1 - clinical and vaccine immunology · 2011-07-13 · 115 69,107 and registered at clintrial.gov...

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Performance of a Rapid and Simple HIV Testing Algorithm in a 1

Multicenter Phase III Microbicide Clinical Trial 2

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Tania Crucitti 1 *, Doug Taylor 2, Greet Beelaert 1, Katrien Fransen 1, Lut 6

Van Damme 2, 3 7

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1 Institute of Tropical Medicine, Antwerp, Belgium 10

2 FHI, NC/VA, USA 11

3 CONRAD, Arlington, VA, USA 12

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* Corresponding author: 14

Tania Crucitti, HIV/STI Reference Laboratory, Institute of Tropical Medicine, 155 15

Nationalestraat, 2000 Antwerp, Belgium, e-mail: [email protected], tel + 32 3 247 65 16

52, fax + 32 3 247 63 33 17

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05069-11 CVI Accepts, published online ahead of print on 13 July 2011

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ABSTRACT 19

A multi-test sequential algorithm based on Rapid/Simple (R/S) assays was 20

applied for the diagnosis of HIV infection among participants in a phase 3 21

microbicide effectiveness trial. HIV testing was performed on finger-prick 22

blood samples from patients after their enrollment in the trial. The specimens 23

were tested in a serial procedure using three different rapid tests (Determine 24

HIV-1/2 (Abbott), SD Bioline HIV 1/2 3.0 (Standard Diagnostics), and Uni-Gold 25

HIV (Trinity Biotech)). In the event of discordant results between the 26

Determine HIV 1/2 and SD Bioline HIV 1/2 3.0 tests, a third assay (i.e., Uni-27

Gold HIV) determined the final outcome. When the final outcome was positive, 28

a second specimen was collected and tested with the same algorithm, only if 29

a positive result was obtained with this sample the participant was informed of 30

her positive serostatus. A total of 5734 post-enrollment specimens obtained 31

from 1398 women were tested. Forty-six women tested positive according to 32

the testing algorithm performed on the first collected specimen. Confirmatory 33

testing results obtained at the ITM confirmed that 42 women were truly 34

infected. Two out of four initial false positives tested negative upon testing of a 35

second blood specimen. The other two tested false positive twice using 36

specimens collected on the same day. A high percentage of specimens 37

reactive with the Determine HIV-1/2 assay only were observed in the study 38

site in Kampala. This result did not appear to be associated with pregnancy or 39

malaria infection. 40

HIV testing algorithms including only R/S assays are suitable for use in clinical 41

trials, provided that adequate quality assurance procedures are in place. 42


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INTRODUCTION 43

Phase 2b/3 trials assessing the effectiveness of products to prevent the 44

heterosexual acquisition of HIV are almost exclusively conducted in African 45

and Asian countries with HIV epidemics and high incidence of infection. Even 46

in such settings, effectiveness trials must be conducted in multiple centers to 47

observe the large number of HIV infection events required to achieve 48

adequate statistical power to detect a treatment effect. 49

50

Study clinics may be located in remote areas, small towns or villages, where 51

well-equipped laboratories are lacking. There is often access only to a small 52

on-site laboratory where rapid and simple (R/S) assays to detect HIV 53

antibodies are performed. Testing using enzyme-linked immunosorbent 54

assays is preferable when large numbers of specimens are to be tested; 55

however, equipment, storage of reagents in refrigerated conditions, and 56

extensive training of laboratory staff is required for these assays. In contrast, 57

R/S assays require little to no equipment and have no storage restrictions (if 58

ambient temperatures are below 27°C). In addition, the use of R/S assays 59

(compared to traditional multiwell immunoassays) has the advantage that 60

results can be provided the day samples are collected; greatly reducing the 61

likelihood that individuals will fail to receive their test results. As with multiwell 62

immunoassays, there exists a time interval between infection and detectable 63

antibody levels (i.e., a window period, often measured in weeks), and false 64

positive results can never be excluded (4). Nonetheless, once patients are out 65


