Early Infant Human Immunodeficiency Virus Type 1 Detection Suitable for Resource-Limited Settings with Multiple Circulating Subtypes by Use of Nested Three-Monoplex DNA PCR and Dried Blood Spots

Sample collection.Serial DBS samples (n = 347) were collected from 151 infants born to HIV-1-infected mothers in four geographically diverse regions of China through the HIV-1-infected mothers' vertical transmission to children prospective cohort study, which was conducted by the China Center for Disease Control and Prevention (China CDC) from January 2005 to December 2006. EDTA-anticoagulated blood specimens were collected, from which 70 to 80 μl of whole blood was applied to filter paper (S&S 903; Whatman, New York, NY) and dried. DBS samples were mailed to the National AIDS Reference Laboratory of the China CDC using the national postal service.

DBS DNA extraction.A 6-mm-sized DBS specimen was detached by an ethanol-flamed punch and transferred into a 1.8-ml screw-cap tube. The DBS specimen was washed twice using deionized water and then was suspended with 10% Chelex 100 resin (lot no. 95577; Sigma). After a 2-h incubation at 56°C, the mixture was boiled for 8 min and centrifuged at 12,500 × g for 3 min. The supernatant was collected and stored at −20°C for PCR amplification.

Primer design and selection.Six pairs of primers were designed on the basis of the conserved regions in HIV-1 env, gag, and pol genes (34, 35, 37). Consensus sequences from the three genomic regions were found to be sufficiently complementary to cover the major HIV-1 subtypes circulating in the world, including those in China (A, B, Thai B [B′], C, D, F, G, CRF01-AE, CRF07-BC, and CRF08-BC). Each pair of primers was validated separately by a panel of plasmids containing cloned HIV-1 env and gag fragments of representative subtypes and CRFs. The plasmids included A1 (PVJ310), AE (PTN238), B2 (PUG280), B1 (PTB132), C (PUG268), D (PK31), F (PBZ162), G (PLBU21-7), H (PUI525), and J (PE1973) and were provided by the NIH AIDS Research and Reference Reagent Program; plasmid stock for CRF07-BC also was used, and it was provided by the National AIDS Reference Laboratory of the China CDC. Primers of the pol fragment were described previously (34). The concentration of the plasmid PBH10 solution containing nearly full-length HIV-1 genomes was quantified by UV-visible spectroscopy. The PBH10 solution was diluted 10-fold to cover a test range of 10¹ to 10⁹ copies per reaction for determining the relevant analytic sensitivities of the selected primers.

N3M-PCR.The N3M-PCR was performed on a GeneAmp PCR system 9600 (Perkin-Elmer, Waltham, MA). The outer PCR mixture consisted of 5 μl of 10× PCR buffer; 3 μl of 25 mM MgCl₂; 1 μl of 10 mM deoxynucleoside triphosphates; 0.5 μl of 5 U/μl Taq polymerase enzyme (Takara Taq; DaLian, People's Republic of China); 1 μl of each (20 mM) outer primer for the gag, env, and internal control β-actin genes; and 28.5 μl of sterile deionized water. Five microliters of outer PCR product was added to 45 μl of the inner PCR mixture, which consisted of 5 μl of 10× PCR buffer, 3 μl of 25 mM MgCl₂, 1 μl of 10 mM deoxynucleoside triphosphates, 0.5 μl of 5 U/μl Taq polymerase enzyme (Takara Taq), 1 μl of each 20 mM outer primer for the gag and env gene, and 33.5 μl of sterile deionized water. The outer and inner PCR master mix of the pol region was almost the same as that for the env and gag regions, except that 2 μl of 25 mM MgCl₂ and 28.5 and 33.5 μl of sterile deionized water were added separately. N3M-PCR products and DNA molecular weight markers were visualized after electrophoresis on an ethidium bromide-stained 1% agarose gel using a UV transilluminator at 300 nm. To test for each extract as an internal control, the human β-actin gene was amplified in a single PCR assay with predesigned primers, β-actin-1 and β-actin-2, under the same conditions as those described above for the outer pol fragments (Table 1). As an external control, N3M-PCRs were performed on DBS-positive and -negative control samples provided by the U.S. Centers for Disease Control and Prevention Proficiency Testing Program for DBS-Based Infant Diagnostics. DBS-positive controls were prepared using an 8E5 cell line (which contains a single integrated copy of proviral DNA) diluted in HIV-1-negative human blood samples to reach positive concentrations. DBS-negative controls were prepared using HIV-1-seronegative blood samples.

Determination of infant HIV-1 infection by N3M-PCR.DBS samples collected from the infants were tested by the N3M-PCR assay. If a β-actin fragment was not present, the PCR result for the sample was scored as indeterminate for HIV-1 and the DNA extraction was repeated. If the β-actin gene was present, the results of PCR were interpreted as follows: (i) if more than two regions of the pol, gag, and env genes were positive (i.e., amplified), the samples were identified as HIV-1 positive; (ii) if only one region was positive, the results were reported as indeterminate for HIV-1; (iii) if none of the three viral regions was amplified, the samples were considered HIV-1 negative. All tests detecting HIV-1 DNA-indeterminate and -positive samples were repeated once by N3M-PCR assay. If there was a discordant result between the original and repeated samples from the same age, the infant had another DBS sample taken for definitive confirmation. Dual concordant results by PCR were classified as HIV infection.

