The document discusses testing for acute HIV infection in North Carolina. It details a pilot study from 2001-2002 where over 8,500 routine HIV tests were pooled and screened for HIV RNA to identify acute infections. This identified 5 cases of acute infection per 10,000 tests, compared to 44 chronic infections. Since 2002, North Carolina has screened over 240,000 HIV samples per year through its STAT program to identify acute infections using nucleic acid amplification tests on pooled samples.
13. The STAT System State Laboratory Laboratory Identification Disease Intervention Specialist Team Notification, Interviews, Confirmatory Testing, Transportation to Clinic UNC Weekly Case-Conference (Surveillance, Lab, DIS, UNC Evaluation Teams) Data collection UNC/Duke Collaborative Free Urgent clinical evaluation Recruitment to studies UNC Acute HIV Program Research Database UNC Specimen Repository -surveillance/research testing
14. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
15. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
16. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
17. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
18. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
19. ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Total 891,210 -- 3,766 0.42% 125 0.014% Sex Male 279,914 31% 2,631 70% 99 79% Female 600,399 67% 1,076 29% 26 21% Median Age (Range) 25 (0-99) 33 (0-84) 26 (16-56) Age ≤ 21 288,998 32% 524 14% 35 28% >21 597,395 67% 3,226 86% 90 72% Race Black 395,825 44% 2,578 68% 79 63% White, Non-Hispanic 284,931 32% 663 18% 34 27% Hispanic 162,901 18% 327 9% 10 8% American Indian 9,536 1% 24 1% 1 1% Missing 9,667 1% 34 1% 1 1% Sexual Risk Group Female 600,399 67% 1,076 29% 26 21% Heterosexual Male 231,575 26% 1,084 29% 22 18% MSM 26,870 3% 1,277 34% 73 58% Unknown Male 21,469 2% 270 7% 4 3%
20. North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 (cont.) No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Site Type CHC/PHC 17,189 2% 109 3% 3 2% Drug Treatment 9,944 1% 31 1% 0 0% Family Planning 156,981 18% 61 2% 0 0% Field Visit 12,819 1% 316 8% 8 6% HIV CTS 62,056 7% 872 23% 23 18% Hosp/PMD 138 0% 3 0% 0 0% Other 43,996 5% 380 10% 15 12% Outreach 24,043 3% 153 4% 1 1% Prenatal/OB 148,996 17% 96 3% 2 2% Prison/Jail 28,836 3% 231 6% 5 4% STD 348,896 39% 1,386 37% 54 43% TB 6,872 1% 14 0% 0 0% Missing 30,439 3% 114 3% 14 11% ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period
21. North Carolina Voluntary Testing and Counseling Testing Population 2003-2008 (cont.) No. HIV Tests‡ Chronic Acute N (%) N (%) N (%) Site Type CHC/PHC 17,189 2% 109 3% 3 2% Drug Treatment 9,944 1% 31 1% 0 0% Family Planning 156,981 18% 61 2% 0 0% Field Visit 12,819 1% 316 8% 8 6% HIV CTS 62,056 7% 872 23% 23 18% Hosp/PMD 138 0% 3 0% 0 0% Other 43,996 5% 380 10% 15 12% Outreach 24,043 3% 153 4% 1 1% Prenatal/OB 148,996 17% 96 3% 2 2% Prison/Jail 28,836 3% 231 6% 5 4% STD 348,896 39% 1,386 37% 54 43% TB 6,872 1% 14 0% 0 0% Missing 30,439 3% 114 3% 14 11% ‡ From NC CTS -- number of HIV Tests performed, previous positives removed. Note: Table excludes 2 Acute cases identified from 11/1/2002-12/31/2002 as the number of HIV tests and chronic cases were not available for this 2 month period
22. Initial HIV Testing Patterns of Acute Patients Stratified by EIA Generation Type 2nd Generation † (2003-2007) 3rd Generation ‡ (2008) N (%) N (%) EIA(-); NAAT(+) 80 85% 17 57% EIA(+); WB(Ind.) 12 13% 10 33% EIA(+); WB(-) 2 2% 3 10% † 2nd Generation EIA: Vironstika HIV-1 (bioMérieux) ‡3rd Generation EIA: HIV-1/HIV-2 Plus O (BioRad)
23. HIV Prevalence in Voluntary Testing and Counseling Testing Population Stratified by Calendar Year No. HIV Tests‡ Chronic Acute N N % N % Calendar Year 2003 106,994 581 0.54% 22 0.021% 2004 118,885 552 0.46% 21 0.018% 2005 130,440 590 0.45% 21 0.016% 2006 145,396 645 0.44% 15 0.010% 2007 175,341 670 0.38% 16 0.009% 2008 214,154 728 0.34% 30 0.014% ‡ From NC CTS -- number of HIV Tests performed, previous positives removed.
