II.18. State of the Art and Trends in Cytomegalovirus Diagnostics. Maria Grazia Revello and Giuseppe Gerna

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1 State of the Art and Trends in Cytomegalovirus Diagnostics II.18 Maria Grazia Revello and Giuseppe Gerna Abstract Diagnosis of human cytomegalovirus (HCMV) infection is required in two clinical situations: the immunocompetent HCMV-seronegative pregnant woman, and the immunocompromised patient. In the case of pregnant women, diagnosis is primarily based on serology, but also on detection of viral DNA in blood. Viral DNA in blood may be quantified by the following assays: viraemia (infectious virus in blood), antigenaemia (leucocytes carrying virus or viral products) or DNAemia. In pregnant women diagnosis of primary infection is mandatory, in view of the possible virus transmission to the fetus. In the fetus, virus infection is diagnosed by performing viral assays on amniotic fluid and, when appropriate, on fetal blood. In newborns, diagnosis of congenital infection may be achieved by virus detection at birth. In immunocompromised transplanted patients virus quantification in blood is also mandatory to determine optimal time to intervene by antiviral treatment, unless a prophylaxis approach is adopted. Repeated antiviral drug courses may cause emergence of drug-resistant HCMV strains harbouring mutations in the UL97 or UL54 genes. Control of HCMV infection is guided by the immune response, which is elicited by viral infection at the level of both humoral and T-cell immunity. Introduction Human cytomegalovirus (HCMV), first detected in the 195s, was somewhat difficult to isolate, since it is strictly host species-specific and cell-associated, and was difficult to cultivate in the laboratory. Initially, pathogenicity appeared to be restricted to newborns with congenital infection, while in healthy individuals the only relatively infrequent clinical disease was HCMV infectious mononucleosis. It became evident early on that congenital infection was mostly related to HCMV primary infection occurring in the mother during pregnancy. With the advent of AIDS and the transplantation era, HCMV was recognized as responsible for the death of a number of immunocompromised patients. This prompted the development of a number of diagnostic procedures as well as specific antiviral drugs. In the immunocompromised patient population, the viral load level in blood was soon related to the appearance of clinical symptoms. Antiviral drugs controlled the infection in most cases, however, in the presence of only the humoral immune response, frequent HCMV reactivation episodes occurred, until the HCMV-specific T-cell response developed or was reconstituted. In this chapter, both aspects of the immune response will be briefly discussed in their relationship with diagnostic implications. Serology Determination of HCMV-specific immunoglobulin (Ig) of the G (IgG) and M (IgM) class represents the most widely used approach for diagnosis of HCMV infection and/or determination of virus-specific immune status. HCMV-specific antibodies can be detected either by their physical binding to viral components or by their biological function. The former approach, which allows for differential determination of virus-specific IgG and IgM, mainly relies on the immunoassay technology, whereas the assessment of biological functions is more complex and requires in vitro neutralization assays. Former assays are mainly used for diagnostic purposes, whereas functional assays are performed mainly for research purposes or for interpretation of the antibody response. Generalities in diagnostic assays for HCMV-specific antibody determination The enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are the most widely used techniques for detection of HCMV-specific antibodies. Traditional ELISA assays use chromogenic

2 Diagnosis of CMV Infection 381 reporters and suitable substrates resulting in a colour or fluorescence change (enzyme-linked fluorescent assay, ELFA) in the reaction well. Newer ELISA-like techniques use chemiluminescent reporters (chemilumiscence immunoassay, CLIA). The great majority of commercially available assays are based on the indirect ELISA/CLIA principle. Briefly, HCMV antigen (represented by cell lysate and/or recombinant proteins) is coated onto a solid phase consisting of polystyrene wells or paramagnetic beads. For IgG determination, test serum is added and incubated for a variable period of time, then a conjugated secondary anti-igg antibody is added. Results are expressed either qualitatively or quantitatively as an index or in arbitrary units as an international standard is not available for HCMV IgG. As for IgM determination either indirect or capture immunoassays can be used. With the indirect immunoassay, test sera are pre-treated with antibodies directed against human IgG (anti-huigg) to prevent binding of IgG-reactive rheumatoid factor IgM that would be detected by secondary conjugated anti-igm antibody, and reduce competition between IgG and IgM for antigen bound to the solid-phase. In capture assays, IgM are immunologically captured by an anti-human IgM monoclonal or polyclonal antibody bound to the solid phase. Capture assays are less prone to nonspecific IgM reactivities and reduce the competition of virus-specific IgG for HCMV antigen. IgM results are usually expressed as an index, consisting of the ratio of test serum and cut-off serum signals. Generally, a single analyte, namely IgG or IgM to HCMV, is measured in each assay. Recently, multiplex flow immunoassay (MFI) technology has emerged as a novel approach to simultaneously assess the serological response to a number of infectious agents, including HCMV (Binnicker et al., 21). A number of manual, semi- or fully automated assays for IgG, IgM, and IgG avidity determination are commercially available. Automated systems are generally preferred (even though they require dedicated instrumentation) because they allow high throughput, and complete automation of HCMV antibody panel determination including reflex testing (i.e. automated avidity determination in case of positive IgM result). Currently available automated assays are listed in Table II It should be emphasized that automated assays, in order to reduce incubation time and increase turnaround activity, require an excess of antigen and conjugate as well as lower test sample dilution. This implies that the performance characteristics of automated assays are generally inferior to those provided by manual assays in which immunological reactions are allowed to occur under equilibrium conditions. HCMV-specific antibody response IgM antibody The IgM antibody response following primary HCMV infection slightly precedes IgG development, reaches a plateau in the first month after onset of infection and then slowly declines in the following 3 6 months (Fig. II.18.1). However, the individual variability and sensitivity of the assay employed may greatly influence these kinetics. Indeed, a very brief IgM antibody response may be observed in some individuals, whereas a lowlevel IgM antibody response has been reported to persist for several