Sputum processing, Cattamanchi et al, UCSF, 20 August, Cattamanchi A a,b*, Davis JL a,b, Hopewell PC a,b, Steingart KR b

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1 Do sputum processing methods increase the accuracy of smear microscopy for diagnosing pulmonary tuberculosis? An updated systematic review and meta analysis Cattamanchi A a,b*, Davis JL a,b, Hopewell PC a,b, Steingart KR b a Division and Critical Care Medicine, San Francisco General Hospital, University of California, San Francisco, USA b Francis J. Curry National Tuberculosis Center, San Francisco, University of California, San Francisco, USA *Correspondence Adithya Cattamanchi, MD Assistant Professor of Medicine San Francisco General Hospital, University of California, San Francisco 1001 Potrero Avenue, Room 5K1, San Francisco, CA acattamanchi@medsfgh.ucsf.edu 1

2 TABLE OF CONTENTS Background.. 4 Methods 4 Results... 8 Discussion Conclusions.. 16 References.. 17 List of Tables Table 1 Characteristics of included studies with culture as the reference standard. 19 Table 2 Processed versus direct microscopy: pooled sensitivity and specificity.. 22 Table 3 Processed versus direct microscopy: pooled sensitivity and specificity differences. 23 Table 4 GRADE Evidence profiles. 24 Table 5 GRADE Summary of findings.. 29 Supplementary Table 1 Characteristics of included studies without a reference standard.. 45 Supplementary Table 2 Pooled Sensitivity and Specificity Differences, Generalized Estimating Equation Model 48 List of Figures Figure 1 Flow of studies 33 Figure 2 Analysis flow chart 34 Figure 3 Bleach centrifugation versus direct microscopy Figure 3A Study quality.. 35 Figure 3B Sensitivity and specificity.. 35 Figure 3C Sensitivity and specificity differences Figure 3D Hierarchical summary receiver operating characteristic curves Figure 4 Bleach sedimentation versus direct microscopy Figure 4A Study quality

3 2Figure 4B Sensitivity and specificity Figure 4C Sensitivity and specificity differences. 38 Figure 4D Hierarchical summary receiver operating characteristic curves.. 38 Figure 5 NALC NaOH centrifugation versus direct microscopy Figure 5A Study quality Figure 5B Sensitivity and specificity Figure 5C Sensitivity and specificity differences. 40 Figure 5D Hierarchical summary receiver operating characteristic curves.. 40 Figure 6 Na OH centrifugation versus direct microscopy Figure 6A Study quality.. 41 Figure 6B Sensitivity and specificity Figure 6C Sensitivity and specificity differences. 42 Figure 6D Hierarchical summary receiver operating characteristic curves. 42 Figure 7 HIV, any processing method versus direct microscopy Figure 7A Study quality.. 43 Figure 7B Sensitivity and specificity Figure 7C Sensitivity and specificity differences. 44 Appendices Appendix A. Literature search strategy Appendix B. Data extraction form 51 Appendix C. QUADAS, a tool for quality assessment

4 BACKGROUND Sputum smear microscopy is the most widely used test for diagnosis of pulmonary tuberculosis and the only test routinely available in high burden countries. In these settings, most laboratories prepare Ziehl Neelsen stained smears from unconcentrated sputum (direct smears). Direct smear microscopy is inexpensive, rapid, and highly specific in tuberculosis endemic settings. However, direct smear microscopy has poor sensitivity (range 20 80%), particularly in HIV co infected patients. Sputum processing by various physical and/or chemical methods has been evaluated as a means to increase the sensitivity of smear microscopy. A systematic review (83 eligible studies) published in 2006 found that sensitivity was increased by approximately 15 20% when microscopy was performed after physical processing of sputum by either centrifugation (14 studies) or overnight sedimentation (4 studies).[1] In addition, the review found moderate evidence supporting increased sensitivity when sputum was processed using bleach and centrifugation (6 studies). Since the publication of this review, the evidence base has grown and approaches to meta analyses of diagnostic tests have changed. In this updated review, we employ state of the art methods to summarize the literature comparing the diagnostic accuracy of direct smear microscopy with smear microscopy performed after chemical and/or physical processing of sputum among patients being evaluated for pulmonary tuberculosis. METHODS We followed standard guidelines and methods for systematic reviews and meta analyses of diagnostic tests.[2 5] Search methods. To update this systematic review, we searched the following electronic databases for primary studies in all languages: PubMed (2005 through 7 June 2009), EMBASE (2005 through 7 January 2009), Biosis (2005 through 7 January 2009), and Web of Science (2005 through 7 January 2009). The search terms included tuberculosis, Mycobacterium tuberculosis, acid fast bacilli, sputum microscopy, bacteriology, sensitivity and specificity, sputum concentration, and direct microscopy. We also searched reference lists of eligible papers and related 4

