GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India

Success of the ambitious “Treatment 2015” initiative by UNAIDS [1] has accelerated the pace of antiretroviral therapy (ART) scale-up globally. The achievement of this initiative is attributed to the international solidarity and responsibilities shared by diverse disciplines. Upto 2014, an estimated 14.9 million people in developing/low- and middle-income countries received ART [2]. The National AIDS Control Programme in India has successfully reduced the incidence of new HIV-1 infections by 57% and the free roll out of ART has covered nearly 0.8 million HIV infected individuals with nearly 67% coverage of those in need of ART [3]. Furthermore, the wider access to ART has resulted in declining the death rate due to AIDS related co-morbidities [4].

Presently, the free ART programme monitors patients on ART clinically and immunologically with six monthly CD4 count and follows a “Targeted Viral Load” approach for treatment failure. However, several studies have demonstrated poor predictive value of the immunological criteria and reported accumulation of HIV drug resistant mutations due to delayed detection of treatment failure [5‐7]. To avoid this, WHO strongly recommends viral load monitoring as the preferred approach to diagnose and confirm treatment failure [8‐11].

Nucleic Acid Testing (NAT) based technologies are considered as the gold standards for HIV-1 viral load estimation because of their high specificity, sensitivity and wide linear range of detection. However, these assays require testing to be performed by well-trained technicians and need at least 8-10 h. With an increasing demand to scale up HIV-1 viral load monitoring, a robust, easy to use and sensitive point-of-care technology (POC) [12] is utmost essential. Numbers of POC viral load assays are in the development pipeline [13, 14] and GeneXpert® HIV-1 Quant Assay (Cepheid Innovations Pvt. Ltd., USA) is one of the recently introduced assays. It is a fully automated integrated system that is routinely used for detecting M. tuberculosis DNA and resistance to Rifampicin [15, 16] as well as other pathogens (MRSA, Norovirus, HBV, HPV [17‐21]) including HIV. It is a rapid assay that delivers results within 90 mins. It uses reverse transcriptase polymerase chain reaction (RT-PCR) technology to achieve high sensitivity over the range of 40 to 10,000,000 copies/ml (1.6 to 7 log₁₀copies/ml) and can quantitate all HIV-1 Group M, N and O subtypes [22]. Furthermore, a reduced turnaround time leads to prompt HIV diagnosis and clinical management in adult and pediatric samples. Apart from this, the assay is very simple and safe to perform; their integrated reagents and consumables are robust in usage and can be stored in refrigerators. Due to minimal requirement of infrastructure, it is very easy to scale up the activity of viral load testing nationally. Considering all these features, the Gene Xpert HIV-1 Quant assay serves as a valuable point of care viral load assay that could be useful for monitoring antiretroviral therapy as well as integrated HIV-TB management in developing countries like India [23]. Therefore in the present study, we compared performance of GeneXpert® HIV-1 Quant Assay with the gold standard, routinely used Abbott m2000rt RealTime HIV-1 (Abbott Molecular, Germany) in the resource limited Indian settings in view of the scale up of viral load testing [24]. The performance evaluation of this new assay will ensure its decentralized utility in the clinical set up and will assist in management of patient care efficiently.

The paired plasma specimens collected from 219 study participants (mean age 37.6 years, SD = 9.4) were tested by Abbott m2000 Real Time PCR and GeneXpert HIV-1 Quant assays.

NAT based real-time quantitative PCR’s are the preferred options for high-throughput HIV-1 viral-load testing in developed and developing countries [31]. However, these assays are time consuming and flow separately through three steps - sample preparation, amplification, detection and require specimen transportation, high-end instrumentation, skilled personnel, and dedicated laboratories [22]. Hence as suggested by the recent WHO guidelines [11] and to generate results with “sample-in/answer-out” approach [32], replacing NAT based assays with partially/fully automated POC assays is crucial.

The National ART programme in India was launched in April 2004 in limited number of hospitals. Subsequently, concentrated efforts resulted in expansion of the programme. Currently, the programme has adopted a public health approach for provision of ART & provides comprehensive prevention, care and treatment services, with a standardized and simplified combination of ART regimen free of cost to more than 8.8 lakhs PLHIVs across 528 ART centers and 1080 Link ART centers across the country which is a second highest number in the world. Wider access to ART has led to 29% reduction in estimated annual AIDS related deaths [33].

However, the success of high-quality ART depends on timely monitoring of viral load and confirmation of ART failure. Introduction of a simple and reliable POC viral load assay will deliver ART service more appropriately and significantly in a decentralized manner. This will potentially reduce the cost of transportation of samples and monitoring patients on antiretroviral therapy without compromising the quality of care required for treatment success. The “ASSURED” criteria set by WHO describes the POC assays as affordable, sensitive, specific, user-friendly, robust/rapid, equipment-free, and deliverable techniques. Many such POC technologies [13] are in development and GeneXpert HIV-1 Quant assay is one of them. The system requires single-use disposable GeneXpert cartridge that are self-contained and could be useful for viral load monitoring in resource limited settings. As the patient load in Indian ART centers is low and variable, the viral load testing requirement can be low every day in smaller in district ART centers. Such small number of viral load tests are manageable with POC GeneXpert platform [33] In certain districts because the RNTCP, some GeneXpert POC machines for TB diagnosis have already been placed [34] The load of TB testing along with viral load estimation on the same instrument is possible. Hence, convergence of both the program’s is possible through integrated testing.

