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For this study, 571 blood samples were collected from injection drug users (IDU) and men who have sex with men (MSM) from Heilongjiang, Jiangxi, and Yunnan province between July 2018 and October 2019.
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Blood samples were collected by venipuncture into EDTA tubes. DBS specimens were prepared using 70 μL of EDTA whole blood per circle and then blotted onto filter paper (Whatman903, UK). The blood spot was allowed to air dry to be used as a DBS for further testing. After DBS preparation, whole blood samples were centrifuged to obtain plasma samples. Plasma and DBS samples were stored at −20 ℃ prior to processing.
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Overall, 571 paired plasma-DBS samples were collected then tested as per the study design (Figure 1).
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For DBS testing, the whole spot was cut out and placed into a 1.5 mL Eppendorf tube. The DBS was then eluted with 500 μL PBS and 0.1% Tween 20 for 4 h at room temperature (18–25 ℃) and DBS eluates was obtained. DBS and plasma samples were tested for anti-HIV, anti-HCV, and anti-TP by ELISA using the Diagnostic Kit for Antibody to Human Immunodeficiency Virus (ELISA) (WANTAI BioPharm, China), the Diagnostic Kit for Antibody to Hepatitis C Virus (ELISA) (WANTAI BioPharm, China), and the Diagnostic Kit for Antibody to Treponema Pallidum (ELISA) (WANTAI BioPharm, China), respectively. All antibody-positive samples in the screening test were further confirmed by the confirmatory recombinant immunobinding assay (RIBA) as follows: Detection Kit for Antibody to HIV (1+2) (RIBA) (WANTAI BioPharm, China); Detection Kit for Antibody to HCV (RIBA) (WANTAI BioPharm, China); and Treponema Pallidum EUROLINE-WB (IgM) (EUROIMMUN, Germany). The assays were performed and interpreted according to the manufacturer’s instructions. Plasma results were used as the reference standard to establish the serological status for HIV, HCV, and TP.
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Nucleic acid extraction: The matching plasma/DBS samples were subjected to nucleic acid extraction if the serological test in plasma showed an antibody positive for HIV, HCV, and TP. Total nucleic acids were extracted from plasma and DBS samples using the optimized QIAamp DNA Blood Mini Kit (Qiagen, Germany), which is an in-house method used for RNA isolation. The DBS samples were eluted using a protocol for nucleic acid extraction from DBSs optimized by our laboratory. Briefly, four disks were punched out with a 6 mm hole punch and nucleic acid was eluted in a 100 μL volume. For plasma, the nucleic acid from the plasma samples was extracted and then eluted to a volume of 120 μL. For the final elution volume, 20 μL was used for the HIV RNA test, 40 μL was used for the HCV RNA test, and 5 μL was used for the TP DNA test.
HIV RNA/DNA test: HIV-1 RNA viral load (VL) was measured in DBS and plasma samples by RT-PCR with the Diagnostic Kit for Quantification of Human Immunodeficiency Virus 1 Type (HIV-1) RNA (PCR-Fluorescence Probing) (DAAN GENE, China), according to the manufacturer’s directions. The results were considered positive if the PCR amplification curve was observed by RT-PCR.
HIV DNA levels were measured in DBSs tested by RT-PCR with Human Immunodeficiency Virus 1 Type (HIV-1) DNA Testing Kit (Kinghawk, China), following the manufacturer’s instructions. In this study, we compared the results with those obtained using a standard plasma HIV RNA assay.
HCV RNA test: HCV RNA VL was measured in DBS and plasma samples tested by RT-PCR using the Diagnostic Kit for Quantification of Hepatitis C Virus (HCV) RNA (PCR-Fluorescence Probing) (DAAN GENE, China), according to the manufacturer’s instructions. The results were considered positive if the PCR amplification curve was observed by RT-PCR.
TP DNA test: The PCR assay used for TP DNA was based on methods described by Heymans et al.[12], Wang et al.[13], and Orle et al.[14] to optimize the assay by adapting it to the available reagents and laboratory conditions in the present study. (Table 1). PCR amplification was performed in a thermal cycler (CFX 96Bio-rad, USA). The reaction mixture included 2 µL of the template DNA, 2 µL each of 10 µmol/L forward and reverse primers, 2 µL of the TaqMan Probe, 10 µL of the PCR Master Mix, and 2 µL of ddH2O. After a denaturation cycle of 5 min at 95 °C, the amplification profile consisted of 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C.
Table 1. TP DNA PCR primer and probe sequences
Target Sequence (bases) tpn47 Forward 5'-TGCGCGTGTGCGAATGGTGGTC Reverse 5'-CACAGTGCTCAAAAACGCCTGCACG TaqMan probe 5’-FAM-AGTAACACCACGATAATGA
CCTCC-MGB-3’polA Forward 5'-GGTAGAAGGGAGGGCTAGTA Reverse 5'-CTAAGATCTCTATTTTCTATAGGTATGG TaqMan probe 5'-FAM-ACACAGCACTCGTCTTCAACTCC-BHQ-3' -
The results from plasma samples were used as the standard for assessment of the performance of DBS testing, and the positive and negative coincidence rates were analyzed. log10 copies/mL transformations of VL values measured in DBS samples and in the paired plasma samples were compared. To evaluate the difference between DBSs and plasma, a Bland-Altman bias plot of the bias versus the average value of DBSs and plasma was generated and the correlation analysis was performed using Spearman’s or Pearson’s correlation coefficient.
