Expert Review of Anti-infective Therapy

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Consolidation of molecular testing in clinical

virology

Carolina Scagnolari, Ombretta Turriziani, Katia Monteleone, Alessandra

Pierangeli & Guido Antonelli

To cite this article: Carolina Scagnolari, Ombretta Turriziani, Katia Monteleone, Alessandra
Pierangeli & Guido Antonelli (2017) Consolidation of molecular testing in clinical virology, Expert
Review of Anti-infective Therapy, 15:4, 387-400, DOI: 10.1080/14787210.2017.1271711

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EXPERT REVIEW OF ANTI-INFECTIVE THERAPY, 2017
VOL. 15, NO. 4, 387–400
http://dx.doi.org/10.1080/14787210.2017.1271711

REVIEW

Consolidation of molecular testing in clinical virology

Carolina Scagnolari, Ombretta Turriziani, Katia Monteleone, Alessandra Pierangeli and Guido Antonelli
Laboratory of Virology, Department of Molecular Medicine, and Istituto Pasteur Italia-Cenci Bolognetti Foundation, ‘Sapienza’ University of Rome,
Rome, Italy

ABSTRACT ARTICLE HISTORY

Introduction: The development of quantitative methods for the detection of viral nucleic acids have Received 3 June 2016
significantly improved our ability to manage disease progression and to assess the efficacy of antiviral Accepted 9 December 2016
treatment. Moreover, major advances in molecular technologies during the last decade have allowed KEYWORDS
the identification of new host genetic markers associated with antiviral drug response but have also Viral load; host genetic
strongly revolutionized the way we see and perform virus diagnostics in the coming years. factors; viral diagnostic;
Areas covered: In this review, we describe the history and development of virology diagnostic syndromic diagnosis; point
methods, dedicating particular emphasis on the gradual evolution and recent advances toward the of care; HIV; HCV; HPV; RSV;
introduction of multiparametric platforms for the syndromic diagnosis. In parallel, we outline the IL28B; ITPA
consolidation of viral genome quantification practice in different clinical settings.
Expert commentary: More rapid, accurate and affordable molecular technology can be predictable
with particular emphasis on emerging techniques (next generation sequencing, digital PCR, point of
care testing and syndromic diagnosis) to simplify viral diagnosis in the next future.

1. Introduction diagnostic virology can begin in the closing years of the nine-
teenth (1892) century with the demonstration made by
Diagnostic virology by definition is that which goes into for-
Guarnieri about the existence of some bodies in the proto-
mulating the diagnosis of viral disease [1]. Both clinical and
plasm of the epithelial cells of variolar and vaccinial lesions [2].
laboratory aspects are part of this discipline. Although discov-
At the beginning, Guarnieri considered that they were spor-
eries contributing to diagnostic virology began to accumulate
ozoa and he gave them the name ‘Cytoryctes vaccinse et
at the turn of the century, diagnosis and management of most
variolse’ [3]. Previously, Renaut (1881) described spherical glo-
of the major viral illnesses have been accomplished during the
bules within the epidermal cells of variolous lesions, but his
past 50 years. In particular, over the past two decades, there
communication was unaccompanied by figures. These bodies
have been substantial improvements in the molecular meth-
were subsequently named ‘Guarnieri’s bodies’ and the pre-
ods used to quantify viral nucleic acid in clinical specimens
sence or absence of the Guarnieri bodies is still considered
and in our understanding of how to use viral load measure-
an important feature in the diagnosis of a vaccinial lesion.
ments in the diagnosis and management of patients suffering
Concomitantly with the description of Guarnieri’s bodies, two
from viral infections. These methods are now integrated into a
botanists Dmitry Ivanovsky (1892) and, later, Martinus
wide range of diagnostic and antiviral treatment guidelines
Beijerinck (1898) published the discovery of the filterable nat-
and commonly deployed in a variety of clinical settings.
ure of the viral agent of tobacco mosaic disease which was
In this review, we provide a personal view about the history
first called by Beijerinck ‘Contagium Vivum Fluidum’ (Lecoq H)
and development of virology diagnostic methods from
[4–6]. In the meantime 1898, Friedrich Loeffler and Paul Frosch
ancient to modern times with the attempt to illustrate the
discovered that causative agent of foot-and-mouth disease is
advances and the evolutions and to show how change toward
filterable (the first animal virus) and in 1901, Walter Reed
qualitative and especially quantitative molecular methods has
discovered Yellow fever virus, the first discovered human
significantly influenced the viral diagnostic field. Moreover, we
virus [7]. Since then, several animal viruses causing viral dis-
discuss the importance of measuring the amount of viral
eases were soon identified. Below are reported some of the
nucleic acids in chronic and acute viral infections as well as
most representative viruses discovered in the twentieth cen-
the new emerging molecular approaches to make diagnosis in
tury: rabies virus (Remlinger, Riffat-Bay, 1903), chicken leuke-
different areas of virology.
mia virus (Ellerman, Bang, 1908), poliovirus (Karl Landsteiner
and E. Popper, 1909), measles virus (Goldberger and Lambert,
2. The history of diagnostic virology 1911), Rous Sarcoma virus (Rous, 1911), human immunodefi-
ciency virus (HIV, Luc Montagnier, and Francoise Barre-Sinousi,
Looking back at some of the key historical discoveries on the
1983),1 and hepatitis C virus (HCV, Choo and Houghton, 1989)
virology field (Figure 1), we believe that the history of

CONTACT Carolina Scagnolari carolina.scagnolari@uniroma1.it Laboratory of Virology, Department of Molecular Medicine, and Istituto Pasteur Italia-Cenci
Bolognetti Foundation, ‘Sapienza’ University of Rome, Viale di Porta Tiburtina 28, 00185, Rome, Italy
© 2016 Informa UK Limited, trading as Taylor & Francis Group
388 C. SCAGNOLARI ET AL.

