DHS COMPARATIVE

REPORTS 39

PERFORMANCE OF ENZYME 

IMMUNOASSAYS FOR HIV SEROLOGY 

IN SURVEYS CONDUCTED BY THE 

DEMOGRAPHIC AND HEALTH 

SURVEYS PROGRAM

March 2016

This publication was produced for review by the United States Agency for International Development (USAID). The report was prepared by

Joy D. Fishel of ICF International and Dean Garrett of PATH.

DHS Comparative Reports No. 39 

Performance of Enzyme Immunoassays for HIV Serology 

in Surveys Conducted by 

The Demographic and Health Surveys Program 

Joy D. Fishel 

Dean Garrett 

ICF International 

Rockville, Maryland, USA 

March 2016 

Corresponding author: Joy D. Fishel, ICF International, 530 Gaither Road, Rockville, Maryland, USA; 

phone: 301-572-0281; fax: 301-407-6501; email: joy.fishel@icfi.com
Acknowledgment: The authors wish to thank Genevieve Dupuis, Guillermo Rojas, and Ladys Ortiz for 

assistance in preparing the data sets used in this analysis, Shireen Assaf for assistance with analysis, and 

Yodit Bekele for compiling information on the test algorithms used in each survey. The authors are also 

grateful to Sunita Kishor, Stephen Delgado, and Bharat Parekh for providing valuable comments. 

Editor: Nancy Johnson 

Document Production: Natalie La Roche 

This study was carried out with support provided by the United States Agency for International 

Development (USAID) through The DHS Program (#AID-OAA-C-13-00095). The views expressed are 

those of the authors and do not necessarily reflect the views of USAID or the United States Government. 

The DHS Program assists countries worldwide in the collection and use of data to monitor and evaluate 

population, health, and nutrition programs. For additional information about The DHS Program, contact 

The DHS Program, ICF International, 530 Gaither Road, Suite 500, Rockville, MD 20850, USA; phone: 

301-407-6500; fax: 301-407-6501; email: reports@dhsprogram.com; Internet: www.dhsprogram.com. 

Recommended citation: 

Fishel, Joy D., and Dean Garrett. 2016. Performance of Enzyme Immunoassays for HIV Serology in Surveys 

Conducted by The Demographic and Health Surveys Program. DHS Comparative Reports No. 39. 

Rockville, Maryland, USA: ICF International. 
iii 

Contents 

Tables and Figures ................................................................................................................................................... v 

Preface ...................................................................................................................................................................... vii 

Abstract ......................................................................................................................................................................ix 

1. Introduction ................................................................................................................................................. 1 

2. Background .................................................................................................................................................. 3 

3. Methods ........................................................................................................................................................ 5 

3.1. Characteristics of the Sample .............................................................................................. 5 

3.2. Specimen Collection and Testing Procedures ..................................................................... 6 

3.3. Data Management and Analysis ......................................................................................... 9 

4. Results ......................................................................................................................................................... 11 

4.1. Agreement between EIA 1 and EIA 2 .............................................................................. 11 

4.2. Signal to Cutoff Values ..................................................................................................... 13 

5. Discussion ................................................................................................................................................... 19 

References ................................................................................................................................................................. 23 

Appendix ................................................................................................................................................................... 25 

v 

Tables and Figures 

Table 1. Description of the sample ........................................................................................ 5 

Table 2. HIV assays used and specimen matrix .................................................................... 7 

Table 3. Agreement of assays .............................................................................................. 11

Table 4. Agreement of repeat EIA testing ........................................................................... 13 

Table 5. Signal to cutoff values ........................................................................................... 14 

Table 6. Final HIV results according to signal to cutoff values on assay 1 ......................... 15 

Table 7. Distribution of final HIV positive specimens by assay results and signal 

to cutoff values ...................................................................................................... 16 

Appendix Table 1.1. Descriptive statistics for signal to cutoff values among weak positive 

specimens, assay 1 ................................................................................................. 25 

Appendix Table 1.2. Descriptive statistics for specimens with weak signal to cutoff values, 

assay 2 ................................................................................................................... 26 

Appendix Table 2. Weak positive signal to cutoff values according to first round test results ........... 27 

Appendix Table 3. Internal quality control .......................................................................................... 28 

Figure 1. DHS HIV testing algorithm ..................................................................................... 3 

vii 

Preface 

The Demographic and Health Surveys (DHS) Program is one of the principal sources of international data 

on fertility, family planning, maternal and child health, nutrition, mortality, environmental health, 

HIV/AIDS, malaria, and provision of health services. 

One of the objectives of The DHS Program is to provide policymakers and program managers in low- and 

middle-income countries with easily accessible data on levels and trends for a wide range of health and 

demographic indicators. DHS Comparative Reports provide such information, usually for a large number 

of countries in each report. These reports are largely descriptive, without multivariate methods, but when 

possible they include confidence intervals and/or statistical tests. 

The topics in the DHS Comparative Reports series are selected by The DHS Program in consultation with 

the U.S. Agency for International Development. 

It is hoped that the DHS Comparative Reports will be useful to researchers, policymakers, and survey 

specialists, particularly those engaged in work in low- and middle-income countries. 

Sunita Kishor 

Director, The DHS Program 

ix 

Abstract 

Since 2001, The Demographic and Health Surveys Program has conducted HIV testing in surveys to 

measure HIV prevalence. Recently, concerns have been raised that the testing strategy used in these surveys 

may result in overestimation of HIV prevalence. This report analyzes existing laboratory data from 20 

recent surveys to assess two indicators of the performance of the enzyme immunoassays (EIAs) used in the 

HIV testing algorithms: agreement between the two EIAs used in the algorithm for each survey, and the 

distribution of signal-to-cutoff (S/CO) values for each assay. S/CO values are a measure of the strength of 

the reaction on an assay. Specimens with high S/CO values are more likely to be true positive specimens 

than those with low S/CO values. However, false positive results can have high S/CO values. 

Overall agreement between the two EIAs reached the optimal value of 99 percent or greater in 6 of the 20 

surveys. Analysis of the S/CO values shows that the majority of specimens with low S/CO values (less than 

5.0) on the first EIA were found negative during later stages of the testing algorithm—reducing, but not 

eliminating concerns about false positive results. In 4 of 20 surveys, the proportion of final positive 

specimens found to have low S/CO values was greater than 20 percent. 

This analysis can provide general insight into the quality of testing and the potential for false positivity in 

HIV test results, but it does not include retesting of EIA positive specimens on a more specific assay, which 

would be required to quantify any over- or underestimation in a survey’s HIV prevalence estimate. 

Nevertheless, the risk of false positive results does appear to vary widely across surveys. In addition, the 

impact that any testing error would have on the accuracy of the prevalence estimate for a survey must be 

considered in the context of other errors affecting this estimate, notably sampling error and selection bias. 

These results suggest the need for more vigilance in the quality of laboratory testing, with even more checks 

built in to every step of the testing process. Finally, testing strategies that include a confirmatory assay 

should reduce false positivity and improve the accuracy of HIV prevalence estimates. 

1 

1. Introduction 

From 2001 to March 2016, The Demographic and Health Surveys (DHS) Program conducted HIV testing 

for 65 surveys in 37 countries. The HIV serology testing for these surveys has relied on centralized 

laboratory testing based on a WHO-recommended testing strategy for the measurement of HIV prevalence 

in population-based surveys (UNAIDS/WHO 2005). Specifically, the HIV testing has employed two 

enzyme immunoassays (EIAs) and a third test, usually a blot assay, to resolve EIA-discordant specimens. 

Recently, concerns have been raised that the testing strategy recommended in the 2005 UNAIDS/WHO 

guidelines may result in overestimation of HIV prevalence. In response to these concerns, The DHS 

Program has undertaken a review of the quality of EIA data from recent DHS and AIS surveys. The review 

is a secondary analysis of existing laboratory data for past surveys. 

This analysis will assess two indicators of the performance of the HIV EIAs: (1) agreement between the 

two EIAs and (2) the distribution of signal-to-cutoff values, which can provide general insight into the 

potential for false positivity in HIV test results. The objective of this analysis is not to quantify any underor overestimation in any specific survey’s HIV prevalence estimate. The only way to measure 

overestimation is to retest positive survey specimens on a more specific confirmatory assay and to compare 

those results with the published survey estimates. Due to funding constraints and issues with the availability 

of blood specimens collected one to five years ago, as well as potentially poor storage conditions, retesting 

specimens for all of these surveys is not feasible. 

3 

2. Background 

Generally, the HIV testing strategy followed by The Demographic and Health Surveys Program has 

consisted of two enzyme immunoassays (EIAs) and a third assay used as a tie-breaker when the two EIAs 

disagree. The first EIA should be the most sensitive assay available, the second EIA should have higher 

specificity, and the two assays should use a different antigen preparation to minimize the risk of shared 

false reactivity (WHO 2015). As shown in Figure 1, if the first EIA result is negative, the specimen is 

rendered negative. If the first EIA is positive, the specimen is also tested with the second EIA. If the second 

EIA result is positive, the specimen is rendered HIV positive with no further testing. If the result of the 

second assay is negative, the specimen is tested on the third assay, and the result of the third assay is 

considered the definitive result for the specimen. In some surveys specimens with discrepant results on EIA 

1 and EIA 2 are retested on both assays in parallel. In these surveys, the specimen is rendered positive if 

both assays are positive and negative if both assays are negative. If the results of the two assays remain 

discrepant after retesting, the specimen is tested on the third and definitive assay. 

To ensure internal quality control (IQC), The DHS Program routinely retests on EIA 2 a random sample of 

5 to 10 percent of specimens that are negative on EIA 1. This testing is conducted as a part of the routine 

EIA 2 testing, and if any IQC specimen has a positive result, the specimen is put back into the testing 

algorithm and either (1) retested on EIA 1 and EIA 2 in parallel if repeat testing of the assays was included 

in the testing algorithm or (2) tested on assay 3 if repeat testing was not included. If the percentage of IQC 

specimens with positive results on EIA 2 is over 1 percent, the testing procedures should be reviewed and 

any problems corrected. Note that this testing strategy is no longer fully in line with more recent WHO 

guidelines for HIV diagnosis and surveillance (WHO 2015) or UNAIDS/WHO guidelines on HIV testing 

in population-based surveys (UNAIDS/WHO 2015). However, the approach does reflect the 

UNAIDS/WHO guidelines for HIV testing in population-based surveys that were in place during the time 

these surveys were conducted (UNAIDS/WHO 2005). 

