Review
Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review

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Summary

Most of the world's tuberculosis cases occur in low-income and middle-income countries, where sputum microscopy with a conventional light microscope is the primary method for diagnosing pulmonary tuberculosis. A major shortcoming of conventional microscopy is its relatively low sensitivity compared with culture, especially in patients co-infected with HIV. In high-income countries, fluorescence microscopy rather than conventional microscopy is the standard diagnostic method. Fluorescence microscopy is credited with increased sensitivity and lower work effort, but there is concern that specificity may be lower. We did a systematic review to summarise the accuracy of fluorescence microscopy compared with conventional microscopy. By searching many databases and contacting experts, we identified 45 relevant studies. Sensitivity, specificity, and incremental yield were the outcomes of interest. The results suggest that, overall, fluorescence microscopy is more sensitive than conventional microscopy, and has similar specificity. There is insufficient evidence to determine the value of fluorescence microscopy in HIV-infected individuals. The results of this review provide a point of reference, quantifying the potential benefit of fluorescence microscopy, with which the increased cost and technical complexity of the method can be compared to determine the possible value of the method under programme conditions.

Introduction

Sputum smear microscopy (henceforth referred to as microscopy) is currently recommended for the diagnosis of pulmonary tuberculosis in low-income and middle-income countries, where more than 90% of tuberculosis cases occur.1, 2, 3, 4, 5 Microscopy is rapid, relatively simple, inexpensive, and highly specific in areas where there is a high prevalence of tuberculosis. In addition, microscopy identifies the most infectious patients and is widely applicable.6, 7 In some studies, microscopy has been reported to have greater than 80% sensitivity for identifying cases of pulmonary tuberculosis;8, 9 however, in other reports, the sensitivity of the test has been relatively low and variable (range 20–60%).10, 11 Microscopy has limited value for the diagnosis of tuberculosis in children and does not, by definition, identify smear-negative tuberculosis, which is more likely in HIV-positive than HIV-negative individuals.1, 3, 12, 13 Smear-negative tuberculosis has been associated with poor treatment outcomes, including death, especially in areas hit hard by the HIV epidemic.14, 15 For these reasons, methods to improve the sensitivity of microscopy are urgently needed, particularly in countries with a high HIV burden. One method, used most commonly in high-income countries and credited with improved sensitivity, is fluorescence microscopy.16, 17, 18

Introduced in the 1930s,19 fluorescence microscopy uses an acid-fast fluorochrome dye (eg, auramine O or auramine-rhodamine) with an intense light source such as a halogen or high-pressure mercury vapour lamp. By comparison, conventional microscopy uses the carbolfuchsin Ziehl-Neelsen or Kinyoun acid-fast stains, and can be used with a conventional artificial light source or reflected sunlight.2 The most important advantage of fluorescence microscopy is that it uses a lower power objective lens (typically 25×) than conventional microscopy (typically 100×), enabling the microscopist to assess the same area of a slide in less time.20, 21 Substantial savings in work effort have been reported with fluorescence microscopy, suggesting it may be cost-effective in some low-income countries.20, 21, 22 Fluorescence microscopy has been credited with increased sensi-tivity,22, 23, 24, 25, 26 and, for this reason, has been proposed by some experts for use in countries with a high prevalence of HIV infection.1, 22 Other advantages include the simplicity of the fluorochrome staining method compared with Ziehl-Neelsen methods.27, 28 A potential shortcoming of fluorescence microscopy is the possibility of false-positive results because inorganic objects may incorporate fluorochrome dyes.29, 30 In the 1970s, Kubica25 assessed the relative value of fluorescence and conventional microscopy in a multicentre study of 61 163 Ziehl-Neelsen and 27 808 fluorescence microscopy specimens from nine countries. Kubica25 found that fluorescence microscopy improved sensitivity, but left a lingering doubt about specificity.

We did a systematic review to summarise the evidence on the accuracy of fluorescence microscopy, according to the guidelines and methods proposed for diagnostic systematic reviews and meta-analyses.31 We specifically addressed the following questions: (1) what is the sensitivity of fluorescence microscopy compared with conventional microscopy? (2) What is the specificity of fluorescence microscopy compared with conventional microscopy? (3) What is the impact of sputum processing on the sensitivity and specificity of fluorescence microscopy? (4) Is there a difference in sensitivity and specificity between auramine O and auramine-rhodamine stains? (5) Does the examination of smears with fluorescence microscopy take less time than with conventional microscopy?

Section snippets

Search strategy and selection criteria

We followed a standard protocol for doing systematic reviews of diagnostic test evaluations.31 We searched electronic databases for primary studies and conference abstracts: PubMed (1950 to May, 2005), BIOSIS (1969 to November, 2004), Embase (1974 to 2004), and Web of Science (1945 to 2004). The search terms used included the following: “tuberculosis”, “Mycobacterium tuberculosis”, “acid-fast AND bacilli”, “sputum AND microscopy”, “bacteriology”, “sensitivity AND specificity”, “fluorescence”,

Description of included studies

Of the 3538 citations identified after literature searches, 30 articles consisting of 45 studies met our eligibility criteria.20, 22, 23, 24, 26, 27, 28, 30, 34, 35, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 We considered most studies to be independent (24, 50). Therefore, no effort was made to account for lack of independence. Figure 1 shows the process for study selection and exclusion. For conventional microscopy, 43 (96%) studies used Ziehl-Neelsen stain

Discussion

Our systematic review of 45 studies comparing fluorescence microscopy with conventional microscopy suggests that: (1) fluorescence microscopy is more sensitive for the diagnosis of pulmonary tuberculosis than conventional microscopy; (2) the specificity of fluorescence microscopy for detection of acid-fast organisms in sputum is similar to that of conventional microscopy; (3) the increased sensitivity of fluorescence microscopy is greater in low-grade positives; (4) fluorochrome-stained smears

Search strategy and selection criteria

Details of the search strategy and selection criteria can be found in the Methods section.

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