(Panel A)
i) First use of the embryonated hen's egg for
virus cultivation (Goodpasture and Burnet)
Discovery of the causative agent of
foot-and-mouth disease (first
animal virus, Loeffler and Frosch) ii) Invention of the electron microscope i) Application of the immunofluorescent
(Ruska and Knoll ) antibody technique in diagnosis of virus
Discovery of Poliovirus (Gardner and Mcquillin)
(Landsteiner and Popper)

ii) First description of an

immunochromatographic
First growth of virus in cell
Discovery of rabies virus technique (Kohn)
culture (Poliovirus, Enders,
(Remlinger and Riffat-Bay) Robbins, Wellers)
Invention of
ultracentrifuge
(Svedberg)

1898 1903 1909 1925 1931 1949 1968

1892 1901 1908 1911 1929 1933 1951 1971

Description of complement-
Discovery of chicken leukemia fixation test for the diagnosis of
virus (Ellerman, and Bang) virus (Bedson and Bland)
i) Discovery of Guarnieri's
bodies (Guarnieri) Development of ELISA
(Engvall and Perlmann)
ii) Descrption of filterable
infectious agent
tobacco mosaic virus (TMV) Plaque assay of poliovirus
(Ivanosky) i) Discovery of measles virus Discovery of human (Dulbecco)
(Goldberger, Lambert) influenza virus (Smith)

Discovery of yellow fever ii) Discovery of Rous sarcoma

virus, the first human virus (Rous)
virus (Reed )

(Panel B) i) Massively Parallel Signature Sequencing

Development of TMA (MPSS) Lynx Therapeutics (USA) Company
(Kacian,and Fultz ) launched the first of the NGS technologies

ii) Development of loop-mediated isothermal

Discovery of the PCR amplicafion technology (Notomi)
(Saiki , Mullis ) First description
of the concept
behind digital US Food and Drug Administration
PCR (Sykes) announced permission for a multiplex
NAT, the xTAG® Gastrointestinal
Pathogen Panel (Luminex Corporation)

Development of DNA sequencing

with chain-terminating inhibitors Discovery of HCV
(Houghton) Development of the method
(Sanger and Gilbert )
of DNA pyrosequencing
(Nyren and Mostafa)

1977 1985 1989 1992 1995 1996 2000 2013

1975 1983 1987 1991 1993 1999 2006 2015

Development of kinetic
PCR analysis: first
real-time PCR
approach (Higuchi)
Discovery of AIDS virus
(Montagnier , Barré-Sinoussi) Description of virome capture
i) Development of sequencing enables sensitive viral
branched DNA First use of the digital diagnosis and comprehensive
amplification method PCR (Kinzler and virome analysis (Briese)
Discovery of hybridoma First appearance in
technology (Kohler and the literature of the for virus detection Vogelstein)
Milstein ) term “viral load” (Urdea)
(Salk)
Development of the commercial digital
ii) Development of PCR system using chip technology
NASBA (Compton) (BioMark™Fluidigm)

Figure 1. Milestones in the history of diagnostic virology. This is a timeline showing the major relevant findings in diagnostic virology from 1892 until now.

[7–9]. After the discovery of animal viruses, it is not surprising In 1929, Bedson and Bland were able to demonstrate spe-
that several attempts have been made to develop and set up cific complement fixation with herpes virus by using an
specific diagnostic tools to allow viral isolation and/or identi- immune guinea-pig serum and an antigen prepared from
fication. Use of the laboratory animal and eggs was the early guinea-pig pads [10]. In the meantime, Ruska and Knoll built
method of virus growth from patient material. However, culti- the first electron microscope (EM) in 1931 and starting from
vation of viruses in laboratory animals was difficult due to the 8 years later (1939), several viruses were visualized by the EM,
need of collecting the correct specimen and finding a suscep- decreeing the settlement of this technology in all the
tible host. branches of virology, including the diagnostic field [11].
Besides the use of eggs and animal for growing viruses, at Perhaps, one of the most significant single event in the devel-
the beginning, some serological reactions (i.e. complement opment of diagnostic virology was Enders and his colleagues’
fixation) were also used as first tool to make diagnosis of success in growing poliovirus in nonneural cell culture in 1949
viral infections [10]. [12,13]. Of relevance in this respect was also the development

of a plaque assay for animal viruses in 1951 by Dulbecco [14].
By the 1960s, the fluorochrome-labeled antibody reagents
were used to visualize viruses in patients specimens and tissue
culture [15,16]. In the early 1970s, Engvall and Perlmann pub-
lished their first paper on enzyme-linked immunosorbent
assay [17]. Moreover, in 1975, the discovery of the hybridoma
technology and the subsequent use of monoclonal antibodies
resulted in the significant improvement of techniques to diag-
nosis of viruses by using immunologic methods [18]. However,
such assays were time consuming, or in the case of determi-
nation of viral antibody response required sera collected 10 or
more days from the onset of infection. In both cases, the tests
frequently provided a diagnosis in retrospect. In the mean-
time, DNA sequencing with chain-terminating inhibitors was
developed by Sanger in 1977 [19]. The discovery of the poly-
merase chain reaction (PCR) in 1985 [20,21] revolutionized
several diagnostic areas, and especially the identification of
viruses benefited from this new genetic technology. After the
discovery of PCR, several methods have been also developed
for the quantification of viral genomes between 1990 and
2000 such as the ligase chain reaction (LCR), branched chain
signal amplification (bDNA), nucleic acid sequence-based
amplification (NASBA), loop-mediated isothermal amplification
(LAMP), transcription-mediated amplification (TMA), and real-
time PCR [22–25]. Through the use of quantitative molecular
methods, we have seen the rise of the concept of ‘viral load’ in
clinical virology, after its first appearance in the literature in
1987 in a report published by Jonas Salk on HIV-1 infection
[26]. In the last years, besides the widely implementation of
quantitative real-time PCR for clinical viral load testing, we
have also seen the introduction into the market of new mole-
cular techniques or assays such as the digital PCR (dPCR) [27],
real-time TMA nucleic acid amplification test for viral mRNA
quantification [28,29], and the novel DNA sequencing techni-
ques, referred to as ‘next-generation’ sequencing (NGS) [30].
Directly related to the latter aspect, a highly multiplexed plat-
form based on NGS technology, namely ‘the virome capture
sequencing platform for vertebrate viruses’ (VirCapSeq-VERT),
has been described in 2015 that may facilitate the transition of
high-throughput sequencing to clinical diagnostic applications
[31]. Indeed, the VirCapSeq-VERT simply and efficiently
enables to simultaneously identify and characterize all
known vertebrate viruses, their genetic variants, and novel
viruses [31].
It is also not surprising that through the significant
advances in molecular technologies, several critical factors
emerged as determinants in ensuring the viral diagnostic
success at the point of care (POC), underlying the need of
new molecular approaches to meet the infrastructure and
workflow limitations in the specific clinical settings in both
the developed and developing world [32].
It must be considered that the need to make point-of-
care testing (POCT) diagnosis emerged just in the last cen-
tury, having been described by Briedigkeit et al. in 1998
[33], as a set of different approaches aimed to offer poten-
tially substantial benefits for the management of a specific
disease. The definition of POCT diagnosis made by
Briedigkeit included laboratory investigation performed: (1)
EXPERT REVIEW OF ANTI-INFECTIVE THERAPY 389
near the patient, (2) with measurement systems that are
rapid and easy to operate, (3) in the context of direct
patient care with therapeutic relevance in patients at risk
of death, (4) within departments for in-patients, (5) out-
patient clinics or special functional areas (e.g. emergency
admissions, operating theater, delivery room, endoscopy
unit, and interventional radiology), and (6) by personnel
who have in general had no detailed training as medical
technical assistants and no experience in laboratory medi-
cine [33]. It is evident that the major advancement of using
POCT diagnosis is to short the time to result and to make
the test available at the bedside or at remote care centers.
Some examples can be made about the use of POCT in viral
diagnosis such as HIV-1 [34], influenza virus [35], viral hepa-
titis [36,37] and also for emerging virus such as Ebola
virus [38].
Of relevance in this context, the US FDA granted in 2015
first CLIA waiver for nucleic acid-based influenza diagnostic
test (Alere i Influenza A & B test) which can now be used by
health-care providers in nontraditional testing sites (e.g. phy-
sician offices, emergency rooms, health department clinics,
pharmacy clinics, and other health-care facilities).
Furthermore, alongside the urgent need of implementing
molecular POCT in viral diagnosis, more recently, the FDA
announced permission in 2013 for a multiplex nucleic acid
test, namely the xTAG® Gastrointestinal Pathogen Panel
(Luminex Corporation, USA), which can be used for the
simultaneous high-throughput detection of multiple viral,
parasitic, and bacterial gastrointestinal pathogens of clinical
and public health importance, indicating inexorably a new
significant change in diagnostic virology toward the new
concept of ‘diagnosis of infectious syndromes.’ A variety of
molecular techniques (e.g. real-time multiplex PCR, DNA
microarray, LAMP) have been examined for diagnosis of
infectious syndromes [39]. Today, several multiplexed in-
vitro diagnostic tests designed for the simultaneous, rapid
detection of common agents of community-acquired menin-
gitis/encephalitis, respiratory, and gastrointestinal tract
infections have been approved by FDA and are commer-
cially available [39–41]. The above emerging diagnostic area
integrated with the POCT approach will inevitably create in
the coming years the beginning of a new era of making
diagnosis.
3. Viral quantification and new molecular
assessment in clinical virology
The ability to detect nucleic acids has had and still has a
major impact on diagnostics in clinical virology. Both
quantitative and qualitative techniques are currently used
routinely in most if not all virology laboratories. The main
clinical utility of nucleic acid test (NAT) in virology field is
illustrated in Table 1.
More importantly, through the introduction of quantita-
tive molecular methods for virus detection, we have wit-
nessed a preponderant consolidation of the importance of
the determination of the amount of viral acid nucleic pre-
sent per volume of clinical specimen (known as the viral
390 C. SCAGNOLARI ET AL.

