Detection of human viral pathogens: conventional versus molecular approaches

The disease burden from acute and chronic infections of viral origin continues to challenge healthcare organizations worldwide. According to the latest Disability-Adjusted Life Year (DALY) estimates by the World Health Organization (WHO), illnesses caused by viruses account for greater than 60% of the burden from all infectious agents.¹ Timely and accurate laboratory diagnosis of viral infections plays a pivotal role in reducing global viral disease burden. In this article, we will review the landmark breakthroughs in technologies that have accomplished monumental improvement in clinical viral testing with respect to sensitivity, specificity, speed, simplicity, and cost.

Cell culture, shell vials, and direct fluorescent antibody staining

The use of mammalian cell cultures six decades ago to propagate the poliovirus in vitro and examine microscopically the cytopathic effect (CPE) on infected cells marked the beginning of the golden era in clinical virology.^2,3 The quality and variety of cell lines have improved dramatically with refined cell monolayers commercially available.⁴ Key examples of cell cultures still commonly used in many clinical laboratories include the primary rhesus monkey kidney cells (RhMK), human lung fibroblasts (MRC-5), human foreskin fibroblasts (HFF), human lung carcinoma cells (A549), and others.

Inoculating a broad range of cell cultures with a clinical specimen provides multiple advantages. For example, it allows the isolation of a wide variety of viruses, which include adenovirus, CMV, RSV, respiratory viruses such as influenza A & B, parainfluenza viruses types 1 to 3, VZV, as well as the Ebola virus, severe acute respiratory coronavirus (SARS-CoV), and human metapneumovirus (hMPV). The main drawbacks of cell culture are the lengthy incubation period and hence the long turnaround time (TAT) and the technical expertise required to recognize CPE in the cell culture monolayers microscopically.

The technological advancement of shell vials and staining with indirect or direct fluorescent-antibody assay (IFA or DFA) dramatically shortens the time to positivity from weeks to less than 48 hours by staining for early antigens of viral infection. Shortly after their successful demonstration in CMV isolation,^5,6 the shell vial/DFA assays have been developed for use in the detection of other viruses including HSV,⁷ influenza virus,⁸ respiratory viruses,⁹ enterovirus,¹⁰ adenovirus,¹⁰ and VZV.¹¹ Further refinements of the DFA assays allow the screening of seven different respiratory viruses (adenovirus; influenza viruses A and B; parainfluenza viruses types 1, 2, and 3; and RSV) by staining the monolayer from the R-mix shell vial after 48 hours of incubation with a sensitivity of 81%.¹² Some HSV kits allow the screening and typing of HSV after 24 hours of inoculation in an MRC-5 cell culture with a sensitivity of 93% and specificity of 95%. The VZV early antigen detection kit allows the detection of VZV directly from a fresh skin lesion specimen with a sensitivity of 100%, compared to 23% from cell culture.¹³ 

Despite some limitations, shell vial/DFA remains the most sensitive non-molecular viral detection method. Nonetheless, the sensitivity and specificity of the shell vial/DFA system are inferior to those of molecular assays. Collectively, more laboratories in developed countries have gradually decided to remove cell culture testing from their laboratory test menu.

Nucleic acid amplification techniques

Nucleic acid amplification techniques (NAATs) for clinical virology have seen rapid development in the past decade. Owing to their low specimen quantity requirement, superior sensitivity and specificity, and dramatically faster turnaround time, NAATs are currently accepted as the gold standard for clinical virology.^14-16 Viruses that do not proliferate in conventional cell culture but cause illnesses with great morbidity and mortality (HIV, HCV, HBV, parvovirus B-19, HHV 6-8, EBV, HPV, and BK virus) can now be detected with clinically meaningful results.