5 reviews to identify additional potentially relevant studies and contacted researchers in the field to identify unpublished or ongoing studies. Selection criteria. As in the previous review, studies were included that compared direct sputum smear microscopy to microscopy following sputum processing by chemical and/or physical methods.[1] The following types of studies were excluded: (1) studies focused on nontuberculous mycobacteria or extrapulmonary tuberculosis; (2) studies in which microscopy was used primarily to monitor treatment response; (3) studies of cost effectiveness that did not report diagnostic performance of direct and processed microscopy; (4) studies using different staining and/or microscopy methods to compare direct and processed sputum smears; (5) studies reporting insufficient data to determine either the smear positive proportion (when no reference standard was used) or sensitivity and specificity (when a reference standard was used); (6) abstracts and reviews. In addition, unlike the previous review, the following studies were explicitly excluded: (7) studies in which the microscopy stain was not reported; (8) studies with fewer than ten participants; (9) studies that only performed processed microscopy when direct microscopy results were negative; and (10) studies that used dithiothreitol for sputum digestion, a chemical not widely available to tuberculosis programs in low income countries. Two reviewers (AC and KRS) independently screened the accumulated citations for relevance and then independently reviewed full text articles using the pre specified eligibility criteria. Disagreements about study selection were resolved by consensus. A list of excluded studies with reasons for exclusion is available from the authors upon request. When studies reported comparison of direct microscopy to more than one sputum processing method, each comparison was considered a unique study. We reviewed and extracted data from studies that did and did not use a reference standard (mycobacterial culture) against which to compare the accuracy of direct and processed microscopy results. However, in this review, we focus our analysis only on studies that used a reference standard. Characteristics of studies that did not use a reference standard are provided in Supplementary Table 1. 5

6 Data extraction. We used a standardized data extraction form that was initially pilot tested with a subset of eligible studies. Two reviewers (AC and KRS) independently extracted data on the following characteristics: study design, methodological quality, sputum collection characteristics, smear preparation, sputum stain, chemical and physical methods for sputum processing, use of a reference standard (mycobacterial culture), and microscopy results. Interreviewer agreement on microscopy results was approximately 100%. We resolved differences in extraction of other data by consensus. Quality assessment. We grouped studies according to their processing method (see Data synthesis and meta analysis below) and assessed study quality separately for each group using a subset of criteria from QUADAS, a validated tool for diagnostic studies.[6] We excluded the following two QUADAS criteria that were not applicable to this review: 1) Acceptable reference standard we only included studies that compared smear microscopy results to the best available gold standard (mycobacterial culture) and 2) Reference standard well described culture was performed on Lowenstein Jensen media in 86% of studies. The following 5 QUADAS criteria were satisfied by all studies included in the review: 1) Acceptable delay between index and reference tests smear microscopy and culture were performed on the same specimens; 2) Partial verification bias the reference standard was performed in all patients or specimens; 3) Differential verification bias the reference standard was performed in all patients or specimens; 4) Incorporation bias smear results were not included in the reference standard test; and 5) Relevant clinical information we assumed laboratories had access to the same information as is routine in clinical practice. In addition, no study reported information to judge whether the reference standard was interpreted without knowledge of index test results. Two reviewers (AC and KRS) assessed the remaining 6 QUADAS items for each study and resolved differences by consensus. Data synthesis and meta analysis. As significant heterogeneity is expected between studies of diagnostic tests and summary estimates of diagnostic accuracy may not be meaningful when heterogeneity is present,[7] we adopted the 6

7 following overall approach. First, we decided a priori to separately synthesize data for each group of at least 4 studies classified according to chemical and physical processing method. Second, for each processing method, we present forest plots to provide a visual assessment of heterogeneity. We also report the amount of variation attributable to heterogeneity (I squared value) and statistically test for heterogeneity (chi squared test). Third, we use a random effects model to calculate pooled estimates of diagnostic accuracy but interpret pooled results cautiously when heterogeneity is present. Finally, when there are sufficient studies, we perform sub group analyses to explore sources of heterogeneity. For each study, we calculated the sensitivity (proportion of positive smear results among patients with positive mycobacterial cultures) and specificity (proportion of negative smear results among patients with negative mycobacterial cultures) of direct and processed microscopy along with their 95% confidence intervals (CI). We generated forest plots to display sensitivity and specificity estimates for each study using Review Manager 5.0 (The Nordic Cochrane Center, Copenhagen, Denmark). We also defined the following effect sizes for each study: 1) the difference in sensitivity between direct and processed microscopy and 2) the difference in specificity between direct and processed microscopy. We calculated a 95% CI for the effect size using McNemar s test for paired proportions. This calculation assumed maximal correlation between direct and processed microscopy results among either culture positive patients (for sensitivity difference) or culture negative patients (for specificity difference). To validate this assumption, we repeated the calculation of pooled sensitivity and specificity differences described below using a generalized estimating equation (GEE) model and obtained nearly identical results (See supplementary Table 2). The GEE model assumes constant correlation across studies and determines the best correlation that fits the data. We used two different approaches to calculate summary estimates of diagnostic accuracy. First, we derived pooled estimates of sensitivity, specificity, likelihood ratio positive [LR+ = sensitivity/(1 specificity) ], and likelihood ratio negative [LR = (1 sensitivty)/specificity)] for direct and processed microscopy using hierarchical summary receiver operating characteristic (HSROC) analysis.[8] The HSROC approach jointly models both sensitivity and specificity, weights studies according to the number of participants, and takes into account unmeasured heterogeneity between studies by using random effects. Next, we performed a random effects meta analysis to pool the sensitivity and specificity 7

