sputum processing, cattamanchi et al, ucsf, august,

Sputum processing, Cattamanchi et al, UCSF, 20 August, 2009

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Do sputum processing methods increase the accuracy of smear microscopy for diagnosing pulmonary tuberculosis? An updated systematic review and meta‐analysis

Cattamanchi Aa,b*, Davis JL a,b, Hopewell PC a,b, Steingart KRb

a Division Pulmonary 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 94110 Email: [email protected]


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TABLE OF CONTENTS

Background……………………………………………………………………………………………………………………….. 4

Methods…………………………………………………………………………………………………………………………… 4

Results………………………........................................................................................................ 8

Discussion…………………........................................................................................................ 14

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

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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………………………………….………………………….. 36

Figure 3D Hierarchical summary receiver operating characteristic curves……….………….. 36

Figure 4 Bleach sedimentation versus direct microscopy

Figure 4A Study quality…………………………………………………………..……………………………….……… 37


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2Figure 4B Sensitivity and specificity……………………………………………..……………………….…… 37

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………………………………………………………….……………………………….. 39

Figure 5B Sensitivity and specificity……………………………………………..…………………………. 39


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……………………………………………..…………………………………. 41


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……………………………………………..………………………………. 43


Appendices

Appendix A. Literature search strategy…………………….…………………………………………………….. 49

Appendix B. Data extraction form…………………………………………………………………………………… 51

Appendix C. QUADAS, a tool for quality assessment …………………………............................. 58


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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


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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.


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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. Inter‐

reviewer 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


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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


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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


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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 95‐100%, I‐squared 46%, p=0.06) but was more variable

for bleach centrifugation microscopy (range 81‐100%, I‐squared 88%, p<0.001). Pooled specificity was high for both


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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 suspects, 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%)


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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 suspects, 2 (25%) studies clearly described patient selection, and 5 (63%)

studies reported that microscopy results were interpreted in a blinded fashion.


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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 79‐100%, I‐squared 50%, p=0.08) but was more variable

for NALC‐NaOH centrifugation microscopy (range 32‐100%, 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 96‐100%) 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 suspects, 2 clearly described patient selection, and

2 reported that microscopy results were interpreted in a blinded fashion.


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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 suspects, 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


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difference (I‐squared 82%, p=0.004) between processed and direct microscopy were inconsistent (Figure 7C). Specificity

was consistent for direct microscopy (range 99‐100%, 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


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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.


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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.


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REFERENCES

1. Steingart KR, Ng V, Henry M, et al. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006 Oct;6(10):664‐74. 2. Deville WL, Buntinx F, Bouter LM, et al. Conducting systematic reviews of diagnostic studies: didactic guidelines. BMC Med Res Methodol 2002 Jul 3;2:9. 3. Gatsonis C, Paliwal P. Meta‐analysis of diagnostic and screening test accuracy evaluations: methodologic primer. AJR Am J Roentgenol 2006 Aug;187(2):271‐81. 4. Leeflang MM, Deeks JJ, Gatsonis C, Bossuyt PM. Systematic reviews of diagnostic test accuracy. Ann Intern Med 2008 Dec 16;149(12):889‐97. 5. Pai M, McCulloch M, Enanoria W, Colford JM, Jr. Systematic reviews of diagnostic test evaluations: What's behind the scenes? ACP J Club 2004 Jul‐Aug;141(1):A11‐3. 6. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development of QUADAS: a tool for the quality assessment of studies of diagnostic accuracy included in systematic reviews. BMC Med Res Methodol 2003 Nov 10;3:25. 7. Lijmer JG, Bossuyt PM, Heisterkamp SH. Exploring sources of heterogeneity in systematic reviews of diagnostic tests. Stat Med 2002 Jun 15;21(11):1525‐37. 8. Rutter CM, Gatsonis CA. A hierarchical regression approach to meta‐analysis of diagnostic test accuracy evaluations. Stat Med 2001 Oct 15;20(19):2865‐84. 9. Daley P, Michael JS, S K, et al. A pilot study of short‐duration sputum pretreatment procedures for optimizing smear microscopy for tuberculosis. PLoS One 2009;4(5):e5626. 10. Angeby KA, Alvarado‐Galvez C, Pineda‐Garcia L, Hoffner SE. Improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis. Int J Tuberc Lung Dis 2000 Jul;4(7):684‐7. 11. Bruchfeld J, Aderaye G, Palme IB, Bjorvatn B, Kallenius G, Lindquist L. Sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of HIV. Trans R Soc Trop Med Hyg 2000 Nov‐Dec;94(6):677‐80. 12. Eyangoh SI, Torrea G, Tejiokem MC, et al. HIV‐related incremental yield of bleach sputum concentration and fluorescence technique for the microscopic detection of tuberculosis. Eur J Clin Microbiol Infect Dis 2008 Sep;27(9):849‐55. 13. Gebre N, Karlsson U, Jonsson G, et al. Improved microscopical diagnosis of pulmonary tuberculosis in developing countries. Trans R Soc Trop Med Hyg 1995 Mar‐Apr;89(2):191‐3. 14. Merid Y, Yassin MA, Yamuah L, Kumar R, Engers H, Aseffa A. Validation of bleach‐treated smears for the diagnosis of pulmonary tuberculosis. Int J Tuberc Lung Dis 2009 Jan;13(1):136‐41. 15. Mutha A, Tiwari S, Khubnani H, Mall S. Application of bleach method to improve sputum smear microscopy for the diagnosis of pulmonary tuberculosis. Indian J Pathol Microbiol 2005 Oct;48(4):513‐7. 16. Wilkinson D, Sturm AW. Diagnosing tuberculosis in a resource‐poor setting: the value of sputum concentration. Trans R Soc Trop Med Hyg 1997 Jul‐Aug;91(4):420‐1. 17. Farnia P, Mohammadi F, Zarifi Z, et al. Improving sensitivity of direct microscopy for detection of acid‐fast bacilli in sputum: use of chitin in mucus digestion. J Clin Microbiol 2002 Feb;40(2):508‐11. 18. Frimpong EH, Adukpo R, Owusu‐Darko K. Evaluation of two novel Ziehl‐Neelsen methods for tuberculosis diagnosis. West Afr J Med 2005 Oct‐Dec;24(4):316‐20. 19. Lawson L, Yassin MA, Ramsay A, et al. Microbiological validation of smear microscopy after sputum digestion with bleach; a step closer to a one‐stop diagnosis of pulmonary tuberculosis. Tuberculosis (Edinb) 2006 Jan;86(1):34‐40. 20. Bahador A, Etemadi H, Kazemi B, Hajabdolbaghi M, Ghorbanzadeh R, Pajand O. A comparison of direct and concentrated flurochrome‐stained smears for the detection of Mycobacterium sp. in clinical respiratory specimens. Journal of Biological Sciences 2006;6(1):103‐5. 21. Cattamanchi A, Dowdy DW, Davis JL, et al. Sensitivity of direct versus concentrated sputum smear microscopy in HIV‐infected patients suspected of having pulmonary tuberculosis. BMC Infect Dis 2009.


