Biology subjects

Drain, P. K.

Publications and source records attributed to Drain, P. K..

3 recordsLinked to original sources

bioRxiv2022

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

microbiology↗

bioRxiv2021

Isolation and Purification of Lipoarabinomannan from Urine of Adults with Active Tuberculosis

Lipoarabinomannan (LAM) is a cell wall component of Mycobacterium tuberculosis that is excreted in the urine of persons with active tuberculosis (TB). Limited diagnostic sensitivity of LAM immunoassays has been due to selecting antibodies against LAM derived from in vitro cultured M. tuberculosis, rather than LAM purified from in vivo clinical urine specimens. Urinary LAM (uLAM) is critical to enable the development of and/or screening of novel uLAM-specific antibodies but is typically dilute and in heterogeneous mixtures with other urine components. We used physical, enzymatic, and chemical processes for the scaled isolation and purification of uLAM. The purified material may then be used to develop more sensitive uLAM diagnostic tests for active TB disease.

microbiology↗

bioRxiv2019

An enzymatic assay to measure long-term adherence to pre-exposure prophylaxis and antiretroviral therapy

Poor adherence to pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) can lead to human immunodeficiency virus (HIV) acquisition and emergence of drug resistant infections, respectively. Measurement of antiviral drug levels provides objective adherence information that may help prevent adverse health outcomes. Gold standard drug-level measurement by liquid chromatography/mass spectrometry is centralized, heavily instrumented, and expensive and is thus unsuitable and unavailable for routine use in clinical settings. We developed the REverse TranscrIptase Chain Termination (RESTRICT) assay as a rapid and accessible measurement of drug levels indicative of long-term adherence to PrEP and ART. The assay uses designer single stranded DNA templates and intercalating fluorescent dyes to measure complementary DNA (cDNA) formation by reverse transcriptase in the presence of nucleotide reverse transcriptase inhibitor drugs. We developed a probabilistic model for the RESTRICT assay by calculating the likelihood of incorporation of inhibitors into cDNA as a function of the relative concentrations of inhibitors and nucleotides. We validated the model by carrying out the RESTRICT assay using aqueous solutions of tenofovir diphosphate (TFV-DP), a measure of long-term adherence to PrEP and ART. We used dilution in water as a simple sample preparation strategy to detect TFV-DP spiked into blood. The RESTRICT assay accurately distinguishes TFV-DP drug levels within the clinical range for adherence and has the potential to be a useful test to identify patients with poor adherence to ART and PrEP.

bioengineering↗