A Comparative Analysis of the different HIV Testing Techniques used in Zambia: Data from a Clinical Performance Study

doi:10.21203/rs.3.rs-3883706/v1

Objective

Early detection of HIV means early identification of people living with HIV, who are immediately linked to care and antiretroviral therapy. This decreases transmission of the virus, which reduces HIV new infection rates. The aim of the study was to investigate the effectiveness of different HIV testing strategies used in Zambia.

Results

A quantitative descriptive approach was used to analyze samples of 2564 participants within Lusaka district. 2564 participants were subjected to OraQuick ADVANCE Rapid HIV-1/2 Antibody Test and Abbot Determine™ HIV-1/2 antibody test, if Reactive, they were tested on Uni-Gold™ Recombigen® HIV-1/2 rapid test as a confirmatory RDT. The samples sent to the Laboratory were tested on fourth generation Abbot ARCHITECT HIV Ag/Ab Combo and then confirmed on Genscreen™ ULTRA HIV Ag-Ab. The Abbot ARCHITECT HIV Ag/Ab Combo fourth generation assay was used as the gold standard to compare the other tests. OraQuick ADVANCE Rapid HIV-1/2 Antibody Test detected 245 reactive tests, Abbot Determine™ HIV-1/2 antibody test detected 249 reactive tests, all reactive tests on the first two RDTs were confirmed on Uni-Gold™ Recombigen® HIV-1/2 rapid test which detected 247 Reactive and 2 Non-Reactive. The fourth generation reference test -ARCHITECT HIV Ag/Ab Combo detected 2297 Non-Reactive and 267 Reactive tests.

Conclusion

The results indicate that the rapid tests false negatives are high and a 4th generation Rapid test to the testing algorithm is vital. This will increase total number of Positive results being missed be accounted for.

According to WHO global HIV summary report, by the end of 2019 there was a total of 38 million people living with HIV, 2.5 million new infections and 1.7 million deaths each year, emphasizing that the global response would have to be sustained for at least several decades in order to curb the HIV epidemic.¹ Despite all the strategies put in place to help end the HIV/AIDS epidemic, there still remains a substantial number of people with HIV who are unaware of their infection and are still infecting others.² The only way to determine a person’s HIV status is to have an HIV test. Globally about 20% of HIV infections are due to transmissions from recently infected individuals.³ The World Health Organization recommends two sequential rapid diagnostic tests (RDTs) for HIV diagnosis, however third generation RDTs detect only HIV antibodies and may miss up to 75% of early acute HIV infections.⁴

There is considerable interest in revising testing guidelines to more accurately reflect new technology and associated challenges.⁵ According to Marks et al the estimated 25% of individuals unaware of their HIV infection are responsible for 54% of new infections. Many HIV health care practitioners believe that individuals’ knowledge of their HIV serostatus is an effective preventive intervention, and this belief is supported by available data⁶.

Point of care (POC) options for the diagnosis of acute HIV are currently limited to third-generation RDTs.⁶ New approaches to the diagnosis of acute HIV such as point-of care testing need more evaluation in the field against agreed definitions of acute HIV in order to realize the public health benefits of making this important diagnosis.⁶An addition of a fourth generation HIV RDT to testing algorithms would help in reducing the number of acute infections being missed by third generation RDTs that only detect antibodies. Identification of acute HIV infection requires detection of HIV nucleic acids or p24 antigens which is possible with 4^th generation laboratory-based assays that detect HIV-1 p24 antigen as well as antibodies to HIV-1/2.^{7, 8, 9}

Recently there has been the development of new fourth-generation point-of-care rapid diagnostic tests (RDTs) that can detect HIV-1 p24 antigen as well as antibodies to HIV-1/2. These RDTs have the potential to meet the above requirements and have been approved for their sale in many settings. Despite the many concerns about the poor diagnostic accuracy in the field for the diagnosis of acute HIV-1,¹⁰many studies have shown that fourth-generation RDTs can still detect a good number of acute HIV infections ¹¹compared to third-generation RDTs.