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Notes de l'éditeur
The timeline of HIV-1 infection as related to the development of symptoms and types of laboratory tests used to detect infection. As shown, symptoms of acute HIV, if present at all, usually don’t appear until about two weeks post infection. If the patient presents during this time, routine antibody tests may still fail to detect the infection due to insufficient antibody level. HIV RNA is the first to appear in the blood and can be detected by nucleic acid amplification tests as early as one week post infection, depending upon the patient’s viral load and the sensitivity of the HIV RNA assay used for detection. P24 antigen appears next around 2 weeks, and the fourth generation ELISA combination assays which detect both p24 antigen and HIV antibodies will be useful in detection of acute HIV infections. These combination assays have been successfully used outside of the US for the past several years and are expected to gain FDA approval later this year for use in the US. The advent of third generation ELISA assays, which detect IgM as well as IgG, have shortened the window for detection by EIA by several days, to under three weeks from time of infection.
In 2001, the NCSLPH participated in a joint pilot study with the University of North Carolina. In this study, all consecutive routine HIV tests that were submitted to the State Lab over a 4 week period from 110 publicly funded counseling and testing sites were tested, around 8500 samples. The initial antibody testing was performed using the Organon Teknika Vironostika HIV-1 Viral Lysate microelisa assay at the State Laboratory. This was followed by the manual pooling of all HIV antibody negative samples in 90:10:1 two-step pooling algorithm. The pools were then sent to UNC labs where they were tested by the Roche Amplicor HIV-1 Monitor assay, both standard and ultrasensitive. Reactive pools were deconstructed and individual positive samples were identified.
Of the 8505 total samples tested, 8005 were pooled for HIV NAAT testing (after subtracting out any confirmed positive HIV infections and those with insufficient volume for further testing). Five samples were identified as HIV RNA positive, of which 4 samples (or patients) were eventually confirmed as true acute infections by the demonstration of seroconversion in follow-up samples. The acute infection rate was calculated at 5 per 10,000, or .05%. Chronic infection rate was 44 cases per 10,000, or .44%. Overall specificity was 99.99% (1 false positive per 8005 samples tested). We estimated the additional cost of NAAT testing per specimen at $2.01 and the total testing costs per additional case diagnosed at around $4,100.
As a result of the 2001 pilot study, NC was able to implement the NC Screening and Tracing Active Transmission, or STAT program in 2002. The STAT program is a statewide collaboration to detect acute HIV infection in NC publically funded testing sites. In 2009, the NCSLPH tested approximately 240,000 HIV samples. Normal turn around time for the complete HIV test algorithm, including NAAT testing, is two to three weeks for negative results. The STAT model includes rapid notification of DIS for NAAT positive patients, and the immediate collection of follow-up samples for confirmation of HIV seroconversion. DIS involvement also includes rapid tracing and prospective screening of partners, based upon risk factors.
