conflict of interest. ⁎ Corresponding author. Tel.: +91-999-605-9545, +91-173-130-4557 (Off). E-mail address: [email protected] (P. Shinu). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.06.021

In the previous years, various methods have been attempted to improve decontamination of sputum specimens for the isolation of Mycobacterium. However, all these methods were either restricted or not evaluated under field conditions (Carricajo et al., 2001; Chakravorty and Tyagi, 2005; Farnia et al., 2002; Ganoza et al., 2008; Thornton et al., 1998). Therefore, the mycobacteriology laboratories require an alternative technique, which is highly effective in decontaminating the clinical specimens. Literatures suggest that the effective liquefaction and decontamination of sputum specimens could be achieved when treated with 1% cetylpyridinium chloride (CPC) and 2% sodium chloride (NaCl) solutions (Pardini et al., 2005; Smithwick et al., 1975). However, no reports are available regarding the digestion and decontamination of clinical specimens (within 24 h) using CPC-NaCl decontamination method. Therefore, the current study was designed to investigate the effect of CPC-NaCl decontamination method and NALC-NaOH decontamination method (reference method) for the recovery of MTB from sputum samples collected from clinically suspected pulmonary TB (PTB) cases. 2. Materials and methods The study was conducted at the Department of Microbiology and Chest and TB department of M.M. Institute of Medical Sciences and Research, Ambala, India, a tertiary care hospital (1050 bedded) that also functions as a peripheral centre for the Revised National Tuberculosis Control Program (RNTCP). All patients, presenting with

P. Shinu et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 150–157

productive cough for more than 2 weeks or longer and who gave a written informed consent to participate in the study and for human immunodeficiency virus (HIV) testing, were recruited prospectively for this test evaluation study. In addition, the other clinical information like persistent low-grade fever (N37 °C for 2 weeks), night sweats, loss of appetite, dyspnoea, chest pain, and haemoptysis was also recorded. However, patients taking or who has taken anti-TB treatment in the last 6 months were excluded. A total of 2 sputum specimens (spot sample at clinic and morning sample at home) from each patient were collected. Further, all the specimens were transported immediately to the microbiology laboratory (Department of Microbiology, M.M.I.M.S.R) and refrigerated (4–8 °C) overnight. After receiving the second sputum samples, both sputum samples, specifically spot sample at clinic and morning sample at home, were mixed by pouring the sample from one bottle to another containing undrilled glass beads (1–2 mm, Himedia, Mumbai, India). The mixing of each specimen was performed to obtain similar quality and adequate quantity for performing all the tests; in fact, during each sampling, most of the patients expectorated insufficient quantity (3–4 mL) of sputum and were inadequate to perform all the techniques. Thereafter, the sputum specimens were subjected to homogenization (using a Pasteur pipette for one min) and vortex for 2 min to ensure the uniform distribution of bacilli. After homogenization, the sputum specimen was divided into 3 parts, the first 0.5 mL was used for direct Auramine ‘O’ smears, the aliquots of 2–3 mL each in a 50-mL and 14-mL Falcon tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for NALC-NaOH and CPC-NaCl decontamination methods, respectively.

151

3.3. CPC-NaCl decontamination method

3. Laboratory methods

The second aliquot of the sputum specimen (2–3 mL) was subjected to CPC-NaCl decontamination method. The decontamination procedure was carried out as described by Smithwick et al. (1975) with minor modifications. To the 2–3 mL of sputum in the 14-mL Falcon tube (Becton Dickinson), an equal volume of CPC working solution [prepared by dissolving 1 g of CPC (Himedia) and 2 g of NaCl (Himedia) in 100 mL of distilled water] was added and vortex for 1 min. The tubes were then incubated at 37 °C for overnight. No sterilization techniques were used to check the contamination as the CPC-NaCl itself is a self-sterilizing solution After incubation, distilled water was added till the brim of Falcon tube. The mixture was vortexed for 1 min and then centrifuged at 3000 × g for 20 min (R-83; Remi, Mumbai, India). The supernatant was carefully discarded into a splash-proof container containing 5% phenol solution. Then, the pellet was re-suspended in 200 μL of distilled water and 0.1 mL re-suspended pellet inoculated on 2 slants of LJ media (one containing glycerol and the other containing sodium pyruvate).Subsequently, the slants were incubated at 37 °C, and the cultures were monitored for MTB growth weekly once for 8 weeks. The identification of mycobacterial isolates was accomplished by positive AFB smear, growth rate, colony pigmentation and morphology, niacin test, nitrate test, catalase test (at 68 °C), and growth on LJ media containing p-nitrobenzoic acid (WHO, 1998a). The quality control of each new lot of LJ media was performed using reference strains [MTB H37Ra (ATCC 25177) and Mycobacterium kansasii (ATCC 12478)].