The reported sensitivity of a test also depends on the sensitivity of the comparator or reference standard.¹²Manufacturers of four commonly studied HIV rapid tests quote sensitivities of 99.3 to 100%, but all were compared with earlier Enzyme Immuno-Assays (EIA) that are less sensitive than the now widely available fourth-generation EIAs.¹² Most current HIV rapid tests detect HIV antibodies only. Fourth-generation EIAs can detect HIV p24 antigen as early as 2 weeks post-infection, as well as being able to detect the antibodies that appear later.¹² These EIAs are recommended as first-line screening tests in the 2014 Centers for Disease Control and Prevention (CDC) HIV laboratory testing algorithm.

Although the proportion of acute infections can be expected to vary across different populations, there are no studies examining the effect of this on the performance of HIV rapid tests.¹³ Health services that have the option of providing either HIV rapid tests or the more sensitive laboratory assays therefore need to know whether there will be a significant loss of sensitivity if they use HIV rapid tests in their clinical population.¹³

Zambia has an adult HIV prevalence of 11.1%¹⁴ meaning if 3^rd generation RDTs are used for first time HIV testing, there will be a lot of missed opportunities. Failure to diagnose HIV in the weeks after an individual has acquired infection negatively impacts the epidemic control by increasing the likelihood of onward transmission.³ According to the Zambia National Guidelines for HIV Counselling and Testing HIV screening is one of the most important activities associated with C&T. It has been established that early detection of HIV infection can result in public health benefit by decreasing risk behaviors that could transmit HIV to uninfected people. On the other hand uninfected people may benefit from HIV testing, if knowing their HIV status assists them in reducing their risk taking behavior. In addition, knowledge of one’s own HIV status is an important influencing factor for preventing acquisition of HIV.

The Zambia Public Health Service HIV testing algorithm has not changed substantially in the past decades, despite the introduction and wide use of new technology. It is very necessary to revise testing guidelines to more accurately reflect new technology and associated challenges.⁶ The Zambian HIV test algorithm uses RDTs that only detect antibodies, which means that most acute infections are missed in the first line tests and may be falsely reported as negative. The HIV rapid test algorithm has been in use for more than a decade, it is the primary screening strategy used at all HIV Testing and counselling sites in Zambia. All clients found negative on first line rapid diagnostic test (RDT), are reported as negative and those positive are subjected to more HIV confirmatory tests. The national RDT algorithm in Zambia consists of a screening test (Determine® HIV 1/2) followed by confirmation of reactive specimens with a second rapid test (Uni-gold HIV 1/2). To test respondents via RDT, a blood sample is collected directly from a finger prick.¹⁴

According to the CDC, guidelines for HIV testing continue to evolve with changes in testing technology and methods to reach persons who can benefit from these services. The ministry of health Zambia recommends the testing algorithm shown in Figure 1 below, which could include the following RDTs

Abbot Determine HIV 1/2 Test
Genie II HIV 1/2 Test
Uni-Gold HIV Test
Bionor HIV 1/2 Test

Study design and setting

This systematic review used quantitative methods by measuring statistical analysis of different variables of the five different HIV tests by using probability methods. The comparison between these diagnostic tests forms the foundation of this comparative evaluation of HIV testing techniques. Serologic test results were divided into Reactive and Non-Reactive findings, as recommend by the manufacturers of the diagnostic tests. This study analyzed data collected from the STAR clinical performance study.¹⁵The analysis is solely dependent on the secondary data from the STAR study. Serologic methodologies for the 5 test methods were evaluated for HIV. The primary purpose of this investigation was to complete a comparative evaluation of 3 HIV rapid diagnostic tests and 2 fourth generation tests.

Test methods

The specimen was collected by trained personnel following participant informed consent and complete assessment. Samples analyzed included oral mucosa transudate using the OraQuick® HIV Self-Test, fingerprick blood was collected and tested on Alere Determine^TM HIV1/2, if positive on Determine it was then confirmed on Unigold^TMHIV1/2 (Trinity Biotech) test following the Zambian national testing algorithm. The results were provided to participants, and data stored in electronic devices, all participants with reactive test results were referred for HIV care and treatment at local health facility.¹⁵

In addition to the three rapid tests performed at Point of Care, 10 mls whole blood was collected from each participant by trained personnel and sent to the Laboratory for further testing on fourth generation assays. Specimens were processed by standardized methods and tested following the manufacturer’s instructions for all the diagnostic test methods. The 10mls blood samples sent to the Laboratory were double-span and plasma was separated and tested on the Abbot Architect HIV Ag/Ab Combo fourth generation instrument as the gold standard for the rapid tests.¹⁵ If result was reactive, it was then run on manual ELISA Genscreen™ ULTRA HIV Ag-Ab to confirm positivity.