8 differences between processed and direct microscopy reported in each study. We performed both the HSROC and random effects meta analyses in Stata IC/10.0 (Stata Corporation, Texas, USA) with the commands metandi and metan, respectively. We obtained HSROC curves using Review Manager 5.0. RESULTS Search results. The initial search yielded 1200 citations (Figure 1). Independent review of the abstracts and titles of these citations identified 67 potentially relevant papers. After independent, full text review, 50 papers were included in the analysis (32 from the prior systematic review). Because some papers reported more than one comparison, there were 77 unique comparisons (referred to as studies) of direct and processed smear microscopy. The remainder of this analysis focuses on the 36 studies that compared smear microscopy results to a reference standard (mycobacterial culture). Study characteristics. There was significant variation in study setting, study design, processing methods, and classification of smear results (Table 1). 18 (50%) studies were conducted in high tuberculosis burden countries and 7 (19%) studies included confirmed HIV infected patients. Three different physical processing methods (centrifugation, sedimentation, and xylene flotation) were evaluated and within these methods, the duration and speed of centrifugation and the duration of sedimentation varied across studies. Seven different chemical processing methods (bleach, N acetyl L cysteine sodium hydroxide [NALC NaOH], sodium hydroxide [NaOH], bleach ammonium sulfate [BAS], hypertonic saline [H S], universal sample processing [USP], and C18 Carboxypropylbetaine [CB 18] were evaluated, and within these methods, the concentration of and the duration of exposure to the chemical agent varied across studies. Data synthesis and meta analysis. Classification of studies according to chemical and physical processing method identified four groups containing at least four studies (Figure 3). Bleach was the most common chemical processing 8

9 agent (14 studies), followed by NALC NaOH (8 studies), and NaOH alone (6 studies). In studies that used bleach, physical processing was performed by either centrifugation (9 studies) or sedimentation (5 studies). All studies that used NALC NaOH or NaOH alone physically processed specimens by centrifugation. The 4 groups identified by combining chemical and physical processing methods were: 1) Bleach centrifugation; 2) Bleach sedimentation; 3) NALC NaOH centrifugation; and 4) NaOH centrifugation. In addition to these 4 groups, we synthesized data separately for the 4 studies that reported diagnostic accuracy estimates among confirmed HIV infected patients. For each group, pooled estimates of sensitivity and specificity are presented in Table 2 and pooled sensitivity and specificity differences in Table 3. Bleach centrifugation (9 studies).[9 16] All studies were of cross sectional design. 5 (56%) studies performed bleach processing using 5% bleach, 5 (56%) studies performed centrifugation at high speed ( 2500 revolutions per minute [rpm] or 2000 g), and 7 (78%) studies examined smears using light microscopy (Ziehl Neelsen stain). Six (67%) studies reported that direct and processed smears were prepared and interpreted in the same laboratory and 4 (44%) studies reported that the laboratory in which microscopy was performed had an external quality assurance system in place. No study met all QUADAS criteria assessed (Figure 3A). Only 3 (33%) studies adequately described patient/specimen selection. The majority of studies satisfied all of the other QUADAS criteria. Sensitivity was inconsistent across studies for bleach centrifugation (range 44 73%, I squared 75%, p<0.001) and direct microscopy (range 31 72%, I squared 87%, p<0.001) (Figure 3B). Estimates of the sensitivity difference between bleach centrifugation and direct microscopy were also inconsistent (I squared 82%, p<0.001) (Figure 3C). Pooled sensitivity was higher for bleach centrifugation (65%, 95% CI 59 71%) than for direct (56%, 95% CI 49 63%) microscopy (Table 2). When sensitivity differences were pooled across studies, bleach centrifugation microscopy was 6% (95% CI 3 10%, p=0.001) more sensitive than direct microscopy (Table 3). Specificity was consistent for direct microscopy (range %, I squared 46%, p=0.06) but was more variable for bleach centrifugation microscopy (range %, I squared 88%, p<0.001). Pooled specificity was high for both 9

10 bleach centrifugation (96%, 95% CI 93 98%) and direct (98%, 95% CI 97 99%) microscopy. However, there was a small but statistically significant decrease in specificity with bleach centrifugation microscopy ( 3%, 95% CI 4% to 1%, p=0.004). The HSROC curves for the two tests crossed and were close together, indicating that neither test was superior (Figure 3D). In sub group analysis, estimates of sensitivity difference were more consistent when studies were stratified by low speed (4 studies, I squared 50%, p=0.12) versus high speed (4 studies, I squared 70%, p=0.02) centrifugation (Figure 3C). Compared to direct microscopy, bleach microscopy was 3% (95% CI 1 6%, p=0.02) more sensitive in studies using low speed centrifugation and 7% (95% CI 1 14%, p=0.002) more sensitive in studies using high speed centrifugation. However, specificity was significantly decreased with high speed ( 6%, 95% CI 11% to 1%, 0.02) but not with low speed ( 1%, 95% CI 3% to +1%, p=0.18) centrifugation. Bleach sedimentation (5 studies).[14, 17 19] All studies were of cross sectional design. Five (40%) studies performed bleach processing using 5% bleach, 3 (60%) studies performed overnight sedimentation, and all studies examined smears using light microscopy (Ziehl Neelsen stain). All studies reported that direct and processed smears were prepared and interpreted in the same laboratory. Two (40%) studies reported that the laboratory in which microscopy was performed had an external quality assurance system in place. Three (60%) studies met all QUADAS criteria (Figure 4A) [14, 19]. Of the remaining 2 studies, 1 did not enroll ambulatory TB, 2 did not adequately describe patient selection, and 1 did not report whether microscopy results were interpreted in a blinded fashion. Sensitivity was consistent across studies for direct microscopy (range 49 51%, I squared 0%, p=0.99), but not for bleach sedimentation microscopy (range 52 83%, I squared 90%, p<0.001) (Figure 4B). Estimates of the sensitivity difference between bleach sedimentation and direct microscopy were inconsistent (I squared 89%, p<0.001) (Figure 4C). Pooled sensitivity was higher for bleach sedimentation (63%, 95% CI 51 74%) than for direct (50%, 95% CI 47 53%) 10