18

22. Ganoza CA, Ricaldi JN, Chauca J, et al. Novel hypertonic saline‐sodium hydroxide (HS‐SH) method for decontamination and concentration of sputum samples for Mycobacterium tuberculosis microscopy and culture. J Med Microbiol 2008 Sep;57(Pt 9):1094‐8. 23. Perera J, Arachchi DM. The optimum relative centrifugal force and centrifugation time for improved sensitivity of smear and culture for detection of Mycobacterium tuberculosis from sputum. Trans R Soc Trop Med Hyg 1999 Jul‐Aug;93(4):405‐9. 24. Peterson EM, Nakasone A, Platon‐DeLeon JM, Jang Y, de La Maza LM, Desmond E. Comparison of direct and concentrated acid‐fast smears to identify specimens culture positive for Mycobacterium spp. J Clin Microbiol 1999 Nov;37(11):3564‐8. 25. Smithwick RW, Stratigos CB. Acid‐fast microscopy on polycarbonate membrane filter sputum sediments. J Clin Microbiol 1981 Jun;13(6):1109‐13. 26. Apers L, Mutsvangwa J, Magwenzi J, et al. A comparison of direct microscopy, the concentration method and the Mycobacteria Growth Indicator Tube for the examination of sputum for acid‐fast bacilli. Int J Tuberc Lung Dis 2003 Apr;7(4):376‐81. 27. Naganathan N, Ganapathy KT, Rajalakshmi R. Evaluation of sputum smears prepared by different methods. Indian J Med Res 1979 Jun;69:893‐900. 28. Van Deun A, Chuquiyauri R, Torrea G, Agapito J, Verdonck K, Gotuzzo E. Yield of fluorescence microscopy versus culture for tuberculosis at a middle‐income country referral hospital. Trans R Soc Trop Med Hyg 2008 Jun;102(6):564‐9. 29. Vasanthakumari R. Concentrated sputum smear microscopy: a simple approach to better case detection in pulmonary tuberculosis. Indian J Tuberc 1988;35(2):80‐2. 30. Schunemann HJ, Oxman AD, Brozek J, et al. Grading quality of evidence and strength of recommendations for diagnostic tests and strategies. BMJ 2008 May 17;336(7653):1106‐10.


19

Table 1. Characteristics of included studies with culture as the reference standard

A. BLEACH CENTRIFUGATION Study Country Stain Chemical

Method Physical Method

Study Population Health Care Setting

Patient Selection

N

EQA Smears Blinded

Smear Positive Criteria

Ängeby (a), 2000

Honduras ZN Bleach 5.25%

Cent 3000 g Pulmonary TB suspects/ patients

Inpatient and outpatient

Convenience 303 NR NR Unclear

Bruchfeld, 2000

Ethiopia ZN Bleach 5%

Cent 3000 g Pulmonary TB suspects

Outpatient Consecutive 510 NR Yes 1

Daley (a), 2009

India AO Bleach Cent 3000 g Pulmonary TB suspects


Consecutive 178 Yes Yes 1

Eyangoh (a), 2005

Cameroon ZN Bleach 1.8%


Outpatient Consecutive 936 Yes Yes 10

Eyangoh (b), 2005

Cameroon AO Bleach 1.8%



Gebre (a), 1995

Ethiopia ZN Bleach 4.4%

Cent speed NR

Pulmonary TB suspects

Outpatient Convenience 100 NR Yes Unclear

Merid (c), 2009




Mutha (b), 2005

India ZN Bleach 5%

Cent 3000 rpm


Outpatient Convenience 297 NR NR Unclear

Wilkinson, 1997

South Africa

ZN Bleach 4‐5%


Inpatient Consecutive 166 NR Yes Unclear

B. BLEACH SEDIMENTATION Study Country Stain Chemical



Patient Selection

N

EQA Smears Blinded


Farnia (b), 2002

Iran ZN Bleach % NR

Sed 12‐15 hours



Convenience 430 NR Yes 1

Frimpong (b), 2005

Ghana ZN Bleach 1%

Flotation Pulmonary TB suspects

Outpatient NR 131 NR NR Unclear

Lawson, 2006 Nigeria ZN Bleach 3.5%

Sed <1h Pulmonary TB suspects

Outpatient Convenience 752 NR Yes 1

Merid (a), 2009


Sed <1h Pulmonary TB suspects


Merid (b), 2009


Sed overnight



C. NALC‐NaOH CENTRIFUGATION Study Country Stain Chemical



Patient Selection

N

EQA Smears Blinded


Bahador, 2006

Iran AR NALC‐NaOH


NR Consecutive 903 NR NR 1


20

Cattamanchi, 2009

Uganda ZN NALC‐NaOH


Inpatient Consecutive 279 Yes Yes 1

Farnia (a), 2002

Iran ZN NALC‐NaOH




Ganoza (a), 2008

Peru ZN NALC‐NaOH


NR Convenience 94 NR Yes 1

Perera, 1999 Sri Lanka ZN NALC‐NaOH


NR Convenience 163 NR NR 3

Peterson (a), 1999

USA AO NALC‐NaOH

Cent 3200 g Specimens Inpatient and outpatient


Peterson (b), 1999

USA AR NALC‐NaOH

Cent 2500 g Specimens Outpatient Convenience 44 NR Yes 3

Smithwick, 1981

USA AP NALC‐NaOH

Cent 2000 g Specimens NR Convenience 916 NR NR 1

D. NaOH CENTRIFUGATION Study Country Stain Chemical



Patient Selection

N

EQA Smears Blinded


Ängeby (b), 2000

Honduras ZN NaOH Cent 3000 g Pulmonary TB suspects/ patients


Convenience 303 NR NR Unclear

Apers, 2003 Zimbabwe ZN NaOH Cent 2000‐3000 g



Consecutive 256 NR NR 1

Mutha (a), 2005

India ZN NaOH Cent 1500 g Pulmonary TB suspects

Outpatient Convenience 297 NR NR Unclear

Naganathan, 1979

India ZN NaOH Cent 4000 rpm


Outpatient Convenience 1499

NR Yes 1

Van Deun, 2008

Peru AO NaOH Cent 3000 g Pulmonary TB suspects


Consecutive 1553

Yes Yes 1

Vasanthakumari, 1988

India ZN NaOH Cent 3000 rpm


Outpatient Consecutive 148 NR NR 3

E. OTHER PROCESSING METHODS Study Country Stain Chemical



Patient Selection

N

EQA Smears Blinded


Biswas (b), 1987

India ZN Bleach % NR

Flotation Pulmonary TB suspects

NR Convenience 102 NR NR Unclear

Chakravorty, 2005

India ZN USP Cent 5000‐ 6000 g


NR Convenience 571 NR NR 1

Daley (b), 2009

India AO USP Cent 3000 g Pulmonary TB suspects


Consecutive 178 Yes Yes 1

Frimpong (a), 2005

Ghana ZN Bleach 1%

Sed 15 hours Pulmonary TB suspects

Outpatient NR 131 NR NR Unclear


21

Ganoza (b), 2008

Peru ZN HS‐NaOH


Outpatient Convenience 94 Yes Yes 1

Haldar, 2007 India ZN USP Cent 5000 g Specimens Outpatient Convenience 148 No Yes 1

Laserson (a), 2005

Vietnam ZN CB‐18 Cent 1818 g Pulmonary TB suspects

Outpatient NR 338 NR NR 1

Laserson (b), 2005

Vietnam A R CB‐18 Cent 1818 g Pulmonary TB suspects

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


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.