Table 1. Detection and quantification of viral genome in clinical practice.

Target Specimen(s) Main techniques used Application(s)
HIV-1 (DNA) Blood, peripheral blood In-house real-time PCR, in- Diagnosis of perinatal infection
mononuclear cells house digital droplet PCR
HIV-1 (RNA) Plasma, seruma Real-time PCR, TMAa Viral load (prognosis, response to ART treatment)
HSV 1, 2 CSF ocular fluid, swabs from PCR, multiplex PCR, real-time Diagnosis of encephalitis, meningitis, retinitis,
mucocutaneous lesions, PCR mucocutaneous lesions, and neonatal infection
blood
VZV CSF, ocular fluid, swabs from Real-time PCR Diagnosis of encephalitis, meningitis, retinitis,
mucocutaneous lesions, mucocutaneous lesions, pulmonary infections,
broncho-alveolar lavage, congenital infections
amniotic fluid
CMV Blood, plasma CSF, ocular PCR, real-time PCR Diagnosis of systemic infection after organ
fluid, amniotic fluid transplantation, encephalitis, radiculomyelitis,
retinitis, congenital infection. Viral load can be used
for the clinical management of transplant recipients
EBV CSF, blood, plasma PCR, real-time PCR Primary CNS lymphoma in AIDS, other CNS infections,
posttransplant lymphoproliferative disorders
HHV-6, 7 Serum, plasma, CSF PCR, real-time PCR Diagnosis of systemic infection and of encephalitis
HHV-8 (Kaposi’s sarcoma virus) Blood, pleural or pericardial Real-time PCR Diagnosis of body cavity lymphoma
fluid
Adenovirus Blood, respiratory secretions PCR, multiplex PCR, real-time Diagnosis and monitoring of disseminated infection in
PCR immunocompromised hosts, diagnosis of respiratory
infection
Parvovirus B19 Serum, amniotic fluid Diagnosis of chronic parvovirus B19 infection, aplastic
crisis, congenital infection
JCV CSF PCR, real-time PCR Diagnosis of progressive multifocal
leukoencephalopathy (PML)
BKV Urine, plasma PCR, real-time PCR Assessment of risk of nephropathy in renal transplant
recipients and of hemorrhagic cystitis in
hematopoiteic stem cell transplant recipients
HPV Anogenital samples, biopsies PCR, liquid hybridization, HPV Diagnosis of HPV infection, primary screening for
mRNA detection cervical cancer
Enteroviruses CSF, blood (whole blood, PCR, real-time PCR Diagnosis of meningitis or systemic infection
serum, or plasma)
Rabies Saliva, CSF PCR Diagnosis of rabies (not commonly used)
Rotavirus Stool PCR Diagnosis of rotavirus infection (not commonly used)
HCV Plasma, serum Real-time PCR, TMA Diagnosis of HCV infection. Viral load is used to assess
response to therapy
HBV Plasma, serum Real-time PCR, TMA Diagnosis of HBV infection when serologic studies are
ambiguous. Recognition of virologically active
infection. Viral load is used to assess response to
therapy and recognize emergence of drug resistance
HEV Serum, stool Real-time PCR, LAMP Diagnosis of HEV infection
Respiratory viruses Respiratory samples PCR, multiplex PCR, real-time Diagnosis of respiratory viral infection
PCR, multiplex real-time
PCR, syndromic panels
DENV Blood, serum, or plasma PCR, real-time PCR Diagnosis of dengue infection (not commonly used)
Yellow fever virus Serum or plasma PCR, real-time PCR Diagnosis of yellow fever infection (not commonly used)
Chikungunya virus CSF, serum, or plasma Real-time PCR Diagnosis of chikungunya infection
Ebola virus Blood, serum, or plasma PCR, real-time PCR Diagnosis of Ebola infection
WNV CSF, serum, or plasma Real-time PCR, TMA Diagnosis of WNV infection (not commonly used);
diagnosis of WNV neuroinvasive disease, screening of
blood units
ZIKV CSF, serum or plasma, PCR, real-time PCR Diagnosis of ZIKV infection
amniotic fluid, urine
a
TMA/SEROM. HIV: human immunodeficiency virus; TMA: transcription mediated amplification; HSV: herpes simplex virus; VZV: varicella zoster virus;
CMV: cytomegalovirus; EBV: Epstein–Barr virus; CNS: central nervous system; AIDS: acquired immunodeficiency syndrome; HHV: human herpes virus;
CSF: cerebrospinal fluid; HPV: human papillomavirus; JCV: John cunningham virus; HCV: hepatitis C virus; HBV: hepatitis B virus; HEV: hepatitis E virus;
DENV: dengue virus; WNV: West Nile virus; ZIKV: Zika virus.