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Samples from 38 anti-HIV-positive samples, 293 anti-HCV-positive samples, and 67 anti-TP-positive samples were obtained through serological testing. Among the antibody-positive samples, 32, 103, and 40 diagnostic samples were available for HIV, HCV, and TP nucleic acid tests, respectively.
Samples were divided according to HIV-1 RNA VL level and HCV RNA VL level in plasma, and detection rates were also analyzed according to different HIV-1 RNA VL and HCV RNA VL thresholds for DBSs, as shown in Table 2. For HIV-1 RNA testing, five samples (5/32) were not detectable in DBSs, while measurable levels were present in plasma (four, between 1.44 to 2.99 log10 copies/mL; one, between 3 to 3.99 log10 copies/mL). All 32 samples with detectable. HIV-1 RNA in plasma had detectable HIV-1 DNA in DBSs. There were two samples (2/94) with undetectable HCV RNA in DBSs, while measurable HCV RNA levels were present in plasma (–5 to 5.99 log10 copies/mL). In all samples, plasma HCV RNA was undetectable and DBS also provided concordant results (n = 9).
Table 2. Concordance analysis of HIV RNA, HIV DNA, and HCV RNA detection results between plasma and DBS among antibody-positive samples
Items VL in plasma No. of samples DBS-positive Detection rate HIV RNA 1.44 to 2.99 log10 copies/mL 14 10 10/14 3 to 3.99 log10 copies/mL 7 6 6/7 4 to 4.99 log10 copies/mL 7 7 7/7 ≥ 5 log10 copies/mL 4 4 4/4 All 32 27 27/32 HIV DNA 1.44 to 2.99 log10 copies/mL 14 14 14/14 3 to 3.99 log10 copies/mL 7 7 7/7 4 to 4.99 log10 copies/mL 7 7 7/7 ≥ 5 log10 copies/mL 4 4 4/4 All 32 32 32/32 HCV RNA N/A 9 0 9/9 2.28 to 3.99 log10 copies/mL 7 7 7/7 4 to 4.99 log10 copies/mL 9 9 9/9 5 to 5.99 log10 copies/mL 12 10 10/12 6 to 6.99 log10 copies/mL 32 32 32/32 ≥ 7 log10 copies/mL 34 34 34/34 All 103 92 92/103 Note. VL, light chain variable region. -
Among paired plasma/DBS samples with detectable HIV-1 RNA, the correlation between the VL determinations from plasma and DBS was assessed using Pearson’s correlation coefficient, as shown in Figure 2A. This demonstrated a moderate, positive correlation (r = 0.683, n = 27), which was statistically significant (P < 0.01).
Figure 2. Correlation and Bland-Altman analysis of agreement between HIV RNA VL in paired plasma and DBS samples. (A) Correlation between HIV VL in paired plasma and DBS samples. (B) Bland-Altman analysis of agreement between HIV VL in paired plasma and DBS samples. The red lines represent ± 1.96 standard deviation. The blue lines represent the mean difference. VL, light chain variable region
The Bland-Altman plot in Figure 2B showed that the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log10 copies/mL, and all samples were within the 1.96-SD limits (–0.97 to –2.97 log10 copies/mL) for DBS. Overall, HIV-1 RNA levels obtained from DBS were lower than those in plasma.
All RT-PCR amplification plots for the HIV-1 DNA test (32) from DBSs are illustrated in Figure 3. All 32 DBS samples for HIV-1 DNA were detectable and the amplification curves of target genes and internal control genes were found to be good.
Figure 3. The amplification curve of the target gene and internal control gene in HIV DNA RT-PCR. The figure shows the HIV DNA test results of 32 DBS samples. The red lines represent the amplification curve of the target gene (FAM probe); the green lines represent the amplification curve of the internal control gene (HEX probe); and the blue circle lines represent the amplification curve of the positive control. If HEX Ct ≤ 15.0 and FAM Ct ≥ 28.0, the reported results were positive. Ct represents the threshold cycle
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Among plasma-DBS pairs with detectable HCV RNA, the correlation between HCV RNA VL values obtained from plasma and DBS was high (Pearson’s correlation coefficient: r = 0.612 (P < 0.01), n = 89, as shown in Figure 4A.
Figure 4. Correlation and Bland-Altman analysis of agreement between HCV RNA VL in paired plasma and DBS samples. (A) Correlation between HCV RNA VL in paired plasma and DBS samples. (B) Bland-Altman analysis of agreement between HCV RNA VL in paired plasma and DBS samples. The red lines represent ± 1.96 standard deviation. The blue lines represent the mean difference. VL, light chain variable region.
