Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis

Abstract

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse® mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >104 fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse® method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 104 and 105 colony forming units (cfu)/mL M.tb. At 103 cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse® method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse® method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study

“…Interestingly, GSCN by itself showed very efficient results in terms of sputum liquefaction, even when combined with the other chemicals. It has been previously shown that concentrated GSCN can be used in sample preparation for the extraction of DNA and protein from a variety of samples [25], and even using an electricity-free protocol [26] or small battery-operated instruments [27]. More importantly, M. tuberculosis was shown to become non-viable after exposure to commercial lysis buffers, which contain concentrated guanidine [8].…”

Closed-type pre-treatment device for point-of-care testing of sputum

“…The sputum specimen pre-treatment procedures and protocols used to diagnose pathogens such as tuberculosis and pneumonia are complex and very labor intensive. The protocol involves liquefaction, homogenization, dissolution, and inactivation 11 , 12 . The pre-treatment methods for infectious sputum specimens currently used in most laboratories require not only daily preparation of reagents but also the process of opening and closing the lid for adding reagents, vortex mixing, and sedimentation using a centrifuge—all of which are done by hand 13 , 14 .…”

Esophageal tuberculosis with coexisting opportunistic infections in a renal allograft transplant recipient

“…It can identify drug resistance and provides early results. The GeneXpert MTB is the most widely used TB nucleic acid amplification testing system, and is recommended for use in areas where TB and multidrug‐resistant TB are prevalent …”