Our results indicated that HIV-1 Quant assay compared well with the Abbott plasma viral load assay [27, 32]. The high percentages of concordance (91.32%), correlation (r = 0.886) and minimum differences in the mean viral loads (0.12 log copies/ml, as measured by Bland-Altman analysis) were observed between the two assays. In comparison to the Abbott assay, the results obtained by the GeneXpert assay for low viral load categories showed an agreement of 79% (19/24) for not detected category, 70%(14/20) for <40 copies/ml category and 100% for 40-200 copies/ml considering number of concordant samples within acceptable limit of 0.5 log copies/ml. Further comparison of the GeneXpert assay showed an agreement of (91, 86 and 83%) in the moderate viral load categories (201-2500, 2501-5000 and 5001-10,000 copies/ml). Whereas in the higher viral load categories (10001-1 L, 100,001-5 L and >5 L copies/ml), the agreement was (87, 82 and 90%). In contrast to the previous studies [35, 36], higher agreements in the viral load categories ranging from 40 to >5 L copies/ml were reported in our studies. Furthermore, it is noteworthy to mention that the LLD (42 copies/ml) obtained in our assays coincided well with the LLD of manufacturer (40 copies/ml).

Assay precision and reproducibility were investigated by spiking the seronegative plasma with high viral load plasma/culture supernatant. Our data indicated that the GeneXpert assay showed excellent intra-assay and inter-assay reproducibility at high viral loads (2000-20,000,00 copies/ml), with %CV ranging between 0.31-1.11, 0.36-1.45 except for inter-assay variation for 20,000 copies/ml (%CV 2.74). However, higher variations were observed at low viral loads, ie; at 200 copies/ml (%CV 3.34, 1.97) respectively which was found to be less than the Abbott assay. Thus, variation observed at low viral load results agrees with previously reported study by Swenson et al., in 2014 where comparisons were based on 4221 paired samples [37], These variations could be attributed to the discrepancies in different testing platforms in quantifying subtype C samples as reported earlier [38]. Apart from variations observed while using such platforms, the parameters like type of sample and its integrity were assessed in one of the study wherein good precision as well as accuracy was reported for another POC (Liat HIV Quant (IQuum) platform) in comparison with other platforms (Roche CAP/CTMv2 and Abbott RealTime HIV-1) [39]. In our study, sero-negative samples were included to assess false positives and the carry over effect, which was found to be 100% specific in classifying negative samples as negative with no carryover effect, even if a negative sample preceded a positive specimen.

The main advantages of the GeneXpert HIV-1 viral load assay lies in its simplicity (ease of performance due to its integrated cartridge based closed system), rapid turnaround time (90 min as compared to other viral load platform that require 8-12 h), compactness (equipment is smaller than other platforms), cost effectiveness (17 USD which is cheaper as compared to other assays) and the large portfolio of different assays to manage patients with different variants of HIV-1, TB, and HIV-TB co-infection in the longer run [13, 15]. As nearly 1.1 lakh PLHIVs are reported to have HIV-TB co-infection. India shares one fourth of the global TB burden and one person dies of TB, every 2 min. Keeping in mind, the morbidity due to HIV-TB co-infections, the Indian government has launched the Revised National TB Control Program (RNTCP) in collaboration with NACO (National AIDS Control Organization) and CTD (Central TB Division) that uses WHO recommended Directly Observed Treatment Short Course (DOTS) strategy that is operational in 632 districts/reporting units. It provides daily anti-TB treatment (ATT) for PLHIV at all ART centers, as a single window approach [23, 33, 34]. Prioritization of rapid molecular Cartridge Based GeneXpert assay for all PLHIV with presumptive TB to ensure early diagnosis of TB will help on convergence of load per day for TB where less number of machines is available at district level. Furthermore, both the activities can be best utilized if two programs converge. However, the decision to use one assay or the other could depend on assay-independent parameters such as laboratory space, number of samples and workflow. As per the study design, the predefined viral load ranges quantified using the gold standard Abbott assay obviate the risk of any potential bias for sample selection as it covers varied viral load ranges from low to high viral copies. Furthermore, the inherent capacity of the system for processing 4 samples at a time could serve as a limitation to provide viral load testing for patient care at ART centers that have higher number of patient visits.

Our results suggest that the GeneXpert cartridge based test as an alternative method for viral load monitoring in resource-limited settings, specifically from the rural and remote parts of the country. Our study ascertains the validity and performance of the GeneXpert HIV-1 Quant assay as a point of care technology which could be helpful as reference to other scientists for validating similar POC instruments. Thus, our study results can be an important tool for clinicians, patients, policy makers etc. to scale up the ambitious UNAIDS 90-90-90 treatment target with timely diagnosis of ART failures as well as for management of HIV-TB patients [40]. Further studies of operational issues to apply these findings within a clinical setting on a large scale will be useful in translating into policy decisions that will benefit India and the global community.

The viral load results of GeneXpert HIV-1 Quant and Abbott HIV-1 m2000 Real Time PCR were comparable. The differences between the HIV-1 viral load estimates for both the assays were within the limits of agreement. Thus, the GeneXpert HIV-1 Quant can be used as a Point of care assay for viral load estimation in resource limited settings. The outcomes from this study appraise the national and international programs for adoption and placement of such integrated platforms for PLHIV and HIV-TB patient care.