load). In particular, viral load is generally defined as number
of virus particles present in the bloodstream, and other
body districts, usually expressed as nucleic acid copies per
milliliter. This measurement helps the clinicians to prognos-
ticate the natural history of a viral infection, in treatment
decisions, and to monitor the efficacy of a treatment.
Viral load measurement is currently considered essential
for the clinical management of the viral hepatitis (e.g. HCV
and hepatitis B virus [HBV]), HIV-1 and for monitoring
citomegalovirus (CMV) and Epstein–Barr virus (EBV) infection
or reactivation in immunocompromised and/or cancer
patients [43–46]. However, interferon (IFN)-free direct-acting
antiviral (DAA) therapy sets the stage to simplify diagnostic
requirements for patients suffering from chronic hepatitis C
and the clinical benefit of measuring HCV load for monitor-
ing the response to antiviral therapy could be reevaluated
with the new pan-genotypic DAAs [47]. Although viral load
testing in viral infections is an early example of how

molecular testing has increased our understanding of a
disease process and improved patient care, its direct appli-
cation to all viral infections is a matter of debate. Indeed,
for instance, viral load determination has been recently
shown to be a potential prognostic factor of the clinical
severity of some respiratory viral infections such as those
associated to respiratory syncytial virus (RSV), or influenza
virus [48–52]. However, the importance of measuring RSV or
influenza RNA load in the clinical setting of respiratory
diseases is still debated and warrants further investigations.
Moreover, viral load impact in infections sustained by
human papillomavirus (HPV) is not clear [53–60].
Below there is a description of two chronic viral infec-
tions, HCV and HIV-1 (RNA viruses), in which viral load is
currently used as a prognostic marker of disease severity,
and it is applicable as indicator for the initiation, monitor-
ing, and modification of antiviral treatment. In addition, the
significance of studies about the use of viral load in HPV
(DNA virus) and RSV (RNA virus) infections as marker of
clinical severity together with the importance of new emer-
ging molecular assessments in clinical virology is also
discussed.
3.1. Human immunodeficiency virus
It has now well established that viremia has important
implications for the clinical outcome of HIV-1 infection as
well as for the therapeutic management of individuals
infected with HIV-1. The association of plasma viral load
with stage of HIV-1 infection was demonstrated in the
early 1990s. In 1996, Mellor by using a bDNA assay to
quantify HIV-1 RNA in plasma demonstrated for the first
time that plasma viral load was a better predictor of pro-
gression to AIDS and death than was the number of CD4+ T
cells [61]. Furthermore, in the same year, it became clear
that viral load could be used to monitor the drug efficacy in
HIV-1-infected patients [62]. To date, viral load measure-
ments are used to determine the risk of disease progression,
monitor response to treatment, and detect viral break-
through as a marker of regimen failure. The goal of antire-
troviral therapy (ART) historically followed the limit of
detection (LoD) of the test used to measure HIV RNA levels.
Following the introduction of viral load monitoring in clin-
ical practice in 1996, the target level of suppression has
been defined by the technical properties of commercial
assays rather than by biological considerations and was
400–500 copies/ml with first-generation tests, and later
50–75 copies/ml with second-generation tests. Currently, in
clinical practice, the cutoff of 50 copies per milliliter is used
[63, European AIDS Clinical Society (EACS, 2015) guidelines].
However, after the introduction of third-generation viral
load assays with increased sensitivity and a LoD below
50 copies/ml, this cutoff has become a matter of discussion
[64]. The third-generation tests, based on real-time PCR
method, show a lower LoD of 40 or 20 copies/ml and also
report qualitative RNA detection below these cutoffs. The
lower LoD of these assays may result in increased measure-
ments of transient and intermittent detectable viral RNA
(‘blips’) in patients that have reached virological suppression
EXPERT REVIEW OF ANTI-INFECTIVE THERAPY 391
[65,66]. There is controversy about the significance and
consequences of viral blips. Some authors did not found
any relationship between isolated blips and virological fail-
ure [67,68]; on the contrary, other authors reported that
patients with viral blips had an higher risk of virological
failure [69,70]. In addition, the introduction of these assay
in clinical practice, capable of detecting HIV RNA also below
LoD, have shown that some patients receiving ART have
detectable RNA in plasma below the clinical cutoff of
50 copies/ml [71,72]. The source and dynamics of persistent
viremia in treated patients remain uncertain. It has been
proposed that low-level viremia may be the result of
ongoing viral replication in patients, which is caused by
incomplete viral suppression during ART. An alternative
hypothesis is that the residual amount of HIV-1 RNA may
be the result of virus release from long-lived latently
infected cells that are activated by stochastic antigen sti-
mulation. Several papers have reported that genetically
homogeneous viral subpopulations can often be observed
in patients on long-term treatment and in the viral popula-
tion that rebounds during treatment interruptions. These
findings further support the concept that persistent low-
level viremia arises from long-lived cells rather than
ongoing viral replication [73,74]. A further line of investiga-
tion has focused on anatomical compartments that may
serve as ‘sanctuary sites,’ such as the central nervous system
and the genital tract, in which HIV-1 replication can occur
unhindered by poorly penetrating antiretroviral agents. The
clinical relevance of residual viremia remains to be defined,
as well. Some authors showed that residual viremia
enhanced the chance of viral rebound increasing the risk
of virological failure [75,76]; others observed an association
between residual viremia and the development of viral blip
and low-level viremia [77]. In addition, a relationship
between residual viremia and the size of viral reservoir has
been reported [78,79]. There is also growing interest in the
chronic inflammatory component of HIV-1 infection, which
can be observed among those who have residual viremia
[80–82]. The persistency of low-level residual viremia repre-
sents a continuous pro-inflammatory stimulus for the
immune system, which underlies chronic immune activation
and inflammation. However, whether residual low-level vir-
emia plays a key role in increasing the inflammatory status
in patients is unclear, and further studies are needed to
better clarify this important issue. Although viral load is
the most important parameters to measure before and dur-
ing ART, HIV laboratory monitoring includes other para-
meters that have to be evaluated to ensure virologic
suppression and assess the safety of treatment. HIV geno-
type should be used to guide the selection of an antiretro-
