Multidisease testing for HIV and TB using the GeneXpert platform: A feasibility study in rural Zimbabwe

Background HIV Viral Load and Early Infant Diagnosis technologies in many high burden settings are restricted to centralized laboratory testing, leading to long result turnaround times and patient attrition. GeneXpert (Cepheid, CA, USA) is a polyvalent near point-of-care platform and is widely implemented for Xpert MTB/RIF diagnosis. This study sought to evaluate the operational feasibility of integrated HIV VL, EID and MTB/RIF testing in new GeneXpert platforms. Methods Whole blood samples were collected from consenting patients due for routine HIV VL testing and DBS samples from infants due for EID testing, at three rural health facilities in Zimbabwe. Sputum samples were collected from all individuals suspected of TB. GeneXpert testing was reserved for all EID, all TB suspects and priority HIV VL at each site. Blood samples were further sent to centralized laboratories for confirmatory testing. GeneXpert polyvalent testing results and patient outcomes, including infrastructural and logistical requirements are reported. The study was conducted over a 10-month period. Results The fully automated GeneXpert testing device, required minimal training and biosafety considerations. A total of 1,302 HIV VL, 277 EID and 1,581 MTB/RIF samples were tested on a four module GeneXpert platform in each study site. Xpert HIV-1 VL testing was prioritized for patients who presented with advanced HIV disease, pregnant women, adolescents and suspected ART failures patients. On average, the study sites had a GeneXpert utilization rate of 50.4% (Gutu Mission Hospital), 63.5% (Murambinda Mission Hospital) and 17.5% (Chimombe Rural Health Centre) per month. GeneXpert polyvalent testing error rates remained lower than 4% in all sites. Decentralized EID and VL testing on Xpert had shorter overall median TAT (1 day [IQR: 0–4] and 1 day [IQR: 0–1] respectively) compared to centralized testing (17 days [IQR: 13–21] and 26 days [IQR: 23–32] respectively). Among patients with VL >1000 copies/ml (73/640; 11.4%) at GMH health facility, median time to enhanced adherence counselling was 8 days and majority of those with documented outcomes had re-suppressed VL (20/32; 62.5%). Median time to ART initiation among Xpert EID positive infants at GMH was 1 day [IQR: 0–1]. Conclusion Implementation of near point-of-care GeneXpert platform for integrated multi-disease testing within district and sub-district healthcare settings is feasible and will increase access to VL, and EID testing to priority populations. Quality management systems including monitoring of performance indicators, together with regular on-site supervision are crucial, and near-POC test results must be promptly actioned-on by clinicians for patient management.


I. Title of Procedure
Quantitative HIV-1 viral load testing using GeneXpert technology

II. Test Summary
State the physiologic and diagnostic reasons for performing the test. Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). It can be transmitted through sexual contact, exposure to infected blood or blood products, prenatal infection of a fetus, or perinatal or postnatal infection of a newborn. HIV diagnostics have evolved significantly in the past two decades and continue to be significantly important in saving lives of millions of HIV infected patients. Today, measurement of blood plasma HIV-1 RNA concentration known as viral load using nucleic acid-based molecular diagnostic assays has been established as standard of care in assessing HIV-positive patient prognosis and response to antiretroviral therapy. Untreated HIV-1 infection is characterized by high-level viral production and CD4 T-cell destruction, despite an often lengthy clinical latency, to significant net loss of CD4 T cells and AIDS. Assessment of viral load levels is a strong predictor of the rate of disease progression and, by itself or in combination with CD4Tcell counts, has great prognostic value. The HIV-1 Quant Assay, performed on GeneXpert® Instrument Systems, is designed for the rapid quantitation of Human Immunodeficiency Virus type 1 (HIV-1) in human plasma from HIV-1 infected individuals over the range of 40 to 10,000,000 copies/mL.