The core technology of NAATs is the thermostable polymerase-based target amplification process that synthesizes several copies of target nucleic acid. The amplicons are detected by two oligonucleotide primers that bind to complementary sequences. The end result is the production of millions of copies of the targeted sequence. The essential components in a standard single target PCR contain target DNA, two oligonucleotide primers, a DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), MgCl2, KCl, and Tris-HCl buffer. The reaction mixture is heated and cooled during several cycles in a programmable thermal cycler, and after n cycles the target sequence can be amplified 2ⁿ-fold. Upon completion of the assay, the amplification products can be evaluated using various techniques such as agarose gel analysis and colorimetric detection.¹⁷

In real-time PCR, the target amplification and detection steps occur simultaneously. The PCR product is detected by using fluorescent dyes that preferentially bind to double-stranded DNA. Special thermal cyclers that can monitor the fluorescence emission from the sample are required. The computer software supporting the thermal cycler monitors the data at every cycle and generates a quantitative amplification plot for each reaction.¹⁸ Real-time PCR decreases the time required to “visualize” nucleic acid amplification products because there are no post-PCR processing steps.

The main advantages of real-time PCR include decreased frequency of contamination and quantitation of the original viral target. One major application of the real-time PCR technology is viral load assays to quantitatively assess the amount of virus in a clinical specimen. HIV, HBV, and HCV are the three most common blood-borne pathogens, causing chronic infections to more than 200 million people worldwide.¹ Findings from the studies of HIV,¹⁹ HBV,²⁰ and HCV²¹ have shown that monitoring viremia is a reliable predictor for prophylaxis or assessment of treatment outcome for these diseases.

It is estimated that acute respiratory disease is responsible for at least 75% of all acute morbidities in developed countries and that 80% of the infections are caused by respiratory viruses.²² With an average of >90% in sensitivity and specificity and turnaround time of no more than six hours, NAATs-based assays are considered the gold standard for rapid and accurate detection of respiratory viruses, compared to shell vial and DFA-based assays (Table 1). Despite the fact that most respiratory viruses do grow in conventional cell culture, they tend to grow very slowly and/or do not exhibit prominent CPE. Since the majority of respiratory viral infections require the administration of antiviral therapy within the first 48 hours of onset of symptoms to be clinically effective, cell culture is not an optimal diagnostic algorithm.

Multiplex PCR

Respiratory viruses account for a high percentage of both pediatric and adult upper respiratory tract infections. In many immune-compromised patient populations, respiratory virus infection shows a high rate of progression to pneumonia and can exhibit increased mortality.²³ Current estimates show that there are at least 26 known respiratory viruses that can cause upper and lower viral respiratory tract infections, and ongoing research is rapidly revealing additional clinically relevant viruses. Running single target PCR assays for all 26 viruses is too expensive and time-consuming for clinical diagnosis.²⁴ Multiplex PCR assays allow for the quantitative detection of multiple nucleic acid targets by assaying with specific probes to different viral targets in a single PCR reaction. These assays are called Respiratory Viral Panels (RVPs). Clinically, an RVP is also able to recognize viral co-infections, though the relevance of these co-infections is still a matter of debate.^25-29 A cost analysis in 2009³⁰ showed that a hospital system could save up to $500,000 per year based on multiplex identification of respiratory viruses compared to shell vial or DFA. Most of the cost savings was associated with decreases in length of hospital stay for pediatric inpatients due to rapid diagnosis of the relevant pathogen.

There are three FDA-approved platforms for multiplex PCR testing of respiratory viruses. One combines multiplex PCR with liquid-phase bead-based array technology and recognizes 19 different viral targets.³¹ The assay is high-throughput (multiple samples can be included in each run), but requires separate specimen nucleic acid extraction and post-amplification handling and takes five to six hours. Comparison studies revealed the assay has 78.8% sensitivity and 99.6% specificity as compared to single target PCR assays. It missed specimens with very low viral loads, or specimens positive for adenovirus.³²

A second platform utilizes multiplex RT-PCR followed by electrochemical detection of hybridized capture probes on gold-plated electrodes and recognizes 14 different viral targets. This assay also offers a high-throughput platform, but requires specimen nucleic acid extraction and post-amplification handling and takes four hours. It showed a 95.4% positive agreement with single target PCR assays.³³