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of the window period, the sensitivity and specificity of the R/S HIV assays are 66

high and comparable to multiwell immunoassays (2, 24, 25, 26). 67

The HIV testing algorithms described in the literature often include the 68

confirmation of reactive rapid or immunoassay tests by supplemental testing 69

such as western blot (WB) (3, 4, 19). Performing WB assays requires 70

additional training of the laboratory staff and specific equipment and is 71

expensive. The UNAIDS/WHO program on HIV/AIDS testing proposes three 72

testing strategies using R/S assays without the requirement for additional 73

confirmatory testing, making them affordable and suitable for application in 74

small laboratories (2, 7, 13, 14). To maximize specificity and positive 75

predictive value, multi-test sequential algorithms have been implemented. 76

However, the sensitivity of any such algorithm can only be as high as that of 77

the first test in the sequence (1). Here, we present the results obtained with a 78

sequential HIV multi-test algorithm based on R/S assays used for the 79

diagnosis of HIV infection among participants in a phase 3 microbicide 80

effectiveness trial. 81

82

MATERIALS AND METHODS 83

Study design 84

The study in which the R/S algorithm was employed has been described 85

elsewhere (23). Briefly, the study was a randomized double blind placebo-86

controlled trial of a candidate vaginal microbicide (cellulose sulfate gel) for HIV 87

prevention. Women were recruited at five sites: a community clinic and a clinic 88


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for sexually transmitted infections in Cotonou, Benin; the Y.R. Gaitonde 89

Center for AIDS Research and Education (Y.R.G. CARE) in Chennai, India; 90

the Medical Research Council in Durban, South Africa; the Mulago Hospital 91

(Makarere University) in Kampala, Uganda; and clinics in Mudhol and 92

Jhamkandi in Karnataka, India (in collaboration with the Karnataka Health 93

Promotion Trust, Bangalore). Eligibility criteria included negative HIV antibody 94

tests at screening and enrollment and agreement to visit the clinic for HIV 95

testing 1, 3, 6, 9, and 12 months after enrollment. 96

97

HIV testing was performed on serum at screening; on plasma at enrollment, 98

the final visit and at any visit at which product use was discontinued; and on 99

whole blood obtained by finger prick at the follow-up visits at 1, 3, 6, and 9 100

months. The sites applied their national or local HIV testing algorithm for the 101

determination of HIV infection at screening and enrollment, but the sequential 102

R/S study algorithm (Figure 1) was applied on all other visits. This paper 103

presents the results and performance of the HIV algorithm used during follow-104

up and at the final visit and does not include the evaluation of the national or 105

local testing algorithm used at screening and enrollment. 106

107

Ethical approval 108

The study was approved by the Institutional Review Board of the Eastern 109

Virginia Medical School and of the Institute of Tropical Medicine and by local 110

ethics committees at each site where women were recruited. All approvals 111


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were granted prior to study initiation. Participants gave written informed 112

consent before screening and enrollment. The trial was conducted under the 113

Food and Drug Administration's Investigational New Drug application number 114

69,107 and registered at clintrial.gov (study number NCT00153777). 115

116

Specimens 117

Whole blood was collected in an EDTA microtainer (Becton Dickinson, 118

Sparks, USA) by finger prick using the Glucolet 2 device (Bayer HealthCare 119

LLC, Mishawaka, USA) at 1, 3, 6, and 9 month follow-up visits. The collected 120

volumes ranged between 250 and 500 microliters. At the final visit, permanent 121

product withdrawal visit or in the event the first finger-prick blood sample was 122

reactive for HIV antibodies, 7 ml of blood was collected in an EDTA blood 123

collection tube and processed within four hours. After centrifugation at 800 x g 124

(± 2000-2200 rpm) at 20°C ± 5°C for 10 minutes, the plasma was transferred 125

to a 15 ml conical tube under sterile conditions. The tube was centrifuged for 126

30 minutes at 1200 x g (± 2200 - 2400 rpm) at 20°C ± 5°C. After HIV R/S 127

testing on site, plasma aliquots were stored at -70°C until shipment on dry ice 128

to the Institute of Tropical Medicine (ITM), Antwerp, Belgium, for additional 129

testing. 130

131

HIV Assays 132


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Post-enrollment specimens were tested according to a sequential HIV R/S 133

algorithm based on the modified WHO/UNAIDS testing approach for strategy 134

III, which was also included as Point Of Contact algorithm 4 in the status 135

report on HIV testing algorithms published by the Association of Public Health 136