Figure 1. DHS HIV-testing algorithm 
4 

Cause for concern over the accuracy of this testing strategy arises from a recent investigation comparing 

EIA-based HIV testing strategies with other strategies, including use of rapid diagnostic tests (RDT) or 

highly specific supplemental assays to confirm dual EIA positive results. These investigations have shown 

that a potential for false positives exists even among specimens with two positive EIA results (CDC 2014). 

As a result, the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA, now 

recommends that all specimens with positive results on two EIAs undergo testing on a third more specific 

assay, such as Western blot, Geenius, or Multispot (CDC 2014). The current UNAIDS/WHO guidelines 

for HIV testing in surveys also recommend the use of a third assay on specimens with positive results on 

assay 1 and assay 2 in settings with prevalence less than 5 percent, and in all settings where assay 1 and 

assay 2 are EIAs (UNAIDS/WHO 2015). 

Enzyme immunoassays for HIV have undergone continual development to detect a broader group of HIV 

strains. For example, first generation EIAs only detected antibodies against group M strains of HIV, 

whereas third generation EIAs detect antibodies against groups M and O and against HIV-2 subtype. Both 

third and fourth generation tests detect HIV infection using synthetic peptides or recombinant protein 

antigens to bind HIV antibodies. These EIAs can detect both IgG and IgM antibodies. Fourth generation 

EIAs also include monoclonal antibodies to bind HIV-1 p24 antigen (CDC 2014; WHO 2015). The standard 

EIA format uses 96-well microtiter plates, with one specimen per well. Upon completion of the assay, 

development of color in a well indicates the presence of HIV antibody or HIV antigen. This color reaction 

is measured and converted into an optical density (OD) by a microplate reader. Optical densities are 

continuous numeric values. The test result is determined by comparing the value of the OD for a specimen 

to a cutoff value. If the OD is greater than or equal to the cutoff, the specimen is HIV positive; if the OD is 

less than the cutoff, the specimen is negative. The method for generating the cutoff value is assay-specific, 

but it is always determined on a plate-by-plate basis, using a formula provided by the kit manufacturer that 

includes the OD values of control specimens from the test kit that are run on each plate. 

EIA results can also be described in terms of a signal-to-cutoff (S/CO) value, calculated by dividing the 

OD value of the specimen by the cutoff value for the plate. An S/CO value of 1.0 or greater indicates the 

specimen is reactive, or positive. The higher the S/CO value, the stronger is the reaction on the assay. In 

general, there appears to be an association between the “strength” of the reaction on the assay and the 

accuracy of the classification of HIV serostatus, with specimens having lower S/CO values being more 

likely to be false positive. In an evaluation of six EIAs, Nkengasong and colleagues describe a 

categorization that has been adopted by others. They describe specimens with S/CO values greater than 5.0 

as “highly reactive,” those with S/CO values of 3.0-5.0 as “moderately reactive,” and those with S/CO 

values of 1.0-3.0 as “weakly reactive” (1999). Specimens rendered positive by an EIA, but with a S/CO 

value of less than 5.0 have been found to be much more likely than those with S/CO values of 5.0 and above 

to be false positive (Nkengasong et al. 1999; Fanmi et al. 2013; Urassa et al. 1999). However, S/CO values 

cannot be used to distinguish between true and false positive results. The ranges of S/CO values for true 

and false positive results can overlap (Urassa et al. 1999), and false positive results with high S/CO values 

have been documented in several studies (Fanmi et al. 2012; Hakim et al. 2016; Zhang et al. 2013). 

Conversely, it is possible for a true positive specimen to produce a weak reaction on an EIA. Specimens 

drawn from individuals early in the seroconversion process and those infected with divergent HIV strains 

can also produce weak positive reactions (Kfutwah et al. 2013). 
5 

3. Methods 

3.1. Characteristics of the Sample 

We attempted to analyze data from all surveys with HIV testing that were conducted after 2010 as part of 

The DHS Program and had results published by August 2015. We were unable to analyze 6 of these 26 

surveys for various reasons. The 2011 Ethiopia DHS, 2010 Burkina Faso DHS, 2013 Liberia DHS, and 

2010-11 Zimbabwe DHS were excluded due to unavailability of the detailed laboratory results. The 2011 

Equatorial Guinea DHS survey data are not available for public use, and all HIV test results for the 2011-

12 Benin DHS have been destroyed due to the inclusion of a community assets questionnaire in that survey, 

which introduced a risk that sampled communities and individuals could be identified even after the dataset 

was anonymized. The list of 20 remaining surveys, including 18 DHS surveys and 2 AIDS Indicator surveys 

(AIS), is included in Table 1. 

Table 1. Description of the sample 

Weighted HIV prevalence among men and women age 15-49, the unweighted total number of respondents tested for HIV (all 

ages), and the unweighted total number of HIV positive respondents, by survey 

 HIV prevalence among women 

and men age 15-49 (weighted)

Total number tested 

(all ages, unweighted) 

Total number HIV 

positive (all ages, 

Survey unweighted) 

Point 

estimate 

95% 

confidence 

interval 

Sub-Saharan Africa 

Burundi DHS 2010 1.4 (1.1, 1.7) 8,588 164 

Cameroon DHS 2011 4.3 (3.9, 4.8) 14,202 649 

Cote d’Ivoire DHS 2011-12 3.7 (3.1, 4.3) 9,008 336 

Democratic Republic of the Congo 

DHS 2013-14 1.2 (0.8, 1.5) 17,638 177 

Gabon DHS 2012 4.1 (3.3, 4.8) 10,992 483 

Gambia DHS 2013 1.9 (1.4, 2.4) 7,769 136 

Guinea DHS 2012 1.7 (1.4, 2.1) 8,380 164 

Malawi DHS 2010 10.6 (9.7, 11.5) 13,910 1,420 

Mali DHS 2012-2013 1.1 (0.9, 1.4) 8,861 97 

Namibia DHS 2013 14.0 (12.8, 15.3) 8,858 1,233 

Niger DHS 2012 0.4 (0.2, 0.5) 8,628 44 

Rwanda DHS 2010 3.0 (2.7, 3.4) 13,248 420 

Senegal DHS 2010-11 0.7 (0.5, 0.9) 9,917 93 

Sierra Leone DHS 2013 1.5 (1.2, 1.8) 14,600 222 

Tanzania AIS/MIS 2011-12 5.1 (4.6, 5.6) 17,745 815 

Togo DHS 2013-14 2.5 (2, 2.9) 9,172 199 

Uganda AIS 2011 7.3 (6.8, 7.9) 21,367 1,495 

Zambia DHS 2013-14 13.3 (12.5, 14.1) 29,006 3,901 

Latin America/Caribbean 

Dominican Republic DHS 2013 0.8 (0.6, 1) 18,614 193 

Haiti DHS 2012 2.2 (1.9, 2.5) 18,531 419 

Note: In most of these surveys, the age ranges for interview and HIV testing are 15-49 for women and 15-59 for men. Exceptions 

include Malawi (women age 15-49 and men age 15-54), Namibia (women and men age 15-64), Tanzania (women and men age 

15-49), and Uganda (women and men age 15-59). Women and men from the entire age range are included in this analysis. 

However, the HIV prevalence estimates in this table are shown for women and men age 15-49 to avoid using an estimate based 

on different ages for women and men, and for consistency across countries. 

The national HIV prevalence among women and men age 15-49 in this group of surveys ranges from 0.4 

percent in Niger to 14 percent in Namibia. The HIV prevalence is near or below 2 percent in around half of 

these surveys. Among all 65 DHS Program surveys that have included HIV testing, the proportion with 

HIV prevalence below 2 percent is also around half. The sample size for the surveys varies widely from 

fewer than 8,000 people tested for HIV in Gambia to nearly 30,000 people tested for HIV in Zambia. Table 
6 

1 also shows the unweighted number of respondents who tested positive in each survey. This number is 

quite low, even at the national level, in the lower prevalence countries. For example, in the 2012 Niger 

DHS, there were only 44 HIV-positive respondents. Many of the analyses in this report are based only on 

the HIV-positive cases, and small sample sizes limit interpretation of the results. 

3.2. Specimen Collection and Testing Procedures 

In most surveys conducted with the assistance of The DHS Program, the blood draw is conducted via a 

finger prick, and drops of blood are collected on a Whatman 903 filter paper card. The blood spots are 

allowed to dry overnight, and the cards are then individually packed in a low-gas permeable Ziploc bag, 

with glassine paper protecting the dried spots (DBS), and with one or two sachets of desiccants and a 

humidity monitor card, which are checked daily and replaced as needed. DBS specimens generally remain 

in the field for 10-14 days before being transported to the central laboratory, where they are stored at -20°C 

or colder until testing begins. In most surveys, the field staff who collect these blood specimens have no 

prior clinical or laboratory training. Training on the collection of these blood specimens usually includes 

2-3 days of classroom instruction and 1-2 days of supervised field practice in households. Classroom 

instruction includes both theory and practice, and covers procedures for informed consent and infection 

prevention as well as proper techniques for pricking the finger, collecting the blood specimen, and 

managing the specimens in the field. Further details can be found in The DHS Program standard biomarker 

training manual (MEASURE DHS 2012). 

Laboratory testing of survey specimens is conducted in the host country. A DHS biomarker specialist 

selects the lab in conjunction with the local implementing agency, ideally after an assessment of at least 

three potential labs. However, countries often do not have multiple laboratories or, at times, even one 

laboratory with the pre-existing capacity to carry out the HIV testing algorithm for the survey. In such 

cases, the DHS Program invests heavily in training laboratory technologists and providing supplies and 

equipment to enable the laboratory to meet the standards required to ensure high quality testing. 