11 microscopy (Table 2). When sensitivity differences were pooled across studies, bleach sedimentation microscopy was 9% (95% CI 4 14%, p=0.001) more sensitive than direct microscopy (Table 3). Specificity was consistent for direct microscopy (range 96 99%, I squared 8%, p=0.36) but was more variable for bleach sedimentation microscopy (range 86 99%, I squared 90%, p<0.001). Pooled specificity was high for both bleach sedimentation (96%, 95% CI 91 99%) and direct (98%, 95% CI 97 99%) microscopy. However, there was a small decrease in specificity with bleach sedimentation microscopy ( 2%, 95% CI 5% to 0%, p=0.05), though this difference was not statistically significant. The HSROC curve for bleach sedimentation microscopy was closer to the upper left corner of the plot and entirely above the curve for direct microscopy (Figure 4D). Though both the summary estimate for sensitivity and the HSROC curve favor bleach sedimentation, positive and negative likelihood ratios were higher for direct microscopy (Table 2). In sub group analysis, estimates of sensitivity difference were more consistent among studies using short term sedimentation (2 studies, I squared 0%, p=0.85) but not overnight sedimentation (3 studies, I squared 90%, p<0.001) (Figure 4C). Compared to direct microscopy, bleach microscopy was 2% (95% CI 1 4%, 0.001) more sensitive in studies using short term sedimentation and 20% (95% CI 3 37%, 0.02) more sensitive in studies using overnight sedimentation. There was no significant difference in specificity with either short term ( 2%, 95% CI 5% to 0%, p=0.05) or overnight sedimentation ( 3%, 95% CI 6% to +1%, p=0.17). NALC NaOH centrifugation (8 studies).[17, 20 25] All studies were of cross sectional design, reported similar NALC NaOH processing methods, and used high speed centrifugation. Four (50%) studies examined smears using light microscopy (Ziehl Neelsen stain). Seven (88%) studies reported that direct and processed smears were prepared and interpreted in the same laboratory and 1 (13%) study reported that the laboratory in which microscopy was performed had an external quality assurance system in place. No study met all QUADAS criteria assessed (Figure 5A). No study reported enrolling ambulatory tuberculosis, 2 (25%) studies clearly described patient selection, and 5 (63%) studies reported that microscopy results were interpreted in a blinded fashion. 11

12 Two studies were excluded when calculating pooled estimates of diagnostic accuracy because data to calculate specificity were not reported. Sensitivity was inconsistent across studies for NALC NaOH centrifugation (range 52 93%, I squared 93%, p<0.001) and direct (range 29 82%, I squared 86%, p<0.001) microscopy (Figure 5B). Estimates of the sensitivity difference between NALC NaOH centrifugation and direct microscopy were also inconsistent (I squared 94%, p<0.001) (Figure 5C). Pooled sensitivity was higher for NALC NaOH centrifugation (78%, 95% CI 62 89%) than for direct (55%, 95% CI 47 62%) microscopy (Table 2). When sensitivity differences were pooled across studies, NALC NaOH centrifugation microscopy was 19% (95% CI 7 32%, p=0.002) more sensitive than direct microscopy (Table 3). Specificity was consistent for direct microscopy (range %, I squared 50%, p=0.08) but was more variable for NALC NaOH centrifugation microscopy (range %, I squared 93%, p<0.001). Pooled specificity estimates were high for both NALC NaOH centrifugation (95%, 95% CI 78 99%) and direct (99%, 95% CI %) microscopy. There was a small decrease in specificity with NALC NaOH centrifugation ( 1%, 95% CI 2% to 0%, p=0.14), but this difference was not statistically significant. The HSROC curve for NALC NaOH centrifugation microscopy was closer to the upper left corner of the plot and entirely above the curve for direct microscopy (Figure 5D). Though both the summary estimate for sensitivity and the HSROC curve favor NALC NaOH centrifugation, positive and negative likelihood ratios were higher for direct microscopy (Table 2). NaOH centrifugation (6 studies).[10, 15, 26 29] All studies were of cross sectional design and performed chemical processing with 4% NaOH. Four (67%) studies used high speed centrifugation and 5 (83%) studies examined smears using light microscopy (Ziehl Neelsen stain). All studies reported that direct and processed smears were prepared and interpreted in the same laboratory and 1 (17%) study reported that the laboratory in which microscopy was performed had an external quality assurance system in place. One study (17%) [27] met all QUADAS criteria assessed (Figure 6A). Of the remaining 5 studies, 3 reported enrolling ambulatory tuberculosis, 2 clearly described patient selection, and 2 reported that microscopy results were interpreted in a blinded fashion. 12