months after a primary infection (Revello and Gerna, 22). Two representative examples of these extremes are shown in Fig. II.18.2A. The specificity of IgM results is of great concern, particularly when IgM determination is carried on in pregnant women in conjunction with IgG determination in order to establish HCMV-specific immune status and detect recent asymptomatic primary infection. Interference with rheumatoid factors, IgM reactive with cellular antigens present in the antigen preparation, crossreactivity or polyclonal stimulation during other viral infections are all potential causes for non-specific results. However, pre-adsorption of sera with anti-huigg, the use of capture rather than indirect immunoassays and the use of control antigen have all contributed to reduce non-specific results. In addition, since the viral proteins pp15 (UL32), pp65 (UL83), pp38 (UL8a), pp52 (UL44), and p13 (UL57) have been shown to be specifically recognized by IgM antibody following primary infection, the use of immunoassays based on the above recombinant proteins was advocated to improve specificity and reduce result variability among different assays. Indeed, the first fully automated IgM assay, developed in 2, was based on a mixture of pp15, pp65, pp52, and pp38 recombinant proteins (AxSYM, Abbott). Although sensitivity was claimed to be very high (Maine et al., 2), specificity was rather low (Lazzarotto et al., 21). Thus, a second generation HCMV IgM assay was developed by Abbott (Architect) which employs a mixture of HCMV lysate and recombinant viral proteins (Table II.18.1). According to available information, all the remaining automated IgM assays, but one, use variably purified and inactivated HCMV lysate as antigen. The only exception is the Elecsys CMV IgM (Roche Diagnostics, Germany) kit in which a cocktail of recombinant proteins is employed (Table II.18.1). In addition to primary infection, HCMV-specific IgM can be detected during recurrent infection. This is well documented in immunocompromised patients whereas, to the best of our knowledge, no study has ever specifically addressed this issue in immunocompetent

3 382 Revello and Gerna Table II.18.1 Characteristics of automated assays commercially available for determination of HCMV-specific IgG, IgM and IgG avidity Manufacturer platform Menu Antigen Technology Incubation time /FDA approval Abbott AxSYM IgG IgM pp15, pp52, pp65, pp38 recombinant proteins Indirect MEIA Indirect MEIA 2 min 2 min /FDA Architect IgG IgM IgG avidity + pp15/pp52 fusion protein Indirect CLIA Indirect CLIA a Indirect CLIA 29 min 29 min 29 min Beckman Coulter Access IgG IgM Indirect CLIA Indirect CLIA a 35 min 55 min BioMérieux VIDAS IgG IgM IgG avidity Indirect ELFA Indirect ELFA a Indirect ELFA 4 min 6 min 4 min /FDA /FDA Bio-Rad BioPlex 22 ToRC IgG ToRC IgM Indirect MFI Indirect MFI a 45 min 45 min /FDA DiaSorin Liaison IgG IgM IgG avidity Indirect CLIA Indirect CLIA a Indirect CLIA 35 min 45 min 45 min /FDA /FDA Ortho Vitros IgG IgM Indirect CLIA Capture CLIA 35 min 68 min /FDA /FDA Roche Elecsys IgG IgM IgG avidity Recombinant proteins Recombinant proteins Recombinant proteins Sandwich ECLIA Capture ECLIA Sandwich ECLIA 18 min 18 min 18 min Siemens Immulite IgG IgM Indirect CLIA Indirect CLIA a 6 min 9 min /FDA b /FDA b Abbreviations:, Conformité Européenne; CLIA, chemiluminescence immunoassay; ECLIA, electrochemiluminescence immunoassay; ELFA, enzyme-linked fluorescence assay; FDA, Food and Drug Administration; MEIA, microparticle enzyme immunoassay; MIF, multiplex flow immunoassay; NA, not available. a Sera are pre-treated with antibody directed against human IgG. b Not approved by FDA for blood donors screening. individuals. Rather, in one study we observed that out of 22 HCMV-seropositive breastfeeding women with positive virus isolation from milk, no subject was IgM-positive as determined by two in-house developed capture ELISAs (Revello et al., 1999). Nevertheless, it cannot be excluded that, even in immunocompetent individuals, transient IgM levels might be detected by highly sensitive assays during recurrent infections. Past and recent studies have reported a wide variability in the performance of HCMV IgM assays. Ultimately, the performance of a given kit in terms of sensitivity and specificity depends on the cut-off selected. In our opinion, the issue of specificity should be a major concern when developing or selecting an assay for HCMV-specific IgM determination in consideration of the following: (i) the target population for HCMV serology is primarily represented by pregnant women, (ii) even though vertical transmission

4 Diagnosis of CMV Infection 383 IgG AU, IgM ratio, log 1 Nt titre, log 1 DNA copies Days after onset of infection Avidity index (%) DNAemia IgM IgG Avidity E-Nt F-Nt Figure II.18.1 Kinetics (median values) of HCMV-specific antibody and DNAemia as determined in 151 sequential serum samples from 43 pregnant women with primary HCMV infection. 15 A 15 IgM ratio 1 5 IgG AU/ml Days after onset of infection Days after onset of infection IgG Avidity Index (%) Days after onset of infection Figure II.18.2 Examples of individual variability in the development of HCMV-specific IgM (A), IgG (B) and IgG avidity maturation (C) response. Serological follow-up of two different pregnant women with primary HCMV infection are shown in A, B, and C, respectively. Commercially available ELISA assays were used for IgM and IgG antibody determination. An in-house developed assay was used for IgG avidity determination. following primary infection is substantial (about 4%), the seroconversion rate in pregnancy is rather low (about 2%), (iii) only a minority of HCMVinfected newborns develop symptoms/sequelae, (iv) no proven treatment is available for preventing intrauterine transmission and (v) interpretation of positive IgM results requires a battery of additional tests which, in turn, need to be interpreted (see also Chapter II.3). IgG antibody The appearance of virus-specific IgG in a previously seronegative subject (seroconversion) represents the most reliable approach to the diagnosis of primary infection. It is recognized that IgG do follow IgM antibody appearance but, unlike IgM, they persist for life (Fig. II.18.1). Indeed, determination of virusspecific IgG is the quickest assay to discriminate between immunity and susceptibility to HCMV in