Specificity and Sensitivity of the five different HIV testing strategies were measured by calculating the percentage of positive results yielded by these tests among all samples that tested positive for HIV based on detection of HIV positive results of the Abbot Architect test.¹⁶ Rapid test positivity was calculated by comparing the characteristics of HIV-positive and HIV-negative results. The Results were reported in accordance with the Standards for Reporting of Diagnostic Accuracy: Sensitivity, specificity, positive and negative predictive values at 95% confidence intervals using SPSS version 25.0. The gold standard serologic tests for HIV are the enzyme immunoassay (EIA). The Determine HIV1/2, product sensitivity and specificity calculations were based on the comparison of test results with an indirect enzyme immunoassay the Abbot Architect and Genscreen HIV-1/2.

Inclusion and exclusion criteria

The study only included samples of participants who gave informed consent to participate in the STAR study by Zambart¹⁵and who were tested on all 5 of the HIV test types being investigated.

Participants who did not give consent or did not give a third sample for Laboratory testing were excluded from the study.

Definition of Terms

Accuracy: Is how close a measurement is to the true or accepted value. It is the description of systematic errors, a measure of statistical bias; low accuracy causes a difference between a result and a ‘true value’.

Antibody: it is a protective protein produced by the immune system in response to the presence of a foreign substance called an antigen.

Antigen: it is a substance that is capable of stimulating an immune response by specifically activating lymphocytes, which are the body’s infection-fighting white blood cells.

Negative Predictive Value (NPV): is the proportion of patients with a negative test who do not have the disease.

Point of Care testing (POCT): it is an investigation taken at the time of the consultation with instant availability of results to make immediate and informed decisions about patient care.

Positive Predictive Value (PPV): is the proportion of patients with a positive test who actually have the disease.

Rapid Diagnostic Test: is a medical diagnostic test that is quick and easy to perform, suitable for preliminary emergency medical screening and for use in facilities with limited resources.

Screening: A screening test (sometimes termed medical surveillance) is a medical test or procedure performed on members of a defined asymptomatic population or population subgroup to assess the likelihood of their members having a particular disease.

Sensitivity: is the percentage of results that will be correctly positive when the disease is actually present. Lower rates of sensitivity will produce more false negative results.

Specificity: is the percentage of results that will be correctly negative when the disease is not present. Lower rates of specificity will produce more false positive results.

Window Period: is the time between the first infection and the time when the infection can be reliably detected.

Statistical analysis

Data was analyzed using Microsoft Excel spreadsheet for broader thematic analysis and SPSS version 25.0. Exact confidence intervals (CIs) were calculated according to the binomial distribution. Variables were summarized as frequencies and percentages with significant level at 0.05 (95%CI). Sensitivity, specificity, positive and negative predictive values were determined using cross-tables and standard formulas of the third generation HIV RDTs, the Abbot fourth generation immunoassay was used as the gold standard. Data from the STAR-Study were entered into a Microsoft excel sheet, checked for duplication and error and then subsequently exported to SPSS version 25 for analysis.

Table 1 below shows Gender, Age Groups and HIV Status on Abbot Architect of the study participants compared to Sites they lived. The study examined specimen of 2564 participants, which were tested on the 5 different HIV tests being investigated. The rapid diagnostic laboratory tests were evaluated based on a comparison test method. The investigation evaluated the test accuracy of five diagnostic assays for the identification of HIV based on sensitivity and specificity measures. Among the 2564 samples analyzed, 1043 (40.7%) were male participants and 1521 (59.3%) female participants. The 2564 samples were from participants of a wide range of ages from the age of 15 to as old as 95 years as show in table 3. All participants below 18 years agreed to participate with guided parental consent.

Specimen of participants who were tested on all the 5 HIV tests were included in the analysis. In addition to the 3 Point of Care rapid tests, a blood sample was collected from each participant and sent to the laboratory for testing on Abbot Architect, all positive results on Abbot were confirmed using Genscreen Biorad ELISA. Variables analyzed include baseline demographics and rapid test results. Measurement of each tests’ sensitivity and specificity were compared and reported with 95% confidence intervals. Sensitivity and specificity of Ora-Quick and Determine was measured compared to HIV positive serostatus. HIV positive serostatus was defined by a positive result on the Abbot Architect Assay.