3.1. Direct smear

4. Statistical analysis

The direct Auramine O–stained smears were prepared as per the standard protocols recommended by RNTCP (RNTCP, 2012), and then the slides were examined using light-emitting diode fluorescent microscope (LED FM, magnification, ×400) (Zeiss primo star iLED, Gottingen, Germany) independently by 2 experienced laboratory technicians, who are routinely involved in examining Auramine O– stained smears. However, the technicians were blinded to the other technician's smear interpretation in order to measure the observer's interreader variability. The RNTCP guidelines were strictly followed while grading the smears (RNTCP, 2012): Neg = No AFB in 40 fields; Scanty = 1–19 AFB in 40 fields; 1+ = 20–199 AFB in 40 fields; 2+ = 5–50 AFB per 1 field on average for 20 fields; 3+ = more than 50 AFB per 1 field on average for 8 fields. Further, as a part of internal quality control, the study laboratory supervisor read all positive smears. In addition, the study supervisor checked 10% of the negative smears selected by systematic random sampling; specifically, only the first slide is selected at random, and subsequently, every fourth slide is selected. At the end of the study, a total of 52 slides were randomly selected, which were further subjected to re-staining using Auramine O and examined by the study laboratory supervisor. Discrepant smear results were resolved by the study supervisor; the supervisor's reading was taken as final.

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the direct AFB smear were calculated and compared with LJ culture. The following formulae were used for calculations; sensitivity was true positives/(true positives + false negatives) × 100; specificity was true negatives/(true negatives + false positives) × 100; PPV was true positives/(true positives + false positives) × 100; and NPV was true negatives/(true negatives + false negatives) × 100. The mean and SD were calculated for age and mass body index. Further, McNemar test (performed using Graphpad Prism) was used to compare the sensitivity of the NALC-NaOH and CPC-NaCl decontamination methods.

3.2. NALC-NaOH decontamination method The first aliquot of the sputum specimen [2–3 mL in 50-mL Falcon tubes (Becton Dickinson)] was mixed with equal volume of NALCNaOH solution (final NaOH concentration 1%) (Kent and Kubica, 1985b). Immediately after the decontamination process, 0.1 mL of processed specimen was inoculated on 2 slants of Löwenstein-Jensen (LJ) medium (one containing glycerol and the other containing sodium pyruvate). Then, the slants were incubated at 37 °C, and cultures were examined once in a week for a maximum period of 8 weeks.

5. Ethics statement Maharishi Markendeshwar University Ethical Committee approved the study protocol, and written informed consent was obtained from all the study participants. 6. Results Between March 2012 and August 2012, a total of 671 out of 796 screened patients were included in the study, and 671 could produce 2 specimens (one sputum in the spot at clinic and the other one at home in the morning), resulting 1342 specimens. The study profile, demographics, and clinical characteristics of the study participants are presented in Figure 1 and Table 1, respectively. Out of 1342 sputum specimens, 1118 specimens (83.31%) were macroscopically purulent or mucopurulent, 195 (14.53%) mucoid, 26 (1.93%) blood tinged, and 3 (0.22%) salivary (Figure 1). The specimens collected (second) in the morning showed significantly better quality (purulent or mucopurulent) (587/671) when compared to the first specimens collected at clinic (531/671). Out of the 671 specimens processed, a total of 149 and 208 isolates were obtained in the LJ culture using NALC-NaOH method (the reference method) and the CPC-NaCl decontamination

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Not recruited;

Total number of patients screened N = 796

Intake of anti tubercular drugs-

Included TB suspects N = 718

in the last six months

=29

Could not produce sputum

=4

Incomplete history

=8

Not fit into symptom criteria

= 37

Not submitted second sputum = 47

Revised included N = 671

Morning sputum (Sample at home)

Spot sputum (Sample at clinic) Purulent = 531(79.3%)

