Population
The study was conducted in three different hospitals (Tamale Teaching Hospital, Tamale Central Hospital and Savelugu Municipal Hospital) in the northern region of Ghana.
Ethnicity
The main ethnic group in Tamale metropolis and Savelugu municipal is Dagomba. However other ethnic groups such as Akan, Frafras, Mamprusi, Moshe, Ewe, can be found in both the metropolis and the municipality [19].
Socio-economic status
The major occupation(s) in these areas (Tamale Metropolis and Savelugu municipality) are farming, craftsmanship and petty trading among women and men. A proportion of the population engages in public services, however, unemployment rate among the youth of the metropolis is high, reflecting the high poverty level in the metropolis.
Participants
Participants were recruited from the Out-Patient Department (OPD), laboratory and Anti-Retro Viral Therapy (ART) centres of these hospitals. They involved: adult HIV patients (≥18 years) who had been on antiretroviral therapy for not more than 10 years; adult OPD patients (≥18 years) with positive or negative HIV results after laboratory testing; patients with normal haematological and biochemical test results after laboratory analysis; and prospective blood donors who tested either positive or negative for HIV, Hepatitis B, Hepatitis C and syphilis. In order to minimize false positive and negative results, all adult HIV patients (≥18 years) who had received antiretroviral therapy for more than 10 years were excluded [11]. Also, all pregnant women who visited the facilities were not considered as participants for this study [14]. Additionally, children less than 18 months born to HIV positive mothers were not recruited [20].
Sample size calculation
The sample size (N) was calculated using the sensitivity (100 %) and specificity (99.5 %) values quoted in the test kit manufacturer’s instruction package insert and the national HIV prevalence (1.3 %). These were imputed into the formula, \(({\text{PPV}} = \frac{{{\text{sensitivity }} \times {\text{prevalence}}}}{{{\text{sensitivity }} \times {\text{prevalence}}}} + (1 - {\text{prevalence}})\) and \({\text{NPV}} = \frac{{{\text{specificity }} \times (1 - {\text{prevalence}})}}{{(1 - {\text{sensitivity}}) \times {\text{prevalence}}}} + {\text{specificity}} \times (1 - {\text{prevalence}})\) to estimate the positive and negative predictive values of the test kit. The positive predictive value was 97 % and the negative predictive value was 100 %. This means there is a 3 % probable positive case that will not be detected by the kit. The predictive values were computed in a sample size calculator to determine the sample size by the formula N = 2 {10.5 × [PPV × (100−NPV) + NPV × (100−PPV)/(PPV−NPV) ^2]} at a power of 80 % and a significance level of 0.05. The sample size was 280 participants after correcting for non-respondents. 15 (5.08 %) participants had incomplete data and were categorized as non-respondents.
Study design
A hospital-based cross-sectional study was conducted on HIV infected and non-infected patients from May 2015 to June 2015. Whole blood and serum samples collected from these participants were tested on First Response HIV-1-2 RDT kit, and ECLIA technology was used as the gold standard assay. The test kit’s sensitivity, specificity, positive and negative predictive values achieved with serum and whole blood specimens were determined and compared.
Testing procedures
Clinical samples collection, processing and storage
The specimens used in this study were fresh samples from HIV infected patients, OPD patients and prospective blood donors. Fresh sets of samples (EDTA-anti-coagulated whole blood, and serum) were collected from 295 patients (This includes participants with incomplete data).
Venous blood collected from each participant was divided into EDTA-anticoagulant and serum separator tubes labelled with the patient’s identification. The anticoagulant prevented the blood from clotting making it possible to obtain whole blood.
The serum separator tubes were centrifuged at 3000 rpm for 15 min to obtain the serum samples. The whole blood samples were stored at refrigeration temperature at 4 °C and the serum samples at −20 °C until used. Testing was done not more than 72 h after sample collection.
HIV antigen (p24)/antibody test
HIV Antigen (p24)/antibody tests were done by automation on Cobas E 411 analyzer using serum samples. Because the sample of choice for this analyzer was serum, whole blood samples could not be analyzed. All 295 serum samples were analyzed to determine their HIV sero-status.
Cobas E 411
Cobas E 411 (Roche Diagnostics GmbH, Mannheim, Germany) is a fully automated immunoassay analyzer which uses electro-chem-iluminescence immunoassay (ECLIA) technology to detect HIV-1 and HIV-2 in human serum. The analyzer had sample loading chambers, a touch-screen monitor and a printer. This study used Elecsys HIV combi PT (Roche Diagnostics GmbH, Mannheim, Germany) as reagents. This reagent is a fourth generation qualitative immunoassay for the determination of HIV-1 p24 antigens and antibodies to HIV-1, including group O, and HIV-2 in serum. Test results were produced within 30 min after loading samples.
Samples with cutoff index (COI) <0.9 were considered non-reactive (negative) and those with COI ≥1.0 considered reactive (positive). There were no observed COI readings in the range ≥0.9 to <1.0 (borderline) among the samples, HIV RNA test was therefore not needed.
HIV antibody test
All HIV antibody tests were performed by applying whole blood or serum on First Response HIV-1-2 test kits.
First Response HIV-1-2 test kit
First Response HIV-1-2 (Premier Medical Corporation Ltd., Kachigam, India) kit is a rapid immuno-chromatographic qualitative test for the detection of antibodies to HIV-1 and HIV-2 in whole blood, plasma or serum. The kit was packaged with a sample pipette and a sample buffer.
The kit had two test-band regions. Region “1” was pre-coated with recombinant HIV-1 antigens (gp 41, including group O and p24) and region “2” was pre-coated with recombinant HIV-2 antigen (gp36). These recombinant antigens were conjugated with colloidal gold particles. There was a region labelled “C”, the inbuilt control line which indicated successful antigen–antibody reaction.
The results were interpreted according to the manufacturer’s instruction document. Red lines at both regions “1” and “C” meant HIV-1 infection and at regions “2” and “C” was indicative of HIV-2 infection.
Legend negative (single red band at control line). Positive (two red bands, one at 1 and the other at the control).
Classifying samples as HIV infected or non-infected
We used ECLIA technology as the gold standard test in this study. The whole blood and serum samples were first tested on First Response HIV-1-2 test kits simultaneously. Both reactive and non-reactive samples were retested on Cobas E 411 analyzer. This enabled us to classify the samples as HIV infected and HIV non-infected. Samples with reactive results for both whole blood and serum on the test kit and reactive serum result on the analyzer were classified positive.
Samples with non-reactive results for both whole blood and serum on the test kit and non-reactive serum result on the analyzer were classified negative. For samples that produced results on the test kit which did not agree with the analyzer (discordant results), a decision was made after consultations with HIV diagnostic experts. A repeated test was done with the test kit. The two results (initial and repeated) were compared and the blood-based specimen type (whole blood or serum) result that agreed with the analyzer was chosen as the final result for classification.
All patient test results and information were treated confidential and the study was approved by the Ethical Review Board of Ghana Health Service (approval number GHS-ERC: 06/02/15).
Data analysis
Data analyses were performed using the statistical software, StataSE (version 13.1, StataCorp, Texas, USA). Variables were summarized as frequencies and percentages with significant level at 0.05. Sensitivity, specificity, positive and negative predictive values were determined using cross-tables and standard formulas {(PPV = sensitivity × prevalence/sensitivity × prevalence + (1−prevalence)} and {(NPV = specificity × 1−prevalence)/(1−sensitivity) × prevalence + specificity × (1−prevalence)}.