Sample Assignments Of Cid

Abstract

Background

Twelve percent of all acute liver failure (ALF) cases are of unknown origin, often termed indeterminate. A previously unrecognized hepatotropic virus has been suspected as a potential etiologic agent.

Methods

We compared the performance of metagenomic next-generation sequencing (mNGS) with confirmatory nucleic acid testing (NAT) to routine clinical diagnostic testing in detection of known or novel viruses associated with ALF. Serum samples from 204 adult ALF patients collected from 1998 to 2010 as part of a nationwide registry were analyzed. One hundred eighty-seven patients (92%) were classified as indeterminate, while the remaining 17 patients (8%) served as controls, with infections by either hepatitis A virus or hepatitis B virus (HBV), or a noninfectious cause for their ALF.

Results

Eight cases of infection from previously unrecognized viral pathogens were detected by mNGS (4 cases of herpes simplex virus type 1, including 1 case of coinfection with HBV, and 1 case each of HBV, parvovirus B19, cytomegalovirus, and human herpesvirus 7). Several missed dual or triple infections were also identified, and assembled viral genomes provided additional information on genotyping and drug resistance mutations. Importantly, no sequences corresponding to novel viruses were detected.

Conclusions

These results suggest that ALF patients should be screened for the presence of uncommon viruses and coinfections, and that most cases of indeterminate ALF in the United States do not appear to be caused by novel viral pathogens. In the future, mNGS testing may be useful for comprehensive diagnosis of viruses associated with ALF, or to exclude infectious etiologies.

pathogen discovery, metagenomic next-generation sequencing, indeterminate ALF, viral hepatitis, SURPI computational pipeline

(See the Editorial Commentary by Fredricks on pages 1486–8.)

Acute liver failure (ALF) is a complex syndrome characterized by rapid deterioration of liver function with hepatic encephalopathy in the absence of a known history of previous liver disease. ALF may progress to multiorgan failure and results in death or transplantation in 57% of cases. Acetaminophen toxicity is the most common documented cause of ALF in the United States, comprising 50% of cases, while infections from hepatitis A virus (HAV) and hepatitis B virus (HBV) combined account for 12% [1]. After standard clinical testing, a clear etiology is not found in approximately 12% of US ALF cases, thus considered indeterminate; an even higher percentage of indeterminate cases (~20%) is seen in developing countries [2]. Infection by a previously unrecognized hepatotropic virus has been posited as a cause for indeterminate ALF [3]. Beyond HAV and HBV, other potential viral etiologies of ALF include hepatitis C virus (HCV), hepatitis E virus (HEV), adenovirus, herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), varicella zoster virus, and parvovirus B19, all of which are rare causes of ALF in the United States [4–7]. Appropriate management of ALF depends on accurate diagnosis, as specific antiviral treatments are sometimes available, and other therapies may be indicated for noninfectious etiologies [7]. Thus, it is imperative to determine the limitations of conventional diagnostic testing for ALF, and whether novel or previously unrecognized viruses may be associated with this condition.

Metagenomic next-generation sequencing (mNGS) is a comprehensive approach for sequence-based identification of pathogenic microbes, especially viruses, in clinical samples [8, 9]. Previous studies have shown that mNGS is useful for viral surveillance [10, 11] and identification of novel viruses circulating in blood [12, 13]. The US Acute Liver Failure Study Group (ALFSG) is a nationwide study established in 1998 to enroll patients with ALF and collect detailed clinical and laboratory information and biosamples for analysis. We hypothesized that mNGS screening of serum samples from 187 indeterminate ALFSG cases from 1998 to 2010 would allow broader identification of infectious causes of indeterminate ALF.

