Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses
2.2. Primers and Probes
2.3. Construction of Plasmids Carrying Viral Targets
2.4. Multiplex qPCR Standardization
2.5. Patients and Clinical Samples
2.6. Viral Detection in Clinical Samples
2.7. Sanger Sequencing
2.8. Statistical Analysis
3. Results
3.1. Specificity of the Primers on a Human DNA Background
3.2. Performance of the Multiplex qPCRs
3.3. Cross-Reactivity Test
3.4. Detection of Beta and Gamma Herpesviruses in Pediatric Transplanted Patients
3.4.1. Analysis of Single and Multiple Infections
3.4.2. Comparison between Viral Loads Detected in Leukocytes vs. Plasma
3.4.3. Persistence of Infections
3.4.4. Association between Viral Infection and Episodes of Graft Rejection
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Tube 1 | Viral/Cellular Target | Gene | F-Q | Sequence 5′→3′ | Amplicon Size (bp) | Ref. |
---|---|---|---|---|---|---|
1 | EBV | BALF5 | FAM-BHQ1a | F CCCTGTTTATCCGATGGAATG R CGGAAGCCCTCTGGACTTC P TGTACACGCACGAGAAATGCGCC | 90 | [19] |
HCMV | UL123 | JOE-BHQ1a | F GCTACAATAGCCTCTTCCTCATCTG R GACTAGTGTGATGCTGGCCAAG P AGCCTGAGGTTATCAGTGTAATGAAGCGCC | 201 | [20] | |
Endogenous human gene | β-actin | Cy5-BHQ3a | F CCAGGCTAACCTCGGAAATCT R CATCGTCATTCCTGTGCAACT P TGGGGTGCCGGCTCTCTGCT | 225 | This study | |
2 | HHV6 | U31 | FAM-BHQ1a | F CGACTCTCACCCTACTGAACGA R GAGGCTGGCGTCGTAGTAGAA P AGCCACAGCAGCCATCTACATCTGTCAA | 121 | [20] and this study 2 |
HHV7 | U57 | JOE-BHQ1a | F CGGAAGTCACTGGAGTAATGACAA R ATGCTTTAAACATCCTTTCTTTCGG P CTCGCAGATTGCTTGTTGGCCATG | 108 | [21] | |
KSHV | LANA | Cy5-BHQ3a | F AGTTATGGGCGACTGGTCTG R GGATGGAAGACGAGATCCAA P AAGTCCGTATGGGTCATTGC | 166 | This study |
Target | E (%) | Predictability (R2) | LOD (Copy Number) | Correlation of Single vs. Multiplex qPCR (R) |
---|---|---|---|---|
EBV | 94 | 0.993 | 21 | 0.9994 |
HCMV | 92 | 0.988 | 18 | 0.9904 |
HHV6 | 96 | 0.988 | 25 | 0.9998 |
HHV7 | 95 | 0.993 | 21 | 0.9981 |
KSHV | 95 | 0.996 | 18 | 0.9999 |
Standard (Plasmid Copies) | Coefficients of Variation (%) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
EBV | HCMV | HHV6 | HHV7 | KSHV | ||||||
Intra 1 | Inter 2 | Intra 1 | Inter 2 | Intra 1 | Inter 2 | Intra 1 | Inter 2 | Intra 1 | Inter 2 | |
106 | 0.31 | 0.96 | 0.73 | 3.12 | 0.44 | 1.89 | 0.54 | 0.49 | 0.31 | 0.66 |
105 | 0.77 | 1.77 | 2.93 | 2.89 | 0.91 | 1.21 | 0.44 | 0.76 | 0.31 | 0.96 |
104 | 0.96 | 1.49 | 1.64 | 2.94 | 0.93 | 1.33 | 0.69 | 0.91 | 1.27 | 1.13 |
103 | 1.43 | 1.75 | 3.30 | 2.35 | 0.50 | 1.95 | 1.05 | 0.85 | 0.67 | 0.57 |
102 | 1.82 | 1.88 | 1.65 | 2.53 | 0.43 | 1.63 | 0.87 | 0.69 | 0.69 | 1.09 |
101 | 2.54 | 2.09 | 3.55 | 3.87 | 0.65 | 0.72 | 1.43 | 2.67 | 1.19 | 2.60 |
Transplanted Organ | Kidney N = 22 | Liver N = 12 |
---|---|---|
Age at transplant | ||