The specific assays used in the HIV testing algorithm are generally determined in coordination with the 

host country; however, the general testing strategy, as shown in Figure 1, tends to be consistent across 

countries. Table 2 includes the testing algorithm and the specimen matrix for each of the 20 surveys 

included in this analysis. HIV testing is typically conducted by three or four laboratory technologists whose 

time is fully dedicated to testing the survey specimens throughout the duration of the project. Training of 

the laboratory technologists includes a visit of 2-3 weeks to the laboratory by a DHS Program biomarker 

specialist. During this visit, the biomarker specialist trains the laboratory technologists to test the survey 

specimens for HIV in accordance with The DHS Program’s standard operating procedures. All of the 

surveys in Table 2, except for the 2011 Uganda AIS, used dried blood spot (DBS) specimens for HIV 

testing. Before testing, the blood must be eluted from the filter paper card. Elution is effected by incubating 

a sample of the blood-soaked filter paper card in a buffer for 18-24 hours at 4o

C. Note that none of the EIAs 

included in the testing algorithms for these surveys have been validated by the manufacturer for DBS, 

though their use with DBS has been common for HIV surveillance. The elution procedures used by the 

DHS Program are recommended by the CDC in Atlanta, Georgia. 
7 

Table 2. HIV assays used and specimen matrix

Name, manufacturer, and generation of assays used in the HIV testing algorithm, and specimen matrix, by survey 

Survey Assay 1 

Assay 1 

generation Assay 2 

Assay 2 

generation 

Assays 1 

and 2 

repeated if 

discrepant Assay 3 

Specimen 

matrix 

Sub-Saharan Africa 

Burundi DHS 2010 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 No Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS 

Cameroon DHS 2011 Murex HIV Ag/Ab 

(DiaSorin) 

4 Genscreen ULTRA 

HIV Ag/Ab (Bio-Rad) 

4 No New Lav Blot 

(Bio-Rad) 

DBS

Cote d’Ivoire DHS 

2011-12 

Enzygnost Integral II 

(Siemens) 

4 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Yes No third assay1

 DBS

Democratic Republic 

of the Congo DHS 

2013-14 

Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 Yes HIV 2.2 Blot (DiaSorin) DBS

Gabon DHS 2012 Genscreen Plus HIV 

Ag/Ab 5PL (Bio-Rad) 

4 Enzygnost Integral II 

(Siemens)

4 No HIV 2.2 Blot (DiaSorin) DBS

Gambia DHS 2013 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 No HIV 2.2 Blot (DiaSorin) DBS

Guinea DHS 2012 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 Yes No third assay1

 DBS

Malawi DHS 2010 Vironostika HIV UniForm II Plus O 

(Biomerieux) 

3 Enzygnost Anti-HIV 

1/2 Plus (Seimens) 

3 Yes Western Blot 

2.2 (Abbott Labs) 

DBS

Mali DHS 2012-13 Vironostika HIV UniForm II Plus O 

(Biomerieux) 

3 Enzygnost Integral II 

(Siemens) 

4 No HIV 2.2 Blot (DiaSorin) DBS

Namibia DHS 2013 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 No Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS

Niger DHS 2012 Vironostika HIV UniForm II Plus O 

(Biomérieux) 

3 Enzygnost Anti-HIV 

1/2 Plus (Seimens) 

3 No Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS

Rwanda DHS 2010 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Murex HIV Ag/Ab 

(DiaSorin)

4 No HIV 2.2 Blot (DiaSorin) DBS

Senegal DHS 2010-11 Vironostika HIV UniForm II Plus O 

(Biomérieux) 

3 Enzygnost Anti-HIV 

1/2 Plus (Seimens) 

3 No Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS

Sierra Leone DHS 

2013 

Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 No Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS

Tanzania AIS/MIS 

2011-12 

Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 Yes HIV 2.2 Blot (DiaSorin) DBS

Togo DHS 2013-14 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 yes Inno-Lia HIV I/II 

Score line 

immunoassay 

(Innogenetics) 

DBS

Uganda AIS 2011 Murex HIV 1.2.0 

(Abbott Labs) 

3 Vironostika HIV UniForm II Plus O 

(Biomérieux) 

3 No Ani Labsystems 

HIVEIA 

Plasma2

Zambia DHS 2013-

2014 

Vironostika HIV Ag/Ab 

(Biomerieux) 

4 Enzygnost Integral II 

(Siemens) 

4 Yes Western Blot 2.2 

(Abbott Labs) 

DBS

Latin America/Caribbean 

Dominican Republic 

DHS 2013 

Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 Yes HIV 2.2 Blot (DiaSorin) DBS 

Haiti DHS 2012 Vironostika HIV Ag/Ab 

(Biomérieux) 

4 Enzygnost Integral II 

(Siemens) 

4 No HIV 2.2 Blot (DiaSorin) DBS

DBS = Dried blood spot

1 The testing algorithms for the 2012 Guinea DHS and the 2013 Cote d’Ivoire DHS did not include a third assay. The few specimens that 

remained discordant on assay 1 and assay 2 were treated as negative in the HIV prevalence calculation. 

2

 Respondents were allowed to provide a DBS specimen from a finger prick if they refused venipuncture. Specimens tested for HIV in the 

2011 Uganda AIS therefore include a mix of plasma and DBS. Less than 2 percent of specimens were DBS. 
8 

Before testing survey blood specimens, it is critical to confirm that the HIV test system—microplate reader 

and washer, DBS control materials, elution procedures, assay method, and data management system—is 

working correctly. To do this, the laboratory prepares at least 40 paired DBS and plasma specimens (i.e., 

DBS and plasma specimens using the blood of the same volunteer) to test the HIV algorithm, equipment, 

and data management system. Each technologist is required to test all specimens on EIA 1 and EIA 2. To 

qualify to test the survey specimens on an assay, each technologist must achieve a minimum expected level 

of agreement between the DBS and plasma results for each matched pair. Specifically, each technician is 

allowed to remove one discrepant pair from their results (if the results for any pair were discrepant), and 

the agreement between the DBS and plasma results on the remaining pairs must be 99 percent or greater. 

This phase is also used to train the technologists to program the plate reader and plate washer for each of 

the two EIAs and to use the data management system. 

The DHS Program uses a data management tool called the CSPro HIV Test Tracking System, or CHTTS, 

to manage HIV test results in the laboratory. This system generates plate maps of specimens to be tested on 

each microplate and captures the optical density (OD) values for each specimen from the plate reader. It 

calculates a cutoff for each plate from the OD values for the kit controls run on each plate using the 

manufacturer guidelines for the particular assay, and assigns each specimen a result of reactive or nonreactive on the assay. Specimens are considered to be HIV negative if their OD value is below the plate 

cutoff and HIV positive if the OD is equal to or greater than the plate cutoff. CHTTS tracks the results of 

each specimen on each assay to ensure that each specimen receives exactly the tests needed to render a final 

HIV status according to the logic of the testing algorithm. Each plate is given a unique identifier which can 

be linked to the technologist who ran the plate. As the results of each plate are read, the technologist must 

review the results and decide whether to save or cancel the plate. If the technologist cancels the results for 

a plate of specimens, the specimens must be retested on that assay. The saved results of each plate must 

then undergo a second step, referred to as validation, before the results for the plate are considered final. 

The laboratory supervisor in charge of testing for the survey is responsible for reviewing, and validating or 

rejecting, the results for each plate. CHTTS is also programmed to produce error messages if the OD values 

of the negative controls do not fall within the range specified by the manufacturer or if the percentage of 

specimens on the plate that are positive exceed a limit set by the DHS for that survey. 

The DHS Program recommends that each technologist process two microplates—roughly 180 specimens, 

depending on the assay—each working day. However, during the first week after training, technologists 

are encouraged to test only one microplate of specimens each day to allow them time to increase their 

confidence and develop proficiency. During the first 2-3 weeks after training, all technologists test 

specimens on EIA 1 in order to accumulate a sufficient number of specimens that need to be tested on EIA 

2. Because fewer specimens are tested on EIA 2, sometimes only one technologist is assigned this task. In 

countries where the HIV testing algorithm includes repeating EIA 1 and EIA 2 in parallel when the results 

are discordant, the repeat testing is also often performed by just one technologist. 

The DHS Program employs multiple strategies for quality assurance throughout the HIV testing process. 

High positive, low positive, and negative dried blood spot control materials provided by the CDC in Atlanta 

are included on each microtiter plate. A subsample of 5-10 percent of EIA 1 negative specimens is randomly 

selected for testing on EIA 2 to check for false negative results on EIA 1. DHS biomarker staff monitor 

testing remotely by reviewing the results of a sample of the plates processed each week and provide 

feedback to the laboratory staff accordingly. In addition, DHS biomarker staff review photographs taken of 

the banding pattern of specimens tested on blot assays such as Western blot or INNO-LIA to confirm the 

final result for the assay. 
9 

3.3. Data Management and Analysis 

An extensive amount of data management was required for this analysis. The DHS Program has not 

formatted or archived the detailed laboratory results files needed for this work for public release. The data 

management system, CHTTS, produces several raw data files, two of which contain data relevant to this 

analysis: a test log file and a plate file. The test log file includes specimens as the unit of analysis. It includes 

the identification information for each specimen, and information used to guide the laboratory technicians 

through the HIV testing algorithm, including the optical density and HIV test result for each EIA. It retains 

data from runs that fail and are not used in the final results and clearly identifies those data as canceled. 

However, this produces a challenge when attempting to use the database analytically because the final 

results for each stage of the testing algorithm can be found in different variables of the data file for different 

specimens. In addition, this file has not included the cutoff values for ascribing seropositivity for each 

specimen for each assay, making the calculation of a signal-to-cutoff value impossible. Cutoff values, as 

well as the complete results for each microplate are included in a second raw data file, which uses the 

microplate as the unit of analysis. 

An application was written in CSPro (1) to restructure the data in the test log file so that the final assay 

results for each stage of the testing algorithm appear in the same variable for each specimen, (2) to merge 

in the cutoff values for each assay for each specimen from the plate file, and (3) to delete specimens that 

could not be linked to a de facto, interviewed individual in the survey. This third step standardized the 

universe of specimens for each survey to those that were included in the HIV prevalence estimate. Each 

survey file was then imported and analyzed using Stata v13. Each file was checked for internal consistency 

and completeness and merged with the questionnaire data to allow linkage with the sex and age of the 

respondent. Analysis consists of simple frequencies and bivariate tabulations. 

The analysis focuses on two variables that can help to characterize the quality of the EIA testing. The first 

indicator is the agreement between EIA 1 and EIA 2. Given the high published sensitivity and specificity 

of EIAs, both EIAs should find concordant results for nearly all specimens. Poor agreement between the 

first and second EIA indicates quality issues in the testing process. The analysis looks at the agreement 

between EIA 1 and EIA 2 in the first round of testing, and after the second round of testing in those surveys 

where specimens with discrepant results on EIA 1 and EIA 2 were tested again on both EIAs. Overall 

agreement of 99 percent or greater is optimal. 