III. Principle
GeneXpert Instrument Systems automate and integrate sample preparation, nucleic acid extraction and amplification, and detection of the target sequence in simple or complex samples using real-time reverse transcriptase PCR (RT-PCR). The systems consist of an instrument, personal computer, and preloaded software for running tests and viewing the results. The systems require the use of single-use disposable GeneXpert cartridges that hold the RT-PCR reagents and host the RT-PCR processes. Because the cartridges are self-contained, crosscontamination between samples is minimized. The HIV-1 VL Assay includes reagents for the detection of HIV-1 RNA in specimens and two internal controls used for quantitation of HIV-1 RNA. The internal controls are also used to monitor the presence of inhibitor(s) in the RT and PCR reactions. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. Version 0 Effective date: 10 June 2014

IV. Specimen Handling and Preparation
o Whole blood should be collected in EDTA, EDTA-PPT, or ACD collection tubes and centrifuged to separate the plasma and red blood cells per the manufacturer's instructions. o A minimum of 1 mL plasma is required for the HIV-1 VL Assay. If using the transfer pipette included in the kit, a minimum of 1.2 mL plasma is required. Alternatively, if using a precision pipette, a minimum of 1 mL plasma is required. o Whole blood may be held at 15-30 °C for up to 8 hours or at 2-8 °C for up to 72 hours, prior to preparing and testing the specimen. After centrifugation, plasma may be held at 15-30 °C for up to 24 hours or at 2-8 °C for up to 6 days, prior to testing. o Plasma specimens are stable frozen (≤ -18 °C and ≤ -70 °C) for six weeks. o Plasma specimens are stable up to three freeze/thaw cycles. o Plasma specimens must be thawed and equilibrated to room temperature prior to transfer to cartridge.

V. Quality Control
Each GeneXpert test cartridge is a self-contained test device with an in-built control for each sample. Normally, no external controls are required. The internal controls enable the system to detect specific failure modes within each for each sample o Instrument system control: Check status-it checks the optics, temperature of the module and the mechanical integrity of each cartridge.

VI. Reagents, Materials, & Equipment
The HIV-1 VL Assay kit contains sufficient reagents to process 10 specimens or quality control samples. The kit contains the following: o Allow samples to come to room temperature prior to loading plasma into the cartridge • Option 1: If using the transfer pipette included in the kit (Figure 1), fill to just below the bulb but above the line to transfer at least 1 mL plasma from the collection tube into the sample chamber of the test cartridge ( Figure 2). Do NOT pour the specimen into the chamber! Version 0 Effective date: 10 June 2014

Note
There is a thin plastic film that covers the inner ring of 13 ports of the test cartridge. This film should not be removed.

Option 2:
If using an automatic pipette, transfer at least 1 mL of plasma into the sample chamber of the test cartridge (Figure xx). Do NOT pour the specimen into the chamber!

Important Start the test within four hours of adding the sample to the cartridge
Loading less than 1 mL plasma into the cartridge will trigger an insufficient volume error (ERROR 2097), preventing the instrument from running the sample

VIII. Interpretation of Results
Click the View Results icon to view results. Upon completion of the test, click the Report button of the View Results window to view and/or generate a PDF report file. The test results take approximately 90minutes to be produced The results are interpreted automatically by the GeneXpert Instrument System from measured fluorescent signals and embedded calculation algorithms and are clearly shown in the View Results window ( Figure 5 and Figure 6). Possible results are shown in Table 1.  •An ERROR result indicates that the Probe Check Control failed or maximum pressure limits were exceeded. An error result indicates that the assay was aborted. Possible causes include: insufficient volume of sample was added, the reaction tube was filled improperly, a reagent probe integrity problem was detected, or the maximum pressure limit was exceeded.
•A NO RESULT indicates that insufficient data were collected. For example, the operator stopped a test that was in progress, a load error occurred, or the software was closed prematurely.

Retest Procedure
For retest of an INVALID, ERROR, or NO RESULT, use a new cartridge (do not re-use the cartridge). 1. Remove a new cartridge from the kit.
2. Section VII on preparing cartridge and starting the test on page 7.

X. Limitations
Good laboratory practices and changing gloves between handling specimens are recommended to avoid contamination of specimens or reagents.
Biological specimens, including used cartridges, should be treated as capable of transmitting infectious agents and should be discarded in hazardous clinical waste bins. Version 0 Effective date: 10 June 2014

Limit of Detection (LoD)
The LOD for the HIV-1 VL Assay is 40 cp/mL for HIV-1 subtype B in EDTA plamsa.

Limit of Quantitation
The LoQ is defined as the lowest concentration of HIV-1 RNA that is determined with acceptable precision and trueness. The LOQ of the HIV-1 VL Assay is 40 cp/mL (1.60 log10).

Linear Range
The HIV-1 VL Assay is linear within a range 30 to 1x10 7 cp/mL

Specificity
The specificity of the HIV-1 VL Assay was evaluated using EDTA plasma specimens from HIV-1 negative blood donors. The HIV-1 VL Assay specificity was demonstrated at 100% (95% CI = 96.7-100.0).