5 384 Revello and Gerna a given subject at any time. Exceptions to this rule are represented by immunocompromised patients in whom passively acquired antibodies or immunosuppression sometimes make serological determination of HCMV-specific immune status unreliable (see below). Studies addressing the ontogeny of IgG response following primary HCMV infection have shown that IgG antibody to structural phosphoproteins (such as pp15, pp65, and pp28) and non-structural proteins (such as p52 and p72) are promptly synthesized, whereas the appearance of glycoprotein-specific antibodies is delayed (Schoppel et al., 1997). Moreover, the kinetics of the appearance of IgG to different antigens is not synchronized and individual variations have been reported (Daiminger et al., 1998). However, individual antibody profiles to different antigens seem to remain qualitatively stable over the years, whereas quantitative variations have been observed. A lysate of HCMV-infected cells (which includes structural and non-structural proteins as well as glycoproteins) is generally used as a solid-phase in diagnostic assays for IgG determination (Table II.18.1). Two representative examples of IgG kinetics observed in two pregnant women are shown in Fig. II.18.2B. Correct determination of HCMV immune status is of critical importance in at least three groups of subjects, i.e. pregnant women (see also Chapter II.3), candidates for solid organ or haematopoietic stem cell donation/transplantation (see also Chapters II.13, II.14 and II.16), and blood donors. In the absence of an International Standard, definition of a cut-off value in a given assay is generally accomplished by comparing its performance with that of a similar commercial assay. The use of neutralization as a reference test to define the best cut-off for optimal discrimination between positive and negative ELISA results has been reported (Gerna et al., 1992a). IgG avidity The terms affinity and avidity are sometimes inappropriately used as synonyms. In fact, although both terms refer to the concept of binding strength, affinity is the strength of binding of an antibody combining site to one corresponding antigen epitope, whereas avidity is the total strength of the antigen-binding capacity of the mixture of polyclonal antibodies. It has long been known that antibodies produced early after an infection react weakly and those produced later react more strongly forming more stable aggregates with the relevant antigen. This phenomenon has been exploited in the laboratory by developing assays in which the strength of antibody-antigen binding is measured by using chemical compounds, such as 6 M urea solution, which disrupts weak antibody antigen complexes, but has little effect on strong antibodyantigen binding. As a result, low avidity antibodies can be easily differentiated from high avidity antibodies. The principle of the assay is rather simple and is based on the dissociation of previously formed IgG- HCMV antigens immunocomplexes by a dissociating agent followed by calculation of the ratio between reactivity (expressed as optical density, relative light units, or relative fluorescence units) in wells treated with dissociating buffer and reactivity of untreated wells (avidity index). An original (and so far unique) approach to determine avidity of HCMV-specific IgG was developed by Abbott for the Architect platform. In this assay (based on the CLIA technology) test serum is pre-treated either with a suspension of HCMV-infected cell lysate (to remove high avidity IgG) or with buffer (control solution). The IgG avidity index is then measured by calculating the ratio of signals obtained with or without the pre-treatment step. Since the original report (Blackburn et al., 1991), a number of studies have confirmed the potential usefulness of IgG avidity determination in distinguishing between primary (with low IgG avidity) and non-primary (with high avidity IgG) HCMV infection, particularly in the pregnancy setting (Grangeot-Keros et al., 1997; Bodeus et al., 1998; Lazzarotto et al., 1999). However, in order to correctly interpret IgG avidity results, one must take into consideration that (i) avidity cannot be reliably measured when IgG level is too low; (ii) individual variation in the rate of avidity maturation does exist; and (iii) different assays are subject to variations. The latter issue is of particular concern given the reported high variability in the performance of commercial assays (Revello et al., 21). Taken together, the above caveats indicate that standardization of avidity assays is lacking, and that IgG avidity determination cannot be used alone for the diagnosis of acute/recent primary HCMV infection. Finally, little is known about avidity maturation to different viral proteins. The use of infected cell lysate in both IgG and IgG avidity determination should obviate major individual variability. Nevertheless, it is not unusual to observe strikingly different kinetics in avidity maturation as exemplified in Fig. II.18.2C. As reported in Table II.18.1, only one recently developed automated kit employs a cocktail of recombinant proteins for avidity determination (Elecsys CMV avidity, Roche Diagnostics, Germany).

6 Diagnosis of CMV Infection 385 Neutralizing and glycoprotein-specific antibody Only a minor fraction of antibodies raised against viral proteins have direct antiviral activity in vitro. These antibodies are referred to as neutralizing antibodies (Nt-Ab) and possess a relatively high affinity and/or avidity for exposed structures on the virus surface. Nt activity and high avidity have been involved in the explanation of the potential benefit of passive immunization (Law and Hangartner, 28). As for Nt-Ab, HCMV could be considered a prototype of viruses that elicit a delayed neutralizing response which favours lifetime persistence in the host and is poorly protective because of its delayed appearance. Indeed, it is well known that (i) recurrent HCMV infections occur in the presence of circulating Nt-Ab; (ii) preconceptional maternal immunity, albeit substantially protective, still does not completely prevent congenital infection; and (iii) the Nt-Ab response until recently has been considered quite delayed following primary infection. With reference to the latter statement, it is important to stress that in vitro determination of Nt activity in human sera, as routinely performed so far, is based on the use of HCMV laboratory strains, such as the AD169 strain, and human fibroblasts. The classical 14-day plaque reduction assay or the more rapid (7- or 3-day) micro-neutralization assay can be used. Nt-Ab titres are expressed as the highest serum dilution causing 5%, 9% or 99% (depending on the end-point used) reduction in the number of plaques or focus forming units (FFU) compared with counts in control wells. Recently, it has been discovered that the UL131A- 128 locus of the HCMV genome is indispensable for the infection of endothelial cells (Hahn et al., 24) and epithelial cells (Wang and Shenk, 25), and that this locus is functionally missing in highly cell culture-adapted HCMV laboratory strains which no longer exhibit either endothelial cell or leucocyte tropism (Gerna et al., 26b). When human sera were tested for NT-Ab on both endothelial and epithelial cells instead of human fibroblasts, and a wild HCMV prototype strain (VR1814) was used, Nt activity was detected much earlier (within 1 days after the onset) and at much higher titres (32 64 ) compared to Nt activity determined with fibroblasts (Gerna et al., 28b). Thus, two quite distinct Nt-Ab responses can be determined in vitro, depending on the HCMV strain and cellular substrate used (Fig. II.18.1). Since the pentameric glycoprotein complex gh/gl/pul is present in the HCMV envelope and is required for infection of both endothelial and epithelial cells, whereas the glycoprotein complex gh/gl/go is required for infection of human fibroblasts (Kinzler et al., 22; Wang