Mehta N, Trzmielina S, Nonyane BA, et al. Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR. PLoS ONE. 2009 Jun 5;4(6):e5819.
Early determination of mother-to-child transmission of HIV requires polymerase chain reaction (PCR)-based nucleic acid amplification and quantification of HIV and for evaluating antiretroviral therapy (ART) efficacy.(1,2,3,4) In resource-constrained areas such tests are not available routinely and not practical given the relatively large volume of blood required for the tests. Currently, infant diagnosis of HIV is not confirmed until antibody testing at age 18 months, the date by which maternal antibodies are no longer circulating in the infants. Lack of early diagnosis can affect decisions regarding infant feeding choices and prevents early ART for infected infants.
To determine the accuracy, reliability, genotype inclusivity and cost of real-time (RT) PCR
The University of Massachusetts Mother-Child HIV program and HEAL Africa, Goma, Democratic Republic of Congo
Prospective comparison of laboratory assays
Participants were HIV-1-positive children presenting for care at the University of Massachusetts Mother-to-Child HIV program (n=33) between May 2005 and September 2008; four HIV-infected adults enrolled in a study of viral kinetics between 1999 and 2000; and 19 infants born to HIV-infected mothers at the HEAL Africa Hospital between February 2008 and January 2009.
Accuracy, reliability, genotype inclusivity, and cost of a real-time PCR assay
At the University, participants gave whole blood, of which 50 µL was placed onto filter paper and left to dry for four hours. Plasma was used in lieu of whole blood from six participants for whom whole blood was not available. In Congo, dried blood spots (DBSs) were collected by heel stick from infants and then stored and shipped to the laboratory. Additional DBSs were made from HIV clades A, C, D, AE, and AG. Genotype clade determination was done using RT-PCR followed by nested amplification of env and/or gag genes. Reference viruses (13 CCR5-tropic and one CXCR4-tropic) were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH which represented clades A, B, C, D, and circulating recombinant forms CRF01_AE and CRF02_AG. The effect of storage time and temperature on DBS was measured using samples from 12 patients. DBS were stored at 1 20° C and 37 C° and extracted on days 1 and 7. Customized DBS standards with known viral loads were prepared to ensure uniformity and reproducibility. RNA and DNA were extracted from DBSs using standard techniques. In addition, RT- PCR amplification of HIV-1 RNA was performed in a one-step, single-tube, closed system of 32 sample format using the LC-32 Roche LightCycler, All the samples were tested in duplicate in a 20-µL total reaction volume containing 16 mL of PCR reaction mix; 0.5 µM each of forward and reverse oligonucleotide primer pairs, and 4 mL of the template. HIV-1 specific amplicons were detected using SYBR Green technology according to the manufacturer's instructions. Linearity of the assay was evaluated using serial dilutions of the customized standard DBS.
The linear dynamic range of the rtLC DBS assay was initially assessed using a 5 log10 dilution series of the customized standard DBS. The assay was shown to be linear over the entire range of 586,000 to 37 copies. A linear regression of the rtLC DBS customized standards copies on true concentrations yielded a correlation coefficient of 0.984 (P<0.001). The sensitivity and specificity were determined using 32 DBS specimens with known infection and viral loads that were above the threshold detection of the Roche bDNA assay. All specimens were positive (100% sensitive). Specificity was measured using HIV-negative specimens from infants and adults, and all were HIV-negative for 100% specificity. Specimens collected from Africa also were tested using RT assays. The range of cell equivalents across these Congo DBS samples was 2,769 to 201,116 cells/DBS when CCR5 copies were determined in the DNA extracts RNA for each sample was spiked into normal human plasma and run on a Roche Amplicor HIV-1 Monitor Ultrasensitive assay. DNA testing determined that 5 of 19 samples were positive for proviral DNA and these samples were positive using the rtLC DBS assay (range 11,084 to 1,123,400) with RNA copy numbers similar to the values obtained on the Roche Amplicor assay. The rtLC DBS assay successfully detected all clades tested. Plasma viral RNA copy numbers determined by the rtLC DBS assay were compared to results obtained using commercial assays (bDNA and Amplicor). Plasma viral RNA copy numbers determined by the rtLC DBS assay were compared to results obtained using commercial assays (bDNA and Amplicor).
The assay results were comparable and correlated well with commercially available viral load assays.
This was a high-quality laboratory comparison study. A gold standard was used for all comparisons. The populations were representative. One limitation, however, was the relatively small size of the sample.
The use of a single tube rapid test to detect HIV RNA from DBSs offers the opportunity for early diagnosis of HIV among infants infected perinatally. This has important implications because infants shown definitively to be HIV infected can be breastfed. In addition, such children may initiate treatment and be monitored, thereby reducing morbidity and mortality.
- Ammann A, Burrowes S. Early Diagnosis of HIV Infection in HIV exposed Infants: Can Complexity and Cost be Overcome in Resource-poor Settings? Women, Children, HIV Resources for Prevention and Treatment, 2007. Accessed 15 December 2008.
- Lambert JS, Harris DR, Stiehm ER, et al. Performance characteristics of HIV-1 culture and HIV-1 DNA and RNA amplification assays for early diagnosis of perinatal HIV-1 infection. J Acquir Immune Defic Syndr 2003;34:512-9.
- Fischer M, Huber W, Kallivroussis A, et al. Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues. J Clin Microbiol 1999;37:1260-4.
- Mofenson LM. Technical report: perinatal human immunodeficiency virus testing and prevention of transmission. Committee on Pediatric AIDS. Pediatrics 2000;106:E88.