Feasibility of magnetic bead technology for concentration of mycobacteria in sputum prior to fluorescence microscopy

Heidi Albert,Stuart Wilson,George Lukyamuzi,Patrick J Ademun,Moses Joloba,Barnabas Nyesiga,Mark D Perkins,Yukari Manabe

doi:10.1186/1471-2334-11-125

Abstract

BackgroundDirect sputum smear microscopy is the mainstay of TB diagnosis in most low and middle income countries, and is highly specific for Mycobacterium tuberculosis in such settings. However it is limited by low sensitivity, particularly in HIV co-infected patients. Concentration by centrifugation has been reported to be more sensitive than direct smear preparation, but is only suitable for referral laboratories. Simpler concentration methods that could be applied in peripheral laboratories are urgently needed.MethodsWe evaluated the feasibility of an early prototype ligand-coated magnetic bead technology to concentrate M. tuberculosis prior to detection by LED-based fluorescence microscopy compared with direct Ziehl-Neelsen microscopy and direct and concentrated fluorescence microscopy in a reference laboratory in Kampala, Uganda. Results were compared with MGIT 960 liquid culture and Lowenstein-Jensen culture.ResultsCompared to culture, concentrated FM had significantly higher sensitivity than direct ZN (74.8% and 51.4%), magnetic bead-FM (65.4%) and direct FM (58.9%). The sensitivity of magnetic bead FM was significantly higher than direct ZN (p < 0.001) but not significantly higher than direct FM (p = 0.210). The specificity of magnetic bead FM and concentrated FM was significantly lower than direct ZN (88.6%, 94.3% and 98.9% respectively) and direct FM (99.4%). There was no significant difference in specificity between magnetic bead FM and concentrated FM. Allowing for blinded resolution of discrepant results, the specificity of magnetic bead FM increased to 93.1%. Direct microscopy was simpler than concentrated FM and Magnetic bead FM which both had a similar number of steps.ConclusionThe sensitivity of the early prototype magnetic bead FM was lower than concentrated FM, similar to direct FM, and significantly higher than direct ZN. Both magnetic bead and concentration by centrifugation led to reduced specificity compared with the direct smear methods. Some magnetic bead FM false positive results were not easily explained and should be further investigated. The prototype version of the magnetic bead procedure tested here was of similar complexity to concentration by centrifugation. As such, if the sensitivity of the magnetic bead FM could be improved in future versions of the technology, this may offer a viable alternative to centrifugation.

Highlights

Direct sputum smear microscopy is the mainstay of TB diagnosis in most low and middle income countries, and is highly specific for Mycobacterium tuberculosis in such settings
This study evaluated the feasibility of using an early prototype ligand-coated magnetic bead technology to concentrate M. tuberculosis prior to detection by LEDbased fluorescence microscopy
The specificity of magnetic bead Fluorescence microscopy (FM) and concentrated FM was significantly lower than direct ZN (p < 0.001 and p = 0.039 respectively) and direct FM (p < 0.001 and p = 0.004 respectively)

Summary

Introduction

Direct sputum smear microscopy is the mainstay of TB diagnosis in most low and middle income countries, and is highly specific for Mycobacterium tuberculosis in such settings. Direct sputum smear microscopy is the mainstay of TB diagnosis in most low and middle income countries, where 95% of TB cases and 98% of deaths associated with TB occur [1] This method is rapid and inexpensive and highly specific for Mycobacterium tuberculosis in such settings. A recent systematic review 1 concluded that processing by a several chemical procedures, followed by centrifugation or overnight sedimentation, were more sensitive than direct microscopy, and that specificity was similar [3]. Many such methods require expensive centrifuges or reagents which would add substantially to the cost and complexity of performing smear microscopy. Fluorescence microscopy (FM) gives an average increase of 10% sensitivity over the ZN method, while retaining specificity [4], a finding which has had little relevance in disease endemic countries until the recent development of inexpensive fluorescence microscopes illuminated by light emitting diodes (LEDs)

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Discussion

Conclusion