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

FIND, Foundation for Innovative New Diagnostics, Kampala, Uganda.
BMC Infectious Diseases (Impact Factor: 2.61). 05/2011; 11(1):125. DOI: 10.1186/1471-2334-11-125
Source: PubMed


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. 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.
We 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.
Compared 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.
The 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.

Download full-text


Available from: Heidi Albert, Sep 17, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Diagnosis represents only one aspect of tuberculosis (TB) control but is perhaps one of the most challenging. The drawbacks of current tools highlight several unmet needs in TB diagnosis i.e. necessity for accuracy, rapidity of diagnosis, affordability, simplicity, and the ability to generate same-day results at point-of-care (POC). When a return visit is required to access test results, time to treatment is prolonged and default rates are significant. However, a good diagnostic tool is also critically dependent on obtaining an adequate biological sample. Here we review the accuracy and potential impact of established and newer potential POC diagnostic tests for TB including smear microscopy, the Xpert MTB/RIF assay (Cepheid) and the Determine TB LAM antigen test (Alere). Novel experimental approaches and detection technologies for POC diagnosis of active TB, including nucleic acid amplification tests, detection of volatile organic compounds or metabolites, mass spectroscopy, microfluidics, SERS, electrochemical approaches, and aptamers amongst others, are discussed. We also discuss future applications, including the potential POC diagnosis of drug-resistant TB and presumed latent TB infection. Challenges to the development and roll-out of POC tests for TB are also reviewed.
    Full-text · Article · Nov 2012 · Respirology
  • [Show abstract] [Hide abstract]
    ABSTRACT: During the last decade there has been a dramatic change in the laboratory approach to tuberculosis (TB) diagnosis in the developing world. This change began with the realization that acid-fast bacillus smear microscopy alone was totally inadequate to deal with the dual problems of human immunodeficiency virus (HIV)-associated TB and drug-resistant TB that threaten to undermine global progress in TB control. Subsequently, increased financial resources for TB laboratory services and the establishment of a systematic process for endorsement of new TB diagnostic tools and approaches by the World Health Organization (WHO) have led to rapid expansion of TB laboratory services and the availability of several new diagnostic tests that have been introduced. These include both commercial automated and noncommercial systems for phenotypic mycobacterial liquid culture and drug susceptibility testing, a simple and inexpensive test for mycobacterial species identification in culture isolates, light-emitting diode fluorescence microscopy, and rapid molecular methods for TB case detection and the diagnosis of drug-resistant TB. The latter methodologies that include line probe assays and an automated cartridge-based real-time polymerase chain reaction (PCR)-based test are being scaled up at an unprecedented pace and are truly revolutionizing the diagnosis of drug-resistant TB. On the other hand, little progress has been made in the quest for a true point-of-care test for TB. Fortunately, this is being addressed in several discovery initiatives that hopefully will provide impetus for the development of rapid, accurate TB diagnostics for the lowest level of the health system.
    No preview · Article · Feb 2013 · Seminars in Respiratory and Critical Care Medicine
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A magnetic-bead protocol and standard centrifugation protocol yielded Mycobacterium tuberculosis in 40/50 sputum specimens in respectively 12±1 days and 11±2 days (p>0.05, Student test). Manipulation took respectively 35 ± 5 min and 45 ± 10 min (p<0.05). Magnetic-bead protocol could advantageously replace centrifugation for culturing M. tuberculosis from sputum.
    Full-text · Article · Feb 2013 · Journal of clinical microbiology
Show more