viral regimen; this test should be performed prior to
initiation of ART and, obviously, when a virological failure
is observed in patients receiving ART. Before initiation of a
CCR5 antagonist or at the time of virologic failure in a
patient receiving a CCR5 antagonist, a viral tropism assay
should be performed. Finally, a screening test for the HLA-
B*5701 allele can assist clinicians to identify patients who
are at risk of developing a hypersensitivity reaction to
392 C. SCAGNOLARI ET AL.
abacavir [62,83, EACS 2015]. Patients who are positive for
the HLAB*5701 haplotype should not be treated with
abacavir.
Although molecular tests are widely used in the monitoring
of HIV infection, it is worth mentioning that NAT are recom-
mended by CDC (Center for Disease Control and Prevention) in
the HIV diagnostic algorithm [84]. Approximately 10 days after
infection, HIV-1 RNA becomes detectable by NAT in plasma and
quantities increase to very high levels. Specimens that are
reactive on the initial antigen/antibody combination immu-
noassay and nonreactive or indeterminate on the HIV-1/HIV-2
antibody differentiation immunoassay should be tested with an
FDA-approved NAT. The results of this algorithm may be used
to identify persons likely to benefit from treatment, to reassure
persons who are uninfected, and for reporting evidence of HIV-
1 infection to public health authorities.
3.2. Hepatitis C virus
HCV RNA can be detected in blood as early as 2–3 weeks after
infection and its levels remain relatively stable over time in
chronically infected patients; indeed, the presence of HCV RNA
at 6 months identifies patients who will not spontaneously
clear infection [85]. Serum HCV RNA levels represent a marker
of HCV replication. In past, the standard of care for HCV
infection consisted in the combination of pegylated IFN
(PEG-IFN) plus ribavirin (RBV) and monitoring of HCV RNA
levels was used to evaluate the efficacy of treatment.
Although first definitions of responses to IFN treatment were
based on the evaluation of serum alanine aminotransferase
levels, the development of PCR-based assays allowed to mea-
sure the viral load in the serum, making HCV RNA levels the
main predictive marker of treatment outcome [86]. The study
of viral dynamics during antiviral therapy in other chronic viral
infections, such as HIV-1 [87,88] and HBV infections [89], has
turned out useful to obtain new insights into the pathogenetic
mechanisms of the viral infections and in guiding clinical
decision-making; then, several studies were designed to ana-
lyze the kinetics of HCV during IFN therapy [90,91]. In particu-
lar, it has been shown that the infection with HCV is highly
dynamic and that a biphasic decline of viral load occurs after
IFNα administration: the first-phase slope is mainly determined
by the free virion clearance rate while the second one is
determined by the infected cell death rate and more affected
by the interindividual variability [91]. Moreover, it was also
reported that the baseline serum viral load does not influence
the rate of early HCV RNA reduction after a single IFNα admin-
istration, although a strong variability in the viremia reduction
was recorded in HCV-positive patients [92]. Altogether these
findings suggested that early monitoring of viral load could
help guide therapy, even if HCV clearance could depend on
still unknown host factors [93]. In this regard, one of the most
revolutionary discovery in the area of HCV infection, within the
past years, has been that single nucleotide polymorphisms
(SNPs) related to IFN lambda (L) genomic region are highly
predictive of both spontaneous clearance of HCV and
response to IFNα and RBV therapy. These SNPs are related to
two different type III IFN subtypes (IFNλ3 or interleukin-28B
and IFNλ4) [94]. Consequently, the molecular evaluation of
IFNL SNPs, in particular IL-28B rs12979860, is recommended
to guide IFNα and RBV treatment for patients with chronic
HCV infections. Interestingly, an association between IL-28B
genotype and early HCV RNA reduction during the treatment
with PEG-IFNα/RBV has been observed [94–96]. The goal of
HCV treatment is to obtain a sustained virological response
(SVR) that was defined as the lack of detectable HCV RNA
24 weeks after the end of the therapy and reflects the eradica-
tion of HCV infection in >99% of cases [97]. The levels of HCV
RNA measured at select time points have been used to define
the likelihood of treatment success and make decisions
regarding the continuation of IFN-containing therapy. First of
all, values of viral load measured at baseline have been shown
to be correlated to response to IFNα and RBV treatment; in
particular, high levels of viral load were associated with low
rates of response [98]. Patients achieving a rapid virological
response (HCV RNA negative at treatment week 4) or an early
virological response (EVR, defined as a reduction of HCV RNA
>2 log10 compare to HCV RNA baseline, at week 12) have a
higher probability of achieving SVR while the lack of EVR
suggests that the patient has virtually no chance of obtaining
a SVR and should stop treatment. Patients were described as
nonresponders when they fail to clear HCV RNA after 24 weeks
of therapy [98]. The development of many new DAAs that
target specific steps of the HCV life cycle and are characterized
by high tolerability and efficacy allowed to simplify and
shorten treatments for HCV infection [99]. Their introduction
led to the need of revising the mathematical model previously
used to describe the HCV RNA decline kinetics observed under
therapy [100]. Indeed, the administration of some DAAs, in
particular protease inhibitors, was associated to a much faster
viral decline than both IFN-based therapy and nucleoside and
nucleotide polymerase inhibitors [101–104] that can be due to
the restoration of innate immune responses within infected
cells and the following loss of intracellular viral RNA [105]. In
this regard, an association between IFNL4 dinucleotide poly-
morphism and HCV kinetics during DAAs treatment has been
also proposed [106]. Although these findings need to be con-
firmed in treatment experienced or cirrothic patients, not
included in clinical trials, the development of a multiscale
model that include both intracellular viral replication and
extracellular spread can be used to gain further information
on viral kinetics [100] and help to define the length of thera-
pies. Furthermore, the utility of HCV RNA assessments in pre-
dicting and monitoring the clinical efficacy of new pan-
genotypic DAA and to guide clinical decisions will be securely
reevaluated and may be lost in the future, due to the high
efficacy of these new anti-HCV regimens [47]. In this regard,
HCV antigen measurement should be considered as a poten-
tial alternative for monitoring treatment response during DAA-
based regimens [47]. Although there is a need of revaluating
the clinical utility of determination of HCV RNA to monitor the
outcomes of new DAAs, CDC has recently included HCV RNA
testing as part of their recommended diagnostic algorithms
[107]. Alongside the HCV RNA testing, there are other mole-
cular assessments which may help clinicians in the manage-
ment of HCV infections. In this context, it is known that