Purulent =587(87.46%)

Mucoid =129(19.22%) Mucoid =66 (9.84 %)

Blood tinged = 8 (1.2%) Salivary =3 (0.45%)

Blood tinged=18(2.68%)

Refrigerated overnight at 4-8o C

Pooled using Pasteur pipette and vortex for 2 min, then divided into three aliquots

Direct smear (LED FM). LED FM direct smear Positive =

122

Negative =

546

Total =

671

NALC-NaOH decontamination Method Culture on LJ MTB

=

149

NTM

=

CPC-NaCl decontamination Method Culture on LJ MTB

=

208

-

NTM

=

-

497

Negative =

446

Contaminated =

25

Contaminated=

17

Total

671

Total

671

Negative

=

=

=

Fig. 1. Study flow diagram/flow chart showing study profile.

methods, respectively. Further, 25 (3.73%) and 17 (2.53%) specimens were contaminated in NALC-NaOH and CPC-NaCl decontamination methods, respectively (Fig. 1). Interestingly, it was observed that all the specimens, which were contaminated in the CPC-NaCl decontamination method, were found to be contaminated in NALC-NaOH decontamination method as well. After exclusion of 25 (3.73%) contaminated culture results (including both NALC-NaOH and CPCNaCl method treated specimens), 23.07% (149/646) patients had a confirmed infection with MTB by culture (processed using NALCNaOH method). However, CPC-NaCl decontamination method could detect 32.19% (208/646) cases from all the clinically suspected PTB cases investigated.

Table 2 depicts the comparison of culture results (NALC-NaOH and CPC-NaCl treated specimens) as indicated by LED FM direct smear. It is apparent from the Table 2 that a significant number (n = 116) of the LED FM direct smear positive specimens (n = 122) demonstrated growth on LJ media when processed with NALC-NaOH decontamination method. However, the 6 specimens, which fail to grow in LJ media, are 2 sputum specimens with smear grades 3+ and1+, respectively; 2 sputum specimens with scanty bacilli; 2 sputum specimens with smear grades 1+ and scanty, respectively (contaminated in both NALC-NaOH and CPC-NaCl decontamination methods), and these specimens were excluded from further analysis. However, the same 4 specimens when processed with CPC-NaCl

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153

Table 1 Demographics and clinical characteristics of the study participants. Characteristics of the patient

n = 646

Demographics characteristics Out patients Gender Male Female Mean age Mean body mass index HIV infected Clinical characteristics Cough more than 2 weeks or longer Persistent low-grade fever (N37 °C for 2 weeks) Night sweats Loss of appetite Dyspnoea Chest pain Haemoptysis

n (%)

612

94.74

425 221 34.79 20.05 18

65.17 34.21 SD (5.6) SD (1.96) 2.79

646 572 486 502 357 336 24

100 88.54 75.23 77.8 55.26 52.01 3.7

decontamination method showed growth on LJ media. Interestingly, we noticed that 33 smear-negative cases (treated with NALC-NaOH decontamination method) showed growth on LJ media. However, CPC-NaCl decontamination method detected significantly higher number of additional cases (n = 59) including all the 33 cases detected using NALC-NaOH decontamination method as well. On the other hand, NALC-NaOH decontamination method could detect 3 additional cases (direct sputum smear grade 2+) when compared to CPC-NaCl decontamination method (Table 2). Further, it is also evident from the Table 2 that 95.9% and 95.08% of the LED FM direct smear-positive specimens (n = 122) have shown growth on LJ media when processed with CPC-NaCl and NALC-NaOH decontamination methods, respectively. However, the 5 specimens, which fail to grow in LJ media, are 3 sputum specimens with smear grade 2+ and 2 sputum specimens with smear grades 1+ and 1 sputum specimen with scanty bacilli. The sensitivity, specificity, PPV, and NPV of all the methods investigated in this study are summarized in Table 3. Further, the results of LED FM direct microscopy were compared with all the culture-positive specimens (both NALC-NaOH– and CPC-NaCl–treated specimens) as well. Of the 122 direct AFB smear-positive specimens, 40 (32.79%), 26 (21.31 %), 36 (29.51%), and 14 (11.46 %) specimens demonstrated confluent, moderate, moderate scanty, and scanty growth in LJ media (using NALC-NaOH decontamination method), respectively. However, among the CPC-NaCl–treated smear-positive specimens (n = 122), 51 (41.80 %), 31 (25.41 %), 28 (22.95%), and 7 (5.74%) specimens demonstrated confluent, moderate, moderate scanty, and scanty growth in LJ media, respectively (Table 2). The data in Table 4 demonstrate the time to detection of MTB and number