23

Table 3. Processed versus Direct Microscopy: Pooled Sensitivity and Specificity Differences Sputum Processing Group Number of

Studies

Sensitivity Difference*, %

(95% CI)

Specificity Difference*, %

(95% CI)

Bleach centrifugation 9 6

(3, 10)

‐3

(‐4, ‐1)

Bleach sedimentation 5 9

(4, 14)

‐2

(‐5, 0)

NALC‐NaOH Centrifugation 6† 19

(7, 32)

‐1

(‐2, 0)

NaOH Centrifugation 5†† 8

(1, 16)

0

(‐1, 1)

HIV, Any Processing Method 3†† 5

(0, 10)

‐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.


24

Table 4. GRADE Evidence profiles 4A.Bleach Centrifugation

Effect (20% prevalence)

No. of studies Design Limitations Directness (Generalizability) Consistency

Imprecise or sparse data

Publication Bias Processed Direct

Absolute Difference

Quality of evidence (GRADE)

1. True positives (Patients with pulmonary TB)

9 studies, 3923 participants 1

Cross‐sectional

Moderate 2 Some uncertainty3

Serious inconsistency4

Serious5 Unlikely 130 per 1000

112 per 1000

18 per 1000

Very low

2. True negatives (Patients without pulmonary TB)


Cross‐sectional



Serious 5 Unlikely 768 per 1000

784 per 1000

‐16 per 1000

Very low

3. False positives (Patients incorrectly diagnosed with pulmonary TB)


Cross‐sectional




16 per 1000

16 per 1000

Very low

4. False negatives (Patients missed with pulmonary TB )


Cross‐sectional




88 per 1000

‐ 18 per 1000

Very low


25

Sensitivity (95% CI): processed 65% (59, 71); direct 56% (49, 63); Specificity (95% CI): processed 96% (93, 98), direct 98% (97, 99)

FOOTNOTES: 1Five (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). 2About 70% of studies considered representative; majority of studies were blinded. Culture is not a perfect reference standard. 3Absence 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. 4Sensitivity 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). 5Wide confidence intervals for sensitivity estimates for both processed and direct microscopy. 4B. Bleach Sedimentation Effect (20% prevalence)




Absolute Difference



5 studies, 2307 participants1

Cross‐sectional

Minor 2 Some uncertainty3



100 per 1000

26 per 1000

Very Low


5 studies 1 Cross‐sectional




784 per 1000

‐16 per 1000

Very Low



Cross‐sectional




16 per 1000

16 per 1000

Very Low


26



Cross‐sectional




100 per 1000

‐ 26 per 1000

Very Low


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 suspects. One study did not report whether microscopy results were interpreted in a blinded fashion. Culture is not a perfect reference standard; 3Absence 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 Effect (20% prevalence)




Absolute Difference




Cross‐sectional

Serious 2 Some uncertainty3



110 per 1000

46 per 1000

Very low



Cross‐sectional




792 per 1000

‐32 per 1000

Very low


27



Cross‐sectional




8 per 1000

32 per 1000

Very low



Cross‐sectional




90 per 1000

‐ 46 per 1000

Very low


FOOTNOTES: 1All 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 suspects. 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; 3Absence 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. 5Wide confidence intervals for sensitivity of processed microscopy. 4D. NaOH Centrifugation Effect (20% prevalence)




Absolute Difference




Cross‐sectional




130 per 1000

26 per 1000

Very Low


28



Cross‐sectional




792 per 1000

0 per 1000 Very Low



Cross‐sectional




8 per 1000

0 per 1000 Very Low



Cross‐sectional




70 per 1000

‐ 26 per 1000

Very Low

Sensitivity (95% CI): processed 78% (61, 89); direct 65% (52, 75); Specificity (95% CI): processed 99% (98 99), direct 99% (98, 99)

FOOTNOTES: 1All 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). 2Only two studies reported that microscopy results were interpreted in a blinded fashion. 3Three studies reported enrolling ambulatory tuberculosis suspects. Culture is not a perfect reference standard; 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. 4Sensitivity 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. 5Wide confidence intervals for sensitivity for both processed and direct microscopy.


29

Table 5. GRADE Summary of Findings

All results are given per 1000 patients tested for a prevalence of 20% and the likelihood ratios shown above. TP – true positive results ‐ important because may lead to treatment initiation TN – true negative results ‐ important because spare patients unnecessary treatment FP – false positive results ‐ important because patients are exposed to unnecessary possibility of adverse effects from drugs FN – false negative results ‐ important because increase the risk of severe disease as a result of delayed diagnosis Sensitivity = TP/TP + FN); specificity = TN/ (FP + TN); LR, likelihood ratios: LR+=sensitivity/(1‐specificity); LR‐ = (1‐sensitivity)/specificity CI, confidence interval § assuming that the reference standard, culture, does not yield false positives or false negatives. + On a 9 point scale GRADE recommends the approach to classify these outcomes as not important (score 1 – 3), important (4 – 6), and critical (7 – 9) to a decision. * Inconclusive results are uninterpretable test results – important because generate anxiety, uncertainty as to how to proceed, further testing and possible negative consequences of either treating or not treating

5A. Should sputum processing by bleach centrifugation versus direct microscopy be used to diagnose pulmonary tuberculosis? Quality of Evidence, Very Low ⊕

Test findings (95% CI) ‐ Processed microscopy

Pooled sensitivity % 65 (59‐71) LR(+) 17 (9‐32)

Pooled specificity % 96 (93‐98) LR(–) .36 (.31‐.43)

Consequences Number per 1000 Importance+

TP 130 9

TN 768 9

FP 32 9

FN 70 9

Inconclusive results* Not calculated – 5

Complications None – 5

Cost Not calculated – 5

Test findings (95% CI) ‐ Direct microscopy

Pooled sensitivity 56 (49‐63) LR(+) 31 (17, 56)

Pooled specificity 98 (97‐99) LR(–) .44 (.38, .52)


TP 112 9

TN 784 9

FP 16 9

FN 88 9





30


5B. Should sputum processing by bleach sedimentation versus direct microscopy be used to diagnose pulmonary tuberculosis? Quality of Evidence, Very Low ⊕ Test findings (95% CI) ‐ Processed microscopy

Pooled sensitivity % 63 (51, 74) LR(+) 17 (6, 46)

Pooled specificity % 96 (91, 99) LR(–) .38 (.27, .53)


TP 120 9TN 768 9FP 32 9FN 80 9Inconclusive results* Not calculated – 5Complications None – 5Cost Not calculated – 5Test findings (95% CI) ‐ Direct microscopy

Pooled sensitivity

50 (47, 53) LR(+) 23 (15, 36)

Pooled specificity

98 (97, 99) LR(–) .51 (.48, .54)


TP 100 9TN 784 9FP 16 9FN 100 9Inconclusive results* Not calculated – 5Complications None – 5Cost Not calculated – 5


31


5C. Should sputum processing by NALC‐NaOH centrifugation versus direct microscopy be used to diagnose pulmonary tuberculosis? Quality of Evidence, Very Low ⊕