Table 1: Gender, Age Groups and HIV Status on Abbot Architect of the study participants compared to Sites they lived.

Gender, Age and HIV results compared to study sites of the participants
	Mtendere		Kanakantampa		Total
Characteristics	n = 1955	%	n = 609	%	n = 2564	%
Gender
Male	719	28.0%	324	12.6%	1043	40.7%
Female	1236	48.2%	285	11.1%	1521	59.3%
Total	1955	76.2%	609	23.8%	2564	100.0%
Age Groups
15 - 17	78	3.0%	30	1.2%	108	4.2%
18 - 25	962	37.5%	189	7.4%	1151	44.9%
26 - 35	554	21.6%	149	5.8%	703	27.4%
36 - 45	228	8.9%	110	4.3%	338	13.2%
46 - 55	78	3.0%	55	2.1%	133	5.2%
56 - 65	41	1.6%	35	1.4%	76	3.0%
66 - 75	10	0.4%	28	1.1%	38	1.5%
76 - 85	4	0.2%	9	0.4%	13	0.5%
86 - 95	0	0.0%	4	0.2%	4	0.2%
Total	1955	76.2%	609	23.8%	2564	100.0%
HIV result
Non-Reactive	1737	67.7%	560	21.8%	2297	89.6%
Reactive	218	8.5%	49	1.9%	267	10.4%
Total	1955	76.2%	609	23.8%	2564	100.0%

The study included participants from two sites: Mtendere, representing the urban population and Kanakantampa the rural population. About 1955 (76.2%) participants were from Mtendere and 609 (23.8%) participants from Kanakantampa. There were more female (63.2%) than male (36.8%) participants in Mtenedere took part in the study, while in Kanakantampa more male (53.2%) participants took part in the study compared to females (46.8%). In both sites the highest participating ages range from 18 to 45 years of Age.

The table 2 below compares gender and Age of participants to HIV test results of the Abbot, used as a reference. As expected, female participants (68.9%) had a higher positivity rate compared to male participants (31.1%). The age range with the highest positive results was 26 to 35 (38.9%) years.

Table 2: Gender and Age categories of Participants compared to the gold standard HIV test results

Gender and Age of Participants compared to ABBOT Results
	Abbot Architect Results (N)
	Non-Reactive		Reactive		Total
Gender of Participant	(N)	%	(N)	%	(N)	%
Male	960	37.4%	83	3.24%	1043	40.7%
Female	1337	52.1%	184	7.18%	1521	59.3%
Total	2297	89.6%	267	10.41%	2564	100.0%
Age Groups
15 -17	102	4.0%	6	0.23%	108	4.2%
18 -25	1087	42.4%	64	2.50%	1151	44.9%
26 - 35	599	23.4%	104	4.06%	703	27.4%
36 - 45	275	10.7%	63	2.46%	338	13.2%
46 - 55	111	4.3%	22	0.86%	133	5.2%
56 - 65	71	2.8%	5	0.20%	76	3.0%
66 - 75	36	1.4%	2	0.08%	38	1.5%
76 - 85	12	0.5%	1	0.04%	13	0.5%
86 - 95	4	0.2%	0	0.00%	4	0.2%
Total	2297	89.6%	267	10.41%	2564	100.0%

The total mean age was 29.71 years (standard deviation 12.18). The mean age of the male participants was 30.89 (standard deviation 12.87) and 28.92 (standard deviation 11.63) for the females.

The blood samples sent to the lab were processed and tested on the Abbot Architect and then all that were positive on Abbot Architect were confirmed using the Genscreen Biorad ELISA test. The table 3 below shows a breakdown of how many tests from each Assay were reactive, non-reactive and not tested.

Table 3: Results of the 5 HIV tests being investigated.