A third assay is a fully integrated system for 17 viruses that includes nucleic acid extraction and nested RT-PCR followed by multiplex-PCR and detection by melt curve analysis. There is no post-amplification handling, but the assay is low-throughput. Only one sample can be analyzed per run and each run takes one hour. This assay showed a combined sensitivity greater than 90% for tested viruses compared to single target PCR.^34,35

It is clear that multiplex PCR assays serve as a cost-effective and sensitive diagnostic method for respiratory viruses. The available assay systems can meet the clinical testing needs for most microbiology labs, but the perfect system would be rapid and high-throughput, with very little post-amplification handling.

Genotyping

Genotypic analysis on the viral genome of certain viruses can provide clinically useful data to predict response to antiviral drug treatment and/or epidemiologic comparison. A combination of PCR and nucleic acid sequencing is used in genotyping. Chronic hepatitis C virus (HCV) infection is the most common blood-borne infection in the United States and can lead to liver cirrhosis and hepatocellular carcinoma. It is estimated that there are 3.2 million persons chronically infected with HCV nationwide and 170 million people globally.^{36 37} 

The current recommendations for chronic HCV infection include interferon and ribavirin³⁸; however, various genotypes respond differently to the therapy. In general, unlike genotypes 2 and 3, genotypes 1 and 4 respond poorly to the therapy.³⁹ Two highly conserved regions on the HCV’s ssRNA genome have been exploited for genotyping by various commercial assays using direct sequence analysis. One of these assays is an amplification- and sequencing-based of the 5′ noncoding region, and another entails an amplification followed by reverse hybridization process using 19 genotype-specific oligonucleotide probes located in the 5’NC region. Both assays detect genotypes 1 through 6 in the 5-UTR. Evaluations on these two assays indicated that sensitivity (variable, 730-4100 IU/ml, genotype-dependent) and specificity (100%) to be comparable at the genotype level.^40-42 However, both assays have limitations in detecting certain genotypes (for example, genotype 6) as well as mixed infections, and neither is recommended for subtyping of HCV.^{42 43} A recently FDA-approved real time genotype assay has improved typing sensitivity (500 IU/ml) and broadened typing spectrum at both the genotype and subtype levels with increased automation and reduced hands-on time. 

Drug resistance among circulating HIV-1 strains is the main reason for antiretroviral treatment (ART) failure. In financially established countries, the HIV-1 genotyping resistance test is considered as a test of care for HIV-1 pretreatment resistance testing as well as for monitoring level of ART resistance. There are two FDA-approved methods for genotypic HIV drug resistance testing. One is RT-PCR and sequencing-based and detects HIV-1 Subtype B viral resistance in plasma samples collected in EDTA with a viral load ranging from 2,000 to 750,000 copies/mL. It also genotypes the entire HIV-1 protease gene from codons 1-99 and two-thirds of the reverse transcriptase (RT) gene from codons 1-335.^44-45 The second is a sequence-based assay targeted at the protease region (codons 1 to 99) and reverse transcriptase region (codon 40 to 247) of the HIV-l genome.^46-47 These regions have been selected because they code for resistance against antiretroviral drugs. Genotyping represents a technological step forward in the diagnosis and treatment of viral disease.

Timely and accurate laboratory diagnosis of viral infections plays a pivotal role in reducing global viral disease burden. Molecular assays represent significant improvements in clinical viral testing with respect to sensitivity, specificity, speed, simplicity, and cost. Real-time PCR, genotyping, and multiplex assays have changed the way clinicians are able to treat both chronic and acute viral illness.

Omai B. Garner, PhD, D(ABMM), is an Assistant Clinical Professor in the Department of Pathology and Laboratory Medicine at UCLA. He serves as the Associate Director of Clinical Microbiology for the UCLA Health System. Max T. Wu, PhD, is an active duty officer in the United States Army Medical Service Corps currently receiving a two-year post-doctoral clinical microbiology fellowship training at UCLA.

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