Validation standard.Infants were determined to be definitively HIV-1 infected when (i) both HIV antibody enzyme immunoassay (Organon Teknika, Boxtel, The Netherlands) and Western blotting (Genelab, Singapore) confirmatory tests were positive for the follow-up infant samples at ≥18 months of age or (ii) if they met rigorous clinical and epidemiologic criteria for pediatric AIDS in the case of infants who died prior to the 18-month PCR assessment. All infants who were breastfed after birth (n = 2) were excluded from our study; China encourages replacement feeding for all infants of HIV-infected women, since formula is supplied and access to clean water is facilitated by the provincial health authorities. For the infants who were retested at ≥18 months of age, any infant with two negative results by enzyme immunoassay was determined to be HIV negative.

N3M-PCR assay reproducibility.DBS samples (n = 32) were randomly selected to be tested in the three genomic regions, and the tests were repeated three times to assess the N3M-PCR assay's reproducibility.

Statistical analyses.All statistical analyses used SPSS/PC+ software, version 13.0 (SPSS Inc., Chicago, IL), and Microsoft Excel (Microsoft Corporation, Redmond, WA). The sensitivity and specificity were calculated from appropriate two-by-two tables.

Ethical considerations.This study was approved by the institutional review boards of the CDC of China and Vanderbilt University. Informed consent was received from all parents of the infants before the samples were collected.

Analytic sensitivity of the N3M-PCR assay.The analytic detection sensitivity of the N3M-PCR assay for each pair of primers based on HIV-1 env, gag, and pol regions reached 10 copies of HIV-1 DNA per reaction. Three sets (Table 1) were able to amplify the targeted regions of HIV-1 subtypes A, B, C, D, F, G, CRF01-AE, CRF08-BC, and CRF07-BC. A panel of plasmids containing cloned full-length HIV-1 or env and gag fragments was amplified by env and gag primers (Fig. 1).

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FIG. 1.

Each pair of primers of the env (left) and gag (right) fragments was validated separately by a panel of plasmids containing cloned HIV-1 env and gag fragments from clinical adult samples representing subtypes A, B(B1), C, D, F, G, H, J, CRF01-AE, and CRF07-BC. The duplex bands in lanes G, F, D, and B1 of the ENV panel were caused by plasmids that were not digested using restriction enzymes before the PCR was performed. The first lane in the left panel and last lane in the right panel are the molecular size markers.

HIV-1 infection status among infants determined by the validation standard.We monitored 106 infants from birth to the age of definitive diagnostic assessment (≥18 months of age), and 4 infants died due to clinically and epidemiologically confirmed AIDS. Sixteen infants were diagnosed with HIV-1 infection by our validation standards, 94 infants seroreverted and were considered HIV-1 negative, and 41 infants who were seen from birth but were <6 months of age at the end of the study were classified as indeterminate for HIV-1 infection. Two mother-infant pairs did not receive any treatment due to their having delivered at home; among the other 14 mother-infant pairs, six mothers did not receive a single dose of nevirapine (or any other antiretroviral drug), while eight mothers and all 14 infants received one dose of nevirapine (L. Wang, unpublished data).

Sensitivity of the N3M-PCR assay.To determine the sensitivity of the N3M-PCR assay, the combined results from the three regions were compared to the results of the validation standard. Of the 16 infants, 10 were sampled at 48 h after birth, 12 at 1 to 2 months of age, and all 16 at 3 to 6 months of age. Among the HIV-infected infants sampled at 48 h after birth, 30.0% (3/10) of the HIV-positive samples were detected by N3M-PCR. For HIV-infected infants sampled at 1 to 2 months of age, 91.7% (11/12) of the samples were positive by N3M-PCR. For HIV-infected infants at 3 to 6 months of age, 93.7% (15/16) of the samples were positive by N3M-PCR (Table 2). The sensitivity of the PCR based on a single region was either lower than or equal to that of the combined results of the three viral regions.

Specificity of the N3M-PCR assay.All 94 HIV-1-negative infants were tested at the same three time points by PCR, and all were confirmed to be HIV-1 negative by the validation standard. When all three regions were used, the specificity of the PCR was 100% (94/94) for the infants at 48 h after birth, at 1 to 2 months, and at 3 to 6 months. This also was true when only one primer set was tested, either env or pol, except that at 1 to 2 months the test with the env primer set gave one false-positive result (98.9%), and at 3 to 6 months the pol primer set gave one false-positive result; both of these results were very weak positives (Table 2).

Reproducibility.The reproducibility of the N3M-PCR assay was 100% at 48 h after birth, 96.7% at 1 to 2 months, and 100% at 3 to 6 months for detecting HIV-1 infection based on the three regions combined (Table 3).