Laboratories (1, 21). Specimens were first tested with the Determine HIV-1/2 137

test (Abbott Laboratories, United Kingdom). If a sample was reactive, it was 138

tested with the SD Bioline HIV-1/2 3.0 test (Standard Diagnostics, South 139

Korea). In the event of a discordant result, the Uni-Gold HIV test (Trinity 140

Biotech, Ireland) was used. The final result of the algorithm scored a sample 141

as positive if two out of the three R/S HIV assays were reactive. In this case, a 142

second specimen was collected from the participant, preferably on the same 143

day, and tested with the same algorithm. The participant was informed of her 144

positive serostatus only if a positive result with the second specimen was 145

obtained. 146

147

The ITM tested additional specimens to exclude the possibility that 148

participants entered or exited the study during their window period. To this 149

end, all enrollment plasma specimens from participants with detectable HIV 150

antibodies within 3 months after enrollment and all plasma specimens 151

collected at the final visits of patients without detectable HIV antibodies were 152

tested using a qualitative HIV-1 RNA PCR assay (COBAS Ampliscreen HIV-1 153

test, Roche Molecular Systems, USA). Due to the large number of specimens, 154

pools of a maximum of 24 final visit plasma samples were tested according to 155

the manufacturer’s specifications. 156


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157

In the context of the quality control established for the study, aliquots of stored 158

plasma specimens from participants who tested HIV positive after enrollment 159

were also tested at the ITM to confirm these results using a different testing 160

algorithm, which is presented in Fig 2. 161

The details regarding guaranteeing the quality of the HIV results obtained at 162

the different study sites have been reported previously (5). In brief, this quality 163

control assessment consisted of the assessment of the study site laboratories, 164

provision of hands-on training, conduction of supervisory visits, distribution of 165

quality and batch control panels, and re-testing of pre-defined specimens. 166

Pregnancy and Malaria testing 167

Participants were tested for pregnancy at all visits using a urine HCG rapid 168

test. When malaria was suspected, a thick blood smear film was examined for 169

the microscopic visualization of Plasmodium parasites. 170

171

Statistical analysis 172

Overall, 38.8% of the potential participants were HIV positive at screening and 173

thus excluded from study participation (Table 1). Here, we summarize the 174

results of the sequential R/S HIV algorithm as applied to 5734 post-enrollment 175

specimens of 1398 enrolled women who had at least one follow-up HIV test 176

specimen as well as confirmatory specimens collected following an initial 177

positive result (specimens collected after a confirmed positive HIV result and 178


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specimens contributed by three women later found to have been in their 179

window period at enrollment are excluded). Descriptive tests of associations 180

between participant characteristics and discordant rapid test results were 181

conducted using logistic regression with generalized estimating equations to 182

account for repeated measures on each enrolled participant. 183

Final confirmation of positive results was performed at the ITM according to 184

the testing algorithm presented in Fig. 2 and/or using the HIV-1 RNA PCR 185

assay. Negative results were confirmed by testing all plasma samples 186

collected at final visits with the HIV-1 RNA PCR assay. 187

188

RESULTS 189

A total of 46 first collected specimens tested positive according to the 190

sequential R/S testing algorithm at the sites (Table 2), with all but one 191

specimen reactive with both Determine HIV-1/2 and SD Bioline HIV-1/2 3.0 192

(Figure 3). Confirmatory results were available for all 46 women, with 42 193

(91.3%) confirmed HIV-1 positive and 4 (8.7%) confirmed HIV negative (one 194

of the 42 confirmed positives became infected after the administrative 195

censoring date of the trial and was excluded from the primary effectiveness 196

analysis reported in (23)). HIV-2 infection was not detected in the post-197

enrollment specimens. According to the confirmed results of a total of 5734 198

specimens tested, the sequential R/S HIV testing algorithm showed an initial 199

sensitivity of 100% (95% CI: 89.6%-100%) and an initial specificity of 99.9% 200

(95% CI: 99.8%-100%). One of the four women with negative confirmatory 201


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results was subsequently lost to follow-up; two of the four had HIV negative 202

results at three or more subsequent visits; and one had a positive HIV result 203

on her first but a negative result on her second collected specimen, followed 204

by negative results at her last two visits. The initial positive results for these 205

four participants were considered false positive. 206

For two of the participants, including the participant with an unconfirmed 207

positive result at her next visit, the final result was false positive. We cannot 208