The overall goal of the STAT program, of course, is to identify acute HIV infections in the routine HIV counseling and testing site population in North Carolina. Health goals for the individual diagnosed with AHI include an improved prognosis with the implementation of early treatment, early entry into care and prevention services, and the opportunity to avoid unwitting transmission to partners. It has been documented that patients who are aware of their HIV infection status are more likely to take necessary steps to prevent transmission. The STAT program also addresses goals that affect overall public health, such as recognizing previously missed infections. The ability to access sexual networks allows us to identify groups that are being actively infected within certain communities and to identify any partners at high proximate risk of infection. These identified infected individuals and groups are actively transmitting the disease in the community, and interventions can be maximized to reduce transmission.
NC DHHS introduced HIV-1 RNA pooling into the routine HIV testing algorithm in November 2002. EIA testing assays and pooling schema have changed during this seven year period, the most dramatic being a change from the 2 nd generation BioMerieux Vironostika HIV-1 EIA to the to 3 rd generation BioRad HIV-1, HIV-2 Plus O EIA in 2008. We have also used different pooling instrumentation and pooling schema over the years; pool size has ranged from 80-96 samples/pool, always in a two-stage pooling design. NAAT assays used include the Nuclisens HIV-1 qualitative assay by BioMerieux for the first year of testing and the GenProbe Procleix HIV-1 assay the second year. When the Procleix assay was withdrawn from the market in 2005, we switched to the BioMerieux Nuclisens Easy Q HIV-1 assay. Again switching back to the GenProbe qualitative HIV-1 RNA assay when it received FDA approval in 2007.
We received nearly 240,000 samples in 2009 for HIV testing, and we are now operating at peak capacity due to laboratory space and staffing limitations. All clients presenting at approximately 135 publicly-funded voluntary testing and counseling sites are tested for HIV RNA using specimen pooling. This excludes those patients who are tested only by a rapid HIV test and result as negative. Serum samples received at the State Lab are screened for antibodies to HIV-1, HIV-2 Plus O using the BioRad assay with EVOLIS automation. We currently have three EVOLIS which are dedicated to HIV testing, running all day five days a week. All serum samples that test negative for HIV antibodies are then pooled using the Hamilton STARlet robotic instrument. Approximately 10-12 pools are prepared each day containing 80 samples each.
This diagram illustrates the pooling schema used at the NC State Lab. Following HIV antibody testing on the EVOLIS instruments, four strip racks holding 20 samples each are placed onto the deck of the STARlet. The instrument pipets 20 ul each of 8 samples into a corresponding well of a deep–well plate. Pipetting continues until each of 10 wells of the plate contain an A pool with 8 samples each. Following a mixing step, the instrument then pipets 120 ul of each A pool into one tube, resulting in a B pool which is subsequently tested for HIV NAAT. It requires about 10 minutes for the robotic construction of each pool. A second pooling program is used for deconstruction of a positive pool, in which the resulting A pools have a sufficient volume for NAAT testing. The advantage of using robotics for pooling is less chance of contamination of samples, greater efficiency, and reduced human error due to fatigue.
We have been using the GenProbe APTIMA HIV-1 NAAT RNA assay since November of 2007. APTIMA was the first FDA cleared HIV-1 RNA qualitative NAAT for plasma, and was subsequently also cleared for the testing of serum. It will detect RNA from the majority of HIV-1 subtypes (Groups M, N, and O). The assay has an excellent analytical sensitivity, manufacturer stated and verified by our laboratory, of 30 copies/ml at 98.5%. It is very important to use a highly sensitive assay when using it for the detection of virus in pooled samples because of the dilution factor. The assay has a clinical specificity claim of 99.83%, which is exceptionally good for any laboratory test. However, it is also important to remember that this translates to a 0.17% false positivity rate, or 17 false positive results per 10,000 tests, and this can be a concern especially in low prevalence testing populations.