The second indicator is the percentage of positive cases with an S/CO value less than 5.0. The choice of 5.0 

is somewhat arbitrary, but this value has been used conventionally to identify specimens that may be more 

likely to be false positive than those with higher S/CO values. For simplicity, specimens are divided into 

just two categories—those with an S/CO value of less than 5.0, and those with an S/CO value of 5.0 or 

greater—rather than using the three categories defined in Nkengasong et al. (1999)—1.0-3.0, >3.0-5.0, and 

greater than 5.0. For this analysis, all positive specimens with an S/CO of less than 5.0 are described as 

“weak” positives. As discussed, S/CO values are difficult to use as an indication of false positivity for an 

individual specimen because it is possible for a true positive specimen to have a weak reaction and for a 

false positive specimen to have a strong reaction. Nonetheless, the distribution of S/CO values for an assay 

across a population can be evaluated as more or less plausible. There is no single optimal value for the 

percentage of positive specimens with an S/CO value of less than 5.0—this will depend on the population 

tested and the specific properties of the assay used. However, in general, the proportion of the HIV positive 

population with weak reactions on an EIA is expected to be relatively low. 

11 

4. Results 

4.1. Agreement between EIA 1 and EIA 2 

The first indicator of quality in the EIA testing, agreement between the two assays, is explored in Tables 3 

and 4. In the HIV testing algorithm used by the DHS Program in these surveys, all samples that test positive 

on the first EIA are retested on a second EIA. The first column in Table 3 shows the percentage of specimens 

with positive results on EIA 1 that are also positive on EIA 2. This percentage varies widely across surveys, 

from 31 percent in Niger to 97 percent in Cote d’Ivoire. Cote d’Ivoire was the only country to use Enzygnost 

as the first assay in the algorithm. The percentage of EIA 1 positive specimens that are also positive on EIA 

2 is also quite low in Guinea, the Democratic Republic of the Congo (DRC), Cameroon, and Uganda (44-

52 percent). Third generation assays were used as EIA 1 in Niger (Vironostika) and Uganda (Murex), while 

fourth generation assays were used as EIA 1 in Cameroon (Murex), DRC, and Guinea (Vironostika for 

both). 

Table 3. Agreement of assays 

Among specimens positive on assay 1, percentage positive on assay 2; among specimens negative on assay 1 and retested on assay 2 

for internal quality control, percentage positive on assay 2; among specimens tested on both assay 1 and assay 2, percentage concordant 

after the first round of testing, and percentage concordant after repeat testing on both assays, by survey [Unweighted] 

Assay 1 positive 

specimens, first round 

of testing 

Assay 1 negative 

specimens retested for 

internal quality control1 

Overall agreement 

between assay 

1 and assay 2 

Survey 

Percentage 

that are 

positive on 

assay 2 

Number of 

specimens 

positive on 

assay 1 

Percentage 

that are 

positive on 

assay 2 

Number of 

specimens 

negative on 

assay 1 

After first 

round of 

testing 

After 

repeat 

testing 

Number of 

samples 

tested on 

assay 1 and 

assay 2 

Sub-Saharan Africa 

Burundi DHS 2010 60.6 259 1.3 830 89.6 na 1,089 

Cameroon DHS 2011 51.4 1,259 0.6 663 68.0 na 1,922 

Cote d’Ivoire DHS 2011-12 97.1 344 0.2 865 99.0 99.7 1,209 

Democratic Republic of the 

Congo DHS 2013-14 

45.4 388 1.5 863 82.0 99.7 1,251 

Gabon DHS 2012 84.1 561 0.2 523 91.7 na 1,084 

Gambia DHS 2013 87.6 153 0.3 749 97.7 na 902 

Guinea DHS 2012 43.8 372 1.2 413 72.7 99.2 785 

Malawi DHS 2010 81.0 1,718 1.0 1,238 88.6 99.0 2,956 

Mali DHS 2012-13 84.2 114 0.6 875 97.7 na 989 

Namibia DHS 2013 87.7 1,387 2.9 421 89.9 na 1,808 

Niger DHS 2012 31.0 100 0.2 863 92.6 na 963 

Rwanda DHS 2010 94.8 442 0.0 1,283 98.7 na 1,725 

Senegal DHS 2010-11 78.2 119 0.0 976 97.6 na 1,095 

Sierra Leone DHS 2013 70.6 269 6.3 1,440 90.1 na 1,709 

Tanzania AIS/MIS 2011-12 84.9 952 3.8 1,678 92.1 99.7 2,630 

Togo DHS 2013-14 82.5 240 1.2 893 95.3 99.1 1,133 

Uganda AIS 2011 51.8 2,629 na na 51.8 na 2,629 

Zambia DHS 2013-2014 78.3 4,741 12.7 2,423 81.3 95.5 7,164 

Latin America/Caribbean 

Dominican Republic DHS 2013 70.1 271 0.1 1,847 96.1 99.9 2,118 

Haiti DHS 2012 74.6 559 0.9 901 89.7 na 1,460 

1

 A random subsample of specimens that are negative on assay 1 in the first round of testing are routinely retested on assay 2 for internal 

quality control. The 2011 Uganda AIS did not include retesting of assay 1 negative samples for internal quality control. 

The percentage of the subsample of EIA 1 negative specimens retested for IQC that are positive on EIA 2 

is shown in column 3 of Table 3. In most countries this percentage is below 1 percent. The percentage of 

IQC specimens found positive is especially high in Sierra Leone (6 percent) and Zambia (13 percent). 
12 

Finally, Table 3 shows the overall agreement between EIA 1 and EIA 2 after the first round of testing on 

each assay, and after the second round of testing on EIA 1 and EIA 2 in surveys where EIA 1 and EIA 2 

were repeated if the results were discrepant in the first round of testing. The denominators for columns 5 

and 6 in Table 3 are shown in column 7 and include all specimens that were positive on EIA 1 as well as 

the EIA 1 negative specimens that were selected for IQC. All of these specimens were retested on EIA 2. 

Agreement is defined as having either a positive result on both EIAs or a negative result on both EIAs. 

Overall agreement of the two EIAs after the first round of testing ranges from 52 percent in Uganda to 99 

percent in Cote d’Ivoire. Because there was no IQC testing in Uganda, this percentage is equivalent to the 

percentage of EIA 1 positive specimens that are positive on EIA 2, shown in the first column. Agreement 

after the first round of testing reaches 99 percent in only one of the 20 surveys. 

In 8 of the 20 surveys included in this analysis, specimens with discrepant results on EIA 1 and EIA 2 were 

tested again on both EIAs. The agreement following the repeat round of testing is calculated as follows: if 

the specimen was retested on EIA 1 and EIA 2, the repeat test results were included; however, if the 

specimens were concordant on EIA 1 and EIA 2 in the first round of testing, then these first round results 

were used. The step of repeating EIA 1 and EIA 2 on specimens with discrepant results improved agreement 

in all eight surveys. Agreement reaches or exceeds the benchmark of 99 percent among all surveys with 

repeat testing on the two EIAs, except for Zambia where agreement is 96 percent. Taking into account the 

results of repeat testing, where conducted, final assay agreement reaches 99 percent in 7 of the 20 surveys. 

Table 4 shows a distribution of those specimens that were discrepant in the first round of testing by the 

results of the repeated tests. These results are broken down by the results of the first round of testing on 

EIA 1 and EIA 2: A1+A2- or A1-A2+ (the IQC specimens). Overall, the vast majority of these discrepant 

samples resolved to negative-negative. In Cote d’Ivoire and Zambia, specimens were less likely than in the 

other countries to resolve to negative-negative. In Cote d’Ivoire, there was a very high level of agreement 

observed between the first and second EIAs—97 percent of specimens that were positive on the first EIA 

were also positive on the second, as shown in Table 3. There were only 12 specimens that were discordant 

on the two EIAs and proceeded to repeat testing. Though it is not clear if this is related to either the high 

level of agreement between the EIAs on initial testing or the lower likelihood of resolving to negativenegative during repeat testing, it should be noted that in Cote d’Ivoire the usual order of EIA 1 and EIA 2 

was reversed, with Enzygnost (fourth generation) serving as the first EIA and Vironostika (fourth 

generation) as the second. By contrast, in Zambia, 22 percent of samples that were found positive on the 

first EIA were found negative on the second EIA. This level of disagreement is comparable to that observed 

during the first round of testing in the Dominican Republic, Malawi, Tanzania, and Togo, and is lower than 

that observed in DRC and Guinea; however, the discrepant specimens in all of those countries were more 

likely to resolve to negative-negative in repeat testing than in Zambia. 

In each survey, the IQC specimens make up a minority of the total number of discrepant specimens, but 

this percentage does vary across surveys from 2 percent in Guinea and the Dominican Republic to 30 

percent in Tanzania. In most of these surveys, the IQC specimens were roughly equally likely or slightly 

more likely to resolve to negative-negative than the A1+A2- specimens. In Togo and Zambia, the IQC 

specimens were more likely than A1+A2- specimens to remain discrepant after repeat testing. It is 

interesting to note that the IQC specimens in Cote d’Ivoire and the Dominican Republic all resolved to 

positive-positive; however, each survey had only two IQC specimens with positive results, limiting the 

interpretation of this finding. 
13 

Table 4. Agreement of repeat EIA testing

Percent distribution of specimens retested on both EIAs according to the results of the repeated tests, by survey [Unweighted] 

Survey 

Resolve to 

negativenegative 

Resolve to 

positivepositive 

Remain 

discordant Total Number 

Sub-Saharan Africa 

Cote d’Ivoire DHS 2011-12 

A1+A2- 60.0 0.0 40.0 100.0 10 

A1-A2+ (IQC) 0.0 100.0 0.0 100.0 2 

All round 1 discrepant specimens 50.0 16.7 33.3 100.0 12 

Democratic Republic of the Congo DHS 2013-14 

A1+A2- 98.1 0.0 1.9 100.0 212 

A1-A2+ (IQC) 100.0 0.0 0.0 100.0 13 

All round 1 discrepant specimens 98.2 0.0 1.8 100.0 225 

Guinea DHS 2012 

A1+A2- 96.7 0.5 2.9 100.0 209 

A1-A2+ (IQC) 100.0 0.0 0.0 100.0 5 

All round 1 discrepant specimens 96.7 0.5 2.8 100.0 214 

Malawi DHS 2010 

A1+A2- 84.7 6.4 8.9 100.0 326 

A1-A2+ (IQC) 100.0 0.0 0.0 100.0 12 

All round 1 discrepant specimens 85.2 6.2 8.6 100.0 338 

Tanzania AIS/MIS 2011-12 

A1+A2- 93.1 2.1 4.9 100.0 144 

A1-A2+ (IQC) 96.8 0.0 3.2 100.0 63 

All round 1 discrepant specimens 94.2 1.4 4.3 100.0 207 

Togo DHS 2013-14 

A1+A2- 90.5 2.4 7.1 100.0 42 

A1-A2+ (IQC) 36.4 0.0 63.6 100.0 11 

All round 1 discrepant specimens 79.2 1.9 18.9 100.0 53 

Zambia DHS 2013-2014 

A1+A2- 67.3 14.0 18.7 100.0 1029 

A1-A2+ (IQC) 44.3 12.7 43.0 100.0 307 

All round 1 discrepant specimens 62.1 13.7 24.3 100.0 1336 

Latin America/Caribbean 

Dominican Republic DHS 2013 

A1+A2- 97.5 1.3 1.3 100.0 79 

A1-A2+ (IQC) 0.0 100.0 0.0 0.0 2 

All round 1 discrepant specimens 95.1 3.7 1.2 100.0 81 

Note: Only 8 of the 20 surveys included in this analysis included repeat testing on both EIAs for samples that had discrepant results 

on EIA 1 and EIA 2. 