Direct AFB smear (LED FM)

Culture positives (%)

Positives (%) n = 122

NALC-NaOH n = 149

107 (87.7)

CPC-NaCl n = 208

131 (87.92)

77 (63.11) 45 (36.89) 35.05 (SD 4.89) 20.12 (SD 1.77) 3 (2.45)

96 53 35.96 20.13 10

122 (100%) 118 (96.72) 105 (86.06) 115 (94.26) 72 (59.01) 70 (57.38) 6 (4.92)

149 138 125 138 89 86 18

186 (89.61)

(64.44) (35.57) (SD 4.73) (SD 1.68) (6.71)

145 63 36.19 20.16 12

(100) (92.62) (83.89) (92.62) (59.73) (57.72) (12.08)

208 191 171 196 123 128 21

(69.71) (30.29) (SD4.22) (SD 1.9) (5.77) (100) (91.82) (82.21) (94.23) (59.13) (61.54 (10.09)

of AFB as indicated by LED FM direct smear scores. Of the 74 (NALCNaOH treated) and 72 (CPC-NaCl treated) culture-positive specimens with higher smear scores (both 3+ and 2+), 51 (68.92%) and 59 (81.94%), respectively, demonstrated growth within 2–4 weeks. However, the sputum specimens with 1+ or scanty smear scores or smear negative cases demonstrated growth on LJ medium [NALCNaOH treated (culture-positive specimens), n = 75 and CPC-NaCl treated (culture-positive specimens), n = 136] after 6 weeks of incubation (Table 4). Further, all the patients, who had culture positive specimens, were traced out and started on treatment. The interreader variability observed in LED FM direct AFB smear (4/122) was found to be low. Further, the study supervisor examined all the positive slides, and no discrepancy was observed. However, of the 52 negative smears (randomly selected) examined. There was no major error, but 3 minor errors were encountered (could detect 3 specimens with scanty bacilli). 7. Discussion Detection of AFB in sputum has vital and epidemiological importance in the diagnosis of TB in both low-prevalent (Migliori et al., 2006) and high-burden countries with limited resources (Keeler et al., 2006). Further, the detection of AFB in sputum implies patients with the greatest potential for MTB transmission (Behr et al., 1999; Grzybowski et al., 1975)]. In overburdened countries, direct microscopy is the most appropriate and most accessible diagnostic tool (WHO, 1998b). However, direct microscopy of sputum has a low sensitivity (analytical sensitivity 10,000 bacilli per mL), often not more than 20–70%, in comparison with culture (Bonnet et al., 2011;

Table 2 Comparison of culture results (obtained after treatment with NALC-NaOH and CPC-NaCl decontamination method) as indicated by LED FM direct smear scores. Direct smear scores 3+ 2+ 1+ Scanty Negative Total a b c d

Culture on LJ media NALC-NaOH decontamination method 3+a

2+b

1+c

Scantyd

19 18 3

9 12 3 2 5 31

3 10 17 6 9 45

2 1 2 9 19 33

40

3+, more than 200 colonies. 2+, 100–200 colonies. 1+, 20–99 colonies. Scanty, less than 20 colonies.

CPC-NaCl decontamination method No growth

3+a

2+b

1+c

Scantyd

No growth

Contaminated

1 1 1 22 25

22 21 6 2 3 54

8 9 8 6 20 51

3 7 10 8 21 49

1 1 2 3 47 54

3 1 1 441 446

1 1 1 14 17

1 2 3 491 497

Total

Contaminated

34 42 28 21 546 671

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Table 3 Comparison of diagnostic accuracy of culture (specimens treated with NALC-NaOH decontamination method) with LED FM direct smear and culture results obtained after treatment with CPC-NaCl decontamination methods. Culturea

Methods

Direct LED FM smear Positive Negative CPC-NaCl decontamination method Positive Negative a

Sensitivity (%)

95% C.I.

Specificity (%)

95% C.I.

PPV (%)

95% C.I.