prediction of anemia, which may have deleterious impact on
SVR, remains important also in the era of DAAs, because these
newer therapies still require RBV. Therefore, the analysis of
two functional SNPs, rs1127354 and rs7270101, within the
inosine triphosphate pyrophosphatase (ITPA) gene that were
strongly associated with protection from RBV treatment-
related hemolytic anemia should be recommended [108]. In
addition, it is important to underline that DAAs can provide
opportunities to reduce laboratory monitoring requirements
over than viral load. Until now, HCV genotyping and in specific
circumstances genotype-1 subtype (1a/1b) determination has
been a precondition for starting old and new anti-HCV treat-
ment, to determine the duration of therapy, and the prospects
of success. Indeed, HCV genotype was relevant to IFN/RBV
treatment and also with the first generation of DAAs (telapre-
vir and boceprevir). If a pan-genotypic DAA combination regi-
men, efficacious against all HCV genotypes, can be developed
and a single treatment duration established, this will remove
the need for genotyping. Lastly, it is important to point out
that a heterogeneity within NS3, NS5A, and NS5B genomic
areas interacting with DAAs has been well documented
between HCV genotypes/subtypes [109]. In this regard, while
the two HCV genotype 1 subtypes appear equally responsive
to PEG-IFN/RBV [110], the response rates and resistance pat-
terns for approved and investigational DAAs are different for
HCV genotype 1a compared with genotype 1b [111]. For
example, HCV NS3 polymorphism Q80K is mainly found in
patients with HCV genotype genotype 1a and has been asso-
ciated with a reduced treatment response to simeprevir [112].
Similarly, L31M and Y93H, key resistance mutations to NS5A
inhibitors, are frequently found (6–12%) among NS5A geno-
type 1 sequences [113]. These differences, however, could be
attenuated with very potent high-resistance barrier combina-
tion. However, even with new IFN-free all-oral regimens, HCV
genotyping/subtyping is likely to remain valuable for the fore-
seeable future, at least for certain subgroups.
3.3. Human papillomavirus
It is well established that a persistent infection with HPV is a
key event in cervical carcinogenesis [114]. HPVs are small,
double-stranded DNA viruses that infect epithelial tissues.
Mucosal HPV genotypes are classified into low-risk types that
cause benign lesions and high-risk (HR) types associated with
anogenital cancers [114,115]. Most HR HPV infections are
thought to be cleared by the host in a period from about 1–
2 years; if immune responses do not succeed in clearing HR
HPV infection during a certain period of time, the risk of
cancer progression increases several folds [116,117]. Clinically
relevant dysplastic changes, classified as cervical intraepithelial
neoplasia (CIN) 1–3, may occur 1–3 years after infection; they
may regress or take another 8–12 years for development of
invasive disease [118]. Molecular tests that detect HR HPV
infections constitute a consolidated approach more sensitive
than cytology alone in cervical cancer screening, with a higher
negative predictive value [119,120]. Moreover, HPV-based
tests are used for management or follow-up of abnormal
cervical cytology or histology. Numerous HPV-DNA assays are
commercially available but only a few are clinically validated
EXPERT REVIEW OF ANTI-INFECTIVE THERAPY 393
and approved for screening or diagnosis. They rely on differ-
ent molecular techniques of template and/or signal amplifica-
tion and fluorescence detection [119] and widely differ in
genotyping information. Many of them test HR HPV types
altogether without genotyping or can provide genotyping
information for a few HR types, mostly HPV16 and HPV18
[119,120]. A complete genotyping is not required in primary
screening and is used in a research context or in epidemiolo-
gical studies. As an unwanted consequence of a wider use of
HPV-DNA based tests, overtreatment of HPV-positive early
dysplastic lesions may have a negative impact on reproductive
outcomes. Therefore, it is very important to use biomarkers
able to identify those HPV-persistent infections with a HR to
develop cervical cancer. As discussed above in this review,
viral load has been established as a clear marker of disease
severity and progression rate in HIV-1, HBV, and HCV infec-
tions. Viral load in HPV infections has been an attractive
candidate as a marker of risk for years and dozens of studies
analyzed its association with cancer risk [53–60]. Positive asso-
ciations of HR HPV DNA load with risk of persistent infection
and cervical precancer lesions have been reported in some
studies [56] but not in others [57–60]. It became evident that
viral load measured at a single time point is a poor predictor
of the HPV infection risk to develop cancer [121,122]; there-
fore, viral load has a limited use for screening or diagnostic
purposes. Different possibilities in molecular diagnostics were
opened by unraveling the complex interactions between HR
HPVs and the infected host. Integration of HR HPV into the
host-cell genome is considered a critical event in the patho-
genesis of cervical neoplasia because of disruption of the HPV
regulatory E2 gene leading to E6/E7 overexpression.
Considering that HPV-DNA testing has high sensitivity but
low specificity in screening, triage of ASCUS (atypical squa-
mous cells of undetermined significance), and in follow-up
after treatment, these processes can be monitored through
biomarkers specific to each of the steps in the progression
toward neoplastic changes. In particular, detection of E6 and
E7 mRNA levels, HPV genome integration status, and cellular
protein detection could help in prediction of premalignant
lesions evolution [114,117,119]. Testing for E6/E7 mRNA
might be more specific than HPV-DNA testing alone; more-
over, collection media for liquid-based cytology can preserve
RNA sufficiently to allow transcript detection, after a first
screening test [119,123,124]. Commercial tests have been
approved for testing HR HPV E6/E7 mRNA, utilizing NASBA or
TMA [119,123–125]. E2/E6 DNA copy number ratio is often
considered a proxy for HPV integration. If only the episomal
form is present, the E2/E6 ratio would be close to 1, and if only
the integrated form was present, the E2/E6 ratios would be
close to zero; if the integrated form and episomal form are
mixed, the E2/E6 ratios would be intermediate. Several studies
considered E2/E6 ratio as a potential biomarker of oncogenic
potential for its correlation with lesion histological grade
[126,127]. However, other study did not support HPV integra-
tion status to be considered as potential biomarkers of cervical
disease [128]. Therefore, before introducing HPV integration as
a biomarker into clinical service, further studies are needed to