Median of years (range) | 15 (6–17) | 6 (4–17) |
Gender | ||
Female | 33% | 58% |
Male | 67% | 42% |
Kind of donor | ||
Cadaveric | 57% | 92% |
Living-donor | 43% | 8% |
EBV serostatus N (%) 1 | ||
Donor+ Recipient+ | 11 (92%) | 6 (55%) |
Donor+ Recipient− | 1 (8%) | 2 (18%) |
Donor− Recipient+ | 0 (0%) | 3 (27%) |
Donor− Recipient− | 0 (0%) | 0 (0%) |
HCMV serostatus N (%) 1 | ||
Donor+ Recipient+ | 13 (62%) | 6 (50%) |
Donor+ Recipient− | 3 (19%) | 3 (25%) |
Donor− Recipient+ | 2 (13%) | 2 (17%) |
Donor− Recipient− | 1 (6%) | 1 (8%) |
Pre-transplant diagnosis N (%) | ESRD of unknown etiology 15 (68%) Focal and segmental glomerulosclerosis 2 (9%) ESRD secondary to complex uropathy 2 (9%) ESRD secondary to hypoplasia JRA 1 (5%) Microscopic polyangiitis 1 (5%) Posterior urethral valves 1 (5%) | Biliary atresia 3 (25%) |
Fulminant hepatitis 2 (17%) | ||
Liver fibrosis 1 (8%) | ||
Neonatal giant cell hepatitis 1 (8%) | ||
Progressive intrahepatic family cholestasis 1 (8%) | ||
Bayler disease 1 (8%) | ||
Alalgille syndrome 1 (8%) | ||
Tyrosinemia 1 (8%) | ||
Cholesterol ester deposition 1 (8%) |
Infection/Coinfection | Kidney | Liver | ||
---|---|---|---|---|
Relative Risk | p Value | Relative Risk | p Value | |
EBV | 2.74 | 0.20 | 2.68 | 0.41 |
HCMV | 4.28 | 0.06 | 40.33 | 0.0013 |
HHV6 | 5.60 | 0.03 | 6 | 0.20 |
HHV7 | 3.04 | 0.12 | 1.27 | 0.87 |
EBV/HCMV | 4.86 | 0.24 | NOT DETECTED | |
EBV/HHV6 | 4.86 | 0.24 | 9.07 | 0.10 |
EBV/HHV7 | 5.60 | 0.03 | 4.46 | 0.29 |
HCMV/HHV6 | 7.32 | 0.12 | NOT DETECTED | |
HCMV/HHV7 | NOT DETECTED | NOT DETECTED | ||
HHV6/HHV7 | 4.86 | 0.24 | NOT DETECTED | |
EVB/HCMV/HHV6 | NOT DETECTED | NOT DETECTED | ||
EBV/HCMV/HHV7 | 7.32 | 0.12 | NOT DETECTED | |
EBV/HHV6/HHV7 | 17.64 | 0.0003 | NOT DETECTED | |
HCMV/HHV6/HHV7 | NOT DETECTED | NOT DETECTED | ||
EBV/HCMV/HHV6/HHV7 | NOT DETECTED | NOT DETECTED |
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Sánchez-Ponce, Y.; Varela-Fascinetto, G.; Romo-Vázquez, J.C.; López-Martínez, B.; Sánchez-Huerta, J.L.; Parra-Ortega, I.; Fuentes-Pananá, E.M.; Morales-Sánchez, A. Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation. Viruses 2018, 10, 730. https://doi.org/10.3390/v10120730
Sánchez-Ponce Y, Varela-Fascinetto G, Romo-Vázquez JC, López-Martínez B, Sánchez-Huerta JL, Parra-Ortega I, Fuentes-Pananá EM, Morales-Sánchez A. Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation. Viruses. 2018; 10(12):730. https://doi.org/10.3390/v10120730
Chicago/Turabian StyleSánchez-Ponce, Yessica, Gustavo Varela-Fascinetto, José Carlos Romo-Vázquez, Briceida López-Martínez, José Luis Sánchez-Huerta, Israel Parra-Ortega, Ezequiel M. Fuentes-Pananá, and Abigail Morales-Sánchez. 2018. "Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation" Viruses 10, no. 12: 730. https://doi.org/10.3390/v10120730