Low-Cost Method to Monitor Patient Adherence to HIV Antiretroviral Therapy Using Multiplex Cathepsin Zymography

Abstract

Monitoring patient adherence to HIV antiretroviral therapy (ART) by patient survey is inherently error prone, justifying a need for objective, biological measures affordable in low-resource settings where HIV/AIDS epidemic is highest. In preliminary studies conducted in Ethiopia and South Africa, we observed loss of cysteine cathepsin activity in peripheral blood mononuclear cells of HIV-positive patients on ART. We optimized a rapid protocol for multiplex cathepsin zymography to quantify cysteine cathepsins, and prospectively enrolled 350 HIV-positive, ART-naïve adults attending the Themba Lethu Clinic, Johannesburg, South Africa, to test if suppressed cathepsin activity could be a biomarker of ART adherence (103 patients were included in final analysis). Poor adherence was defined as detectable viral load (>400 copies/ml) or simplified medication adherence questionnaire, 4-6 months after ART initiation. 86 % of patients with undetectable viral loads after 6 months were cathepsin negative, and cathepsin-positive patients were twice as likely to have detectable viral loads (RR 2.32 95 % CI 1.26-4.29). Together, this demonstrates proof of concept that multiplex cathepsin zymography may be an inexpensive, objective method to monitor patient adherence to ART. Low cost of this electrophoresis-based assay makes it a prime candidate for implementation in resource-limited settings.

Keywords: AIDS; Cysteine protease; Infectious disease; Monitoring; Sub-Saharan Africa; Zymography.

Figure 1. Patients on antiretroviral therapy have reduced cathepsin activity in their peripheral blood mononuclear cells in cohorts from Johannesburg and Addis Ababa

A) PBMCs isolated from whole blood of HIV-positive patients attending the Helen Joseph Clinic in Johannesburg, South Africa were lysed and equal amounts of protein (10 μg) were loaded for multiplex cathepsin zymography and quantified by densitometry. Patients on ART for different lengths of time did not have cathepsin activity. B) HIV-positive patients attending Black Lion Hospital in Addis Ababa, Ethiopia multiplex cathepsin zymography. Data normalized to the HIV-negative controls for each gel. There was statistically significant reduction in cathepsin activity in patients on either tenofovir (TDF) or efavirenz (EFV) (n=7, **p<.05,). Neg- HIV-negative, NVP-nevirapine, AZT-zidovudine, d4T-didanosine, PI-protease inhibitor, ART Naïve- HIV-positive patients.

Figure 2. Efavirenz (EFV) and tenofovir (TDF) suppress cathepsin activity in monocytes, but lamivudine (3TC) does not

THP-1 monocytes were incubated with 100 μM of either EFV, TDF, or 3TC for 24 hours or with appropriate vehicle control (veh); DMSO for TDF and 3TC, methanol for EFV. Then cells were lysed, and equal protein amounts were examined by multiplex cathepsin zymography and quantified by densitometry (n=3, * p<0.05 or 0.001).

Figure 3. Six months of ART reduces cathepsin activity in PBMCs as measured by multiplex cathepsin zymography

A) Representative zymograms (of more than 30 individual gels run and imaged) are shown from baseline when patients were ART naïve and B) after 6 months of ART. Arbitrary numbering of wells in the gels between baseline and 6 months, not patient matched. Zymo-negative signal indicated by bold, underlined number, and zymo-positive signal is not).