determine how often E2 is disrupted during the natural history
of HPV infection [129]. In addition to viral genes, biomarkers of
394 C. SCAGNOLARI ET AL.
the cellular counterpart may be utilized as indicative for the
presence of proliferative lesions. Overexpression of cyclin-
dependent kinases 4 and 6 (CDK4/CDK6) inhibitor p16
(p16INK4a)-protein, and of the proliferation marker Ki-67,
points to HPV-induced deregulation of the cell cycle; their
immunohistochemical detection as a surrogate marker of
oncogenic HPV process entered clinical use long ago but it’s
not as sensitive as most molecular-based methods [130].
Recent improvements include contesting of p16 and Ki-67
proteins within the same cervical samples used for conven-
tional or liquid-based cytology [131]. New technologies could
be the next step in the search of better molecular markers.
Methylation profiles in cell and viral genes [132], genomic
changes (losses and/or gains in specific chromosomal regions)
[133], miRNAs aberrant expression [134], and telomerase activ-
ity deregulation are presently understudy for determining
association with lesion grade [135]. Molecular markers either
alone or in combination with more traditional techniques
seem to have the potential to further improve diagnostics,
management, and treatment of cervical dysplasia and cervical
cancer. A remarkable improvement would be HPV detection
and genotyping tests applicable in POC settings, eventually
together with other Sexually Transmitted Infections [136].
3.4. Respiratory syncytial virus
Viral load may provide important information about the
interaction between the infective agent and the host. The
information about the possible correlation between viral
load, epidemiological characteristics, and the severity of
respiratory disease is scarce if compared to that available
for chronic viral infections. Therefore, the best clinical man-
agement of conventional and emerging respiratory viral
infections is currently limited to the rapid and accurate
detection of the viral genome in clinical specimens by
single and multiplexed PCR techniques [137,138]. In the
last few years, several multiplex molecular assays have
been developed for the detection and quantification of
respiratory viruses directly from clinical specimens [139].
Moreover, the need to perform broad-spectrum analysis
through multiple microbial diagnosis test panels for patho-
gens in patients with respiratory tract infections is becom-
ing more relevant as the number of potential infectious
agents is still increasing. Some multiparametric platforms
for the diagnosis of respiratory syndrome are already com-
mercially available. An examples is represented by the
TrueScience™ RespiFinder® Identification Panels developed
by Applied biosystem which use a multiplex PCR test that
can detect up to 19 of the most common respiratory patho-
gens (14 RNA viruses, 1 DNA virus, and 4 bacteria) including
influenza virus, RSV, parainfluenza virus, and Bordetella per-
tussis. Alongside the classical and new diagnostic approach
for detection of respiratory viruses, viral load at diagnosis
has been emerging as an important indicator of disease
severity in some respiratory viral infections such as those
sustained by influenza virus (H5N1 and H1N1) [48–51], and
RSV (see below). The latter represents the most common
cause of bronchiolitis [140], a frequent pathology associated
with lower respiratory tract infection in newborns and
young infants. Severe bronchiolitis early in life is also asso-
ciated with an increased risk of asthma, especially after
rhinovirus or RSV bronchiolitis, and an increased risk of
asthma may persist into early adulthood. The clinical sever-
ity of RSV bronchiolitis is likely to be influenced by both
host and viral factors. In particular, besides well-known risk
factors for severe RSV disease such as prematurity, lung or
heart disease, and immunodeficiencies, a pivotal role for
viral load has been recently proposed [140–146]. However,
the influence of RSV RNA levels on determining the bronch-
iolitis severity is not fully addressed, even if several studies
reported a significant association between disease severity
and RSV load measured in nasopharyngeal secretion of
infants with primary respiratory tract infection [141–146].
We also found that in previously healthy infants with high
levels of RSV RNA in their nasopharyngeal washes, there
was a tendency to have more signs of severe respiratory
distress [52]. Moreover, viral load levels were associated
with high airway levels of IFN-stimulated gene (ISG)56 [52],
suggesting that RSV replication may influence the magni-
tude of airway innate immune activation which in turn can
determine the clinical course of bronchiolitis. Interestingly,
an association between viral load, IFNλ, and recurrent
wheezing 36 months after the first episode of RSV bronch-
iolitis was also observed [147]. In particular, higher RSV RNA
load and higher levels of IFNλ2/3 were found in children
with recurrent wheezing at 36-month follow-up, suggesting
that chronic epithelial reactivity changes in the still imma-
ture and developing lung of young infants and that IFNλ
activation at the time of admission for bronchiolitis was
ineffective in controlling the high viral load [147,148].
These observations indicate that the measurement of RSV
load in respiratory clinical specimens could be useful to
improve the clinical management of bronchiolitis and recurrent
respiratory diseases. However, besides the increased evidences
about the existence of an association between RSV RNA
amounts and respiratory disease severity in children, other
authors failed to detect such an association [50,149–151].
Altogether, these findings highlight the need of implementing
molecular diagnostic tools for a rapid, sensitive, and quantita-
tive RSV detection. Further larger scale studies are also urgently
needed to support the predictive value of measuring RSV load
on the clinical course of bronchiolitis and to establish the actual
‘cutoff value’ of RSV load associated to disease severity.
4. Expert commentary
It is known that the main goal of a clinical virology laboratory
is to assist clinicians in the diagnosis and treatment of viral
diseases and to support infection control specialists in their
tasks. Therefore, it is not surprising that over the last decade,
there was need to provide simple methods for a rapid identi-
fication of the etiological agents. The answer came from the
fast development of NAT. These molecular techniques are
currently used in the diagnosis and clinical management of a
wide array of viral infectious diseases. Technological improve-
ments, from automated sample isolation to real-time PCR plat-
form, have given the ability to develop and introduce systems
for most viruses of clinical interest and to obtain clinical