13 One study was excluded when calculating pooled estimates of diagnostic accuracy because data to calculate specificity were not reported. Sensitivity was inconsistent across studies for NaOH centrifugation (range 45 94%, I squared 95%, p<0.001) and direct (range 47 80%, I squared 94%, p<0.001) microscopy (Figure 6B). Estimates of the sensitivity difference between NaOH centrifugation and direct microscopy were also inconsistent (I squared 95%, p<0.001) (Figure 6C). Pooled sensitivity was higher for NaOH centrifugation (78%, 95% CI 61 89%) than for direct (65%, 95% CI 52 75%) microscopy (Table 2). When sensitivity differences were pooled across studies, NaOH centrifugation microscopy was 8% (95% CI 1 16%, p=0.03) more sensitive than direct microscopy (Table 3). Specificity was consistent for NaOH centrifugation (range 95 99%, I squared 0%, p=0.66) and direct (range 95 99%, I squared 53%, p=0.08) microscopy. Pooled specificity was high for both NaOH centrifugation (99%, 95% CI 98 99%) and direct (99%, 95% CI 98 99%) microscopy. There was no significant difference in specificity between the two methods (0%, 95% CI 1% to +1%, p=0.53). The HSROC curve for NaOH centrifugation microscopy was closer to the upper left corner of the plot and entirely above the curve for direct microscopy (Figure 6D). In addition, NaOH centrifugation had a higher positive but not negative likelihood ratio compared to direct microscopy (Table 2). HIV, any processing method (4 studies).[11, 12, 21] Direct microscopy was compared to microscopy following bleach centrifugation in 3 studies and NALC NaOH centrifugation in 1 study. Three (75%) studies examined smears using light microscopy (Ziehl Neelsen stain). Three (75%) studies reported that direct and processed smears were prepared and interpreted in the same laboratory and 3 (75%) studies reported that the laboratory in which microscopy was performed had an external quality assurance system in place. One study (25%) [11] met all QUADAS criteria assessed (Figure 6A). Of the remaining 3 studies, none reported enrolling ambulatory tuberculosis, none clearly described patient selection, and 2 reported that microscopy results were interpreted in a blinded fashion. One study was excluded when calculating pooled estimates of diagnostic accuracy because data to calculate specificity were not reported. Sensitivity was consistent across studies for processed (range 52 55%, I squared 0%, p=0.88) and direct (range 47 51%, I squared 0%, p=0.68) microscopy (Figure 7B). However, estimates of the sensitivity 13

14 difference (I squared 82%, p=0.004) between processed and direct microscopy were inconsistent (Figure 7C). Specificity was consistent for direct microscopy (range %, I squared 0%, p=0.78) but was more variable for processed microscopy (range 89 99%, I squared 84%, p=0.002). Pooled estimates for sensitivity and specificity could not be calculated as HSROC analysis requires a minimum of 4 studies that report data to calculate both sensitivity and specificity. However, when sensitivity and specificity differences were pooled across studies, there was a small increase in sensitivity (5%, 95% CI 0 10%, p=0.04) and decrease in specificity ( 3%, 95% CI 6% to 0%, p 0.10) with processed microscopy (Table 3). GRADE Evidence Profiles and Summary of Findings GRADE stands for the grading of recommendations assessment, development and evaluation.[30] The GRADE approach can be used to grade the quality of evidence and strength of recommendations of diagnostic tests and strategies. As recommended, the quality of evidence (Table 4A 4D) and summary of findings (Table 5A 5D) are presented for each comparison of direct and processed microscopy. The overall quality of evidence was graded as very low for all comparisons, indicating that any estimate of effect is uncertain. DISCUSSION Principal findings In this systematic review, we found that four sputum processing methods to improve the diagnostic accuracy of smear were commonly reported in the literature: 1) Bleach centrifugation; 2) Bleach sedimentation; 3) NALC NaOH centrifugation; and 4) NaOH centrifugation. With the exception of NALC NaOH centrifugation, these sputum processing methods led to small (6 9%) increases in the sensitivity of microscopy compared to direct smear examination. Though NALC NaOH centrifugation resulted in a larger increase in sensitivity (19%), the confidence interval around this point estimate was wide (7 32%) and the studies in this group had serious quality limitations. We also found that sputum 14

15 processing resulted in small decreases (1 3%) in specificity, though this finding was only statistically significant with bleach centrifugation. As in the previous review, compared to direct microscopy, microscopy using sputum treated with bleach and other chemicals and concentrated by centrifugation was found to be more sensitive. However, in comparison to the findings in the previous review, the magnitude of increase in sensitivity was smaller. Possible explanations for the differences in our findings include: 1) Inclusion of additional studies published since the prior review; 2) use of random rather than fixed effects modeling; and 3) pooling of sensitivity differences using meta analytic methods rather than simple weighted averages. Strengths and limitations of the systematic review Our systematic review had several strengths. We used a standard protocol for doing the systematic review, including a comprehensive search strategy to retrieve both published and unpublished relevant studies. Two reviewers independently carried out all data extraction. We used rigorous methods for data analysis and attained similar results with several different approaches to data analysis (HSROC analysis, standard random effects meta analysis, and generalized estimating equation modeling). The major limitation of our systematic review was the considerable heterogeneity in diagnostic accuracy estimates for both direct and processed smear microscopy in most comparisons. Heterogeneity was expected given the variation in processing methods, study design, study settings, and patient selection. For bleach centrifugation, we found that centrifugation speed accounted for some of the heterogeneity in study results. However, there were either insufficient studies or we were unable to identify sources of heterogeneity for other processing methods. Due to the significant heterogeneity, the pooled estimates reported in our systematic review should be interpreted with caution. However, the similar findings obtained with multiple different analyses lend support to our overall conclusions. 15