METHODS

Study Patients

A total of 204 patients with ALF (187 patients with indeterminate ALF and 17 as controls) were selected from the ALFSG cohort (Supplementary Methods) to undergo further mNGS testing for viral infection. Clinical and laboratory data for the 204 ALF patients, including available liver biopsy data and history of injection drug and alcohol use, are provided in Supplementary Tables 1–4. The 187 indeterminate ALF cases were consecutive (collected during enrollment years 1998–2010), provided a broad geographic representation across all study sites in the United States, and comprised 61% of the overall cohort to date (n = 307). Notably, 11 of the indeterminate samples tested positive for viruses at the study site, including human immunodeficiency virus (HIV) (n = 2), chronic HCV (n = 3), mixed HBV, HCV, and hepatitis D virus (HDV) (n = 1), combined EBV and cytomegalovirus (CMV) (n = 1), and HBV (n = 4). The 17 control patient samples included 8 samples from ALF-associated hepatitis A or B cases (n = 4 each) and 9 samples corresponding to noninfectious negative control groups: acetaminophen (N-acetyl-para-aminophenol [APAP]) toxicity (n = 3), autoimmune hepatitis (n = 2), drug-induced liver injury (n = 3), and hepatic ischemia (n = 1).

Metagenomic Next-Generation Sequencing and Analysis

Serum samples from each patient were processed in a blinded fashion for metagenomic sequencing individually or in pools of 2–6 (Supplementary Table 5), using an approach demonstrating high sensitivity for unbiased virus detection [14]. Individual samples or pools were first treated with a cocktail of Turbo DNase (Ambion) and Baseline-ZERO DNase (Epicentre) prior to nucleic acid extraction. Pretreatment with DNase biases mNGS testing toward viral detection as microbial and human host DNA is depleted whereas encapsidated (“protected”) viral nucleic acid is preserved [15]. With analysis of serum, there is also a minor limitation with respect to potential decreased sensitivity of detection of integrated proviruses (eg, HIV type 1 [HIV-1]), episomal viruses (eg, herpesviruses), or strongly cell-associated viruses (eg, human T-cell lymphotropic virus 1) [16]. Nucleic acid extraction was performed on the Qiagen EZ1 Advanced XL automated system using the EZ1 Virus Mini Kit version 2.0 (Qiagen). Extracted nucleic acid was amplified with random hexamers to generate a complementary DNA (cDNA) library as previously described [17]. In brief, 23 pools and 12 individual samples were initially processed using a modified TruSeq protocol (Illumina) [18], and 116 individually prepared samples were later processed using the NexteraXT protocol (Illumina). Samples were sequenced on 10 Illumina HiSeq lanes, and sequencing reads were analyzed using sequence-based ultra-rapid pathogen identification (SURPI), a computational pipeline for comprehensive pathogen identification from mNGS data by comparison to microbial sequences in the National Center for Biotechnology Information (NCBI) nucleotide (NT) database [9]. SURPI+, the clinical version of SURPI used for analysis [18, 19], employs taxonomic classification according to a lowest common ancestor algorithm for more accurate read assignments. As the first set of TruSeq libraries used only single-end barcode indexes (vs paired-end barcodes for the NexteraXT libraries), a predetermined threshold of >25 reads and >2% genome coverage was used for assessment of positive viral signatures by mNGS in these single-end barcoded samples given the potential for cross-contamination. Detected reads from viruses of unknown pathogenicity that constitute part of the normal viral flora circulating in blood, such as anelloviruses and human pegivirus 1/GB virus C [20, 21], were not considered as causes of ALF (Supplementary Table 6). Manual analyses for bona fide reads corresponding to nonviral pathogens (bacteria, fungi, and parasites) revealed only the presence of known laboratory/environmental contaminants or false-positive identifications due to misannotations in the NCBI NT database.

Details regarding genome assembly, genotyping, and identification of resistance mutations, as well as sequencing data deposition into the NCBI Sequence Read Archive, are provided in the Supplementary Methods.