Pooled sensitivity % 78 (62‐89) LR(+) 17 (3, 86)



TP 156 9TN 570 9FP 30 9FN 44 9Inconclusive results* Not calculated – 5Complications None – 5Cost Not calculated – 5Test findings (95% CI) ‐ Direct microscopy

Pooled sensitivity

55 (47, 62) LR(+) 89 (15, 542)

Pooled specificity

99 (96, 100) LR(–) .45 (.38, .54)


TP 110 9TN 594 9FP 6 9FN 90 9Inconclusive results* Not calculated – 5Complications None – 5Cost Not calculated – 5


32

All results are given per 1000 All rAll results are given per 1000 patients tested for a prevalence of 20% and the likelihood ratios shown above. TP – true positive results ‐ important because may lead to treatment initiation TN – true negative results ‐ important because spare patients unnecessary treatment FP – false positive results ‐ important because patients are exposed to unnecessary possibility of adverse effects from drugs FN – false negative results ‐ important because increase the risk of severe disease as a result of delayed diagnosis Sensitivity = TP/TP + FN); specificity = TN/ (FP + TN); LR, likelihood ratios: LR+=sensitivity/(1‐specificity); LR‐ = (1‐sensitivity)/specificity CI, confidence interval § assuming that the reference standard, culture, does not yield false positives or false negatives. + On a 9 point scale GRADE recommends the approach to classify these outcomes as not important (score 1 – 3), important (4 – 6), and critical (7 – 9) to a decision. * Inconclusive results are uninterpretable test results – important because generate anxiety, uncertainty as to how to proceed, further testing and possible negative consequences of either treating or not treating

5D. Should sputum processing by NaOH centrifugation versus direct microscopy be used to diagnose pulmonary tuberculosis? Quality of Evidence, Very Low ⊕


Pooled sensitivity % 78 (61, 89) LR(+) 68 (45, 101)



TP 156 9TN 594 9FP 6 9FN 44 9

Inconclusive results* Not calculated – 5Complications None – 5


Test findings (95% CI) ‐ Direct microscopy

Pooled sensitivity

65 (52, 75) LR(+) 44 (31, 63)

Pooled specificity

99 (98, 99) LR(–) .36 (.26, .50)


TP 130 9TN 594 9FP 6 9FN 70 9





33

FIGURE 1. Flow of Studies

* Some comparisons were reported using a reference standard and others reported without using a reference standard in 3 studies.

Full papers retrieved for more detailed evaluation

67

Papers (comparisons) included 50 (77)

Papers (comparisons) using a reference standard

25* (36)

1146 Excluded Citations Reasons: Duplicate publication: 421 Relevance: 725

13 papers added from reference review and contact with experts

49 Excluded Papers Reasons: Abstract 7 Comparison lacking 4 Could not obtain 1 Data insufficient 2 Direct microscopy missing 8 Direct smear negatives only 1 Editorial, commentary 4 Not diagnostic study 1 Not pulmonary TB 2 Relevance 17 Review 2

32 papers added from 2006 systematic review

Papers (comparisons) not using a reference standard

28* (41)

Titles/abstracts identified and screened for retrieval

1200


34

FIGURE 2. Analysis Flow Chart

What chemical processing method was used?

What physical processing method was used?Abbreviations: CENT, Centrifugation; SED, Sedimentation; NALC, N-Acetyl-L-Cysteine; NaOH, Sodium Hydroxide; USP, Universal Sample Processing; H-S, Hypertonic Saline; CB-18, C18-Carboxypropylbetaine; BAS, Bleach Ammonium Sulfate

Total Number

N=36

Bleach

N=14

NaOH

N=6

CENT

N=9

SED

N=5

CENT

N=6

NALC

N=8

CENT

N=8

1 H-S2 CB-182 Xylene Flotation

EXCLUDED (N=8)

3 USP


35

FIGURE 3. Bleach Centrifugation versus Direct Microscopy

3A. Study Quality

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Withdrawals explained?

Uninterpretable results reported?

Index test results blinded?

Index tests described?

Selection criteria described?

Representative spectrum?

Yes Unclear No

3B. Forest Plot: Sensitivity and Specificity

Direct microscopy

StudyAngeby (a) 2000Bruchfeld 2000Daley 2009Eyangoh (a) 2008Eyangoh (b) 2008Gebre (a) 1995Merid (c) 2009Mutha (a) 2005Wilkinson 1997

TP269126

289290

16118

1138

FP2

114420854

FN207710

139138

36113

651

TN255331138504506

48258275

73

Sensitivity0.57 [0.41, 0.71]0.54 [0.46, 0.62]0.72 [0.55, 0.86]0.68 [0.63, 0.72]0.68 [0.63, 0.72]0.31 [0.19, 0.45]0.51 [0.44, 0.58]0.65 [0.38, 0.86]0.43 [0.32, 0.54]

Specificity0.99 [0.97, 1.00]0.97 [0.94, 0.98]0.97 [0.93, 0.99]0.99 [0.98, 1.00]1.00 [0.99, 1.00]1.00 [0.93, 1.00]0.97 [0.94, 0.99]0.98 [0.96, 0.99]0.95 [0.87, 0.99]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1Bleach centrifugation

StudyAngeby (a) 2000Bruchfeld 2000Daley 2009Eyangoh (a) 2008Eyangoh (b) 2008Gebre (a) 1995Merid (c) 2009Mutha (a) 2005Wilkinson 1997

TP30

10624

312308

36147

1139

FP9

1411

514

05116

2

FN166212

116120

1684

650

TN248328131503494

48215264

75

Sensitivity0.65 [0.50, 0.79]0.63 [0.55, 0.70]0.67 [0.49, 0.81]0.73 [0.68, 0.77]0.72 [0.67, 0.76]0.69 [0.55, 0.81]0.64 [0.57, 0.70]0.65 [0.38, 0.86]0.44 [0.33, 0.55]

Specificity0.96 [0.93, 0.98]0.96 [0.93, 0.98]0.92 [0.87, 0.96]0.99 [0.98, 1.00]0.97 [0.95, 0.98]1.00 [0.93, 1.00]0.81 [0.76, 0.85]0.94 [0.91, 0.97]0.97 [0.91, 1.00]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1

Abbreviations: TP, true positives; FP, false positives; FN, false negatives; TN, true negatives


36

3C. Forest Plot: Sensitivity and Specificity Differences

3D. Hierarchical Summary Receiver Operating Characteristic Curves

Eyangoh SI (a) (2008)* Eyangoh SI (b) (2008)* Mutha A (b) (2005)* Wilkinson D (1997)* Angeby KA (a) (2000)† Bruchfeld J (2000)† Daley P (a) (2009)† Merid Y (c) (2009)† Gebre N (a) (1995)††

Study

0.05 (0.03, 0.08)0.04 (0.02, 0.06)0.00 (-0.06, 0.06)0.01 (-0.02, 0.04)0.09 (-0.02, 0.19)0.09 (0.04, 0.14)-0.06 (-0.16, 0.05)0.13 (0.08, 0.17)0.38 (0.23, 0.54)

Sensitivity Difference(95% CI)