	Ora-quick		Determine		Uni-Gold		Abbot Architect		BIORAD-ELISA
	N	%	N	%	N	%	N	%	N	%
Non-Reactive	2319	90.4	2311	90.1	6	0.2	2297	89.6	1	0.0004
Reactive	245	9.6	253	9.9	247	9.6	267	10.4	266	10.4
Not Tested	0	0	0	0	2311	90.1	0	0	2297	89.6
Total	2564	100	2564	100	2564	100	2564	100	2564	100

Interpretation of Table 3:

Ora-Quick: Of the 2564 samples tested on this test, 2319 (90.4 %) were non-Reactive and 245 (9.6%) were Reactive. All samples had a result.

Determine: all samples tested on Ora-quick were tested on Determine, out of the 2564 samples tested, 2311 (90.1%) were non-Reactive and 253 were Reactive.

Uni-Gold: Since this was used as a confirmatory test, only samples Reactive on the Determine test were tested on Uni-Gold. 253 samples Reactive on Determine were tested on Uni-gold, 247 were Reactive and 6 samples were non-Reactive. 2311 were not tested because they were Non-Reactive on Determine.

Abbot Architect: The Abbot Architect was the gold standard test were all above tests were compared to. All 2564 samples were tested on this test, 2297 samples were non-Reactive, and 267 samples were Reactive.

Genscreen Ultra ELISA: This was a confirmatory test for the Abbot Architect test. Only samples Reactive on Abbot were tested on this test. Of the 267 samples tested, 266 were Reactive and 1 which had a low signal to cut off on Abbot Architect tested Negative. This 1 discordant result was tested on Geenius confirmatory test, and it was Non-Reactive, showing that the Genscreen ultra ELISA is slightly more sensitive than the Abbot Architect test.

Table 4 below shows the Abbot Architect test was the reference standard, it had 2297 negative results and 267 positive results, when compared to the other Test result. When OraQuick results were compared to the gold standard, it was observed that of the 267 reactive results, OraQuick only detected 245 (91.8%) positive results and missed 22 (8.2%). When compared to Abbot Architect results, Determine 249 (93.3%) reactive and 18 tests negative on Determine but positive on ABBOT. The 249 Reactive results on Determine were confirmed on the UniGold test kit, of the 249 tested on UniGold, 247 were reactive and 2 gave false positive results. Compared to Abbot Architect results, UniGold was Reactive on 247 (92.5%) tests and gave 20 (7.5%) non-reactive results.

Table 4: Comparison of the results of OraQuick, Determine and UniGold to the gold standard.

Results of 3 Rapid Diagnostic tests compared to Abbot fourth generation test
RAPID DIAGNOSTIC TEST RESULTS		Abbot Architect Results
RAPID DIAGNOSTIC TEST RESULTS		Non-Reactive	Reactive	Total
OraQuick Result	Non-Reactive	2297	22	2319
OraQuick Result	Reactive	0	245	245
	Total	2297	267	2564
Determine Result	Non-Reactive	2293	18	2311
Determine Result	Reactive	4	249	253
	Total	2297	267	2564
Unigold Result	Non-Reactive	4	2	6
	Reactive	0	247	247
	Not done	2293	18	2311
	Total	2297	267	2564

Interpretation of table 4:

OraQuick Results: Figure 2 below shows the tests that were Non-Reactive on both OraQuick and ABBOT were 2297(89.6%), 245(9.6%) were Reactive on both tests. 22(0.9%) tests were Reactive on ABBOT and non-reactive on OraQuick, meaning that ABBOT picked up 22(0.9%) more positive tests that OraQuick tested as non-reactive. 4(0.2%) tests were Non-reactive on Oraquick but reactive on Determine and Unigold.

OraQuick Rapid Test breakdown with reference to ABBOT test results

Determine Results: in Figure 3 below 2293(89.4%) tests were non-reactive on both determine and Abbot Architect. 249 (9.7%) tests were reactive on both Abbot Architect and Determine. 18(0.7%) were Reactive on Abbot Architect and Non-reactive on Determine, showing that Abbot Architect has a higher sensitivity compared to Determine.¹⁷There were 4 tests that were reactive on Determine and non-reactive on Abbot Architect the reference test, showing a 0.2% low specificity rate of Determine.

Determine Rapid Test breakdown with reference to ABBOT test results

UniGold Results: figure 4 below shows the confirmatory test for the Determine rapid tests, therefore only the tests reactive on Determine were tested on UniGold. Of the 253(9.9%) tests tested on UniGold, 2(0.9%) were Non-Reactive on Unigold but reactive on Determine and Abbot Architect.