relevant information to be used for the management of
patients. In this regard, it is well established that qualitative
virus detection can reveal merely the presence or absence of a
viral pathogen and this might be not sufficient in a variety of
clinical situations. Indeed, in many instances, the rate of dis-
ease outcome was shown to be related directly to the viral
DNA or RNA levels detected in clinical specimens. Therefore, a
number of quantitative molecular procedures, especially those
based on the real-time PCR technology, have been developed
to allow accurate and rapid quantitative detection of viral
nucleic acids in biological fluids. Through these molecular
quantitative assays, our understanding of the natural history
HIV-1, HBV, and HCV infections has been greatly facilitated by
accurate determinations of viral and infected cell burden.
Moreover, quantitative determination of viral load in infected
individuals has also been useful to monitor the antiviral ther-
apy efficacy and to predict treatment failure and the emer-
gence of drug-resistant viruses. It is important to underline
that alongside the consolidate association between disease
progression and HIV-1 or hepatitis viruses viremia, the need
to quantitatively monitor infections with CMV and EBV has
been long also appreciated in immunosuppressed patients
[43–46]. The clinical relevance of a number of other viral
infections in this setting is less well established, but there is
a growing body of evidence indicating that viruses such as
adenovirus [152,153], human herpes virus 6 [154], varicella–
zoster virus [155], and parvovirus B19 [156] merit also careful
monitoring in these patients. Another clinically significant
application of viral load detection is the possibility of distin-
guishing between latent infection and reactivation. Indeed,
the detection of viral pathogens causing persistent infection
by qualitative PCR causes consistently positive results and it
may not be determinant to follow the clinical outcome in
particular situations such as those characterized by an immu-
nosuppression; consecutive assessments of the viral genome
levels seen to play an important role in the diagnosis and
prognosis of patients with viral reactivation by providing a
basis for timely initiation of appropriate treatment [157].
Therefore, molecular diagnostic assays for quantification of
viral load are of increasing clinical importance and impact.
5. Five-year view
What about the future? Besides the consolidation of quan-
titative detections methods for most of the persistent virus
infections as clearly documented by the transition from the
use of laboratory-developed tests to FDA-cleared tests to
measure viral load, future work will surely delineate whether
the detection of viral RNA and DNA molecules may be
useful for the clinical management of other human viral
infections. Indeed, for many viral diseases, the clinical sig-
nificance of a viral load reading detected at an anatomical
site at a particular time point in a patient’s infection
remains unclear. Furthermore, more rapid and accurate
molecular technology can be anticipated, with particular
emphasis on new emerging NGS and dPCR technologies.
Such developments have already produced key advances
in the clinical virology but also have the potential to
strongly modify the way we see and perform virus
EXPERT REVIEW OF ANTI-INFECTIVE THERAPY 395
diagnostics in the coming years. New and more effective
antiviral treatment, more commercial kits, and better quality
control as well as office and home testing can be surely
expected. Certainly, the recent availability of multiple com-
mercial dPCR platforms has led to further increased interest
in low viral load detection and low abundance mutant
detection, where dPCR could be superior to traditional
quantitative real-time PCR. Furthermore, new sequencing
methods have enabled metagenomics, including virome,
microbiota, and trascriptome studies, and are of increasing
interest in the field of diagnostics. However, concerns
regarding sensitivity, especially in high-host-background
settings, cumbersome and time-consuming sample proces-
sing, and cost pose hurdles that would need to be over-
come in order to realize the potential of NGS. Additional
emphasis is also placed in the POCT diagnosis of viral
infectious diseases. It is clear that further evolution of viral
diagnosis will include the integration of the concepts of
POCT together with the emerging approach of syndromic
diagnosis. Indeed, in the last few years, we have also wit-
nessed growing interest in the need to consolidate in diag-
nostic area the concept of ‘differential diagnosis’ through
the development of rapid, sensitive molecular assays
designed to detect in the same specimens a wide range of
microorganisms causing the same disease. There are already
several examples of syndrome-based panels of pathogen
tests available for the detection of respiratory, gastrointest-
inal, neurological syndrome, and sexually transmitted patho-
gens regardless that they are viruses, bacteria, or parasites
[158]. These modern diagnostic methods have the potential
benefit of improving not only the diagnostic area but also
patient treatment and infection control and they can also
significantly reduce the economic burden of health-care
systems. What do we need to implement the use of new
diagnostic approaches combining POCT diagnosis of all
possible agents suspected in a specific clinical setting?
Surely, the use of these systems would have increased
even more, were it not that their cost can hardly be covered
in medical practices. Second, to reach this goal, the devel-
opment of serology-based and/or molecular-based microar-
rays/multiplexed tests based on modern technology and
new microfluidic devices will be needed. In this regard,
one of the most technical challenges is to provide sensitive
and specific diagnosis in complex biological fluids in an
easy-to-use format. Third, it will be imperative to continue
training on the use and potentiality of POCT, and the need
to develop systems for quality control of POCT if they are
going to achieve maximum potential. Furthermore, there is
need to spend more resources for these technologies to be
applied to infectious diseases, increase efforts to lower the
barriers to market entry through streamlined and harmo-
nized regulatory approaches, faster policy development for
adoption of new technologies, and novel financing mechan-
isms to enable countries to scale up implementation [159].
All these changes in turn will set new courses and directions
in the laboratory diagnosis of infectious diseases. However,
we firmly believe that most of the traditional diagnostic
methods invented and perfected during the last century
can’t be totally replaced by the only use of molecular
396 C. SCAGNOLARI ET AL.
techniques but they must be integrated depending on the
specific clinical setting.
Key issues
● The high sensitivity/specificity related to the use of mole-
cular assays has greatly improved the field of viral disease
diagnostics. The molecular assays are currently completely
automatized and for these reasons provide clinicians with
results that are both accurate and rapidly obtained.
● Real-time PCR has revolutionized the 21st century diagnos-
tic virology due to its remarkable application mainly in:
qualitative detection of viruses in biological samples; quan-
titative measurements of viral load; identification of the
viral variants; detection of polymorphism in host genes
related to the mechanism of defense against viruses;
● Viral load (defined as number of copies of nucleic acids of a
virus/ml of biological sample) measurement can help moni-
tor the efficacy of antiviral drugs;
● Viral load at diagnosis has been emerging as an important
indicator of disease severity and its measurement may help
to predict the outcome of some viral diseases;
● Viral load measurement is currently considered essential for
the clinical management of human immunodeficiency
virus-1, chronic hepatitis, cytomegalovirus, and Epstein-
Barr virus although the clinical significance of hepatitis C
virus load measuring need to be redefined with the repla-
cement of interferon/ribavirin-regimens with the new
highly potent direct-acting-antivirals;
● Host genetic factors may be important determinants in the
outcome of an infection and their definition may help the
clinical and diagnostic management of viral infectious
diseases;
● The impact of viral load measurement in other infections,
such as respiratory syncytial virus or human papillomavirus,
is still a matter of debate;
● New multi-panel platforms designed for the simultaneous
detection of multiple pathogens that cause a disease are
rapidly changing the diagnosis scenario in the coming years
through the introduction of a new diagnostic approach,
generally called ‘syndromic diagnosis’.
Notes
1. A group directed by Robert C. Gallo published related findings in
the same issue of Science and later confirmed the discovery of the
virus and demonstrated that it caused AIDS [42].
Funding
This paper was not funded.
Declaration of interest
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial con-
flict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or pending, or
royalties.
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