Molecular diagnosis of suspected tuberculosis from archived smear slides from the Balimo region, Papua New Guinea

Abstract

Undetected smear-negative TB is occurring in the Balimo region of PNG, as well as some unnecessary empirical treatment. Molecular methods of diagnosis could greatly reduce the frequency of inappropriate clinical assessment, as well as providing point-of-care diagnosis. This may provide substantial patient and programmatic benefits, including lowering the economic burden on patients from rural areas seeking medical diagnosis in Balimo.

Diversity of Mycobacterium tuberculosis in the Middle Fly District of Western Province, Papua New Guinea: microbead-based spoligotyping using DNA from Ziehl-Neelsen-stained microscopy preparations

“…A total of 345 smeared sputa were collected from June 2012 to March 2014, of which 206 showed evidence of mycobacterial DNA based on combined TaqMan qPCR assays (see 14 for details). Out of those 206 qPCR-positive samples, the spoligotyping and resistance analyses focused on the ones with the lowest Cq values after qPCR, with a cutoff of 36 chosen from preliminary spoligotyping results.…”

“…Sputum samples are collected routinely for diagnostic purposes from patients with clinical signs of TB, and are ZN-stained and examined for AFB (acid-fast bacilli) by light microscopy, with slides then archived and stored. Each sputum sample was smeared on several slides, with up to five slides per sputum sample (see 14 for details). Archived slides were transferred to James Cook University, in Townsville, Australia, for DNA extraction from the sputum smears.…”

“…DNA extracts were obtained using a modified version of a previously described Chelex DNA extraction method in biosafety level 2 laboratories 21 ; the modified protocol has been published elswhere 14 . The presence of MTBC in the extracted DNA was analyzed using a TaqMan real-time PCR 51 and the molecular diagnosis results have been published elsewhere 14 . Regarding duplicate sputum smears (i.e.…”

“…Spoligotyping from smear slides has previously shown better results with AFB-positive sputa quantified as '2+' and '3+' because of higher DNA loads 55 . However, instead of using microscopy results, we took into account Cq values obtained from qPCR targeting MTBC to select samples showing high DNA loads (see 14 for detailed qPCR results). The Cq cutoff to be used was chosen from preliminary spoligotyping results.…”

“…BDH does not have facilities for performing Mtb culture and is not equipped with a GeneXpert (Cepheid) instrument, so TB diagnosis relies primarily on clinical presentation and direct sputum smear microscopy, based on the PNG National Tuberculosis Management Protocol 13–15 . DNA extracted from Ziehl-Neelsen (ZN) stained sputum smears has already proven useful in molecularly confirming the microscopy diagnosis of TB in this region of PNG 14 , and spoligotyping has been undertaken successfully on DNA extracted from cultures 10,16 , from decontaminated sputa 17 , as well as from ZN-stained sputum smears 18–21 . Direct sputum smear microscopy is routinely performed in diagnostic laboratories worldwide, including in developing countries, and stained slides can be stored and transported at room temperature, which makes them a prime target for spoligotyping of Mtb samples collected in remote settings 21 .…”