Uni-Gold™ Recombigen® HIV-1/2 Test breakdown

Interpretation of results of the 3 rapid diagnostic tests and ABBOT assay

When all 3 rapid tests were compared to Abbot Architect, we see that OraQuick gives 22(0.86%) false negatives, UniGold gives 20(0.78%) false negatives and Deterimine had 18(0.70%) false negatives and 4(0.2%) false positives. Of the 3 RDTs, Determine is the most accurate with the highest Sensitivity (93.3%), then UniGold with 92.5% and lastly OraQuick with 91.7% sensitivity, which show a similar result pattern to a study by Kashoshi.

Plasma of the 2564 participants was tested on the Abbot Architect analyzer, 2297 (89.6%) were negative and 267 (10.4%) were positive as shown in the table 5. 2,311 (90.5%) had both a negative OraQuick and Determine test result. 245 (91.8%) participants had both a positive Determine and OraQuick test result. The sensitivity and specificity of the OraQuick test were 91.7 (95%CI) and 100 (95%CI), respectively when compared to HIV positive ABBOT test (Table 5). The Sensitivity and Specificity of Determine test were 93.3% and 99.8% respectively.

Table 5: Sensitivity, Specificity, Positive predictive value (PPV) and Negative predictive value (NPV) of the 4 Assays.

Test (Assay)	OraQuick	UniGold	Determine	Abbot Architect
No of positive results(a)	245	247	249	267
No of Negative results(d)	2319	2313	2311	2297
False Negative results©	22	20	18	0
False Positive results(b)	0	0	4	0
Sensitivity (%)	91.8	92.5	93.3	100
Specificity (%)	100	100	99.8	100
PPV (%)	100	100	98.4	100
NPV (%)	99.1	99.1	99.2	100

Discordant results

There was a total of 26(1%) discordant Determine and OraQuick test results. 18 (69.2%) were Determine negative and Abbot Architect positive, 8 (30.8%) were negative on Oraquick and positive on Determine, of the 8, 4 (15.4%) were Determine positive but negative on Abbot Architect and Oraquick and the other 4 (15.4%) were Oraquick negative and positive on both Determine and Abbot. Uni-GoldTM test had less positivity when compared to the total true positive tests. Of the 267 total reactive tests, Unigold detected 247 (92.5%) positives whereas Determine detected 249 (93.3%) positives and Oraquick detected 245 (91.8%).

Overall Description of the Analyzed Data

Of the 2564 participants who gave a sample, 1521 (59.4%) were female with a mean age of 28.9 years (95% CI: 28.3 – 29.5). 1043 (40.6%) were male with a mean age of 30.9 years (95% CI: 30.1 – 31.6). 267/2564 samples (10.4%) were found to be HIV-positive by the 4th generation ABBOT Architect HIV Ag/Ab assay. In contrast, only 245/2564 (9.6%), 249/2564 (9.7%), and 247/2564 (9.6%) samples tested positive by OraQuick ADVANCE® Rapid HIV-1/2 Antibody Test, Abbot Determine™ HIV-1/2 antibody test and Uni-Gold™ Recombigen® HIV-1/2 respectively (Table 5). When compared to the 4th generation Abbott Architect HIV Ag/Ab Combo assay, there were 22 HIV+ samples that were missed (“false negative”) by all three RDTs under evaluation, but only 4 samples had a reactive result (“false positive”) by Alere Determine HIV-1/2 which were negative on and Uni-Gold HIV which was not confirmed by the 4th generation Abbott Architect HIV Ag/Ab Combo assay.