NPV (%)

95% C.I.

6 491

77.85

70.17–84.06

98.79

97.26–99.51

95.08

89.15–97.99

93.7

91.17–95.56

62 435

97.99

93.77–99.48

87.53

84.22–90.23

70.19

63.41–76.21

99.32

97.84–99.82

Positive

Negative

116 33 146 3

Number of specimens positive (149) and negative (497) after treatment with NALC-NaOH decontamination method.

sweats (75.23%) and loss of appetite (77.8%), dyspnoea (55.26%), chest pain (52.01%), and haemoptysis (3.7%) were comparable with earlier reports (Bonnet et al., 2011; Daley et al., 2009). The other important features of our study design was that the overnight refrigeration of spot sputum specimen followed by mixing with second day morning sputum specimen. This mixing of specimens (both spot and morning sputum) assisted to obtain an adequate quantity of sample to perform the 2 decontamination methods from the same specimen. Thereby, minimized the bias in quality of sputum samples, most of the spot samples were mucoid or salivary, but when it mixed with early morning sample (most of the morning samples were purulent), improved the sputum quality. Further, mixing of sputum specimen using Pasteur pipette and vortexing of the specimen for 2 min with glass beads in a Falcon tube facilitated lysis of purulent portion of the sputum and equal distribution of tubercle bacilli, which subsequently yielded satisfactory culture results. In the current study, culture could detect 149 (after treatment with NALC-NaOH decontamination method) and 208 (after processed with CPC-NaCl decontamination method) MTB isolates when compared to LED FM direct microscopy (n = 122). However, none of the decontamination methods (both NALC-NaOH and CPC-NaCl decontamination method) detected NTM in LJ culture. This total absence of NTM infection among the study population could be due to decreasing incidence of NTM infections in the this geographical region (Shinu et al., 2011, 2013) or it may also be due to the excellent laboratory sterilization techniques (proper sterilization of biosafety cabinets after carrying out the smear and culture techniques) adopted in the current study, which must have in turn reduced the laboratory crosscontaminations (it is known that NTMs are ubiquitous in environment and may cross contaminate the laboratory LJ cultures) (De Groote and Huitt, 2006). Further, in the current study, the overall contamination rate was found to be very low for both the NALC-NaOH (3.73%) and CPC-NaCl (2.53%) decontamination methods, and the difference between 2 decontamination methods was not statistically significant (P = 0.6842). These results were consistent with the previous studies reported by Pardini et al wherein the contamination rate (for specimens treated with CPC-NaCl and NALC-NaOH method) was found to be 2.2% and 3.6%, respectively. Further, the reduced contamination rate among the NALC-NaOH treated specimens could

Shinu et al., 2013). Further, the increasing frequency of smearnegative PTB cases could be due to the low sensitivity of the direct AFB smear microscopy. On the other hand, culture detects 10–100 bacilli/ mL of clinical specimens and considered as gold standard for the diagnosis of TB (Biotec Laboratories, 2000). However, satisfactory culture results could be achieved using efficient decontamination methods. The specimen decontamination methods used to alleviate the isolation rate of mycobacteria also kills mycobacteria, and the percentage of organisms killed varies depending on the method used and the mycobacterial species present in the specimens (Ratnam et al., 1987). The most widely used NALC-NaOH procedure uses 2% NaOH as the decontaminant (final specimen concentration of 1%) and is a relatively mild decontamination method (Kubica et al., 1963; Kubica et al., 1964). However, Ratnam et al reported that even using this procedure, the loss of mycobacteria could be as much as 10 4 depending on the species present. Further, various decontamination methods have been attempted in the last 2 decades (Carricajo et al., 2001; Chakravorty and Tyagi, 2005; Farnia et al., 2002; Ganoza et al., 2008; Thornton et al., 1998.). However, these methods were either restricted or not evaluated in the field conditions. The above issues demand the need of an efficient reagent for the isolation of MTB from clinical specimens. Therefore, the present study was designed to evaluate the performance of CPC-NaCl decontamination method with NALC-NaOH decontamination method (the reference method) to diagnose PTB suspected cases. In the present study, clear descriptions of all enrolled patients were recorded for the reader to compare the TB suspects in our settings with other settings as well (Table 1). Of the total 646 patients, male patients (n = 425) comprised nearly double the number of females (n = 221) confirming the usual sex distribution of PTB in the Southeast Asia region. However, the age distribution showed a peak case detection among the average age of 35.05 (SD 4.89) comparable with previous reports published by WHO (WHO, 2005). Further, a body mass index of 20.05 (SD 1.96) was recorded among the clinically suspected TB patients indicating the risk factor for TB (Lönnroth et al., 2010). However, the HIV prevalence among the study population was found to be low as noticed by the previous investigators (Arora et al., 2004). The other clinical characteristics such as cough more than 2 weeks or longer (100%), persistent low-grade fever (88.54%), night

