Ngo-Giang-Huong N, Khamduang W, Leurent B, et al. Early HIV-1 diagnosis using in-house real-time PCR amplification on dried blood spots for infants in remote and resource-limited settings. JAIDS 2008 Dec;49(5):465-71.
To assess the performance of a low-cost, in-house, real-time polymerase chain reaction (PCR) assay to detect HIV-1 DNA in infant dried blood spots (DBSs).
Cross-sectional comparison of the in-house, real-time PCR to the Roche Amplicor HIV-1 DNA test as the gold standard
A total of 1,319 DBSs were collected from 932 infants for the primary comparison, and serum specimens were collected from 1240 infants to compare the performance of the two assays using serum. The probe for in-house, real-time PCR was designed to be able to detect the two main subtypes circulating in Thailand: CRF01_AE and B. The DBSs for the Roche Amplicor test were shipped at room temperature to the New England Newborn Screening Program (Boston, MA) laboratory for HIV-1 DNA PCR testing. Diagnosis HIV serology was performed on-site using either enzyme-linked immunosorbent assay (ELISA) or gelatin particle agglutination (GPA) test kits. Children with a negative serology result at any age were considered HIV uninfected. All tests were performed independently from each other and blinded to clinical data.
For validation of the in-house real-time PCR: Investigators enrolled 287 infants born in the context of the Perinatal HIV Prevention Trial study (PHPT-2) between July 2002 and November 2002, and 645 infants enrolled between September 2004 and September 2006 in the pilot phase of a program to provide free access to early HIV-1 diagnosis in 31 public hospitals throughout Thailand. At the time of testing, the median age was 4.1 months (10th-90th percentiles: 1.7-7.2 months). The 4-month samples were chosen because the Roche test can detect virtually all perinatal HIV-1 infections by this age in other epidemiological contexts and on liquid blood.
For validation of the Roche Test: Investigators also enrolled 1,240 of 1,409 non-breast-fed infants born to HIV-1-infected mothers between January 1998 and July 2000 in the context of another perinatal HIV prevention trial study (PHPT-1). Only these 1,240 had a blood sample available at the 4-month and 18-month visits. For this analysis, the samples from the 4-month visit were tested by Roche Amplicor PCR using the HIV serology results at the 18-month visit as reference.
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV)
Comparison between the in-house real-time PCR and Amplicor: The detection of HIV-1 by the two techniques was concordant in all 1,319 DBSs analyzed. In the subset restricted to the first sample drawn in each of the 932 infants (median age: 3.5 months, 10th-90th percentiles: 1.5-6.7 months), the concordance rate was 100% (95% confidence interval [CI]: 94.6%-100%) for the 66 positive samples and 100% (95% CI: 99.6%-100%) for the 866 negative samples. The earliest positive tests were obtained at birth.
Comparison between Amplicor on DBS and HIV serology: For the specificity and sensitivity analyses, three cases were excluded. The sensitivity of Amplicor was 98.6% (95% CI: 92.6%-100.0%), and specificity was 99.7% (95% CI: 99.2%-99.9%). Using the reported rate of HIV-1 transmission in Thailand (3.9%), the estimated PPV would be 94.0% and the NPV 99.9%. There were four discordant results. Three children tested positive by Roche DNA PCR at 4 months of age but had a negative HIV serology result at 18 months, and 1 child tested negative by DNA PCR at 4 months of age with a positive HIV serology result at 18 months. Three discordant results were suspected to be due to human error. In the fourth case, three sequential HIV-1 DNA PCR-positive results were followed by two negative DNA PCR results and a negative serology result. Excluding the three discordant cases believed to be due to human error, the sensitivity and specificity were 100% and 99.91%, respectively, with a PPV of 97.9% and NPV of 100%.
Cost: The authors calculated that the cost per reportable test was USD$20 for the in-house assay and USD$42 for Amplicor.
In-house real-time PCR performed as well as Amplicor in detecting HIV-1 DNA on DBSs in Thailand. Combined use of DBSs and real-time PCR assays is a reliable and affordable tool to expand access to early HIV-1 diagnosis in remote and resource-limited settings, enabling timely treatment for HIV-1-infected infants.
There are no quality rating scales for studies of this type. Three of the four discordant results of the Roche test compared to HIV serology were deemed due to human error, emphasizing the importance of blood sample identification and labeling to ensure reliable diagnosis and the need for confirmation testing.
This is the first report describing the use of an in-house real-time HIV-1 DNA PCR test on DBSs for the early diagnosis of HIV-1 infection in a very large number of non-breastfed infants born to HIV-1-infected mothers. The results using this test were identical to those obtained using Amplicor on DBSs. Furthermore, although the Roche DNA test has been widely validated and is the reference test for HIV-1 diagnosis in infants born to HIV-1-infected mothers, it had not previously been validated with the main subtype in Thailand, CRF01_AE. These results are consistent with those reported by a study from infants in Thailand using a larger quantity of blood(1) and by a study of HIV-1 DNA PCR on DBS from infants in South Africa.(2)
WHO recommends that all HIV-infected infants initiate therapy in the first year of life due to high risk of mortality. Assays based on real-time PCR display very high sensitivity and specificity, provide results more rapidly than classical PCR, and avoid crossover PCR contamination, and thus do not require separated amplification and detection rooms. Moreover, equipment costs have dramatically reduced with increasing competition and improvement in detection system: standard real-time machines, priced USD$90,000 in 1995, are now priced from USD$20,000. The use of low-cost, in-house, real-time HIV-1 DNA PCR tests that can be used on DBSs will facilitate access to early diagnosis of HIV-1 infection for all infants born to HIV-1-infected mothers, including those in rural and remote settings.
- Young NL, Shaffer N, Chaowanachan T, et al. Early diagnosis of HIV-1-infected infants in Thailand using RNA and DNA PCR assays sensitive to non-B subtypes. J Acquir Immune Defic Syndr 2000;24:401-7.
- Sherman GG, Cooper PA, Coovadia AH, et al. Polymerase chain reaction for diagnosis of human immunodeficiency virus infection in infancy in low resource settings. Pediatr Infect Dis J 2005;24:993-7.