Accuracy of the Different Tests

As shown in Table 5, OraQuick ADVANCE® Rapid HIV-1/2, Alere Determine HIV-1/2 and Uni-Gold Ultra HIV, at 95% CI had Sensitivities of: 91.8%, 93.3% and 92.5% respectively, of which when compared to the sensitivities reported by the manufacturers is quite low despite manufacturers reports of high sensitivity and specificity for these RDTs.¹⁸ Generally, the performance of HIV rapid diagnostic testing in a population is influenced by Positive and Negative Predictive values of the RDT kits used. Thus sensitivity and specificity results obtained from test kit evaluation studies to licensing and marketing of the kit will not necessarily be achieved in practice.¹⁹

The specificities of OraQuick ADVANCE® Rapid HIV-1/2 and Uni-Gold HIV were the same (100.0%; 95% CI: 98.8 -100.0) but slightly different from Alere Determine HIV-1/2 (99.8%). Negative predictive values (at 95% CIs) were 99.1, 99.2 and 99.1 for OraQuick ADVANCE® Rapid HIV-1/2, Alere Determine HIV-1/2, and Uni-Gold Ultra HIV respectively similar to Kashoshi study findings.¹⁷

The results shown in table 5 of the reference test-4th generation Abbot Architect HIV Ag/Ab assay demonstrating high sensitivity and maximum positive predictive values. Kappa statistic showed almost perfect agreement between the 4th generation Abbot Architect HIV Ag/Ab and three RDTs: OraQuick ADVANCE® Rapid HIV-1/2 (k = 0.952; 95% CI: 0.932 – 0.972), Alere Determine HIV-1/2 (k = 0.953; 95% CI: 0.933 – 0.972), and Uni-Gold Ultra HIV (k = 0.957; 95% CI: 0.938 – 0.976).

This study has established that the three 3rd generation HIV RDTs used at Point of Care for HIV testing though quite sensitive, still fail to detect a significant number of acute infections. About 1 in every 10 people tested using 3rd generation RDTs goes home with false negative results. The identification of persons with acute HIV infection represents a significant challenge, owing to the absence of antibodies in the earliest stages, limitations of standard rapid tests to detect p24 or HIV RNA, and logistical and cost issues with p24 antigen and HIV RNA.¹⁹ In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections.²⁰

Given the brief window of acute HIV infection, the acute HIV prevalence in a population is very low at any given point in time, even among relatively high-risk groups. Consequently, a test must have exceptional performance characteristics to be useful, especially without additional confirmatory testing.¹⁹ The Determine®HIV-1/2 Ag/Ab Combo rapid test has been marketed as such a test, because it is relatively inexpensive and can be administered at the point of care.¹⁹ Although the performance characteristics of the Determine®HIV-1/2 Ag/Ab Combo rapid test were insufficient for use in clinical setting, field evaluation in other settings may be warranted to assess whether the test performs better. The Determine®HIV-1/2 Ag/Ab Combo rapid test has been previously evaluated with stored serum or plasma specimens.²¹

The study is consistent with the findings by Kashosi who found that when compared to the laboratory-based ELISA HIV Ag/Ab assay, the currently used 3rd generation HIV RDTs showed poor field accuracy.¹⁷ According to their Manufacturers, RDTs have high sensitivity and specificity for licensing purposes and subsequent World Health Organization (WHO) prequalification.¹⁸ Despite this, HIV RDTs have some limitations since they cannot identify persons who are within the window period of an acute HIV infection because these individuals have not yet developed HIV-specific antibodies.^{22, 23} Such individuals are highly infectious due to concurrent high plasma as well as vaginal and semen HIV-1 viral load.^{3, 24}

The study found an unacceptably low sensitivity yet high specificity, and a moderate agreement for all three HIV RDTs evaluated, when compared to the 4th generation Abbot Architect HIV Ag/Ab assay. These results are in line with findings from Ethiopia by Dessie and Bossuyt in which Alere Determine HIV-1/2 showed low sensitivity (60.5%). ^25,26

This study highlights the important issue of possible false-negative HIV test results, which in part may be explained by the failure of these RDTs to diagnose HIV during the window.¹⁷ During this period, HIV infection can be detected only by tests detecting also viral antigen such as the laboratory-based 4th generation ELISA.²² The baseline HIV prevalence in the population influences the negative and PPV and needs to be considered in the interpretation of HIV RDT results. On the other hand, it is not certain that the currently available 4th generation HIV RDTs would perform better based on recent published data showing that the HIV p24 antigen detection component of some 4th generation RDTs also lacks analytical and diagnostic sensitivity.^{26, 27}

According to Kashosi it seemed unlikely that the false-negative samples missed by HIV RDTs in the study, but which were positive for p24 antigen and/or HIV antibodies by the 4th generation ELISA, might represent very early infections (p24 antigen only) or somewhat later ones with antibodies at a low level, not detectable by the RDTs.¹⁷ Given the qualitative results generated by the laboratory-based 4th generation ELISA test used and the lack of resources for p24 antigen or nucleic acid testing (NAT) in Eastern DRC, Kashosi could not resolve this uncertainty.¹⁷