and Shenk, 25; see also Chapter I.17), it is reasonable to infer that neutralizing activity in endothelial/epithelial cells is preferentially mediated by antibodies directed against the pentameric complex, whereas antibodies to gb and gh are primarily involved in HCMV neutralization in fibroblasts. The IgG response to the gh/gl/pul protein complex during primary and recurrent HCMV infection was recently investigated by both immunofluorescence and ELISA using epithelial cells infected with one or more adenoviral vectors, each expressing one of the five proteins of the complex (Genini et al., 211). ELISA-IgG seroconversion to the complex was detectable in all cases of primary infection examined, although slightly later (17 68, median 55.5 days) compared to the neutralizing antibody response as determined with HCMV (VR1814)-infected ARPE-19 epithelial cells, but much earlier than neutralizing activity as determined with HCMV (VR1814)-infected fibroblasts. The same delay observed in vitro for the Nt-Ab response when measured with HCMV laboratory strains has been reported to occur also with glycoprotein (gb, gh)-specific antibodies (Schoppel et al., 1997). From a diagnostic point of view, this delayed appearance of Nt-Ab is considered a valuable aid for the interpretation of HCMV serology in pregnant women, particularly when only a single serum sample is available (Eggers et al., 1998). Viral detection Introduction to diagnostic assays Following its discovery in the second half of the 195s, HCMV was isolated and identified in HFF cell cultures, where it grew slowly as a cell-associated virus spreading from cell-to-cell and eventually resulting in slow-expanding cell foci (or plaques). Initially, in the presence of low virus amount in the sample examined, it took up to 2 3 weeks for the virus to develop a CPE focus revealing the presence of virus in the sample. In the mid 197s, IHC, such as immunoperoxidase and immunofluorescence, mostly using convalescent-phase human sera, as well as ISH techniques, allowed for faster HCMV identification. However, it was in the mid 198s that the development of the hybridoma technology allowed a significant improvement in the diagnosis of HCMV infection. Overall, mabs recognizing different viral proteins were employed to develop rapid diagnostic

7 386 Revello and Gerna assays allowing virus detection within 3 6 to 24 hours either in clinical sample cells or in cell cultures following sample inoculation. Among the rapid assays developed in cell cultures, the most useful was the so-called shell-vial assay. This assay was based on the use of the shell vial technique (Gleaves et al., 1984) and allowed detection of HCMV-infected cells prior to CPE appearance. It was generally applied to detection of infectious virus in peripheral blood leucocytes (polymorphonuclear leucocytes and monocytes) of subjects with primary HCMV infection, newborns with congenital HCMV infection, or immunocompromised transplanted patients with disseminated HCMV infection. The shell-vial assay (or viraemia assay) was performed by inoculating an HFF monolayer with a test sample and visualizing infected cell nuclei with an IHC technique hours after inoculation (Gerna et al., 199) using a p72/ie1-specific mab. Subsequently, a pool of p72 mabs was used to overcome problems of virus identification due to mutations in the IE region (Gerna et al., 23a). A major breakthrough in the diagnosis of disseminated HCMV infection was achieved at the end of 198s with the introduction of the antigenaemia assay, which was developed simultaneously by two groups of researchers in Groningen, The Netherlands, and in Pavia, Italy (van der Bij et al., 1988; Revello et al., 1989). This assay was aimed at detecting the presence of HCMV pp65 protein in nuclei of blood leucocytes by using a pool of pp65-specific mabs (Gerna et al., 1992b) and an IHC technique. Antigenaemia offers a greater sensitivity than viraemia and a shorter turnaround time, requiring about 2 hours to complete. The assay is relatively easy to perform, but it may not be possible to perform with a neutrophil count less than 1 cells/µl blood, and should be completed within 6 8 hours of collection to prevent a decrease in sensitivity. However, the major achievement of the last two decades has been the development of PCR and RT-PCR techniques for detection of HCMV genomic DNA and RNA transcripts in clinical samples following amplification of target sequences by a cyclic enzymatic procedure. In addition, methods for detecting HCMV DNA through signal amplification have been developed using either branched DNA probes (Kolberg et al., 1996) or RNA probes (Hebart et al., 1998). A different amplification technique (nucleic acid sequence-based amplification, NASBA) was also developed for detection of HCMV transcripts in blood (Gerna et al., 1999). More recently, automatic extraction procedures and a real-time readout format have improved the standardization of PCR conditions. HCMV quantification Viraemia assay Quantification of viral load in blood was found to possess a high positive predictive value for the development of HCMV disease (Grossi et al., 1995) and to correlate with virus replication. However, the shell vial assay, which was based on the finding that a single PBL was able to infect a single HFF cell (i.e. the number of p72-positive HFF nuclei correlated with the number of leucocytes carrying infectious virus) lacked sensitivity (Gerna et al., 21). Antigenaemia assay A major advance in the quantification of viral load was the introduction of the antigenaemia assay. Following twice a week monitoring of antigenaemia levels in solid-organ transplant recipients (SOTR), threshold values associated with appearance of HCMV disease were identified (Grossi et al., 1995). Thus, antigenaemia levels preceding the appearance of clinical symptoms were identified to start presymptomatic (pre-emptive) therapy in transplanted patients (Locatelli et al., 1994; Grossi et al., 1995; Boeckh, 1999). The quantification of HCMV antigenaemia also allowed monitoring of antiviral treatment, including the emergence of drugresistant HCMV strains (Baldanti et al., 1998, 24). In addition, in the late 199s, the in vitro transfer of HCMV pp65 into PBL allowed standardization of the assay (Gerna et al., 1998a; Revello et al., 1998a). However, standardization of the assay could not avoid two major limitations: one, is that it cannot be used in HSC transplant recipients during engraftment (Limaye et al., 1997); and the other, is that a paradoxical rise in antigenaemia levels during GCV treatment may occur independently of viral replication (Gerna et al., 1998b, 23b, 25). PCR-based molecular assays Pitfalls of the antigenaemia assay were overcome by the introduction of PCR-based molecular assays (Gerna et al., 1991; Humar et al., 1999; Emery et al., 2; Razonable et al., 23), which have been rapidly replaced by real-time PCR techniques. These techniques are more precise and rapid, possess a broader linear range and a higher output, and entail a lower risk of carryover contamination (Mengelle et al., 23). Although viral DNA can be quantified in a number of clinical samples, including different blood fractions (leucocytes or plasma) or whole blood (Gerna et al., 1994), whole blood is now accepted as the specimen of choice for disseminated infections since it allows earlier

