Samples
The study group consisted of 27 patients with ALF. The protocol was approved by the Ethics Committee of the Oswaldo Cruz Institute (Protocol no. 440.614). The serum and liver samples were collected after liver transplantation. Serum samples for testing were obtained from all patients and liver samples acquired from 14 patients. Samples had been previously determined as negative for hepatitis A, B, C, E and of unknown etiology. All patients were recruited from the hepatic transplantation service at the Federal Hospital of Rio de Janeiro, Brazil.
The written informed consent was signed by the patients or the responsible person.
Samples were examined via real-time multiplex PCR and confirmed using singleplex, qualitative PCR and nucleotide sequencing.
Extraction of DNA
Viral DNA was extracted from 200 mL aliquots using the QIAamp DNA Blood Mini Kit for serum samples and QIAamp DNA Tissue Mini Kit for liver samples (Qiagen, Germany), according to the manufacturer’s protocols. DNA samples were stored at -70ºC until processing.
Multiplex qPCR for betaherpesviruses
Betaherpesviruses were detected and quantified according to the method of Sassenscheidt and colleagues (15). Oligonucleotide probes for detection of betaherpesviruses target highly conserved regions in the viral genome of published strains. For amplification of HCMV, we used UL54 and the U56 regions were used for HHV-6 A/B. This assay does not allow differentiation between consensus HHV-6 types A and B in clinical samples (15). For detection of HHV-7, the U37 region were used as described earlier (16). Primers and oligonucleotide TaqMan probes are presented in Table 1. Multiplex qPCR was performed in a reaction mixture comprising 1 µL 25x PCR Enzyme (Mix Life Technologies, California, USA), 2.5 µL of each oligonucleotide (3 µM), 2 µL probe (0.4 µM), 12.5 µL of 1x PCR Buffer (Life Technologies, California, USA) and 5 µL DNA. Absolute quantification of DNA virus was performed with the aid of a synthetic standard curve designed for this study, ranging from 5 to 5 x 108 genome copies/uL (Table 1).
Singleplex qPCR for betaherpesviruses
We employed the protocol described by Sassenscheidt et al. (15) using the same primers, oligonucleotide probes and amplification regions. For each reaction, a mixture comprising 1 µL 25x PCR Enzyme (Mix Life Technologies, California, USA), 2.5 µL each oligonucleotide (1 µM), 2.0 µL probe (0.4 µM) and 12.5 µL of 1x PCR Buffer (Life Technologies, California, USA) was used according to the manufacturer's instructions. Absolute quantification was performed based on the synthetic standard curve designed for this study.
Specificity analysis
For analysis of specificity, different targets of the Herpesviridae family were used. Serum samples stored in the LADTV sample database positive for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) and Epstein-Barr virus (EBV) were examined via previously standardized multiplex standard qPCR using betaherpesvirus targets as the positive control.
Reference Control
The TaqMan RNase P Control Reagent kit (Applied Biosystems Foster City, USA , Catalog number 4316844) was used as an internal reference control for analysis of human clinical samples to ensure the quality of DNA and exclude the possibility of false-negative results due to the presence of PCR inhibitors or low DNA integrity. TaqMan RNase P probe was 5’-labeled with VIC fluorophore and 3’-labeled with NFQ-MGB. The reference control was used in qPCR singleplex assays and quality of samples monitored based on Ct values. The reaction mixture for RNase P control comprised 6 μL DNase/RNase-free water, 1 μL of 25X qPCR enzyme mix, 12.5 μL of 2X qPCR buffer (including the reference dye ROX) and 1 μL RNase P mix (containing oligonucleotides for this region and specific buffers; Applied Biosystems, California, USA) in 96-well plates. In addition, 5 μL extracted DNA was subsequently added to the mix on the plate under similar conditions to those used in qPCR for betaherpesvirus detection.
Qualitative Pan herpesvirus-PCR
Samples showing positivity in qPCR were additionally examined via qualitative Pan-PCR to confirm diagnosis. For simultaneous detection of betaherpesviruses, we employed the Pan herpesvirus technique based on amplification via nested PCR (Pan-PCR) of the Dpol gene, a highly conserved region in the herpesvirus genome, and species identification via sequencing of the viral genome. The protocol for herpesvirus detection and specific oligonucleotides were designed based on the report by Ehlers et al. (1).
Sequencing
Reactions were conducted in the DNA sequencing platform of the Technological Development Program in Health Supplies /PDTIS (Oswaldo Cruz Foundation, Rio de Janeiro, Brazil). Nucleotide sequences obtained were analyzed in the BioEdit 7.2.5 program and compared to other sequences deposited in GenBank with the BLAST tool (Basic Local Alignment Search Tool) to identify the betaherpesvirus species detected using nested PCR.
Biochemical analysis of samples
Biochemical analyses were performed via spectrophotometric determination of pyruvic transaminase (ALT), oxaloacetic transaminase (AST) and alkaline phosphatase. Spectrophotometry procedures were performed using commercial kits (Abbott, Illinois, United States of America). For analysis of biochemical markers, samples were divided into three groups: betaherpesvirus-positive ALF samples (ß-Herpesvirus positive), betaherpesvirus-negative ALF samples (ß-Herpesvirus negative) and healthy controls. Diagnosis of ALF was performed based on grade of encephalopathy along with prothrombin time (PT) and international normalized ratio (INR).