4.2. Signal to Cutoff Values 

Table 5 examines the S/CO values of specimens that were found positive on the first round of testing on 

EIA 1 and EIA 2. The percent of EIA 1 positive specimens that have an S/CO value of less than 5.0 ranges 

from 3 percent in Cote d’Ivoire to 58 percent in Guinea. The percentage of EIA 1 positive specimens with 

an S/CO value below 5.0 exceeds 20 percent in roughly half of the surveys included in this analysis (9 out 

of 20). There are some differences noted by assay type. The percentage of EIA 1 positive specimens with 

an S/CO value of less than 5.0 is over 20 percent in 1 of 4 surveys using a third generation Vironostika as 

EIA 1, compared with 6 of 12 surveys using a fourth generation Vironostika as EIA 1. The percentage of 

EIA 1 positive specimens with a S/CO value of less than 5.0 is over 20 percent in both surveys using Murex 

as EIA 1, with this percentage being higher in Gabon, which used a fourth generation Murex, than in 

Uganda, which used a third generation Murex. It is important to note, however, that the percentage of EIA 

1 positive specimens with an S/CO value of less than 5.0 in surveys using a fourth generation assay as EIA 

1 is not high across the board. For example, this percentage was only 3 percent in Cote d’Ivoire, which used 

fourth generation Enzygnost, and 5 percent and 8 percent in Rwanda and Gambia, respectively, both of 

which used fourth generation Vironostika. 
14 

Table 5. Signal to cutoff values 

Among specimens positive on assay 1 in the first round of testing, percentage with a signal to cutoff (S/CO) value of less than

5.0; among specimens positive on assay 2 in the first round of testing, percentage with an S/CO of less than 5.0, by survey 

[Unweighted] 

Assay 1 positive specimens Assay 2 positive specimens 

Survey 

Percentage with 

S/CO <5.0 Number 

Percentage with 

S/CO <5.0 Number 

Sub-Saharan Africa 

Burundi DHS 2010 28.2 259 7.7 168 

Cameroon DHS 2011 49.5 1,259 2.0 651 

Cote d’Ivoire DHS 2011-12 2.9 344 0.9 336 

Democratic Republic of the Congo DHS 2013-14 51.8 388 12.2 189 

Gabon DHS 2012 12.1 561 4.2 473 

Gambia DHS 2013 7.8 153 5.9 136 

Guinea DHS 2012 57.5 372 7.1 168 

Malawi DHS 2010 15.7 1,718 12.1 1,404 

Mali DHS 2012-13 10.5 114 9.9 101 

Namibia DHS 2013 10.5 1,387 1.9 1,229 

Niger DHS 2012 46.0 100 78.8 33 

Rwanda DHS 2010 5.4 442 2.4 419 

Senegal DHS 2010-11 11.8 119 21.5 93 

Sierra Leone DHS 2013 13.8 269 28.8 281 

Tanzania AIS/MIS 2011-12 13.3 952 12.7 871 

Togo DHS 2013-14 16.7 240 6.2 209 

Uganda AIS 20111 36.9 2,629 3.6 1,360 

Zambia DHS 2013-2014 25.7 4,741 24.3 4,018 

Latin America/Caribbean 

Dominican Republic DHS 2013 21.8 271 0.0 192 

Haiti DHS 2012 24.9 559 3.1 425 

1 The 2011 Uganda AIS includes one sample with a missing signal-to-cutoff value for assay 2. 

Descriptive statistics on the distribution of weak positive specimens by S/CO value indicate that in many 

surveys there is a clustering of these specimens at lower S/CO values, close to the cutoff for seropositivity 

(Appendix Table 1). The percentage of positive specimens with S/CO values less than 5.0 is also lower 

among specimens with positive results on both EIA 1 and EIA 2 than among specimens that are EIA 1 

positive and EIA 2 negative (Appendix Table 2). 

Table 5 also shows the percentage of EIA 2 positive specimens with an S/CO value less than 5.0. As a 

reminder, specimens tested on EIA 2 in the first round of testing include all specimens that were positive 

on EIA 1 and a portion of those that tested negative on EIA 1. The proportion of EIA 2 positive specimens 

that comes from the IQC process varies by survey from 0 percent in Rwanda and Senegal to 32 percent in 

Sierra Leone (see Appendix Table 2).1

 The proportion of EIA 2 positive specimens with an S/CO value less 

than 5.0 is lower in most surveys than the proportion of EIA 1 positive specimens. In the majority of 

surveys, this percentage is less than 10 percent. The highest percentage of EIA 2 positive specimens with 

an S/CO value less than 5.0 is observed in Niger (79 percent). Senegal (22 percent), Sierra Leone (29 

percent), and Zambia (24 percent) also had exceptionally high percentages of EIA 2 positive samples with 

weak S/CO values. 

Assessing differences by assay is difficult because 14 of the 20 surveys used the same assay as EIA 2: 

fourth generation Enzygnost. Among surveys using this assay as EIA 2, the proportion of EIA 2 positive 

 

1

 The percentage of EIA 2 positive specimens that come from IQC in Table 5 should not be confused with the 

percentage of specimens with discrepant results on EIA 1 and EIA 2 that come from IQC, as discussed in Table 4. In 

Table 4, the percentage of specimens from IQC is equivalent to A1-A2+/(A1+A2- or A1-A2+). By contrast, in Table 

5, the percentage of specimens from IQC is equivalent to A1-A2+/(A1+A2+ or A1-A2+). 
15 

specimens with an S/CO value less than 5.0 exceeds 20 percent in only two. The percentage of EIA 2 

positive specimens with an S/CO value less than 5.0 exceeds 20 percent in two of the three surveys using 

third generation Enzygnost as EIA 2. Among the two surveys that used Vironostika as EIA 2, the percentage 

of EIA 2 positive specimens with an S/CO value less than 5.0 is 4 percent in Uganda, which used the third 

generation assay, compared with 1 percent in Cote d’Ivoire, which used the fourth generation assay. 

In general, the percentage of EIA 2 positive specimens with an S/CO value less than 5.0 is higher among 

IQC specimens than specimens with positive results on both EIA 1 and EIA 2 (see Appendix Table 2). 

However, it does not appear that the portion of EIA 2 positive specimens that come from IQC explains 

much of the variation across surveys in the proportion of EIA 2 positive specimens with weak reactions. 

On the one hand, the proportion of EIA 2 positive specimens with S/CO values less than 5.0 (29 percent) 

and the proportion of EIA 2 positive specimens that come from IQC (32 percent) are both high in Sierra 

Leone. On the other hand, in Senegal, where 22 percent of the EIA 2 positive specimens have S/CO values 

less than 5.0, none of the EIA 2 positive specimens were from IQC. 

Given the high percentage of HIV positive specimens with an S/CO value less than 5.0 in the first round of 

testing, and the potential for false positive results among them, Table 6 presents the proportion of EIA 1 

positive specimens that are assigned a final result of HIV positive after the completion of the testing 

algorithm, according to S/CO value. EIA 1 positive specimens are much more likely to be assigned a final 

result of HIV positive if they have an S/CO value of 5.0 or greater. The percentage of EIA 1 positive 

specimens with S/CO values of 5.0 or greater that are rendered positive after completion of the HIV testing 

algorithm ranges from 78 percent in Niger to 99 percent in Cameroon. On the other hand, the majority of 

specimens with S/CO values less than 5.0 on the first round of testing on EIA 1 are rendered negative 

through later stages of the HIV testing algorithm. The percentage of specimens with weak positive results 

on the first round of EIA 1 testing that remained positive after completion of the testing algorithm ranges 

from less than 1 percent in DRC to 38 percent in Zambia. In 13 of the 20 surveys, the percentage of EIA 1 

weak positives that are ultimately rendered positive is less than 10 percent. 