-1 0 1

Favors Bleach Centrifugation

Eyangoh SI (a) (2008)*Eyangoh SI (b) (2008)*Mutha A (b) (2005)*Wilkinson D (1997)*Angeby KA (a) (2000)†

Bruchfeld J (2000)†

Daley P (a) (2009)†

Merid Y (c) (2009)†

Gebre N (a) (1995) ††

Study

-0.00 (-0.01, 0.00)-0.02 (-0.04, -0.01)-0.04 (-0.07, -0.01)0.03 (-0.02, 0.07)-0.03 (-0.05, -0.00)-0.01 (-0.02, 0.00)-0.05 (-0.09, -0.01)-0.16 (-0.21, -0.11)0.00 (-0.02, 0.02)

Specificity Difference(95% CI)

-1 0 1Favors Bleach Centrifugation

00.10.20.3 0.40.5 0.60.7 0.80.910

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

SPECIFICITY

SENSITIVITY

Bleach Centrifugation Microscopy

Direct Microscopy

* Low speed centrifugation (< 2500 revolutions per minute or 2000 g)† High speed centrifugation (≥ 2500 revolutions per minute or 2000 g) †† Centrifugation speed not recorded


37

FIGURE 4. Bleach Sedimentation versus Direct Microscopy 4A. Study Quality

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%







Yes Unclear No 4B. Forest Plot: Sensitivity and Specificity

Direct microscopy

StudyFarnia (b) 2002Frimpong (b) 2005Lawson 2006Merid (a) 2009Merid (b) 2009

TP3630

222118118

FP53388

FN3631

230113113

TN35367

297258258

Sensitivity0.50 [0.38, 0.62]0.49 [0.36, 0.62]0.49 [0.44, 0.54]0.51 [0.44, 0.58]0.51 [0.44, 0.58]

Specificity0.99 [0.97, 1.00]0.96 [0.88, 0.99]0.99 [0.97, 1.00]0.97 [0.94, 0.99]0.97 [0.94, 0.99]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1Bleach sedimentation

StudyFarnia (b) 2002Frimpong (b) 2005Lawson 2006Merid (a) 2009Merid (b) 2009

TP6043

233123133

FP337

1836

FN1218

21910898

TN35567

293248230

Sensitivity0.83 [0.73, 0.91]0.70 [0.57, 0.81]0.52 [0.47, 0.56]0.53 [0.47, 0.60]0.58 [0.51, 0.64]

Specificity0.99 [0.98, 1.00]0.96 [0.88, 0.99]0.98 [0.95, 0.99]0.93 [0.90, 0.96]0.86 [0.82, 0.90]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1



38



Lawson L (2006)*

Merid Y (a) (2009)*

Farnia P (b) (2002)†

Frimpong EH (b) (2005)†

Merid Y (b) (2009)†

Study

0.02 (0.01, 0.04)

0.02 (-0.00, 0.04)

0.33 (0.21, 0.46)

0.21 (0.09, 0.33)

0.06 (0.03, 0.10)


-1 0 1

Favors Bleach Sedimentation

Lawson L (2006)*

Merid Y (a) (2009)*

Farnia P (b) (2002)†

Frimpong EH (b) (2005)†

Merid Y (b) (2009)†

Study

-0.01 (-0.03, 0.00)

-0.04 (-0.06, -0.01)

0.01 (-0.00, 0.02)

0.00 (-0.01, 0.01)

-0.11 (-0.15, -0.06)


-1 0 1

Favors Bleach Sedimentation

Direct microscopy

Bleach sedimentation microscopy

00.10.2 0.30.40.5 0.60.70.8 0.910

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

SPECIFICITY

SE

NS

ITIVITY

* Short‐term sedimentation (< 1 hour) † Overnight sedimentation (15‐24 hours)


39

FIGURE 5. NALC‐NaOH Centrifugation versus Direct Microscopy

5A. Study Quality

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%








Direct microscopy

StudyBahador 2006Cattamanchi 2009Farnia (a) 2002Ganoza (a) 2002Perera 1999Peterson (a) 1999Peterson (b) 1999Smithwick 1981

TP3787366

85863642

FP11506002

FN1583361550

1218

34

TN850108353732200

838

Sensitivity0.71 [0.57, 0.83]0.51 [0.43, 0.59]0.50 [0.38, 0.62]0.29 [0.11, 0.52]0.63 [0.54, 0.71]0.42 [0.35, 0.49]0.82 [0.67, 0.92]0.55 [0.43, 0.67]

Specificity1.00 [0.99, 1.00]0.99 [0.95, 1.00]0.99 [0.97, 1.00]1.00 [0.95, 1.00]0.79 [0.59, 0.92]

Not estimableNot estimable

1.00 [0.99, 1.00]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1NALC-NaOH Centrifugation

StudyBahador 2006Cattamanchi 2009Farnia (a) 2002Ganoza (a) 2002Perera 1999Peterson (a) 1999Peterson (b) 1999Smithwick 1981

TP46896414

1241544145

FP3

1259

19008

FN6

8187

11533

31

TN84897

35364900

832

Sensitivity0.88 [0.77, 0.96]0.52 [0.45, 0.60]0.89 [0.79, 0.95]0.67 [0.43, 0.85]0.92 [0.86, 0.96]0.74 [0.68, 0.80]0.93 [0.81, 0.99]0.59 [0.47, 0.70]

Specificity1.00 [0.99, 1.00]0.89 [0.82, 0.94]0.99 [0.97, 1.00]0.88 [0.78, 0.94]0.32 [0.16, 0.52]

Not estimableNot estimable

0.99 [0.98, 1.00]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1



40



Bahador A (2006)Cattamanchi A (2009)Farnia P (a) (2002)Ganoza CA (a) (2008)Perera J (1999)Smithwick RW (1981)

Study*

-0.00 (-0.01, 0.00)-0.10 (-0.17, -0.04)0.00 (-0.00, 0.00)-0.12 (-0.21, -0.03)-0.46 (-0.68, -0.24)-0.01 (-0.01, -0.00)


-1 0 1Favors NALC-NaOH Centrifugation

Bahador A (2006) Cattamanchi A (2009) Farnia P (a) (2002) Ganoza CA (a) (2008) Perera J (1999) Smithwick RW (1981)

Study*

0.17 (0.05, 0.30)0.01 (-0.01, 0.03)0.39 (0.26, 0.52)0.38 (0.13, 0.64)0.29 (0.21, 0.37)0.04 (-0.02, 0.10)

Sensitivity Difference (95% CI)

-1 0 1Favors NALC-NaOH Centrifugation

Direct microscopy

NALC-NaOH Centrifugation

00.10.20.30.4 0.50.60.70.80.9 10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

SPECIFICITYS

EN

SITIVITY

* All studies performed high speed centrifugation (≥ 2500 revolutions per minute or 2000 g)


41

FIGURE 6. NaOH Centrifugation versus Direct Microscopy

6A. Study Quality

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%








Direct microscopy

StudyAngeby (b) 2000Apers 2003Mutha (a) 2005Naganathan 1979Van Deun 2008Vasanthakumari 1988

TP26

15811

4418785

FP215

24120

FN20766

10810063

TN255

21275926

13540

Sensitivity0.57 [0.41, 0.71]0.68 [0.61, 0.73]0.65 [0.38, 0.86]0.80 [0.77, 0.84]0.47 [0.39, 0.54]0.57 [0.49, 0.66]