8 Diagnosis of CMV Infection 387 and higher viral load detection compared to plasma. Leucocytes are no longer used owing to the cumbersome procedure and somewhat unrealistic evaluation of viral load. The use of real-time PCR does not avoid the risk of cross-contamination between clinical samples. To reduce this risk, suitable automated extraction platforms are available. As for target amplification, oligonucleotide primers and probes for amplification and detection of nucleic acid, respectively, are selected from conserved nucleotide sequences within a viral gene; these products constitute the first level of sensitivity and specificity for quantitative real-time PCR. Together with other components, this assay is subsequently adjusted to permit the polymerase enzyme to function optimally and to produce sensitive and specific signals from labelled probes that are proportional to the amount of the target DNA present in the blood sample (Espy et al., 26). In recent years, a variety of commercial and laboratory-developed assays have been utilized for CMV DNA quantification. To assess interlaboratory variability in quantitative HCMV viral load testing, a panel of samples was distributed to 33 laboratories in the USA, Canada and Europe, where testing was performed using commercial reagents or laboratory-developed assays (Pang et al., 29). Variations observed in reported results for individual samples ranged from 2. log 1 (minimum) to 4.3 log 1 (maximum), and variation was greater with low viral load values. The use of commercially available reagents and procedures was associated with less variability compared with laboratory-developed assays. Another study evaluated 15 Italian laboratories belonging to different transplantation centres who performed testing using five commercial reagents and two laboratory-developed assays (Lilleri et al., 29). The variability range was wide (about 2 log 1 ) for sample containing low amounts of HCMV DNA (< 1 copies/ml), but it decreased with increasing concentrations of HCMV DNA. For HCMV DNA levels 5 copies/ml, the different methods provided results within a ±.5 log 1 variability range, while the 8% range (range in which 8% of the results obtained will fall) was within ±.3 log 1 or less. If we consider that the precision of quantitative nucleic acid amplification techniques (QNAT) used to test viral load is such that changes in values should be greater than 3-fold (.5 log 1 ) to represent biologically important changes in viral replication (Caliendo et al., 21), an acceptable level of variability was reached among different methods for HCMV DNA quantitation in samples containing a clinically significant viral DNA amount. Intralaboratory variance was negligible. Just a few months ago the first WHO International Standard for Human Cytomegalovirus DNA quantification, (NIBSC code 9/162, version 3.. dated 3 November 21) was made available. It is intended to be used in the standardization of NAT-based assays for HCMV. The standard consists of a whole virus preparation of the HCMV Merlin strain (Dolan et al., 24) formulated in an universal buffer containing Tris-HCl and human serum albumin. The material is lyophilized in 1. ml aliquots and stored at 2 C. The standard was evaluated in a worldwide collaborative study involving 32 laboratories performing a range of NAT-based assays for HCMV (Freyer et al., 21). The standard has been assigned a concentration of 5,, International Units (IU) when reconstituted in 1. ml nuclease-free water. Once reconstituted, it should be diluted in the matrix appropriate to the material being calibrated and should be extracted prior to HCMV DNA measurement. Another limitation to the optimal use of real-time PCR is the genomic variability of HCMV. In this regard, not all primer probe combinations are equally efficient in amplifying viral DNA. We observed in our system that in 1 2% of samples tested, figures provided by different assays may exceed the.5 log 1 range. In this respect, sequence analysis of the real-time target region (US8) showed a substitution (from GG to AA) of the 3 nucleotides (nt position ) of the genome sequence recognized by the TaqMan probe (Lilleri et al., 29). A variety of commercial molecular assays have been developed (Table II.18.2). One of the more widely used is COBAS Amplicor CMV Monitor test (Roche Diagnostics, Indianapolis, IN), an end-point detection PCR-based assay that amplifies a 365 base pair region of the HCMV polymerase gene. The dynamic range of the assay using a plasma-based standard is reported to be between 4 and 5, copies/ml of plasma (Caliendo et al., 21). The assay has been designed for use with plasma, leucocytes, and whole blood specimens. Another commonly used assay is the Hybrid Capture System CMV DNA test (version 2; Digene Corporation, Gaithersburg, MD), a signal amplification method using an RNA probe that targets 17% of the HCMV genome. The target is detected by employing antibodies that specifically bind RNA:DNA hybrids. The dynamic range is 14 56, copies/ ml (Wattanamano et al., 2). The assay has been designed for whole blood specimens. Recently, several analyte-specific reagents (ASRs) for HCMV have become available. One example is Artus CMV ASR (Hamburg, Germany; primers, probes, internal control, standards targeting a 15-base pair region of the major IE coding sequence), which is distributed in one version by Abbott Molecular

9 388 Revello and Gerna Table II.18.2 Real time PCR for HCMV detection in blood [37] Manufacturer Assay Target Specimen Range (copies/ml) Automation Roche COBAS Ampliprep/ UL54 Plasma a Complete COBAS TaqMan CMV Abbott Real-Time CMV Unknown Whole blood Complete Plasma Complete QIAGEN Artus CMV qs-rgq UL122 Plasma Complete Argene- BioMérieux CMV r-gene UL83 Whole blood plasma Up to 1 8 Up to 1 8 Nanogen Q-CMV Real-Time UL123 Not specified ASR Cepheid Affigene CMV trender Unknown Whole blood plasma ASR ASR ASR ASR Abbreviation: ASR, analyte-specific reagent. a Conversion to International Units (IU)/ml available, corresponding to [35] (Des Plaines, IL), and in another version by QIAGEN (Valencia, CA). In a recent study (Caliendo et al., 27), the performance characteristics of the Abbott test and the QIAGEN test compared to Amplicor CMV Monitor and Hybrid Capture assays were evaluated. For plasma specimens, the Abbott test had a limit of detection of 2.3 log 1 copies/ml and a linear range up to at least 6. log 1 copies/ml. Viral load obtained from plasma specimens tested by Abbott and QIAGEN tests were in close agreement (mean difference.144 log 1 copies/ml). These data support the concept that Abbott and QIAGEN reagents provide laboratories with additional tools that can be used reliably for HCMV viral load testing. Diagnosis of HCMV infection in different clinical settings Pregnancy Pregnant women represent the most important and critical target for diagnosis of HCMV infection given the risk of fetal infection associated with primary HCMV infection in