Confirmation by Research Nucleic Acid Testing

Individual samples or all samples comprising a pool were selected for research nucleic acid testing (NAT) confirmation if reads from a human viral pathogen (HAV, HBV, HCV, HDV, HEV, CMV, HIV, parvovirus B19, and human papillomavirus virus type 159 [HPV-159]) were detected by mNGS. Total nucleic acid was extracted using the Qiagen Ultrasens Virus Kit (Qiagen), followed by construction of cDNA libraries using random hexamers and Superscript III Reverse Transcriptase (Invitrogen). Libraries were screened by polymerase chain reaction (PCR) (Qiagen OneStep RT-PCR Kit) using previously published primer sets and conditions for detection of individual viruses (Supplementary Table 7).

Confirmation by Clinical Nucleic Acid Testing

In parallel, NAT was independently performed on 0.5 mL serum from samples testing positive by mNGS and with sufficient volume available using the Procleix Ultrio assay on the Tigris platform (Grifols Diagnostic Solutions and Hologic). This US Food and Drug Administration–approved clinical assay is employed in the United States and internationally for screening blood donors for simultaneous detection of HIV types 1/2 and HBV and HCV nucleic acids [22, 23]. The analytic sensitivities of Ultrio for detection of HIV, HCV, and HBV are 10–15 copies or IU/mL at a 95% lower limit of detection.

RESULTS

Clinical Testing

Serum samples from 204 patients were investigated in a blinded fashion using mNGS, of which 187 (92%) were classified as indeterminate, 8 (4%) were positive controls, and 9 (4%) were negative controls (Table 1). The extent of primary clinical testing for hepatitis viruses at the study sites was variable, with HCV serology and HBV antigen testing being performed >80% of the time, and HDV or HEV PCR testing <10% of the time (Figure 1).

Table 1.

Cause of Acute Liver Failure at Time of Hospital Discharge for Patients in the Study (N = 204)

Cause of Acute Liver FailureaSamples, No. (%) 
Indeterminate 187 (91.7) 
Hepatitis A virus infectionb4 (2.0) 
Hepatitis B virus infectionb4 (2.0) 
Acetaminophen toxicityc3 (1.5) 
Autoimmune hepatitisc2 (1.0) 
Drug-induced hepatitisc3 (1.5) 
Hepatic ischemiac1 (0.5) 
Cause of Acute Liver FailureaSamples, No. (%) 
Indeterminate 187 (91.7) 
Hepatitis A virus infectionb4 (2.0) 
Hepatitis B virus infectionb4 (2.0) 
Acetaminophen toxicityc3 (1.5) 
Autoimmune hepatitisc2 (1.0) 
Drug-induced hepatitisc3 (1.5) 
Hepatic ischemiac1 (0.5) 

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Figure 1.

Clinical diagnostic testing for viral infections in study patients with indeterminate acute liver failure. The percentage of patients in the study positive for a given viral infection (y-axis) is plotted against the specific laboratory test that was ordered by the clinical site (x-axis). Abbreviations: anti-HBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; HAV, hepatitis A virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, hepatitis E virus; HIV, human immunodeficiency virus; IgM, immunoglobulin M.

Figure 1.

Clinical diagnostic testing for viral infections in study patients with indeterminate acute liver failure. The percentage of patients in the study positive for a given viral infection (y-axis) is plotted against the specific laboratory test that was ordered by the clinical site (x-axis). Abbreviations: anti-HBc, hepatitis B core antibody; anti-HBs, hepatitis B surface antibody; HAV, hepatitis A virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, hepatitis E virus; HIV, human immunodeficiency virus; IgM, immunoglobulin M.

Metagenomic Next-Generation Sequencing Results

Serum samples collected at the earliest available time point after clinical presentation of ALF were analyzed (median, 7 days [range, 2–38 days] from symptom onset to sample collection; Table 2

Элементы! - воскликнул.  - Мы говорим о математике, а не об истории. Головы повернулись к спутниковому экрану. - Танкадо играет с нами в слова! - сказал Беккер.  - Слово «элемент» имеет несколько значений.

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