Table 6. Final HIV results according to signal to cutoff values on assay 1

Among specimens that are positive on assay 1 in the first round of testing, percentage that remain positive after all HIV testing, according 

to the signal to cutoff (S/CO) value on assay 1, by survey [Unweighted] 

Survey 

Percentage HIV 

positive after 

all testing 

Number of assay 

1 positive samples 

with S/CO <5.0 

Percentage HIV 

positive after 

all testing 

Number of assay 

1 positive samples 

with S/CO ≥5.0 

Sub-Saharan Africa 

Burundi DHS 2010 2.7 73 86.6 186 

Cameroon DHS 2011 2.7 623 99.4 636 

Cote d’Ivoire DHS 2011-12 20.0 10 97.6 334 

Democratic Republic of the Congo DHS 2013-14 0.5 201 94.1 187 

Gabon DHS 2012 2.9 68 97.6 493 

Gambia DHS 2013 8.3 12 95.0 141 

Guinea DHS 2012 3.7 214 98.7 158 

Malawi DHS 2010 3.7 269 97.3 1,449 

Mali DHS 2012-13 8.3 12 94.1 102 

Namibia DHS 2013 8.9 146 97.9 1,241 

Niger DHS 2012 2.2 46 77.8 54 

Rwanda DHS 2010 29.2 24 98.8 418 

Senegal DHS 2010-11 14.3 14 86.7 105 

Sierra Leone DHS 2013 21.6 37 92.2 232 

Tanzania AIS/MIS 2011-12 11.0 127 97.1 825 

Togo DHS 2013-14 2.5 40 99.0 200 

Uganda AIS 2011 14.1 970 81.9 1,659 

Zambia DHS 2013-2014 37.8 1,217 96.5 3,524 

Latin America/Caribbean 

Dominican Republic DHS 2013 1.7 59 89.6 212 

Haiti DHS 2012 5.8 139 97.9 420 
16 

Table 7 shows the distribution of all specimens that were rendered positive after the completion of the HIV 

testing algorithm according to EIA agreement and S/CO value.2

 The S/CO values of EIA 1 and EIA 2 are 

taken from the first round of testing unless the specimens were retested on EIA 1 and EIA 2, in which case 

the S/CO values from EIA 1 and EIA 2 from the repeat round replaced the S/CO values from EIA 1 and 

EIA 2 from the first round. As a reminder, EIA 1 and EIA 2 were repeated only if (1) the results on the two 

assays were discrepant in the first round of testing and (2) repeat testing of the EIAs was included in the 

survey’s testing algorithm (as noted in Table 2). Over 85 percent of positive specimens within each country 

have S/CO values of 5.0 or higher on both EIAs in all surveys except Niger (16 percent), Senegal (77 

percent), Sierra Leone (71 percent), and Zambia (78 percent). The proportion of positive specimens with a 

high S/CO value on one EIA and a low S/CO value on the other EIA is less than 5 percent in most countries. 

Higher percentages (9-25 percent) were observed in Burundi, Malawi, Senegal, Sierra Leone, and Zambia. 

The especially high percentage observed in Niger (55 percent) is related to the high proportion of weak 

positive specimens on the second EIA, which was Enzygnost (79 percent, as shown in Table 5). This pattern 

was not observed in the other two surveys that used the third generation Enzygnost assay as EIA 2. The 

percentage of positive specimens with S/CO values less than 5.0 on both EIAs is 1 percent or less in most 

surveys. Guinea, Sierra Leone, and Uganda have slightly higher percentages, 2-3 percent. However, 

Zambia, with 12 percent, is a notable outlier. 

Table 7. Distribution of final HIV positive specimens by assay results and signal to cutoff values 

Among specimens rendered positive after the completion of all HIV testing, percent distribution by EIA results and signal to cutoff 

(S/CO) values, by survey [Unweighted] 

Survey 

High 

S/CO on 

both EIA 

S/CO high on 

one EIA, low 

on the 

other EIA 

Low 

S/CO on 

both EIA

High S/CO on 

one EIA, 

negative on the 

other EIA 

Low S/CO on 

one EIA, 

negative on the 

other EIA Total 

Number 

final 

positive 

specimens

Sub-Saharan Africa 

Burundi DHS 2010 86.6 9.1 0.0 4.3 0.0 100.0 164 

Cameroon DHS 2011 96.8 1.7 1.1 0.5 0.0 100.0 649 

Cote d’Ivoire DHS 2011-12 96.4 3.0 0.6 0.0 0.0 100.0 336 

Democratic Republic of the 

Congo DHS 2013-14 

93.8 5.1 0.6 0.6 0.0 100.0 177 

Gabon DHS 2012 93.6 4.1 0.0 2.1 0.2 100.0 483 

Gambia DHS 2013 93.4 4.4 0.7 1.5 0.0 100.0 136 

Guinea DHS 2012 93.3 4.3 2.4 0.0 0.0 100.0 164 

Malawi DHS 2010 87.7 11.4 0.4 0.4 0.1 100.0 1,420 

Mali DHS 2012-13 93.8 5.2 0.0 0.0 1.0 100.0 97 

Namibia DHS 2013 96.0 2.5 0.2 1.3 0.0 100.0 1,233 

Niger DHS 2012 15.9 54.5 0.0 25.0 4.5 100.0 44 

Rwanda DHS 2010 96.9 1.9 1.0 0.0 0.2 100.0 420 

Senegal DHS 2010-11 77.4 21.5 1.1 0.0 0.0 100.0 93 

Sierra Leone DHS 2013 70.7 12.2 2.7 14.4 0.0 100.0 222 

Tanzania AIS/MIS 2011-12 92.8 5.9 0.9 0.2 0.2 100.0 815 

Togo DHS 2013-14 98.0 1.5 0.5 0.0 0.0 100.0 199 

Uganda AIS 20111 87.1 1.7 2.2 2.7 6.3 100.0 1,494 

Zambia DHS 2013-2014 77.9 9.8 12.1 0.2 0.0 100.0 3,901 

Latin America/Caribbean 

Dominican Republic DHS 2013 99.5 0.0 0.5 0.0 0.0 100.0 193 

Haiti DHS 2012 96.9 1.7 1.0 0.5 0.0 100.0 419 

Notes: High S/CO defined as ≥ 5.000. Low S/CO defined as 1.000-4.999. The assay results and S/CO reported in this table are taken 

from the repeat round of EIA testing if a specimen was subjected to repeat testing, and from the first round of testing if the specimen 

was tested on EIA 1 and 2 only once. 

 

2

 Table 7 includes specimens from the IQC process to the extent that they were rendered positive after the completion 

of all HIV testing. Across all surveys, the number of specimens that re-enter the testing algorithm due to a positive 

result on the IQC test and that are assigned a final status of HIV positive is few, if any (Appendix Table 3). 
17 

The last two columns in the distribution include specimens that were positive on one EIA and negative on 

the other. These specimens were rendered positive by virtue of a positive result on the third assay in the 

testing algorithm. In most countries this assay was a Western blot or a line immunoassay, such as INNOLIA. Uganda is the only survey that used a third EIA as the tie-breaker assay. The percentage of positive 

specimens with a high positive result on one EIA and a negative result on the other ranges from 0 to 4 

percent in most countries, but reaches 14 percent in Sierra Leone and 25 percent in Niger. The percentage 

of positive specimens with a weak positive result on one EIA and a negative result on the other is 1 percent 

or less in every survey except Niger (5 percent) and Uganda (6 percent). 

This analysis also examined the test results of specimens selected for IQC to assess the potential for false 

negative classification of specimens in this group of surveys. The percentage of IQC specimens (i.e., the 

subsample of specimens with negative results on EIA 1 that were selected for retesting on EIA 2) that were 

found to be positive on EIA 2 is shown in the third column of Table 3. This percentage is very low in most 

surveys. Specimens found to be positive through IQC were either retested on EIA 1 and EIA 2 (if these 

assays are repeated when discrepant in the survey’s algorithm) or immediately tested on a third assay to 

break the tie between EIA 1 and EIA 2. An analysis of the final serostatus of the specimens found to be 

positive on EIA 2 during the IQC process shows that the vast majority—and, in many cases, all—of these 

specimens are rendered negative by the third assay (Appendix Table 3). 

19 

5. Discussion 

The results of this analysis cannot say definitively if there has been any over- or underestimation of HIV 

prevalence in these surveys, or how great any error may be. However, it appears likely that testing error 

associated with false positivity is present to some degree in the surveys included in this analysis. With 

regard to the two quality indicators measured—agreement between EIA tests and proportion of positive 

specimens with an S/CO value less than 5.0—the analysis shows that they vary widely across surveys. 

Upon completion of the testing algorithm, agreement between the first and second EIA was high in most 

surveys, but only 7 of the 20 surveys reached the desired benchmark of at least 99 percent. Cameroon (68 

percent) and Uganda (52 percent) had the lowest levels of agreement on EIA 1 and EIA 2. Agreement in 

the remaining 11 surveys ranged from just under 89 percent to just under 99 percent. It is important to 

examine possible reasons why some of the surveys in this analysis have lower than desired levels of 

agreement. 

A principal factor contributing to lower levels of agreement is a low percentage of EIA 1 positive specimens 

being found positive on EIA 2. The percentage of EIA 1 positive specimens that are found positive on EIA 

2 after the first round of testing varies by survey, but is below 80 percent in 11 of the 20 countries and 

below 90 percent in all but two surveys. Published literature indicates that EIAs are highly sensitive. In 

several studies, EIAs have been found to detect every antibody-positive infection, resulting in no false 

negative results (Fanmi et al. 2012; Hakim et al. 2016; Urassa et al. 1999; Zhang et al. 2013). In the 20-

country analysis presented in this report, EIA 1 positive specimens found negative on EIA 2 tended to have 

weak reactions on EIA 1. This finding supports evidence from the published literature that where the 

percentage of EIA 1 positive specimens that are positive on EIA 2 is low, much if not all of the disagreement 

is likely due to false positive results on EIA 1. 

This analysis shows that repeat testing of discrepant specimens on EIA 1 and EIA 2 improved the level of 

agreement between the two assays. The improvement in agreement is achieved primarily through resolution 

of the discrepant results to negative-negative. In each of the eight surveys with repeat testing most, though 

not all, of the discrepant results resolve to negative-negative. Further, the benefit of repeat testing seems to 

be stronger where the first round test results are weaker. For example, Guinea improved from 73 percent 

agreement after the first round of testing to over 99 percent agreement after the second round of testing—

on par with Cote d’Ivoire, in which assay agreement was 99 percent after the first round of testing. Though 

the reasons for this improvement are not clear, two observations can be made. First, in the sequence of steps 

in the laboratory testing for these surveys, the repeat testing on specimens with discrepant results on EIA 1 

and EIA 2 is usually conducted after completing the first round of testing on EIA 1 and EIA 2 for all 

specimens. By this point, technologists may have become more proficient in their techniques on these 

assays and in working with the DBS specimens. Second, the repeat testing is often conducted by fewer, 

more experienced technologists. 

According to the testing strategy followed in these surveys, specimens with discrepant EIA results were 

rendered positive if the tie-breaker assay was positive. However, according to the HIV testing strategies 

currently recommended by CDC (2014) and WHO (2015), all of these specimens would be treated as 

negatives for the purpose of prevalence calculation. Specimens with discrepant EIA results make up less 

than 1 percent of positive specimens in 12 of the 20 surveys in this analysis, 1-5 percent in five surveys, 9 
20 

percent in Uganda, 14 percent in Sierra Leone, and 30 percent in Niger.3

 It should also be noted that in most 

of these surveys, only a small percentage of all EIA discrepant specimens are positive on the third assay 

(data not shown). Therefore the specimens with discrepant EIA results that are included among the final 

positive specimens are a small percentage of all specimens with discrepant EIA results. 