Table 4 Comparison of growth detection time of MTB (obtained after treatment with NALC-NaOH and CPC-NaCl decontamination methods) as indicated by LED FM smear scores. Growth detection time (weeks)

LED FM direct smear scores as per RNTCP guidelines 3+

2+

2 4 6 8 No growth Total

8 15 7 3 1 34a

16 12 9 4

a b

41a

NALC-NaOH decontamination method. CPC-NaCl decontamination method.

1+

7 8 10 2 27a

Scanty

5 12 3 20a

Negative

7 26 491 524a

3+

2+

1+

Scanty

Negative

10 18 5 1

18 13 5 2 3 41b

1 8 9 8 1 27b

2 16 1 1 20b

34 57 433 524b

34b

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be due to the addition of NaOH (final concentration 1%). However, the contamination rate among CPC-NaCl–treated specimens was lower than NALC-NaOH–treated specimens. This probably could be due to the inhibitory action of CPC on normal commensal organisms (Tazir et al., 1979). The mode of action of CPC-assisted decontamination is poorly understood, but structural relatedness to quaternary ammonium compounds suggests that CPC may act as membrane active agents and thereby lowering the cellular surface tension, resulting in disruption of the bacterial cell membrane and loss of selective permeability of bacterial cell, eventually results in bacterial cell death (Hiom et al., 1993; McDonnell and Russell, 1999). In general, it is known that mycobacterial species (including MTB) use glycerol as the carbon source and produce eugonic or abundant growth (Ratledge, 1982; Tepper 1965, 1968) on LJ medium. However, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti are unable to utilize glycerol as a sole source of carbon, and therefore, on medium containing glycerol, M. bovis is “dysgonic” or sparse colonies. This difficulty can be overcome by addition of sodium pyruvate (act as sole source of carbon) to the LJ media (Stonebrink, 1958; Wayne and Kubica, 1986). Therefore, 2 LJ slants were used (one containing glycerol and the other one with sodium pyruvate) for inoculating sediments from each decontamination methods. However, no M. bovis species was isolated in the current study. The absence of M. bovis infection was probably due to the awareness of population regarding the usage of domestic animals and their products (particularly about the consumption of boiled milk of cows/buffalos). In 2010, the WHO recommended to replace the conventional fluorescent microscope (CFM) with LED FM in all settings where CFM is used, and LED microscopy to be placed as an alternative for conventional Ziehl-Neelen microscopy (WHO, 2009). In the current study, the LED FM was used for direct microscopic examination of sputum, and it could obtain a sensitivity and specificity of 77% and 98%, respectively, when compared to culture. These data are comparable with earlier reports wherein the sensitivity and specificity of direct microscopy were found to be 70% and 95%, respectively (Bonnet et al., 2011). LED FM direct microscopy could detect 5 (in specimens treated with CPC-NaCl decontamination method) and 6 (in specimens processed with NALC-NaOH decontamination method) additional cases when compared to culture. This false-positive smear results could be attributed to the fact that the actual rate of recovery of viable mycobacteria from clinical samples depends on the technique of specimen processed, individual species, and the degree of vulnerability of different mycobacterial strains for alkali (NaOH toxicity in the NALC-NaOH method) (Ratnam and March, 1986). Further, it may also be due to loss of AFB while decanting the supernatants (mycobacteria have a low specific gravity and may remain buoyant during centrifugation) (Sommers and Good, 1985). Furthermore, the sputum specimens of patients treated with anti-TB drugs will reduce the number of AFBs in the sputum specimens, irrespective of the duration of treatment. In addition, the specimens with varying durations of treatment may respond different to sputum processing methods. Considering these issues, in this study, we have excluded patient who were treated with antitubercular drugs in the last 6 months. However, the LED FM direct microscopy also possesses few limitations; for instance, LED FM failed to detect 27 (detected using NALC-NaOH decontamination method) and 86 (detected using CPC-NaCl decontamination method) TB culture-positive cases, indicating the poor sensitivity of direct microscopy in diagnosis of paucibacilllary TB. Further, among HIV-positive culture-confirmed cases (NaOH-treated specimens, n = 18), the LED FM direct microscopy could detect 33.3% cases. This low detection rate is probably due to the low sensitivity of direct microscopy to diagnose HIV-positive PTB (Elliott et al., 1993). It was also noted in the current study that the LED FM direct AFB smear grades had an effect on growth detection time as well (Table 4). Of the sputum specimens with higher smear grades (3+ and 2+) processed, culture could