The HIV Testing Algorithm used at the NC State Lab is as follows: All samples are initially screened by EIA. Antibody negative samples are pooled in an 80:10:1 schema. All 80 samples in a pool with a NAAT negative result are resulted as “RNA not detected” and negative for HIV. A NAAT positive pool is deconstructed and the NAAT positive individual sample (or samples) is resulted as “RNA detected” and is considered to represent an unconfirmed acute infection. Samples that are repeatedly reactive by EIA but fail to confirm by Western Blot (negative or Indeterminate) are further tested by an individual RNA test, and if NAAT positive are considered to represent unconfirmed acute infection. If these samples are NAAT negative, repeat HIV testing is recommended to rule out a false positive EIA. We also test these samples with the BioRad Multispot rapid discriminatory assay to rule out a possible HIV-2 infection. EIA repeatedly reactive samples that are Western Blot reactive are, of course, considered to be established HIV infections.
As soon as the laboratory identifies a patient sample as a possible acute case, a call is immediately placed to the Disease Intervention Specialist Team, who notifies the patient, conducts interviews, draws blood for follow-up confirmatory testing, and arranges for transportation to the clinic. Weekly conference calls involve all interested parties (surveillance, lab, DIS, and UNC evaluation teams). Data is collected on individual cases. Aliquots of the samples from newly identified acute cases are sent to UNC for further research and surveillance testing. The UNC/Duke collaborative offers free urgent clinical evaluation to get the identified cases into treatment as soon as possible, and also offers patients opportunities for study participation.
Some results of the STAT program over a six year period, from 2003-2008. Figures from 2009 are missing, because we’re still in the process of compiling those figures: We identified 125 acute HIV infections and 3,766 chronic HIV infections in six years of testing. NAAT testing increased case identification by 3.2% over standard antibody testing alone.
Although more females presented for testing at VCT sites (67% of tests vs. 31% for men), men accounted for the largest percentage of both acute and chronic HIV infections. Among the 26 female AHI cases, median age was 30 years, with a range of 16-48, 73% were African American, and 3 were pregnant. Risk factors for women with AHI: 37% were incarcerated or had a partner who was incarcerated, 33% of the female cases were crack users, and 27% were commercial sex workers. Sex partners of 6 women were previously diagnosed with HIV. 50% of women were more likely to have an STI within 8 weeks of AHI diagnosis, compared to 23% of men.
35 of the acute cases were adolescents of whom about half were identified from 2007-2008. Adolescent acutes were predominately MSM of color (74%), compared to only 23% of adult acutes.
Racial disparities clearly exist for both chronic and acutes cases. It is interesting to note that there is a slight decrease in acute cases vs. chronic cases in Blacks, but a significantly higher percentage of acute cases than chronic cases in White, Non- Hispanic populations.
Chronic and acutes cases were approximately the same for females compared to heterosexual males.
But clearly the highest number of acute cases were seen in the MSM population. In general, acutes were younger, more likely to be men, and more likely to be MSM compared to chronic HIV cases.
When we look at where the majority of acute cases are being seen, most are from patients screened at HIV CTS sites and STD clinics.
Very few acute cases have been detected in patients from Family Planning, Prenatal, or OB clinics. This stratification of cases by site type makes a strong argument for targeted screening strategies that are population specific, such as larger pool sizes for low incidence groups in a effort to reduce testing costs.
This slide illustrates the improved ability to detect acute HIV infection at the initial antibody screening when using a more sensitive 3 rd generation EIA. With the 2 nd generation EIA assay, 85% of our acute cases were detected by NAAT only following a negative antibody result. After switching to the 3 rd generation assay in 2008, 43% of our acute cases were EIA repeatedly reactive, but Western Blot negative or indeterminate. Confirmation of acute AHI status with a positive NAAT increased the number of AHI cases with EIA Reactive results at diagnosis.
HIV prevalence in the VCT population declined from 2003 to 2008 in both acute and chronic groups. This decline may be due to increased testing in low-risk populations. For example, family planning and prenatal/OB sites accounted for 33% of all tests in 2003, rising to 38% in 2008. The increase in acute cases in 2008 compared to previous years may be due to various reasons, including the change to the more sensitive APTIMA assay for NAAT testing. Preliminary figures for 2009 and 2010 acute cases are following this pattern.
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