The corresponding Bland-Altman plot illustrated the agreement between plasma and DBS in Figure 4B, which indicated that the mean difference (± SD) was 0.15 ± 1.08 log10 copies/mL and 94.38% (84/89) results were within ± 1.96 SD (–1.96 to 2.67 log10 copies/mL). Moreover, HCV RNA levels were, on average, slightly higher in plasma than in DBS.
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In this study, we attempted to develop a triple nucleic acid test for HIV, HCV, and TP to allow for the simultaneous detection of three infections using one DBS sample. We also evaluated the performance of the TP DNA test in plasma and DBS samples. The tolulized red unheated serum test was performed in anti-TP samples (24/67). All samples were undetectable for TP DNA (24) in plasma and DBS.
Results showed a failure to detect TP DNA in the plasma and DBS. Although TP can spread systemically after infection, the concentration of TP is too low to be detected and the volume of blood in DBSs is generally small (50–100 µL), which complicates the use of DBSs in detection of TP DNA[2, 15].
doi: 10.3967/bes2021.034
Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot
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Abstract:
Objective The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. Method A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson’s correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. Results Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log10 copies/mL). There were two samples (2/94) with undetectable HCV RNA in DBS, while measurable HCV RNA levels were present in plasma (−5 to 5.99 log10 copies/mL). The correlation between HIV-1 RNA light chain variable region (VL) values obtained from plasma and DBS showed that r = 0.683 (P < 0.01), n = 27 and r = 0.612 (P < 0.01), n = 89 in HCV RNA. Bland-Altman analysis revealed that in HIV-1 RNA, the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log10 copies/mL, and all samples were within ± 1.96 SD (−0.97 to 2.97 log10 copies/mL) for DBS. The mean difference (± SD) in HCV RNA was 0.15 ± 1.08 log10 copies/mL, and 94.38% (84/89) were within ± 1.96 SD (−1.96 to 2.67 log10 copies/mL). Overall, HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma. HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma. HIV-1 DNA RT-PCR using a DBS showed acceptable performance. Conclusion The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access. -
Key words:
- Dried blood spot (DBS) /
- Correlation /
- Bland-Altman /
- HIV-1 RNA /
- HIV-1 DNA /
- HCV RNA
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Figure 2. Correlation and Bland-Altman analysis of agreement between HIV RNA VL in paired plasma and DBS samples. (A) Correlation between HIV VL in paired plasma and DBS samples. (B) Bland-Altman analysis of agreement between HIV VL in paired plasma and DBS samples. The red lines represent ± 1.96 standard deviation. The blue lines represent the mean difference. VL, light chain variable region
Figure 3. The amplification curve of the target gene and internal control gene in HIV DNA RT-PCR. The figure shows the HIV DNA test results of 32 DBS samples. The red lines represent the amplification curve of the target gene (FAM probe); the green lines represent the amplification curve of the internal control gene (HEX probe); and the blue circle lines represent the amplification curve of the positive control. If HEX Ct ≤ 15.0 and FAM Ct ≥ 28.0, the reported results were positive. Ct represents the threshold cycle
Figure 4. Correlation and Bland-Altman analysis of agreement between HCV RNA VL in paired plasma and DBS samples. (A) Correlation between HCV RNA VL in paired plasma and DBS samples. (B) Bland-Altman analysis of agreement between HCV RNA VL in paired plasma and DBS samples. The red lines represent ± 1.96 standard deviation. The blue lines represent the mean difference. VL, light chain variable region.
Table 1. TP DNA PCR primer and probe sequences
Target Sequence (bases) tpn47 Forward 5'-TGCGCGTGTGCGAATGGTGGTC Reverse 5'-CACAGTGCTCAAAAACGCCTGCACG TaqMan probe 5’-FAM-AGTAACACCACGATAATGA
CCTCC-MGB-3’polA Forward 5'-GGTAGAAGGGAGGGCTAGTA Reverse 5'-CTAAGATCTCTATTTTCTATAGGTATGG TaqMan probe 5'-FAM-ACACAGCACTCGTCTTCAACTCC-BHQ-3' Table 2. Concordance analysis of HIV RNA, HIV DNA, and HCV RNA detection results between plasma and DBS among antibody-positive samples
Items VL in plasma No. of samples DBS-positive Detection rate HIV RNA 1.44 to 2.99 log10 copies/mL 14 10 10/14 3 to 3.99 log10 copies/mL 7 6 6/7 4 to 4.99 log10 copies/mL 7 7 7/7 ≥ 5 log10 copies/mL 4 4 4/4 All 32 27 27/32 HIV DNA 1.44 to 2.99 log10 copies/mL 14 14 14/14 3 to 3.99 log10 copies/mL 7 7 7/7 4 to 4.99 log10 copies/mL 7 7 7/7 ≥ 5 log10 copies/mL 4 4 4/4 All 32 32 32/32 HCV RNA N/A 9 0 9/9 2.28 to 3.99 log10 copies/mL 7 7 7/7 4 to 4.99 log10 copies/mL 9 9 9/9 5 to 5.99 log10 copies/mL 12 10 10/12 6 to 6.99 log10 copies/mL 32 32 32/32 ≥ 7 log10 copies/mL 34 34 34/34 All 103 92 92/103 Note. VL, light chain variable region. -
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