pregnancy (see also Chapter II.3). Routine HCMV testing of pregnant women is not recommended in any country. However, determination of HCMV immune status is de facto performed in a large proportion of pregnant women at least in some European countries such as Italy, Belgium, Germany and France. This information is based on (i) personal experience relevant to Italy; (ii) available literature data; and (iii) the fact that more than 11 million HCMV IgG/IgM tests are sold every year in Europe for about 27,, euros specifically for HCMV antibody testing of pregnant women (personal communication). Primary infection Since maternal infections are mostly asymptomatic, it is common practice to test pregnant women for the presence of both IgG and IgM in order to detect subclinical infections. Such an approach, however, carries the potential risk of detecting HCMV-specific IgM in an higher number of women than those actually experiencing an acute/recent primary infection. Indeed, in our experience as well as in the experience of others, only about 2% of positive IgM results are actually due to an acute/recent primary HCMV infection (Revello and Gerna, 22). A correct and quick interpretation of any IgM positivity is therefore mandatory in order to avoid anxiety and unnecessary pregnancy termination (Guerra et al., 27). It is beyond the scope of this chapter to discuss the technicalities involved in performing a correct diagnosis of primary HCMV infection (Revello and Gerna, 22). However, some general rules may be of help. Diagnosis of acute/recent primary infection can be reliably achieved only when two or more of the following parameters are concomitant: (i) IgG seroconversion; (ii) presence of virus-specific IgM; (iii) presence of low IgG avidity; (iv) presence of DNAemia; (v) absence of neutralizing antibody (as determined in HFF). In no instance, should a diagnosis of acute/recent primary HCMV infection be based on the presence of only one of the above criteria for the following reasons: (i) passively acquired IgG antibody, specimen mix-up or technical problems in assay performance (particularly when fully automatized systems are used), may result in false seroconversions; (ii) IgM positivity may be due to interfering factors, such as cross-reactivity, polyclonal activation due to other infections or IgM persistence; (iii) low/intermediate IgG avidity may be detected for

10 Diagnosis of CMV Infection 389 many months after the onset of infection depending on individual variability and the assay used (Revello et al., 21); (iv) the diagnostic value of DNAemia in immunocompetent individuals has to be defined in each laboratory since, contrary to our long-standing experience of exclusive detection in immunocompetent subjects undergoing primary infection (Revello et al., 1998b, 21), circulating DNA has been recently detected also in healthy seropositive women (Arora et al., 21); (v) Nt-Ab may be absent either in the early phase of primary infection or in seronegative individuals. From a diagnostic standpoint, determination of IgG avidity, DNAemia and neutralizing antibodies are considered indispensable adjunctive tests for interpretation of positive IgM results. Only reference laboratories with an array of additional commercial and non-commercial assays, and virologists with specific experience and competence in interpreting laboratory data should attempt to interpret correctly a positive IgM result. Once diagnosis of primary infection is confirmed, defining its presumed onset is important for prognosis, management of pregnancy as well as counselling. Anamnestic data and/or clinical history will help in timing the onset as long as they fit chronologically with serological/virological data (Revello and Gerna, 22). Gestation timing at the first testing is crucial for defining whether primary infection occurred before or during pregnancy (Revello et al., 22). More specifically, pregnant women should be tested as early as possible (ideally no later than 12 weeks of gestation). First testing after 2 weeks gestation should be avoided unless suggested by clinical symptoms. Preconceptional testing represents the best way to assess HCMV immune status of women in child-bearing age. Recurrent infection Recurrencies include reactivations of the endogenous HCMV strain(s) as well as reinfection with new HCMV strains. From the diagnostic standpoint, a recurrent infection is diagnosed in an immunocompetent IgGseropositive individual, whenever infectious HCMV or viral DNA is recovered from bodily fluids (urine, saliva, genital secretions, human milk, etc.) in the absence of serological markers of recent primary infection (presence of IgM antibody and/or low-intermediate IgG avidity). It must be emphasized that the presence of IgM antibody does not substantiate per se diagnosis of recurrent HCMV infection. In fact, as already mentioned above, the issue of whether recurrent HCMV infections elicit an IgM response in the immunocompetent host (in pregnant women, in particular) has never been properly addressed. Similarly, an increase in the IgG antibody level cannot be considered a marker of an ongoing recurrence in the absence of virus detection/ isolation. Therefore, serological monitoring of HCMV IgG-seropositive pregnant women has no clinical value. Recent reports seem to indicate that reinfections rather than reactivations are more likely to be responsible for symptomatic congenital infection (Yamamoto et al., 21). Reactivations and reinfections cannot be distinguished by using routine serological/virological techniques. The detection of IgG antibodies with new antigenic specificities to the surface glycoproteins H and B has been proposed as a tool for diagnosing reinfections (Novak et al., 29). However, at the moment, such an approach is restricted to investigational purposes. Finally, the search for HCMV in bodily fluids in pregnant women has no utility either for diagnosis or management of HCMV infection and is therefore not recommended. Maternal prognostic markers of in utero transmission Maternal markers that could reliably identify pregnant women at risk of transmitting the HCMV infection to the fetus have not been found yet. In the past, a limited number of studies have been performed to determine whether specific deficits in the antibody response were associated with intrauterine transmission with variable and sometimes contrasting results (Table II.18.3). A recent study performed by our group failed to show a significant difference in the kinetics of antibodies neutralizing the infection of endothelial cells (HUVEC) between a group of 18 transmitter and a group of 23 non-transmitter mothers (Revello et al., 21). On the other hand, major differences between women who transmitted the infection and those who did not were observed when HCMV-specific cell-mediated immunity was investigated. In fact, a significantly lower and delayed lymphoproliferative response was reported in transmitter compared to non-transmitter mothers (Revello et al., 26; Lilleri et al., 27a). Furthermore, it was found that a higher percentage of HCMV-specific effector memory T-cells that revert to the RA isoform of CD45 (CD45RA + CCR7 + ) during the first months after infection, was associated with a lower risk of transmitting HCMV infection to the fetus (Lilleri et al., 28, Fornara et al., 211) Thus, the development of the cell-mediated immune response appears to be crucial for the control of vertical transmission of HCMV infection. Unfortunately, none of the parameters listed above