16 In addition to inconsistent results across studies, the quality of evidence was also downgraded due to limitations in study design. At least one QUADAS criterion was not satisfied by a majority of studies in every group. CONCLUSIONS We found very low quality evidence that sputum processing by bleach centrifugation, bleach sedimentation, NALC NaOH centrifugation, or NaOH centrifugation results in small increases in sensitivity compared to direct microscopy for the diagnosis of pulmonary tuberculosis. 16

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19 Table 1. Characteristics of included studies with culture as the reference standard A. BLEACH CENTRIFUGATION Study Country Stain Chemical Method Ängeby (a), 2000 Honduras ZN Bleach 5.25% Physical Method Cent 3000 g Study Population TB / patients Health Care Setting Inpatient and outpatient Patient Selection N EQA Smears Blinded Smear Positive Criteria Convenience 303 NR NR Unclear Bruchfeld, 2000 Ethiopia ZN Bleach 5% Cent 3000 g TB Outpatient Consecutive 510 NR Yes 1 Daley (a), 2009 India AO Bleach Cent 3000 g TB Inpatient and outpatient Consecutive 178 Yes Yes 1 Eyangoh (a), 2005 Cameroon ZN Bleach 1.8% Cent 400 g TB Outpatient Consecutive 936 Yes Yes 10 Eyangoh (b), 2005 Cameroon AO Bleach 1.8% Cent 400 g TB Outpatient Consecutive 936 Yes Yes 10 Gebre (a), 1995 Ethiopia ZN Bleach 4.4% Cent speed NR TB Outpatient Convenience 100 NR Yes Unclear Merid (c), 2009 Ethiopia ZN Bleach 5% Cent 3000 g TB Outpatient Consecutive 497 Yes Yes 1 Mutha (b), 2005 India ZN Bleach 5% Cent 3000 rpm TB Outpatient Convenience 297 NR NR Unclear Wilkinson, 1997 South Africa ZN Bleach 4 5% Cent g TB Inpatient Consecutive 166 NR Yes Unclear B. BLEACH SEDIMENTATION Study Country Stain Chemical Method Farnia (b), 2002 Iran ZN Bleach % NR Physical Method Sed hours Study Population TB Health Care Setting Inpatient and outpatient Patient Selection N EQA Smears Blinded Smear Positive Criteria Convenience 430 NR Yes 1 Frimpong (b), 2005 Ghana ZN Bleach 1% Flotation TB Outpatient NR 131 NR NR Unclear Lawson, 2006 Nigeria ZN Bleach 3.5% Sed <1h TB Outpatient Convenience 752 NR Yes 1 Merid (a), 2009 Ethiopia ZN Bleach 5% Sed <1h TB Outpatient Consecutive 497 Yes Yes 1 Merid (b), 2009 Ethiopia ZN Bleach 5% Sed overnight TB Outpatient Consecutive 497 Yes Yes 1 C. NALC NaOH CENTRIFUGATION Study Country Stain Chemical Method Bahador, 2006 Iran AR NALC NaOH Physical Method Cent 3000 g Study Population TB Health Care Setting Patient Selection N EQA Smears Blinded Smear Positive Criteria NR Consecutive 903 NR NR 1 19

20 Cattamanchi, 2009 Uganda ZN NALC NaOH Cent 3000 g TB Inpatient Consecutive 279 Yes Yes 1 Farnia (a), 2002 Iran ZN NALC NaOH Cent 3000 g TB Inpatient and outpatient Convenience 430 NR Yes 1 Ganoza (a), 2008 Peru ZN NALC NaOH Cent 3400 g TB NR Convenience 94 NR Yes 1 Perera, 1999 Sri Lanka ZN NALC NaOH Cent 4000 g TB NR Convenience 163 NR NR 3 Peterson (a), 1999 USA AO NALC NaOH Cent 3200 g Specimens Inpatient and outpatient Convenience 207 NR Yes 3 Peterson (b), 1999 Smithwick, 1981 USA AR NALC NaOH USA AP NALC NaOH Cent 2500 g Specimens Outpatient Convenience 44 NR Yes 3 Cent 2000 g Specimens NR Convenience 916 NR NR 1 D. NaOH CENTRIFUGATION Study Country Stain Chemical Method Ängeby (b), 2000 Physical Method Study Population Honduras ZN NaOH Cent 3000 g TB / patients Health Care Setting Inpatient and outpatient Patient Selection N EQA Smears Blinded Smear Positive Criteria Convenience 303 NR NR Unclear Apers, 2003 Zimbabwe ZN NaOH Cent g TB Inpatient and outpatient Consecutive 256 NR NR 1 Mutha (a), 2005 India ZN NaOH Cent 1500 g TB Outpatient Convenience 297 NR NR Unclear Naganathan, 1979 India ZN NaOH Cent 4000 rpm TB Outpatient Convenience NR Yes 1 Van Deun, 2008 Peru AO NaOH Cent 3000 g TB Inpatient and outpatient Consecutive Yes Yes 1 Vasanthakum ari, 1988 India ZN NaOH Cent 3000 rpm TB Outpatient Consecutive 148 NR NR 3 E. OTHER PROCESSING METHODS Study Country Stain Chemical Method Biswas (b), 1987 India ZN Bleach % NR Physical Method Flotation Study Population TB Health Care Setting Patient Selection N EQA Smears Blinded Smear Positive Criteria NR Convenience 102 NR NR Unclear Chakravorty, 2005 India ZN USP Cent g TB NR Convenience 571 NR NR 1 Daley (b), 2009 India AO USP Cent 3000 g TB Inpatient and outpatient Consecutive 178 Yes Yes 1 Frimpong (a), 2005 Ghana ZN Bleach 1% Sed 15 hours TB Outpatient NR 131 NR NR Unclear 20