Specificity0.99 [0.97, 1.00]0.95 [0.77, 1.00]0.98 [0.96, 0.99]0.97 [0.96, 0.98]0.99 [0.98, 1.00]

Not estimable

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1NaOH Centrifugation

StudyAngeby (b) 2000Apers 2003Mutha (a) 2005Naganathan 1979Van Deun 2008Vasanthakumari 1988

TP35

20416

42485

134

FP3167

120

FN11301

12510214

TN254

21274943

13540

Sensitivity0.76 [0.61, 0.87]0.87 [0.82, 0.91]0.94 [0.71, 1.00]0.77 [0.73, 0.81]0.45 [0.38, 0.53]0.91 [0.85, 0.95]

Specificity0.99 [0.97, 1.00]0.95 [0.77, 1.00]0.98 [0.95, 0.99]0.99 [0.98, 1.00]0.99 [0.98, 1.00]

Not estimable

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1

Abbreviations: TP, True Positives; FP, False Positives; FN, False Negatives; TN, True Negatives


42



Apers L (2003)*

Mutha A (a) (2005)*

Angeby KA (b) (2000)†

Naganathan N (1979)†

Van Deun A (2008)†

Study

0.00 (-0.05, 0.05)

-0.00 (-0.01, 0.01)

-0.00 (-0.02, 0.01)

0.02 (0.01, 0.03)

0.00 (-0.00, 0.00)

Specificity Difference

(95% CI)

-1 0 1

Favors NaOH Centrifugation

Apers L (2003)*

Mutha A (a) (2005)*

Angeby KA (b) (2000)†

Naganathan N (1979)†

Van Deun A (2008)†

Study

0.20 (0.14, 0.25)

0.29 (0.02, 0.57)

0.20 (0.06, 0.33)

-0.03 (-0.05, -0.01)

-0.01 (-0.03, 0.01)

Sensitivity Difference

(95% CI)

-1 0 1

Favors NaOH Centrifugation

Direct microscopy

00.10.20.30.40.50.60.70.80.9 10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

SPECIFICITY

NaOH Centrifugation

SE

NS

ITIVITY

* Low speed centrifugation (< 2500 revolutions per minute or 2000 g)† High speed centrifugation (≥ 2500 revolutions per minute or 2000 g)


43

FIGURE 7. HIV‐Infected Patients, Any Processing Method versus Direct Microscopy

7A. Study Quality

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%








Direct microscopy (HIV)

StudyBruchfeld 2000Cattamanchi 2009Eyangoh (a) 2008Eyangoh (b) 2008

TP37878787

FP0100

FN59839898

TN0

108233233

Sensitivity0.39 [0.29, 0.49]0.51 [0.43, 0.59]0.47 [0.40, 0.54]0.47 [0.40, 0.54]

SpecificityNot estimable

0.99 [0.95, 1.00]1.00 [0.98, 1.00]1.00 [0.98, 1.00]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1Processed microscopy (HIV)

StudyBruchfeld 2000Cattamanchi 2009Eyangoh (a) 2008Eyangoh (b) 2008

TP4889

100102

FP0

1216

FN48818583

TN0

97232227

Sensitivity0.50 [0.40, 0.60]0.52 [0.45, 0.60]0.54 [0.47, 0.61]0.55 [0.48, 0.62]

SpecificityNot estimable

0.89 [0.82, 0.94]1.00 [0.98, 1.00]0.97 [0.94, 0.99]

Sensitivity

0 0.2 0.4 0.6 0.8 1

Specificity

0 0.2 0.4 0.6 0.8 1

Abbreviations: TP, True Positives; FP, False Positives; FN, False Negatives; TN, True Negatives


44


Eyangoh SI (a) (2008)*

Eyangoh SI (b) (2008)*

Cattamanchi A (2009)†

Study

0.07 (0.03, 0.11)

0.08 (0.04, 0.13)

0.01 (-0.01, 0.03)


-1 0 1

Favors sputum processing

Eyangoh SI (a) (2008)*

Eyangoh SI (b) (2008)*

Cattamanchi A (2009)†

Study

-0.00 (-0.02, 0.01)

-0.03 (-0.05, -0.00)

-0.10 (-0.17, -0.04)

Specificity Difference (95% CI)

-1 0 1

Favors sputum processing

* Bleach centrifugation (< 2500 revolutions per minute or 2000 g)† NALC‐NaOH centrifugation (≥ 2500 revolutions per minute or 2000 g)


45

Supplementary Table 1. Characteristics of included studies without a reference standard