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demonstrate growth within 4 weeks in most of the cases. However, in the case of 1+ and scanty, culture showed growth after 6 weeks or more. This can be substantiated due to the fact that the burden of TB bacilli in the 3+ and 2+ clinical specimens is high enough to yield the growth within the specified time. However, sputum with low bacillary loads (1+ and scanty sputum specimens), the detection time is prolonged due to the paucibacillary nature of these specimens. Various decontamination methods have been developed to improve the sensitivity and timely detection of AFB in sputum (Carricajo et al., 2001; Chakravorty and Tyagi, 2005; Farnia et al., 2002; Ganoza et al., 2008; Thornton et al., 1998). Among these, most procedures use a concentration technique and a chemical decontamination step. In NALC-NaOH, the most widely used decontamination method, NALC (0.5%), acts as mucolytic agent, and sodium hydroxide (1%) acts as decontamination agent (Kubica et al., 1963; Kubica et al., 1964). In the current study, NALC-NaOH–treated specimens could demonstrate growth in 27 (18.12%) smear-negative cases. This can be substantiated due to the fact that the culture is more sensitive (analytical sensitivity 10–100 bacilli/mL) than direct microscopy. Further, NALC-NaOH decontamination method could detect 3 additional cases when compared to CPC-NaCl decontamination method. This probably could be due to the different chemical or physiological characteristics of the sputum specimens of TB patients to various specimen decontamination methods. However, NALCNaOH possesses few limitations as well; for instance, the mixture containing NALC has to be prepared daily and must be discarded if not utilized (Kent and Kubica, 1985a). In contrast, the CPC-NaCl solution can be kept at room temperature for several days without any change (Selvakumar et al., 2003). Further, a relatively prolonged growth detection time (Table 4) and decreased colony count (Table 2) were also observed for NALC-NaOH–treated specimens when compared with CPC-NaCl–treated specimens. This prolonged growth detection time and decreased colony count of NALC-NaOH–treated specimens could be likely due to the reduction in the bacillary load of clinical specimens (due to the NaOH toxicity), which subsequently reduced the number of parent cells (yielding progenies) and thereby increase the duration of colony formation (each colony consist of millions of bacteria formed from a single parent cell) and size of mycobacterial colonies. In addition, while performing decontamination using NALCNaOH method, the investigators should also be aware of the duration of exposure of TB bacilli to NaOH (should not exceed 25–30 min) as it may be injurious to some mycobacterial species (Ratnam et al., 1987). In addition, the NALC-NaOH method requires careful processing of the specimen at different steps that are time consuming and also require treatment with phosphate buffered saline (helps to slightly neutralizes the alkali and there by reduces the detrimental effect of NaOH to certain mycobacterial species) to enhance the isolation of rate of mycobacteria (Kubica et al., 1964). Literatures suggest that the treatment of sputum specimens with a solution of 1% CPC and 2% NaCl effectively liquefy and decontaminates the sputum specimens (Smithwick et al., 1975). In the present study, the CPC-NaCl–treated specimens could achieve a sensitivity and specificity of 97.99% and 87.53%, respectively, when compared to NALC-NaOH decontamination method (Table 3). This significantly higher sensitivity (McNemar test P b 0.0001) could be attributed to the low inhibitory action of CPC on mycobacterial species (Pardini et al., 2005) The data in the Table 2 illustrate that 3+, 2+, or 1+ smear-positive and CPC-NaCl– or NALC-NaOH–treated specimens do not significantly differed in culture positivity. However, for sputa reported as negative or scanty, culture positivity was significantly higher for CPC-NaCl–treated (n = 110) than for NALC-NaOH–treated (n = 50) sputa (Fisher's exact text, P = 0.0247). Therefore, all these patients obtained the advantage of diagnosis of PTB and immediate treatment, which otherwise would have remained undiagnosed. Further, these observations indicate that the CPC preserves the viability of MTB (in specimens containing relatively low bacillary