21 Ganoza (b), 2008 Peru ZN HS NaOH Cent 3400 g TB Outpatient Convenience 94 Yes Yes 1 Haldar, 2007 India ZN USP Cent 5000 g Specimens Outpatient Convenience 148 No Yes 1 Laserson (a), 2005 Laserson (b), 2005 Vietnam ZN CB 18 Cent 1818 g TB Vietnam A R CB 18 Cent 1818 g TB Outpatient NR 338 NR NR 1 Outpatient NR 338 NR NR 1 Abbreviations: AO, Auramine O; AP, Auramine Phenol; AR, Auramine Rhodamine; AR, Cent, Centrifugation; NR, NALC NaOH, N acetyl L cysteine sodium hydroxide solution; HS, hypertonic saline; NR, Not Reported; Sed, Sedimentation; TB, tuberculosis; USP, universal sample processing solution; ZN, Ziehl Neelsen 21

22 Table 2. Processed versus Direct Microscopy: Pooled Sensitivity and Specificity Sputum Processing Group No. of Studies Sensitivity* % (95% CI) Specificity* % (95% CI) Likelihood Ratio + Likelihood Ratio Processed Direct Processed Direct Processed Direct Processed Direct Bleach Centrifugation 9 65 (59, 71) 56 (49, 63) 96 (93 98) 98 (97, 99) 17 (9, 32) 31 (17, 56).36 (.31,.43).44 (.38,.52) Bleach Sedimentation 5 63 (51, 74) 50 (47, 53) 96 (91, 99) 98 (97, 99) 17 (6, 46) 23 (15, 36).38 (.27,.53).51 (.48,.54) NALC NaOH Centrifugation 6 78 (62, 89) 55 (47, 62) 95 (78, 99) 99 (96, 100) 17 (3, 86) 89 (15, 542).23 (.13,.42).45 (.38,.54) NaOH Centrifugation 5 78 (61, 89) 65 (52, 75) 99 (98, 99) 99 (98, 99) 68 (45, 101) 44 (31, 63).22 (.11,.42).36 (.26,.50) * Pooled estimates calculated using hierarchical summary receiver operating characteristic analysis 2 studies excluded (data to calculate specificity not reported) 1 study excluded (data to calculate specificity not reported) Abbreviations: CI, confidence interval; NALC NaOH, N acetyl L cysteine Sodium hydroxide; NaOH, Sodium hydroxide. 22

23 Table 3. Processed versus Direct Microscopy: Pooled Sensitivity and Specificity Differences Sputum Processing Group Number of Sensitivity Difference*, % Specificity Difference*, % Studies (95% CI) (95% CI) Bleach centrifugation 9 6 (3, 10) Bleach sedimentation 5 9 (4, 14) NALC NaOH Centrifugation 6 19 (7, 32) NaOH Centrifugation 5 8 (1, 16) HIV, Any Processing Method 3 5 (0, 10) 3 ( 4, 1) 2 ( 5, 0) 1 ( 2, 0) 0 ( 1, 1) 3 ( 6, 0) * Positive difference favors processed microscopy; pooled estimate calculated using random effects meta analysis 2 studies excluded (data to calculate specificity not reported) 1 study excluded (data to calculate specificity not reported) Abbreviations: CI, confidence interval; NALC NaOH, N acetyl L cysteine Sodium hydroxide; NaOH, Sodium hydroxide. 23

24 Table 4. GRADE Evidence profiles 4A.Bleach Centrifugation No. of studies Design Limitations Directness (Generalizability) Consistency Imprecise or sparse data Effect (20% prevalence) Publication Bias Processed Direct Absolute Difference Quality of evidence (GRADE) 1. True positives (Patients with pulmonary TB) 9 studies, 3923 participants 1 Crosssectional Moderate 2 Some uncertainty 3 Serious Serious 5 Unlikely 130 per inconsistency per 18 per Very low 2. True negatives (Patients without pulmonary TB) 9 studies, 3923 participants 1 Crosssectional Moderate 2 Some uncertainty 3 Serious Serious 5 Unlikely 768 per inconsistency per 16 per Very low 3. False positives (Patients incorrectly diagnosed with pulmonary TB) 9 studies, 3923 participants 1 Crosssectional Moderate 2 Some uncertainty 3 Serious Serious 5 Unlikely 32 per inconsistency 4 16 per 16 per Very low 4. False negatives (Patients missed with pulmonary TB ) 9 studies, 3923 participants 1 Crosssectional Moderate 2 Some uncertainty 3 Serious Serious 5 Unlikely 70 per inconsistency 4 88 per 18 per Very low 24