Other possible causes that may have caused false-negative results in this study include divergent HIV sub-types, human error- such as the addition of insufficient specimen or too much buffer when procedures are handled by non-trained or unsupervised staff.²⁸ transportation or storage of test kits outside of recommended conditions, leading to possible denaturation of reagents or test devices, and the use of expired reagents or test devices.¹¹

This study went out to establish the possibility of unacceptably low sensitivities of the RDTs Alere Determine HIV-1/2 and Uni-Gold HIV, using blood and, OraQuick ADVANCE® Rapid HIV-1/2 using mouth swabs in Zambia. This raises serious concerns about rapid test dependability and calls for stringent control measures as well as the need for 4th generation Ag-Ab RDTs and a cross-evaluation of all Rapid diagnostic tests results by a laboratory-based antigen testing, whenever feasible.

An alternative HIV testing algorithm, which includes a fourth-generation Determine® HIV-1/2 Ag/Ab Combo rapid test with a better sensitivity and specificity, is necessary¹⁹ .Developing and introducing a point-of-care test with adequate sensitivity and specificity for acute HIV detection is extremely challenging but very feasible. The Determine®HIV-1/2 Ag/Ab Combo rapid test was designed to provide a marginal reduction in the HIV window period by testing for both Antibodies and Antigens at point of care settings.¹⁹

The results of this study have several important clinical and public health implications, for example in the era of test-and-treat strategies, there is a need to use and scale-up highly sensitive and specific HIV RDTs to optimize the first and second steps in the HIV care cascade (diagnosis and linkage to care).¹⁷

The Determine®HIV-1/2 Ag/Ab Combo rapid test is one example of many other 4th generation tests that have been invented for the main purpose of detection both Antibodies and Ag P24 at point of care settings. The introduction of such a test to the HIV testing algorithm will significantly increase total Positives caught in early stages.¹⁷

The use and scale-up of emerging low cost and point-of-care tests, such as the highly sensitive and easy-to-handle molecular HIV diagnostic tests Xpert HIV-1 Qualitative and Xpert HIV-1 Viral Load Test (e.g., Alere qHIV1/2 detect), would represent a significant step forward in our efforts towards early HIV diagnosis.¹⁷ These automated HIV diagnostic platforms do not require much infrastructure and can be used at or near point-of-care. However, they are more expensive, and efforts need to be made in the future through public-private partnership to make them affordable and accessible in sub-Saharan Africa where they are badly needed.^{29, 30}

Limitations

The data used in this study was secondary, and collected from another study, access to the data was not easy.

Ethical approval and consent to participate

Ethical approval was granted by the University of Lusaka, medical Ethics Committee. All methods were carried out in accordance with relevant guidelines and regulations with relevant authorizations from Ministry of Health. Consent to participate was obtained. For participants with age less than 16 years, informed consent was obtained from their parent(s)/guardian(s) as well In addition, and Informed consent was obtained from participants and management before the study.

Consent to publish

Not applicable

Availability of data and materials

All data generated or analyzed during this study are included in this published Article. For other data, these may be requested through the corresponding Author.

Competing interest

The authors declares that they have no competing interests.

Funding

Not applicable

Authors’ Contributions

LM and AT conceived the study. LM and AT conducted the data collection and drafting of Manuscript. LM, AT and MZ contributed to data analysis and led the writing of the manuscript. PS, MZ and PJC reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Special acknowledgements to ZAMBART for the opportunity to use the STAR data for this study.

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No competing interests reported.

A Comparative Analysis of the different HIV Testing Techniques used in Zambia: Data from a Clinical Performance Study

Status:

Version 1

Abstract

Objective

Results

Conclusion

Figures

Introduction

Methods

Study design and setting

Test methods

Definition of Terms

Statistical analysis

Results

Interpretation of Table 3:

Table 4: Comparison of the results of OraQuick, Determine and UniGold to the gold standard.

Interpretation of table 4:

Discordant results

Overall Description of the Analyzed Data

Accuracy of the Different Tests

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1