exclude that the false positive results were partially caused by the use of 209

deteriorated Determine HIV-1/2 test strips. Indeed, we observed that the 210

Determine HIV-1/2 test strips were not stored properly and were exposed to 211

humidity at this test site. In addition, the SD Bioline HIV-1/2 3.0 assay had a 212

false reaction leading to a final false positive result. 213

There were 124 specimens from 98 women with a reactive result in the 214

Determine HIV-1/2 test only (i.e., both SD Bioline HIV-1/2 3.0 and Uni-Gold 215

HIV were not reactive; Table 3). Five of the 98 women were not tested again, 216

2 were HIV antibody positive according to the R/S algorithm at their next study 217

visit and 91 were subsequently tested but never diagnosed with HIV. Of the 218

91, 22 (24.1%) were reactive only in the Determine HIV-1/2 test on multiple 219

occasions (Table 4). 220

221

The proportion of specimens that were only reactive in the Determine HIV-1/2 222

assay was significantly higher in Uganda (7.2%) than in the study as a whole 223

(2.2%; p-value<0.001). Given this observation, we sought to explore the 224


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possible influences of pregnancy and malaria on the performance of the 225

Determine HIV-1/2 assay. Pregnancy and malaria were both highly prevalent 226

in Uganda, and there is evidence in the literature to suggest that these 227

conditions may impact the specificity of the Determine HIV-1/2 assay (9, 10, 228

11, 15,18, 28). However, we found no significant difference in the proportion 229

of specimens in which only the Determine HIV-1/2 test was reactive between 230

pregnant and non-pregnant women (3.6% reactive among women testing 231

positive for pregnancy, 2.1% for women not testing positive for pregnancy; p-232

value=0.24). Malaria was only diagnosed in Benin and Uganda. Restricting 233

analysis to those two sites, we likewise found no significant difference in the 234

proportion of specimens in which only the Determine HIV-1/2 test was reactive 235

according to malaria status (4.7%, regardless of previous malaria diagnosis; 236

p-value=0.61). 237

238

Having a reactive result only in the Determine HIV-1/2 assay was significantly 239

and positively associated with the probability of obtaining a reactive result only 240

on the Determine HIV-1/2 test at a subsequent visit (p-value=0.014), but this 241

result is confounded by the effect of site (20 of the 22 women with repeat 242

samples only reactive in the Determine HIV-1/2 test were from Uganda). 243

Given these results, we are unable to explain the high prevalence of 244

specimens reactive only in the Determine HIV-1/2 test in Uganda. 245

246


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Using the qualitative HIV-1 RNA PCR assay on stored plasma specimens, 247

three participants were found to be HIV infected at enrollment. These three 248

participants were from Uganda, Benin, and South Africa; sites which used a 249

different testing algorithm at enrollment than at follow-up and hence do not 250

provide information on the utility of the R/S algorithm presented here. 251

Another participant was found to be recently infected at her final visit. 252

253

DISCUSSION 254

HIV testing plays a crucial role in HIV prevention clinical trials as it determines 255

the eligibility of potential participants and is used to identify the primary study 256

endpoints. 257

258

The use of R/S HIV tests provides the opportunity for small or field-based 259

laboratories to deliver HIV results on the same day as the study visit. 260

Moreover, R/S tests do not require refrigeration as long as temperatures are 261

below 27°C, nor do they require special equipment, processing of specimens 262

in batches, or highly skilled laboratory staff. 263

264

In general, multiwell immunoassays are considered to be superior to R/S tests 265

for the detection of HIV antibodies (16). However, all but one infection taking 266

place during this study were detected using the sequential R/S HIV algorithm. 267

The single missed infection was detected at the patient’s final visit using a 268