Though agreement of around 99 percent between two EIAs is expected when testing is of high quality, 

these high levels of agreement do not rule out the possibility of false positive results—it is still possible for 

dual EIA positive specimens to be false positive. The percent of dual EIA positive specimens that are 

confirmed positive, or the positive predictive value (PPV), varies in the published literature, but low values 

have been observed. While the PPV of two positive EIAs was found to be 100 percent in Urassa et al. 

(1999) and 99.9 percent in Nkengasong et al. (1999), a PPV of only 90.8 percent was observed by Hakim 

et al. (2016), and 84.0 percent by Zhang et al. (2013). It is important to note that the prevalence in the study 

by Zhang et al. was only 0.03 percent and that PPV is expected to be lower where prevalence is lower. 

Although false positives do occur at high S/CO values, the PPV of EIAs generally increases with S/CO 

value. Therefore, this analysis also examined the distribution of S/CO values on the two EIAs. The 

percentage of specimens with an S/CO value less than 5.0 on EIA 1 exceeds 20 percent in 9 of the 20 

surveys. As noted in the methods section, it is difficult to know what an optimal distribution of S/CO values 

would look like for these populations; however, it is highly unlikely that the percent of positive specimens 

with true weak positive reactions would be as high as 20 percent. This finding indicates that there is a risk 

of a notable number of false positive specimens in the first round of testing on EIA 1. 

Combining the S/CO values of the two EIAs, dual EIA positive specimens were categorized into three 

groups: specimens with low S/CO values (<5.0) on both EIAs, specimens with a low S/CO value on one 

EIA and a high S/CO value on the other, and specimens with high S/CO values on both EIAs. The 

proportion of positive specimens with low S/CO values on both EIAs in this 20-survey analysis is below 1 

percent in most surveys, compared with a high of 12 percent in Zambia. Evidence from other studies 

indicates that the positive specimens with low S/CO values on both EIAs are the least likely of the dual 

EIA positive specimens to be true positives. In an investigation of six EIAs, Nkengasong et al. (1999) found 

that only around half of specimens with S/CO <3.0 and 73 percent of specimens with S/CO <5.0 on 

individual EIAs were confirmed positive. Only 4 percent of specimens with S/CO <5.0 were confirmed 

positive on Enzygnost Integral II in a study by Fanmi et al. (2012). Zhang et al. (2013) and Urassa et al. 

(1999) found no true positive specimens among those with S/CO values on both EIAs of <5.0 and <3.0, 

respectively. 

The percentage of positive specimens with a low S/CO value on one EIA and a high S/CO value on the 

other varies across the 20 surveys in this analysis. The percentage of positive specimens with “mixed” S/CO 

values is below 5 percent in 11 surveys, 5-10 percent in five surveys, and above 10 percent in the remaining 

four surveys. In the published literature, specimens with a low S/CO value on one EIA and a high S/CO 

value on the other appear to be uncommon, and there is little evidence of their PPV. There were no 

specimens with an S/CO value <5.0 on one EIA and ≥5.0 on the other in the study by Zhang et al. (2013). 

In the study by Nkengasong et al. (1999), 9 of 12 specimens that had S/CO values <3.0 on one or both EIAs 

were confirmed positive, for a PPV of 75 percent. All dual EIA positive specimens were confirmed positive 

 

3

 If all discrepant specimens with a positive result on the third assay were to be considered negative rather than positive, 

the HIV prevalence estimates would remain unchanged in 15 of the 20 surveys, and would decrease by 0.1-0.2 

percentage points in four surveys and by 0.6 percentage points in Uganda (data not shown). 
21 

in the study by Urassa et al. (1999), but it is unclear how many of these, if any, may have had mixed S/CO 

values. 

The percentage of final positive specimens with S/CO values of 5.0 or greater on both EIA 1 and EIA 2 is 

over 85 percent for 16 of the 20 surveys in this analysis, and is over 95 percent in 7 surveys. Though it 

cannot be assumed that all are positive, some studies do show that the group of specimens with S/CO values 

greater than 5.0 have much higher PPV than do all positives as a whole. For example, the PPV of Enzygnost 

Integral II increased from 84.3 percent among all positive specimens to 98.9 percent among specimens with 

S/CO >5.0 (Fanmi et al., 2012). No false positives were found among specimens with S/CO >3.0 on both 

EIAs in Nkengasong et al. (1999). On the other hand, in the study by Zhang et al. (2013), the group of 

positive specimens with S/CO >5.0 had a PPV of 90.8 percent, a relatively small improvement over the 

PPV of 84.0 percent for all positives. Again, the low HIV prevalence in the study by Zhang et al. and its 

association with lower PPV should be noted. 

The results of this 20-survey analysis also show that the vast majority of specimens with discrepant results 

on the two EIAs, as well as specimens with low S/CO values on EIA 1, are rendered negative in the later 

stages of the testing algorithm. However, the literature on the specificity of EIA tests provides evidence 

that some false positives are likely to remain among dual EIA positive specimens and across all S/CO 

values. 

A few factors that further limit the applicability of results from published studies to the surveys included in 

this analysis should be noted. The EIAs used in most of these studies were third generation tests. In this 

analysis, on average, surveys that used a fourth generation Vironostika assay as EIA 1 have a higher 

percentage of positive specimens with an S/CO value of less than 5.0 than do surveys that used a third 

generation Vironostika assay. Though this finding is inconclusive, it may be associated with a lower PPV 

for the fourth generation tests. In addition, with the exception of the study by Hakim et al. (2016), all other 

studies cited were performed on serum or plasma specimens. By contrast, the specimen matrix was DBS in 

19 of the 20 surveys included in this analysis. In at least one published study, assays run on DBS specimens 

have been found to have slightly lower PPV, and thus a higher risk of false positive results, compared with 

assays run on serum specimens from the same individual—99.5 percent vs. 100 percent (de Castro, et al. 

2008). Finally, variation in the two quality indicators across surveys does not appear to be strongly 

associated with differences in either the level of HIV prevalence or the specific assays used. This finding 

suggests that the most likely explanation for differences across surveys may be related to operator 

performance. This finding, as well as the variation in the PPV of EIAs observed in the published literature, 

make it difficult to make specific estimates about the percentage of specimens in the 20 surveys in this 

analysis that may be false positive. Further testing of specimens on a more specific assay would be 

necessary to measure the magnitude of false positive classification that may have occurred in these surveys. 

Though no conclusions as to magnitude can be drawn from this analysis, it appears that the amount of 

testing error could vary notably by survey. Taking into account the lower percentages of final positive 

specimens with S/CO values of 5.0 or greater on both EIA 1 and EIA 2, relative errors are likely to be 

greatest in Niger, Sierra Leone, Senegal, and Zambia. Taking into consideration both relative error and the 

magnitude of the HIV prevalence, absolute errors are likely to be most notable in Malawi, Uganda, and 

Zambia. 

To understand in context the impact that any error introduced through inaccurate test results may have on 

the validity of the survey HIV prevalence estimates, it is helpful to consider other sources of error for the 

surveys. These sources include both sampling and non-sampling errors. Sampling error, driven exclusively 

by sample size and design, determines the precision of the HIV prevalence estimate. The precision of an 
22 

estimate is often described by the relative standard error (RSE), which measures the statistical uncertainty 

in the estimate as a proportion of the value of the estimate. Across this group of surveys, the RSE for the 

estimated HIV prevalence ranges from 3 to 20 percent, and tends to be higher where the HIV prevalence is 

lower. For the sake of comparison, HIV prevalence would have to be overestimated by at least a relative 6 

percent to result in a revised HIV prevalence estimate falling outside the confidence interval on any of these 

surveys. For most of these surveys, relative overestimation of HIV prevalence would need to exceed 15 

percent to fall outside the confidence interval. 

Additionally, in about half of the surveys included in this analysis, the HIV prevalence is below 2 percent. 

Even a relative 10 percent overestimation of HIV prevalence, for example, would result in an absolute 

overestimation of just 0.1 to 0.2 percentage points. Thus, in very low prevalence countries testing error is 

likely to be small in absolute terms and relative to the precision afforded by the sample design. However, 

the consequences of overestimation are of greater concern in surveys in the high prevalence countries, 

because the absolute magnitude of any error would be greater and more important relative to sampling 

error. 

Misclassification of specimens due to testing error is one of many types of non-sampling errors that can 

affect the validity of HIV prevalence estimates in population-based surveys. Selection bias, that is, 

differential participation in the survey HIV test by survey respondents according to their likelihood of 

having HIV, may be an important non-sampling error in HIV prevalence estimates in population-based 

surveys. The Heckman-selection method for removing selection bias has been applied to DHS surveys, and 

the results show that HIV prevalence may be higher among respondents who do not participate in the survey 

HIV test than among respondents who do, creating a potential for underestimation of HIV prevalence 

(McGovern, et al. 2015). In assessing the risk of under- or overestimation of HIV prevalence in populationbased surveys, it is important to take into account the direction and magnitude of all potentially meaningful 

sources of error. 

These results suggest the need for more vigilance in the quality of laboratory testing, with even more checks 

built into every step of the testing process. In addition, use of a testing strategy, including repeat testing of 

two EIAs when the results are discrepant, improves assay agreement and appears to improve EIA specificity 

to some degree. Finally, the HIV testing strategies currently recommended by both UNAIDS/WHO (2015) 

and CDC (2014) include a more specific assay to confirm all EIA positive results, regardless of the level of 

HIV prevalence. Testing strategies, including a confirmatory assay such as Western blot, Geenius, or 

INNO-LIA, should reduce false positivity and improve the accuracy of HIV prevalence estimates. 
23 

References 

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“Evaluation of the Human Immunodeficiency Virus Type 1 and 2 Antibodies Detection in Dried Whole 

Blood Spots (DBS) Samples.” Revista do Instituto de Medicina Tropical de São Paulo 50:151–6. 

Centers for Disease Control and Prevention (CDC), Division of Global HIV/AIDS (DGHA). 2014. DGHA 

Recommendations for HIV Serology Testing in Surveillance. Atlanta: CDC. 