25 Sensitivity (95% CI): processed 65% (59, 71); direct 56% (49, 63); Specificity (95% CI): processed 96% (93, 98), direct 98% (97, 99) FOOTNOTES: 1 Five (56%) studies performed bleach processing using 5% bleach, 5 (56%) studies performed centrifugation at high speed ( 2500 revolutions per minute [rpm] or 2000 g), and 7 (78%) studies examined smears using light microscopy (Ziehl Neelsen stain). 2 About 70% of studies considered representative; majority of studies were blinded. Culture is not a perfect reference standard. 3 Absence of direct evidence about patient important outcomes: false positives, likely detriment from unnecessary treatment and may halt further diagnostic work up; false negatives, likely increased morbidity from delayed treatment. 4 Sensitivity was inconsistent across studies for bleach centrifugation (range 44 73%, I squared 75%, p<0.001) and direct microscopy (range 31 72%, I squared 87%, p<0.001). 5 Wide confidence intervals for sensitivity estimates for both processed and direct microscopy. 4B. Bleach Sedimentation No. of studies Design Limitations Directness (Generalizability) Consistency Imprecise or sparse data Publication Bias Effect (20% prevalence) Processed Direct Absolute Difference Quality of evidence (GRADE) 1. True positives (Patients with pulmonary TB) 5 studies, 2307 participants 1 Crosssectional Minor 2 Some uncertainty 3 Serious Serious 5 Unlikely 126 per inconsistency per 26 per Very Low 2. True negatives (Patients without pulmonary TB) 5 studies 1 Crosssectional Minor 2 Some uncertainty 3 Serious Serious 5 Unlikely 768 per inconsistency per 16 per Very Low 3. False positives (Patients incorrectly diagnosed with pulmonary TB) 5 studies, 2307 participants 1 Crosssectional Minor 2 Some uncertainty 3 Serious Serious 5 Unlikely 32 per inconsistency 4 16 per 16 per Very Low 25

26 4. False negatives (Patients missed with pulmonary TB ) 5 studies, 2307 participants 1 Crosssectional Minor 2 Some uncertainty 3 Serious Serious 5 Unlikely 74 per inconsistency per 26 per Very Low Sensitivity (95% CI): processed 63% (51, 74); direct 50% (47, 53); Specificity (95% CI): processed 96% (91, 99), direct 98% (97, 99) FOOTNOTES: 1 Five (40%) studies performed bleach processing using 5% bleach, 3 (60%) studies performed overnight sedimentation, and all studies examined smears using light microscopy (Ziehl Neelsen stain). 2 One study did not enroll ambulatory TB. One study did not report whether microscopy results were interpreted in a blinded fashion. Culture is not a perfect reference standard; 3 Absence of direct evidence about patient important outcomes: false positives, likely detriment from unnecessary treatment and may halt further diagnostic work up; false negatives, likely increased morbidity from delayed treatment. 4 Sensitivity was consistent across studies for direct microscopy (range 49 51%, I squared 0%, p=0.99), but not for bleach sedimentation microscopy (range 52 83%, I squared 90%, p<0.001). 5 Relatively wide confidence intervals for sensitivity of processed microscopy. 4C. NALC NaOH Centrifugation No. of studies Design Limitations Directness (Generalizability) Consistency Imprecise or sparse data Publication Bias Effect (20% prevalence) Processed Direct Absolute Difference Quality of evidence (GRADE) 1. True positives (Patients with pulmonary TB) 6 studies, 2785 participants 1 Crosssectional Serious 2 Some uncertainty 3 Serious Serious 5 Unlikely 156 per inconsistency per 46 per Very low 2. True negatives (Patients without pulmonary TB) 6 studies, 2785 participants 1 Crosssectional Serious 2 Some uncertainty 3 Serious Serious 5 Unlikely 760 per inconsistency per 32 per Very low 26

27 3. False positives (Patients incorrectly diagnosed with pulmonary TB) 6 studies, 2785 participants 1 Crosssectional Serious 2 Some uncertainty 3 Serious Serious 5 Unlikely 40 per inconsistency 4 8 per 32 per Very low 4. False negatives (Patients missed with pulmonary TB ) 6 studies, 2785 participants 1 Crosssectional Serious 2 Some uncertainty 3 Serious Serious 5 Unlikely 44 per inconsistency 4 90 per 46 per Very low Sensitivity (95% CI): processed 78% (62, 89); direct 55% (47, 62); Specificity (95% CI): processed 95% (78, 99), direct 99% (96, 100) FOOTNOTES: 1 All studies reported similar NALC NaOH processing methods and used high speed centrifugation; four (50%) studies examined smears using light microscopy (Ziehl Neelsen stain). 2 No study reported enrolling ambulatory TB. Only 2 (25%) studies clearly described patient selection; 5 (63%) studies reported that microscopy results were interpreted in a blinded fashion. Culture is not a perfect reference standard; 3 Absence of direct evidence about patient important outcomes: false positives, likely detriment from unnecessary treatment and may halt further diagnostic work up; false negatives, likely increased morbidity from delayed treatment. 4 Sensitivity was inconsistent across studies for NALC NaOH centrifugation (range 52 93%, I squared 93%, p<0.001) and direct (range 29 82%, I squared 86%, p<0.001) microscopy. 5 Wide confidence intervals for sensitivity of processed microscopy. 4D. NaOH Centrifugation No. of studies Design Limitations Directness (Generalizability) Consistency Imprecise or sparse data Publication Bias Effect (20% prevalence) Processed Direct Absolute Difference Quality of evidence (GRADE) 1. True positives (Patients with pulmonary TB) 6 studies, 4056 participants 1 Crosssectional Serious 2 Some uncertainty 3 Serious Serious 5 Unlikely 156 per inconsistency per 26 per Very Low 27