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PCR RNA test and was most likely in the window period. This recent infection 269

would thus probably not have been detected using a third generation 270

immunoassay either. 271

272

The advantage of most R/S assays is that they can be performed on finger-273

prick blood, in contrast to multiwell immunoassays, for which venous blood 274

has to be collected. We chose to collect 250 - 500 µl of whole blood from 275

finger pricks in an EDTA microtainer because the procedure is less invasive 276

and often more acceptable to participants and provides sufficient volume to 277

perform the 3 R/S assays included in the testing algorithm. 278

279

Testing of a second specimen following an initial positive HIV algorithm result 280

was done to exclude transcription errors and to control for possible specimen 281

mix-ups (20). This procedure is routine practice in Belgium but not in other 282

countries. The first specimen tested was blood obtained by finger prick, which 283

in some instances may influence the reliability of the visual reading of the R/S 284

test (12). Among the four participants with HIV false positive results, one 285

participant had substances in her blood which cross-reacted at all testing 286

occasions with the SD Bioline HIV-1/2 3.0 assay. For this participant only, the 287

sequence of the rapid assays was changed to the Determine HIV-1/2 test first 288

and in the event of a reactive result, the Unigold HIV test was performed 289

instead of the SD Bioline HIV-1/2. 290

291


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We found 124/5734 (2.2%) specimens that were only reactive in the 292

Determine HIV-1/2 test, with a much higher rate (7.2%) in Uganda than in any 293

other site. The lack of specificity of the Determine HIV-1/2 assay with 294

specimens from Uganda has been reported previously (6, 12, 22). We 295

hypothesize that in Kampala, Uganda, in particular, participants had 296

undiagnosed endemic non-HIV infections or circulating substances in their 297

blood that may have cross-reacted with the Determine HIV-1/2 test (12). 298

Cross-reactivity of HIV assays is not uncommon; a recent publication reported 299

cross-reactivity of the Murex HIV Ag/Ab combination EIA with S. haematobium 300

IgG and a study from our group showed cross-reactivity of the Determine HIV-301

1/2 test with Trypanosoma brucei gambiense (8, 17). The 124 results came 302

from 98 women, and only two were ultimately diagnosed with HIV during the 303

trial. This counters the impression expressed by laboratory staff in the field 304

that the Determine HIV-1/2 test is more sensitive at detecting recent 305

infections. 306

It should be noted that the Determine HIV 1/2 rapid test was initially 307

developed by Abbott; however, in May 2005 Abbott entered an agreement to 308

sell the rapid test to Inverness Medical Innovation. Currently, the rapid test is 309

manufactured by Alere Medical in Japan. Per the new manufacturer, the 310

performance characteristics of the assay has not changed with the change in 311

manufacturer. 312

However, we note that some of the false reactive results obtained with the 313

Determine HIV-1/2 assay could be attributed to the deterioration of the test 314

strips that was observed at the start of the study and the over-interpretation of 315


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reddish ‘shadows’ or ‘ghost lines’ especially when finger-prick blood was 316

tested (15). Re-training on the storage and handling of the strips and test 317

procedure, including the interpretation of ‘reactivity,’ was provided (5). It 318

should be noted that according to the guidelines of the World Health 319

Organization, the discordance of test results between first and second or 320

tiebreaker tests should not exceed 5% (27). Although the number of false 321

reactive Determine HIV-1/2 assays obtained in Kampala decreased following 322

training and thorough supervision, it remained approximately 5%. This 323

observation reinforces our hypothesis that interfering blood substances played 324

a role in the observed false reactive Determine HIV-1/2 results. In addition, 325

this finding emphasizes the need for and importance of assay and algorithm 326

validation in the populations and geographical areas in which assays will be 327

used (7, 12). 328

Although rapid assays are simple, training in the test procedure and reading of 329

the test devices is still necessary (5, 12). Likewise, quality control and 330

assurance procedures should be put into place for any clinical trial. First, the 331

quality of the test devices should be assessed when received by the 332

laboratory. Once the test devices pass batch-entry control, internal controls 333

should be run at regular intervals. The staff administering the tests should also 334

be assessed at a pre-defined frequency by testing quality control panels (20). 335