Fanmi, a N., C. Ramière, J. C. Tardy, and P. André. 2013. “Real-life Evaluation of a Human 

Immunodeficiency Virus Screening Algorithm Using a Single Combined p24 Antigen-Antibody Assay.” 

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a Regional Laboratory in Kagera, Tanzania.” Journal of Clinical Virology 14:25-29. 

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25 

Appendix 

Appendix Table 1.1. Descriptive statistics for signal to cutoff values among weak positive specimens, assay 1

Median, 25th and 75th percentiles, mean, and standard deviation of the S/CO values for specimens that are positive on assay 1 with 

signal to cutoff values of 1.000-4.999 on the first round of testing, by assay type and generation and by survey [Unweighted] 

Survey Median 

25th 

percentile 

75th 

percentile Mean 

Standard 

deviation Number 

Enzygnost 4 

Cote d’Ivoire DHS 2011-12 2.8 2.5 3.3 3.1 1.0 10 

Genscreen 4 

Gabon DHS 2012 1.8 1.3 3.5 2.4 1.2 68 

Murex 3 

Uganda AIS 2011 1.6 1.2 2.5 2.0 1.0 970 

Murex 4 

Cameroon DHS 2011 1.2 1.1 1.3 1.3 0.5 623 

Vironostika 3 

Malawi DHS 2010 1.4 1.1 2.0 1.8 0.9 269 

Mali DHS 2012-13 1.5 1.1 2.0 1.7 0.8 12 

Niger DHS 2012 1.6 1.2 2.3 1.9 0.8 46 

Senegal DHS 2010-11 2.4 1.4 3.5 2.5 1.3 14 

Vironostika 4 

Burundi DHS 2010 1.5 1.1 2.1 1.8 1.0 73 

Democratic Republic of the Congo DHS 2013-14 1.3 1.1 1.8 1.6 0.8 201 

Dominican Republic DHS 2013 1.6 1.3 2.2 1.9 0.9 59 

Gambia DHS 2013 3.6 2.5 3.8 3.2 1.2 12 

Guinea DHS 2012 1.2 1.1 1.4 1.4 0.5 214 

Haiti DHS 2012 1.1 1.0 1.3 1.4 0.8 139 

Namibia DHS 2013 1.3 1.1 1.9 1.6 0.8 146 

Rwanda DHS 2010 2.0 1.3 3.2 2.3 1.1 24 

Sierra Leone DHS 2013 1.2 1.1 2.0 1.8 1.2 37 

Tanzania AIS/MIS 2011-12 1.2 1.1 1.9 1.7 0.9 127 

Togo DHS 2013-14 1.5 1.2 2.0 1.7 0.8 40 

Zambia DHS 2013-2014 1.6 1.2 2.3 1.8 0.8 1,217 

 
26 

Appendix Table 1.2. Descriptive statistics for specimens with weak signal to cutoff values, assay 2 

Median, 25th and 75th percentiles, mean, and standard deviation of the S/CO values for specimens that are positive on assay 2 with 

signal to cutoff values of 1.000-4.999 on the first round of testing, by assay type and generation and by survey [Unweighted] 

Survey Median 

25th 

percentile 

75th 

percentile Mean 

Standard 

deviation Number 

Enzygnost 3 

Malawi DHS 2010 4.0 2.7 4.5 3.6 1.2 170 

Niger DHS 2012 2.3 1.9 3.5 2.7 1.2 26 

Senegal DHS 2010-11 3.5 3.2 4.1 3.5 0.9 20 

Enzygnost 4 

Burundi DHS 2010 4.3 4.0 4.8 4.2 0.8 13 

Democratic Republic of the Congo DHS 2013-14 1.5 1.1 2.9 2.1 1.3 23 

Dominican Republic DHS 2013 na na na na na 0 

Gabon DHS 2012 4.1 3.0 4.5 3.6 1.3 20 

Gambia DHS 2013 3.2 2.3 3.9 3.1 1.3 8 

Guinea DHS 2012 1.4 1.1 1.6 1.6 1.1 12 

Haiti DHS 2012 1.3 1.2 1.5 1.6 1.0 13 

Mali DHS 2012-13 2.1 1.4 3.4 2.3 1.1 10 

Namibia DHS 2013 3.5 2.5 4.0 3.2 1.1 23 

Rwanda DHS 2010 4.0 3.2 4.8 3.8 1.1 10 

Sierra Leone DHS 2013 2.8 1.3 4.2 2.8 1.4 81 

Tanzania AIS/MIS 2011-12 1.3 1.1 2.5 1.9 1.2 111 

Togo DHS 2013-14 1.4 1.1 2.4 1.8 0.9 13 

Zambia DHS 2013-2014 1.6 1.2 2.5 2.0 1.0 978 

Genscreen 4 

Cameroon DHS 2011 2.1 1.1 4.0 2.6 1.5 13 

Vironostika 3 

Uganda AIS 2011 1.7 1.4 2.7 2.0 0.9 49 

Vironostika 4 

Cote d’Ivoire DHS 2011-12 3.3 2.4 4.0 3.2 0.8 3 

na = Not applicable 

 
27 

Appendix Table 2. Weak positive signal to cutoff values according to first round test results 

Among specimens positive on assay 1 in the first round of testing, percentage with an S/CO value of less than 5.0 according to the first round 

results of assays 1 and 2; and among specimens positive on assay 2 in the first round of testing, percentage with an S/CO value of less than 

5.0 according to first round results of assays 1 and 2, and percentage that are negative on assay 1, by survey [Unweighted] 

Assay 1 positive specimens Assay 2 positive specimens 

 A1+A2+ A1+A2- A1+A2+ A1-A2+ (IQC) 

Percentage 

of A2+ that 

Survey are A1- (IQC)

Percentage with 

S/CO <5.0 Number

Percentage with 

S/CO <5.0 Number

Percentage with 

S/CO <5.0 Number

Percentage with 

S/CO <5.0 Number

Sub-Saharan Africa 

Burundi DHS 2010 1.3 157 69.6 102 8.3 157 0.0 11 6.5 

Cameroon DHS 2011 2.8 647 98.9 612 1.4 647 100.0 4 0.6 

Cote d’Ivoire DHS 2011-12 2.4 334 20.0 10 0.9 334 0.0 2 0.6 

Democratic Republic of the 

Congo DHS 2013-14 

0.6 176 94.3 212 5.7 176 100.0 13 6.9 

Gabon DHS 2012 0.2 472 75.3 89 4.0 472 0.0 1 0.2 

Gambia DHS 2013 0.7 134 57.9 19 5.2 134 50.0 2 1.5 

Guinea DHS 2012 4.9 163 98.6 209 4.3 163 100.0 5 3.0 

Malawi DHS 2010 0.6 1,392 80.1 326 11.8 1,392 50.0 12 0.9 

Mali DHS 2012-13 0.0 96 66.7 18 5.2 96 100.0 5 5.0 

Namibia DHS 2013 1.1 1,217 78.2 170 1.8 1,217 8.3 12 1.0 

Niger DHS 2012 0.0 31 66.7 69 77.4 31 100.0 2 6.1 

Rwanda DHS 2010 1.4 419 78.3 23 2.4 419 na 0 0.0 

Senegal DHS 2010-11 2.2 93 46.2 26 21.5 93 na 0 0.0 

Sierra Leone DHS 2013 4.2 190 36.7 79 16.3 190 54.9 91 32.4 

Tanzania AIS/MIS 2011-12 1.5 808 79.9 144 6.1 808 98.4 63 7.2 

Togo DHS 2013-14 0.5 198 92.9 42 2.0 198 81.8 11 5.3 

Uganda AIS 20111 3.2 43 73.1 927 3.6 1,360 na na 0.0 

Zambia DHS 2013-2014 10.7 3,711 79.7 1,030 19.4 3,711 84.4 307 7.6 

Latin America/Caribbean 

Dominican Republic DHS 2013 0.0 190 72.8 81 0.0 190 0.0 2 1.0 

Haiti DHS 2012 1.9 417 92.3 142 1.7 417 75.0 8 1.9 

Note: The 2011 Uganda AIS did not include retesting of assay 1 negative samples for internal quality control. 

A1 = assay 1 

A2 = assay 2 

1 The 2011 Uganda AIS includes one sample with a missing S/CO value for assay 2. 

 
28 

Appendix Table 3. Internal quality control 

Number of specimens tested for HIV, number found HIV positive after completion of the HIV testing algorithm, number of specimens tested for 

internal quality control (IQC), number of IQC specimens found positive on assay 2, and the number of IQC specimens that remain positive after 

completion of the HIV testing algorithm, by survey [Unweighted] 

All specimens tested Specimens included in IQC testing 

Survey 

Number of 

specimens 

tested for HIV 

Number of final 

positive 

specimens 

Number of 

specimens 

tested for IQC 

Number of ICQ 

specimens 

found positive 

on assay 2 

Number of IQC 

specimens that 

remain positive 

after all testing

Sub-Saharan Africa 

Burundi DHS 2010 8,588 164 830 11 1 

Cameroon DHS 2011 14,202 649 663 4 0 

Cote d’Ivoire DHS 2011-12 9,008 336 865 2 2 

Democratic Republic of the Congo DHS 2013-14 17,638 177 863 13 0 

Gabon DHS 2012 10,992 483 523 1 0 

Gambia DHS 2013 7,769 136 749 2 1 

Guinea DHS 2012 8,380 164 413 5 0 

Malawi DHS 2010 13,910 1,420 1,238 12 0 

Mali DHS 2012-13 8,861 97 875 5 0 

Namibia DHS 2013 8,858 1,233 421 12 5 

Niger DHS 2012 8,628 44 863 2 1 

Rwanda DHS 2010 13,248 420 1,283 0 na 

Senegal DHS 2010-11 9,917 93 976 0 na 

Sierra Leone DHS 2013 14,600 222 1,440 91 0 

Tanzania AIS/MIS 2011-12 17,745 815 1,678 63 0 

Togo DHS 2013-14 9,172 199 893 11 0 

Zambia DHS 2013-2014 29,006 3,901 2,423 307 39 

Latin America/Caribbean 

Dominican Republic DHS 2013 18,614 193 1,847 2 2 

Haiti DHS 2012 18,531 419 901 8 0 

Note: The 2011 Uganda AIS did not include retesting of negative specimens for IQC.