A. BLEACH CENTRIFUGATION Study Country Stain Chemical


Study Population

Health Care

Setting

Patient Selection

N Smears Blinded

DS Positive Proportio

n

PS Positive Proportio

n Abdurahman (b),

2000 Ethiopia ZN Bleach Cent 800‐

3000 g Pulmonary TB suspects

Outpatient Consecutive 100 Yes .22 .41

Ängeby (c), 2000 Honduras ZN Bleach 5.25%

Cent 3000 g

Specimens NR Convenience 971 NR .08 .10

Ängeby (d), 2000 Honduras ZN Bleach 5.25%

Cent 3000 g

Specimens Outpatient Convenience 1422 NR .02 .03

Aung, 2001 Myanmar ZN Bleach % NR

Cent 3000 g

Pulmonary TB patients

Outpatient Convenience 948 NR .26 .31

Cameron a, 1945 USA ZN Bleach % NR

Cent 3000 rpm

Pulmonary TB suspects and patients

NR NR 329 NR .18 .33

Cameron b, 1945 USA ZN Bleach % NR

Cent 3000 rpm


NR NR 211 NR .22 .33

Cameron, 1946 USA ZN Bleach % NR

Cent 3000 rpm


NR NR 397 NR .21 .25

Contijo Filho (b), 1979

Brazil ZN Bleach % NR

Cent 1200 g


NR Convenience 122 NR .34 .36

Gebre (b), 1995 Ethiopia ZN Bleach 4.4%

Cent Speed NR


Outpatient Convenience 500 Yes .08 .14

Gebre (c), 1995 India ZN Bleach 4.4%

Cent Speed NR


Outpatient Convenience 103 Yes .16 .34

Gebre‐Salassie (b), 2003

Ethiopia ZN Bleach 5% Cent 3000 g


Outpatient Consecutive 200 NR .09 .30

Habeenzu, 1998 Zambia ZN Bleach 4‐5%

Cent 3000 g


NR Convenience 488 No .14 .24

Miörner (a), 1996 Ethiopia ZN Bleach 5% Cent 3000 g


NR Convenience 545 Yes .17 .21

Rasheed (a), 2008 Ethiopia ZN Bleach 5% Cent 3000 g



Saxena, 2001 India ZN Bleach 4‐5%

Cent 1500 rpm




46

Selassie (b), 2001 Ethiopia ZN Bleach % NR

Cent 3494 g


NR Consecutive 200 NR .13 .31

B. BLEACH SEDIMENTAION Study Country Stain Chemical


Study Population

Health Care

Setting

Patient Selection

N Smears Blinded


n


n Abdurahman (a),

2000 Ethiopia ZN Bleach

4‐5% Sed

overnight Pulmonary TB suspects


Bonnet, 2008 Kenya ZN Bleach 3.5%

Sed 15 hours



Contijo Filho (a), 1979

Brazil ZN Bleach % NR

Sed <1 hour



Douthwaite, 2007 Ethiopia ZN Bleach 5% Sed <1 hour



Gebre‐Salassie (a), 2003

Ethiopia ZN Bleach 5% Sed 16 hours



Lawson, 2007 Nigeria ZN Bleach 3.5%

Sed <1 hour



Miörner (b), 1996 Ethiopia ZN Bleach 5% Sed 15‐18 hours



Pandey (a) 2009 India ZN Bleach 5% Sed 12‐15 hours



Rasheed (b), 2008 Ethiopia ZN Bleach 5% Sed overnight



Van Deun, 2000 Bangladesh ZN Bleach % NR

Sed overnight

Specimens Outpatient Consecutive 3287 Yes .16 .17

Yassin, 2003 Ethiopia ZN Bleach 5% Sed <1 hour



C. NALC‐NaOH CENTRIFUGATION Study Country Stain Chemical


Study Population

Health Care Setting

Patient Selection

N Smears Blinded


n


n Pandey (b) 2009 India ZN NALC‐

NaOH Cent 3000

rpm Pulmonary TB suspects


Smithwick , 1979 USA AO NALC‐NaOH

Cent Speed NR


D. NaOH CENTRIFUGATION Study Country Stain Chemical


Study Population

Health Care Setting

Patient Selection

N Smears Blinded


n


n


47

Biswas (a), 1987 India ZN NaOH Cent speed NR



Cameron a, 1945 USA ZN NaOH Cent 3000 rpm


NR NR 329 NR .22 .27

Cameron b, 1945 USA ZN NaOH Cent 3000 rpm


NR NR 211 NR .18 .27

Gopinathan (a), 1984

India ZN NaOH Cent 3000 g

Pulmonary TB patients


Harries, 1998 Malawi ZN NaOH Cent 3000 g



Kochhar, 2002 India ZN NaOH Cent 3000 g


Tech, 1965 The Philippines

ZN NaOH Cent Speed NR

Specimens Inpatient and

outpatient

NR 581 No .28 .20

E. OTHER PROCESSING METHODS Study Country Stain Chemical


Study Population

Health Care Setting

Patient Selection

N Smears Blinded


n


n Contijo Filho (c),

1979 Brazil ZN Bleach %

NR Flotation Pulmonary

TB suspects and patients


Gopinathan (b), 1984

India ZN Bleach % NR

Flotation Pulmonary TB patients


Rattan, 1994 India ZN Bleach 1% Flotation Specimens NR Random 100 Yes .33 .38

Selassie (a), 2001 Ethiopia ZN None Cent 3494 g


NR Consecutive 200 NR .13 .23

Vincy, 2008 India ZN Bleach ammoniu

m sulphate

Sed overnight

Specimens NR NR 190 Yes .22 .24

Abbreviations: AO, Auramine‐O; Cent, Centrifugation; DS, Direct smear: NR, Not Reported; PS, Processed smear; Sed, Sedimentation; TB, tuberculosis; ZN, Ziehl‐Neelsen


48

Supplementary Table 2. Pooled Sensitivity and Specificity Differences – Generalized Estimating Equation Model*

Group Number of

Studies

Sensitivity Difference†,

% (95% CI)

Specificity Difference†,

% (95% CI)

Bleach centrifugation 9 7 (4, 11) -3 (-7, 0)

Bleach sedimentation 5 7 (0, 13) -3 (-7, 1)

NALC-NaOH Centrifugation 6†† 17 (2, 32) -2 (-4, 1)

NaOH Centrifugation 5‡ 4 (-8, 16) 1 (-1, 2)

HIV, Any Processing Method 3‡ 6 (2, 10) -3 (-7, 1)

* Parameters of Generalized Estimating Equation Model: Clustering variable – study; Predictor – microscopy type (processed verus. direct); Outcome – number of true positive smear results (for sensitivity difference) or true negative smear results (for specificity difference); Outcome distribution – binomial; Link – logit; Correlation structure – exchangeable with robust standard errors † Positive difference favors processed microscopy †† 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.


49

Appendix A. LITERATURE SEARCH, SPUTUM PROCESSING, RUN DATE JANUARY 7, 2009

PubMed

Search Most Recent Queries Time Result

#2 Search #1 Limits: Publication Date from 2005 to 2008 14:03:26 564

#1 Search (tuberculosis[MeSH] OR mycobacterium tuberculosis[MeSH] OR tuberculosis[ti]) AND (microscopy[MeSH] OR (sputum[MeSH] AND smear*) OR acid‐fast[TI] OR (AFB[TIAB] AND smear*) OR (AFB[TIAB] AND sputum) OR (sputum smear*[TI]) OR (smear examination*[TI]) OR ("sputum microscopy"[TI]) OR (bacteriolog*[TI] AND tuberculosis[TI]) OR (direct microscop*[TI]) OR (sensitivity[TI] AND microscopy[TI]) OR (microbiolog*[TI] AND tuberculosis[TI]))

14:03:05 3151

No. Query Results Date

#1 (('tuberculosis'/exp OR 'mycobacterium tuberculosis'/exp OR 'acid fast bacterium'/exp OR tuberculosis:ti OR (('acid-fast':ti OR 'acid fast':ti) AND (bacill*:ti OR bacteri*:ti))) AND ('sputum'/exp OR 'sputum examination'/exp OR sputum:ti OR smear*:ti) AND ('microscopy'/exp OR microscop*:ti)) OR (('tuberculosis'/exp OR 'mycobacterium tuberculosis'/exp OR 'acid fast bacterium'/exp OR tuberculosis:ti OR (('acid-fast':ti OR 'acid fast':ti) AND (bacill*:ti OR bacteri*:ti))) AND ('sputum'/exp OR 'sputum examination'/exp OR sputum:ti OR smear*:ti) AND (sensitiv*:ti OR bacteriolog*:ti OR 'direct microscopy':ti OR 'direct microscopic':ti OR microbiolog*:ti))

675 07 Jan 2009

#2 #2 AND [01-12-2004]/sd NOT [31-12-2008]/sd

Date limit: Dec 1, 2004 – Dec 31, 2008

187 07 Jan 2009

Web of Science®

Search History

# 2 286 #1

Databases=SCI-EXPANDED Timespan=2005-2008

# 1 1,043 Topic=(tuberculosis) AND Title=(sputum or smear or smears or microscop* or bacteriolog* or afb or acid-fast)

Databases=SCI-EXPANDED Timespan=All Years


50

BIOSIS Previews®

Search History

# 2 156 #1

Databases=PREVIEWS Timespan=2005-2008

# 1 1,648 Topic=(tuberculosis) AND Title=(sputum or smear or smears or microscop* or bacteriolog* or afb or acid-fast)

Databases=PREVIEWS Timespan=All Years


51

Appendix B: DATA EXTRACTION FORM, SPUTUM PROCESSING

Title of Article:_________________________________________________________________

Title of Journal:_________________________________________________________________

PART 1: PUBLICATION INFORMATION 1. Publication #:

2. Study # (a, b, c, etc):

3. First author: 4. Data extracted by: (initials)

5. Year of Publication:

6. Type of publication:

� full paper

� short communication

� unpublished document

Other: ___________________________________

7. Country where study was done

� NR

� Name of country: ___________________

PART 2: STUDY DESIGN AND POPULATION CHARACTERISTICS

8. Study design � Cross‐sectional

� Case‐control

� Longitudinal

� Other (specify):______________________________________

8a. Study population � PTB suspects

� PTB patients

� PTB patients and suspects

� Specimens

� Other (specify):______________________________________

9. Data collection � NR � Prospective � Retrospective


52

10. Healthcare setting � NR � In‐patient (hospitalized) � Out‐patient � Both

11. Did the study involve patients or specimens?

� Patients � Specimens

12. Patient selection � NR

� Consecutive sample

� Random sample

� Convenience sample

� Other (specify): _______________________________________

13. Age category � NR � Adults only (≥15 years) � Children Only � Both

14. Did the study include patients with HIV?

� NR � Yes � No

14a. If Yes, % HIV positive:______

15. Were eligibility criteria clearly reported? � Yes � No

16. Did all patients receive same reference standard (regardless of smear result)?

� Yes � No

17. Was the processed smear method described in sufficient detail to permit replication?

� Yes � No

18. Were smears interpreted without knowing culture results?

� NA

� NR

� No

� Direct smears only

� Processed smears only

� Both Direct and Processed smears


53

19. Were DS and PS interpreted blindly (without knowing result of other)?


Comments on study design and quality:

PART 3: LABORATORY DETAILS 20. Number of specimens examined per patient

� NR

� Specify:_________________

21. Sputum type � NR

� Spontaneous (expectorated)

� Induced

� Both

22. Smear preparation technique � NR

� Single specimen used for both DS and PS

� Separate specimens used for DS and PS

� Pooled specimens used for PS after taking part for DS

� Other (specify):_______________________________

23. Did a peripheral lab perform DM?


24. Did a peripheral lab perform PM?


25. Were DM and PM performed in the same lab?


26. Were microscopists reported as being trained?


27. EQA program in place? � NR � Yes � No


54

PART 4: SPUTUM PROCESSING AND MICROSCOPY DETAILS 28. Chemical digestion/ decontamination method

� NR � PhAS

� No chemical method used � NALC‐NaOH

� NaOCl (bleach) < 5% � NaOH (Petroff)

� NaOCl (bleach) ≥ 5% � Other (specify):___________

� NaOCl (bleach) % NR

29. Physical processing method

� NR (go to q30)

� No physical processing (go to q30)

� Centrifugation < 2500 rpm or 2000g (go to q29a)

� Centrifugation ≥ 2500 rpm or 2000g (go to q29a)

� Centrifugation (speed NR) (go to q30)

� Sedimentation < 1 hour (go to q29c)

� Sedimentation ≥ 1 hour (go to q29c)

� Sedimentation (time NR) (go to q30)

� Other (specify): __________________________________

29a. Centrifugation speed

Specify: ___________ � rpm � g

29b. Centrifugation time

Specify (in minutes): ___________

29c. Sedimentation time

Specify (in hours): ___________

30. Stain used: � NR � Auramine‐Rhodamine (AR)

� Ziehl‐Neelsen (ZN) � Auramine‐Phenol (AP)

� Kinyoun (KN) � Other: ____________________

� Auramine‐O (AO)


55

31. How was a positive smear defined?

(if guideline referenced, look up guideline)

NR

≥ ________ bacilli per _______ high power fields

(fill in both blanks)

32. Gold standard used:

� NR � YES (Go to q33) � NO (Go to q36)

33. If gold standard was used, which method?

� NR

� Solid culture media (e.g., LJ, Ogawa, Middlebrook 7H10)

� Liquid culture media (e.g., Middlcbrook 7H9, BACTEC, MGIT)

� Other: ______________________

PART 5: RESULTS: 2X2 TABLES FOR SENSITIVITY/SPECIFICITY (when gold standard used) or SMEAR POSITIVE PROPORTION (when no gold standard used)

34. Direct Smear Results (Fill in numbers for each cell if gold standard used)

Culture

Direct S

mear + ‐

+ 34a._______ 34c._______

‐ 34b._______ 34d._______

35. Processed Smear Results (Fill in numbers for each cell if gold standard used)

Culture


56

Processed

Smear

+ ‐

+ 35a._______ 35c._______

‐ 35b._______ 35d._______

Smear positive proportion (Fill only when no gold standard used)

36. Direct Microscopy

37. Processed Microscopy

# Positive

36a. _______________

37a. _______________

# Tested

36b. _______________

37b. _______________

FILL THE FOLLOWING SECTION ONLY IF RESULTS FOR HIV‐INFECTED PATIENTS REPORTED SEPARATELY

38. Direct Smear Results in HIV‐infected patients (Fill in numbers for each cell if gold standard used)

Culture

Direct S

mear + ‐

+ 38a._______ 38c._______

‐ 38b._______ 38d._______

39. Processed Smear Results in HIV‐infected patients (Fill in numbers for each cell if gold standard used)

Culture

Processed

Smear

+ ‐

+ 39a._______ 39c._______

‐ 39b._______ 39d._______


57

Smear positive proportion in HIV‐infected patients (Fill only when no gold standard used)

40. Direct Microscopy

41. Processed Microscopy

# Positive

40a. _______________

41a. _______________

# Tested

40b. _______________

41b. _______________


58

APPENDIX C

QUADAS: A TOOL FOR THE QUALITY ASSESSMENT OF STUDIES OF DIAGNOSTIC ACCURACY 1. Was the spectrum of patients representative of the patients who will receive the test in practice?

YES NO UNCLEAR

2. Were selection criteria clearly described? YES NO UNCLEAR

3. Is the reference standard likely to correctly classify the target condition?

YES NO UNCLEAR

4. Is the time period between reference standard and index test short enough to be reasonably sure that the target condition did not change between the two tests?

YES NO UNCLEAR

5. Did the whole sample or a random selection of the sample, receive verification using a reference standard of diagnosis?

YES NO UNCLEAR

6. Did patients receive the same reference standard regardless of the index test result?

YES NO UNCLEAR

7. Was the reference standard independent of the index test (i.e. the index test did not form part of the reference standard)?

YES NO UNCLEAR

8. Was the execution of the index test described in sufficient detail to permit replication of the test?

YES NO UNCLEAR

9. Was the execution of the reference standard described in sufficient detail to permit its replication?

YES NO UNCLEAR

10. Were the index test results interpreted without knowledge of the results of the reference standard?

YES NO UNCLEAR

11. Were the reference standard results interpreted without knowledge of the results of the index test?

YES NO UNCLEAR

12. Were the same clinical data available when test results were interpreted as would be available when the test is used in practice?

YES NO UNCLEAR

13. Were uninterpretable/intermediate test results reported? YES NO UNCLEAR

14. Were withdrawals from the study explained? YES NO UNCLEAR

Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development of QUADAS: a tool for the quality assessment of studies of diagnostic accuracy included in systematic reviews. BMC Med Res Methodol, 3, 25 (2003)

sputum processing, cattamanchi et al, ucsf, august,

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