11 39 Revello and Gerna [38] Table II.18.3 HCMV-specific antibody response in transmitter (TR) and non-transmitter (non-tr) mothers Reference Parameter Assay Finding Alford et al. (1988) IgG RIP More intense in TR Boppana and Britt (1995) IgM RIA Higher titres in TR Lazzarotto et al. (1998) IgM IB Higher number of reactive bands in TR Revello et al. (26) IgM ELISA No difference Boppana and Britt (1995) IgG to gb RIA increased in TR Eggers et al. (21) IgG to gb IB Increased in TR IgG to gh IB No difference Boppana and Britt (1995) Nt-Ab NTA on fibroblasts Increased in non-tr Revello and Gerna (21) Nt-Ab NTA on HUVEC No difference Boppana and Britt (1995) Avidity RIA Lower in TR Revello et al. (26) Avidity ELISA No difference Abbreviations: HUVEC, human umbilical vein endothelial cell; IB, immunoblot; NTA, neutralization assay; RIA, radioimmunoassay; RIP, radioimmunoprecipitation. can be used for counselling on an individual basis since results partially overlap between transmitter and nontransmitter mothers. Fetus Diagnosis of congenital infection during fetal life may be accomplished only by a search for virus or viral components in amniotic fluid (AF) following amniocentesis or in fetal blood (FB) following cordocentesis. Amniocentesis HCMV presence in AF can be detected by conventional, rapid and molecular assays. HCMV isolation from AF has represented for quite some time the only approach to virus detection in AF, and is considered the gold standard for prenatal diagnosis. As mentioned above, by using mabs to the major IE protein p72 (Gerna et al., 23a) and the shell vial technique (Gleaves et al., 1984), HCMV can be detected within 24 hours of sample collection. However, virus isolation is still considered a very valuable assay for confirmation of results obtained by molecular techniques. Molecular techniques (PCR, nested PCR and realtime PCR) have greatly improved the sensitivity of HCMV DNA detection in AF. Studies conducted in large series of pregnancies have reported sensitivities ranging around 8 9% (Liesnard et al., 2; Enders et al., 21; Revello and Gerna, 22). Importantly, specificity has been reported to be very high (98 1%), thus indicating that, provided that these highly sensitive assays are performed properly, even small amounts of viral DNA in AF correlate with congenital infection at birth. The advent of the real-time PCR methodology (which avoids the risk of false-positive results due to carryover products) represented a great improvement in the specificity of prenatal diagnosis results. However, it must be kept in mind that even when the most sensitive techniques are used and invasive procedures are performed at the most opportune gestation time, a small number of false-negative prenatal diagnosis results cannot be avoided. In our experience, 8% of negative prenatal diagnosis results are not confirmed at birth (Revello et al., 211). Delayed transmission, i.e. transmission occurring after amniocentesis represents an objective limitation of prenatal diagnosis and, as such, it should be discussed with the woman/couple during counselling sessions in order to avoid recriminations, in case of negative results at amniocentesis which are not confirmed at birth. Finally, it must again be emphasized that prenatal diagnosis is a very delicate task leading to irrevocable decisions and, thus, it should never be based on the result of a single assay. Cordocentesis In general, examination of FB does not improve the sensitivity of prenatal diagnosis of fetal infection with respect to virus detection in AF. In fact, determination of virus-specific IgM in fetal blood has a limited diagnostic value due to its low (25% to 75%) sensitivity (Revello et al., 1999). Similarly, low sensitivities have been reported for detection of virus or viral components in fetal blood. Only determination of DNAemia seems to provide sensitivity levels comparable to those obtained in AF (92% to 1%) (Enders et al., 21;

12 Diagnosis of CMV Infection 391 Transmitting women (n=2) HCMV-specific CD4 + T-cells HCMV-specific CD8 + T-cells T cells/ l blood CDI T cells/ l blood CDI IFN TNF IL2 Perforin CD45RA + LPR Days after onset Days after onset P <.1 Non-transmitting women (n=26) P=.3 l blood T cells/ HCMV-specific CD4 + T-cells P=.4 CDI T cells/ l blood HCMV-specific CD8 + T-cells CDI IFN TNF IL2 Perforin CD45RA + LPR Days after onset Days after onset Figure II.18.3 Comparison of different immunological parameters between a group of pregnant women transmitting and a group of women not transmitting the virus to the fetus. Two parameters were significantly different between the two groups: lymphoproliferative response (LPR, dotted purple line) for CD4 + (P =.4) T-cells, and CD45RA + T-cells (white line) for HCMV-specific CD4 + (P =.7), and CD8 + (P =.3) T-cells. CDI, cell division index. [39] Benoist et al., 28), when cordocentesis is performed after 3 weeks gestation. However, examination of fetal blood may confirm results obtained with AF, and when tests are performed quantitatively, may provide important prognostic information (see below). In addition, other haematological, biochemical and immunological non-specific parameters may be assessed in fetal blood and may contribute to a more comprehensive prognostic evaluation of fetal infection. Fetal prognostic markers of congenital disease Once diagnosis of fetal infection is made, the main issue is whether it is possible to predict which infected fetuses will be symptomatic at birth or later in life. The clinical significance of HCMV load in AF has been repeatedly investigated over the years by a number of researchers. Apart from initial reports from a single group showing that high DNA levels were predictive of poor outcome, other studies have clearly indicated that other variables such as gestational age at time of amniocentesis and the time elapsed since maternal infection could influence viral load irrespective of fetal outcome. In particular, while a low viral load in AF was consistently found to be associated with asymptomatic congenital infection, it was observed that high viral load in AF was associated with either symptomatic or asymptomatic congenital infection. The delicate issue of predicting fetal outcome has been recently addressed by our group in a retrospective study, in which a cohort of pregnant women who suffered from primary HCMV infection at different times during gestation and who underwent amniocentesis and cordocentesis was examined (Fabbri et al., 21). A panel of non-viral and viral assays was performed on fetal blood samples, and the results were compared between infected and non-infected, and between symptomatic and asymptomatic fetuses. The best non-viral factor for differentiating symptomatic from asymptomatic congenital infections was β 2 -microglobulin