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load) considerably higher than that of NALC-NaOH decontamination method (Bobadilla-del-Valle et al., 2003; Pardini et al., 2005). Furthermore, a relatively reduced growth detection time (Table 4) and enhanced colony count (Table 2) were observed for CPC-NaCl– treated specimens when compared with NALC-NaOH–treated specimens. This reduced growth detection time and enhanced colony count of CPC-NaCl–treated specimens could be attributed to the ability of CPC-NaCl to preserve viability of TB bacilli than NALC-NaOH decontamination method (Pardini et al., 2005). The other advantages of CPC-NaCl decontamination method include stability of reagent at room temperature if kept tightly capped, easy to prepare, inexpensive [NALC (10 g) and CPC (100 g) powders ~ costs 710 and 736 , respectively], autoclaving of solution not necessary as CPC-NaCl solution is self-sterilizing, the specimens need a single centrifugation and washing with distilled water before inoculation, thereby sparing technologists' time. Further, CPC-NaCl decontamination method helps to eliminate pathogenic fungi from the sputum specimens (Phillips and Kaplan, 1976). This technique, however, possesses few limitations; for example, it requires overnight incubation of the sputum specimens with CPC-NaCl and formation of crystals while working solution, or specimens with CPC-NaCl were kept below 22 °C (Smithwick et al., 1975), reduces the detection rate of AFB (by Ziehl-Neelsen method) in sputum samples preserved in CPC solution (Selvakumar et al., 2004). Further, a negative effect of CPC-treated specimen (probably involving the levels of fluorescence) in the Bactec MGIT 960 system was reported and also recommended to discourage the use of CPC-NaCl decontamination method where the MGIT system is used (Sankar et al., 2009). However, literatures recommend the use of a liquid media (Bactec MGIT) together with a solid media (LJ media) for better recovery of Mycobacterium (Hillemann et al., 2006; Shinu et al., 2011). Therefore, even the Bactec MGIT provides falsenegative signals (for specimens treated with CPC-NaCl decontamination method); the growth may be demonstrated on LJ media. Thus, the detection rate of Mycobacterium species from clinical specimens is enhanced, which otherwise would have remained as an undiagnosed case of TB (if the specimens were treated with NALC-NaOH decontamination method). Further, it is also reported that the negative effect of CPC-treated specimens in the Bactec MGIT can be minimized by washing with phosphate buffer before inoculation. Furthermore, it is advised to prepare a smear from the culturenegative MGIT tubes before declaring a culture report as negative (Sankar et al., 2009). Considering these rationales, the use of CPC-NaCl decontamination method has to be encouraged in the mycobacteriology laboratories to improve the detection rate of MTB. In summary, CPC-NaCl decontamination method effectively detected significantly more number of (MTB) isolates particularly in sputum with scanty bacilli and smear-negative cases, indicating the potential of this method to preserve paucibacillary cases more efficient than NALC-NaOH decontamination method. Acknowledgments We appreciate the laboratory technicians Mr Gulabh Singh and Mr Manoj Sharma for their excellent technical support and the nontechnical staff for assisting in sputum collection and transportation. Finally, we are thankful to all the patients for their support. References Arora DR, Gautam V, Gill PS, Arora B, Gupta V. Haryana state in India, still a low HIV prevalence state. Sex Transm Infect 2004;80:325–6. Behr MA, Warren SA, Salamon H, Hopewell PC, Ponce de Leon A, Daley CL, et al. Transmission of Mycobacterium tuberculosis from patients smear-negative for acidfast bacilli. Lancet 1999;353:444–9. Bobadilla-del-Valle M, Ponce-de-León A, Kato-Maeda M, Hernández-Cruz A, CalvaMercado JJ, Chávez-Mazari B, et al. Comparison of sodium carbonate, cetylpyridinium chloride, and sodium borate for preservation of sputa for culture of Mycobacterium tuberculosis. J Clin Microbiol 2003;41:4487–8.

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