Improved Tool Concentrates Tubercle Bacilli

The novel technique, which involves vacuum filtering a sputum sample treated with household bleach and other simple chemicals through a small filter, could dramatically improve TB diagnoses globally, particularly in settings where the disease is common and resources are limited.

A lung disease specialist, now at the University of Florida (UF; Gainesville, FL, USA) with colleagues at the Federal University of Espírito Santo (Vitória, Brazil), used the traditional microscope-based diagnosis method as a starting point, and devised a way to identify more cases of the Mycobacterium tuberculosis infection. The most widely used way to confirm TB infection is to use a microscope to identify and count disease-causing bacteria in sputum smeared onto a glass slide. Although the method has been in continuous use for more than a century, it can be unreliable, on average catching cases only about half of the time.

Part of the problem is that sometimes sputum samples do not contain many bacteria, making it hard to detect TB. Concentrating bacteria onto a small area could help improve detection accuracy, and although previous efforts have led to improvements, they tend to require expensive equipment or technical knowhow. In some cases, gains were offset by loss of sample or safety concerns. Therefore, the quest for a low-cost, simple, effective method led back to the trusty microscope. The scientists decided to try 13 mm polycarbonate membrane filters with 1.0 µm pores, that fit neatly onto microscope slides and that concentrated the bacteria even more.

For the 314 patients in Brazil suspected as having TB, but not yet in treatment, the team modified small-filter method by adding 95% ethanol and 1% Triton X detergent to the bleach, using filters with an 0.8 µm pore size, and prefiltering the sputum digests through 25 mm polypropylene prefilters with 30 µm pores (Millipore, Billerica, MA, USA). This method detected 89% of cases, compared with 60 % detection when samples were concentrated by the currently used method of rapid spinning, and 56% detection when sputum smears were looked at directly under the microscope. Furthermore, the small-filter method identified almost three-quarters of TB-positive cases that had been incorrectly reported as negative based on the traditional technique.

Kevin Fennelly, MD, MPH, an associate professor in the UF College of Medicine, said, "A point-of-care dipstick that can say yes or no is the Holy Grail, but we're a long way from there. ‘Microscope’ has become a dirty word in the TB diagnostics world, but almost every clinical laboratory in the world has one.” The method is being tested in western Uganda, where many people have both TB and HIV. They are comparing it again with the traditional direct-smear method and with a sophisticated DNA-based test that can also detect whether bacteria are drug resistant. The study was published in the June 2012 issue of the Journal of Clinical Microbiology.

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University of Florida
Federal University of Espírito Santo
Millipore