13 392 Revello and Gerna to make an informed decision regarding termination/ continuation of pregnancy, after diagnosis of fetal infection. A B Figure II.18.4 (A) Antigenaemia assay, showing the presence of HCMV pp65 in the nucleus of some peripheral blood leucocytes. Indirect immunofluorescence. (B) Viraemia assay, showing the presence of p72/ie1-positive cells in the fibroblast cell monolayer, following inoculation of leucocytes carrying infectious virus from a patient with disseminated HCMV infection. followed by platelet count, whereas the best virological markers were IgM antibody and DNAemia. When two virological markers at established cutoffs (3. for IgM ratio and 3, DNA copies/ml blood for DNAemia level or per 1 7 peripheral blood leucocytes), along with β 2 -microglobulin at the established cutoff (11.5 mg/l, and platelet count at the established cutoff of 5,/ μl blood) were analysed in combination (and when at least three out of four were abnormal), the diagnostic efficacy was very acceptable with 1% specificity, 1% positive predictive value, 94% negative predictive value and 86% sensitivity. In another recent study, low platelet count determination was shown to be an independent indicator of poor outcome (Benoist et al., 28). Therefore, fetal blood sampling appears justified whenever additional information on fetal conditions are requested by the pregnant woman/couple in order Newborn Examination of newborns for congenital HCMV infection is required to confirm a prenatal diagnosis as well as for investigating transmission to fetus following primary infection in the mother. Specimens must be collected within the first 2 weeks of life in order to exclude peri-postnatal infection acquired via contacts with infected vaginal secretions or breast milk. Virus detection The gold standard diagnostic assay is virus recovery from urine or saliva samples. PCR for HCMV DNA detection in urine has been reported to be 1% sensitive and specific when compared with conventional virus isolation (Demmler et al., 1988). However, urine is not an easy sample to collect for screening purposes. A recent study conducted on 34,989 infants reported that real-time PCR performed on saliva (either liquid or dried) specimens was 1% and 97.4% sensitive, respectively, and 1% specific when compared to rapid culture (Boppana et al., 211). Some potential problems with the use of saliva for screening for congenital HCMV infection may arise from oral contamination by virus present in the maternal genital tract or breast milk. However, collection of saliva samples on the second day of life should minimize the chance of false-positive results. HCMV DNA detection in blood Detection of HCMV DNA in neonatal blood is an additional approach potentially useful for screening purposes since viral DNA has been detected by PCR both in serum and in peripheral blood leucocytes of both symptomatic and asymptomatic HCMV-infected newborns (Nelson et al., 1995; Revello et al., 1999). Moreover, since dried blood spot (DBS) are routinely collected in the first 3 5 days of life in many countries for neonatal screening of metabolic and hereditary diseases, the search for viral HCMV DNA in DBS has attracted considerable interest. Despite the potential advantages of such an approach (DBSs can be stored for years, thus they may provide an important potential tool for retrospective diagnosis), the actual sensitivity and specificity of HCMV DNA detection in DBSs remains to be assessed. In fact, sensitivities varying from 71% to 1% and specificities around 1% have been reported (reviewed in Barbi et al., 26), whereas

14 Diagnosis of CMV Infection 393 a recent study performed prospectively on 2,448 infants reported very low sensitivity ( % when a single or two primers were used, respectively) when compared with saliva rapid culture (Boppana et al., 21). On the other hand, specificity was excellent (99.9%). Finally, IgM antibody determination at birth is of limited value given its low sensitivity (Revello et al., 1999). Immunocompromised patients Diagnostic and therapeutic impact of HCMV DNA quantification The quantification of HCMV DNA has allowed the identification of threshold levels associated with onset of clinical symptoms and, thus, prevention of onset of HCMV disease by adopting therapeutic interventions prior to symptom onset (pre-emptive therapy). This approach is advantageous as compared with universal prophylaxis (treatment of all patients from the day of transplant for a period of 3 6 months) as a smaller number of patients are treated for a shorter period of time. Other advantages of pre-emptive therapy include savings in terms of drug toxicity and patient management costs (Kusne et al., 1999), and, for some authors, this approach has an efficacy comparable to that of prophylaxis in preventing the indirect effects of HCMV infection, such as graft failure/rejection and fungal/ bacterial infections (Singh et al., 25, 26). Today, several transplantation centres (and particularly HSCT centres) still do not use any cut-off for initiation of pre-emptive therapy, thereby starting antiviral treatment upon first virus detection in blood; and cut-offs used for HSCTR and SOTR in different centres vary greatly. Using continuous monitoring of viral load (twice a week), we adopted a cut-off of 3, DNA copies/ml whole blood before starting pre-emptive therapy in SOTR, while we use a cut-off of 3, DNA copies/ml whole blood in HSCTR. In SOTR, the above reported DNAemia cut-off was eventually selected after a trial in which it was compared with a previously selected antigenaemia cut-off of 1 pp65- positive/2x1 5 leucocytes (Gerna et al., 27). Results obtained on a large number of patients showed that: (i) the selected DNAemia cut-off significantly reduced the number of patients receiving treatment compared with antigenaemia; (ii) was capable of guiding pre-emptive therapy of both primary and reactivated HCMV infections; and (iii) did not significantly alter the overall duration of treatment. Notwithstanding the results of our study, several transplantation centres still use a differential approach to preventative treatment of primary and reactivated infections. Recent guidelines published A B Figure II.18.5 A. Primary HCMV infection: acute-phase serum not reactive with epithelial cells expressing the HCMV gh/gl/pul pentamer complex. B. Convalescent-phase serum from the same patient staining epithelial cells expressing the pentamer complex. In both panels, ARPE-19 cells were infected with five adenovirus vectors, each carrying a single gene of the pentamer complex. by The Transplantation Society (TTS) of America suggested prolonging prophylaxis from 3 to 6 months in D + /R SOT patients (Kotton et al., 21). Since our cut-off was determined on a large number of SOTR, and as we did not encounter problems with HCMV disease, we continue to use our cut-off which is higher than those used by other transplantation centres. Based on previous observations in HSCTR, where the use of a quantitative, rather than qualitative, DNAemia cut-off appeared suitable to increase the negative predictive value without altering the positive predictive value compared to qualitative