Although the assays in this study do not require refrigeration, they cannot be 336

stored at temperatures higher than 27°C or 30°C, and the tests should not be 337

performed in conditions exceeding those temperatures. In addition, the test 338

devices are susceptible to humidity and need to be stored in sealed pouches 339

containing a desiccant. 340


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341

With the use of a qualitative HIV RNA PCR assay, we were able to more 342

accurately determine the time of infection and exclude three participants from 343

the analysis who were found to be HIV infected but still in their window period 344

at enrollment. We were also able to detect one early/recent infection in a 345

participant who did not develop HIV antibodies at her last study visit and who 346

was likely in her window period. Not only could this endpoint be included in 347

the data analysis and improve the precision of the estimated treatment effect, 348

the use of PCR on the final visit sample allowed the participant to be referred 349

for further HIV infection management. Excluding this recent infection detected 350

at a final visit, no additional infections other than those determined by the HIV 351

testing algorithm were detected using HIV RNA PCR. This observation 352

confirms the sensitivity of the HIV testing algorithm and its suitability in clinical 353

trials. 354

355

In conclusion, our results demonstrate that HIV algorithms using only rapid 356

and simple HIV assays with results confirmed on a second specimen provide 357

reliable HIV results and can easily be implemented in HIV prevention trials 358

conducted in resource-limited areas. If more accurate time point 359

determinations of HIV infection are required for outcome analysis, we 360

recommend using HIV RNA PCR in a look-back procedure on stored 361

specimens. 362

363


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Acknowledgements 364

The authors thank all study participants and study staff, especially the 365

laboratory staff, at the study sites for their dedication. 366

We thank Marjan Mangelschots, Mirriam Van Rooy, and Sergio Garcia for the 367

HIV testing and Wendy Thys for her data entry work. 368

This study was supported by grants from the United States Agency for 369

International Development and the Bill & Melinda Gates Foundation. 370

No potential conflict of interest relevant to this article was reported. 371

372

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virus rapid serum screening in a predominantly Hispanic prenatal 484

population. J. Parasitology 24:743-747. 485

486


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Figures 487

488

Figure 1: HIV testing algorithm used in the trial during follow-up visits and at 489

final visits. 490

491

Figure 2: HIV testing algorithm used for re-testing and final confirmation of 492

HIV seroconversion at the Institute of Tropical Medicine, Belgium. 493

494

Legend to Figure 2 495

EIA 1: Enzygnost® Anti-HIV 1/2 Plus, Dade Behring GmbH, Duitsland; EIA 2: 496

Vironostika® HIV Uni-Form II plus O, Organon Teknika, France; LIA: INNO-497

LIATM HIV I/II Score, Innogenetics, Belgium; Ag: INNOTEST® HIV Antigen 498

mAb, Innogenetics, Belgium; IND: indeterminate ; +:positive; -: negative, 499

500

Figure 3: HIV test results on the first specimens collected 501

502


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TABLE 1 HIV prevalence rates in women who presented for screening at the 503

5 study sites 504

Site

Number of women

tested HIV positive (%)

Benin, Cotonou 464 129 (27.8)

South Africa, Durban 1432 723 (50.5)

Uganda, Kampala 571 185 (32.4)

India, Bangalore 72 36 (50.0)

India, Chennai 377 60 (15.9)

Pooled 2916 1133 (38.8)

505

506

507

508

509

510


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TABLE 2 Positive HIV rapid test results of first specimens collected at follow-511

up 512

Site

Number of first

specimens tested HIV Positive (%)






Pooled 5734 46 (0.8) a,b

513

a All but one of the 46 specimens were Determine reactive/SD Bioline reactive 514

(HIV-1); one specimen was Determine reactive/Unigold reactive 515

b 42 of the 46 were confirmed positive at the ITM 516

517

518


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TABLE 3 Specimens reactive only in the Determine HIV-1/2 assay 519

Site

Number of first

collected specimens Reactive (%)






Pooled 5734 124 (2.2) a

520

a contributed by 98 women 521

522

523


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TABLE 4 Women with reactive results only in the Determine HIV-1/2 assay 524

Women (% enrolled) with

reactive results only in

the Determine assay

Number of study visits

with reactive results

Site 1 2 3 4

Benin, Cotonou 5 (2.2) 5 0 0 0

South Africa, Durban 8 (1.5) 7 1 0 0

Uganda, Kampala 84 (27.7) 64 17 2 1

India, Bangalore 0 (0.0) 0 0 0 0

India, Chennai 1 (0.4) 0 1 0 0

Pooled 98 a (7.0) 76 19 2 1

525

a Five were not tested again; 2 tested positive for HIV according to the R/S 526

algorithm at their next study visit; and 91 had subsequent test visits but were 527

never diagnosed with HIV 528

529

530

531


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1 - clinical and vaccine immunology · 2011-07-13 · 115 69,107 and registered at clintrial.gov...

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