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Journal of Clinical Virology 46 (2009) 254–258

Contents lists available at ScienceDirect

Journal of Clinical Virology

journa l homepage: www.e lsev ier .com/ locate / j cv

uantification of cytomegalovirus DNA levels in intestinal biopsies as aiagnostic tool for CMV intestinal disease

ina Ganzenmuellera,∗, Cornelia Henke-Gendoa, Jerome Schluéb, Jochen Wedemeyerc,abine Huebnera, Albert Heima

Institute of Virology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, GermanyInstitute of Pathology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, GermanyDepartment of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany

r t i c l e i n f o

rticle history:eceived 11 August 2009ccepted 13 August 2009

eywords:ytomegalovirusMV intestinal diseaseuantitative PCRut biopsyiral load

a b s t r a c t

Background: CMV intestinal disease (CMV-ID) is a serious complication in immunocompromised patientsand mainly diagnosed by clinical, endoscopic and histopathologic findings, whereas qualitative CMV-PCRin tissue samples is not recommended for diagnosis due to its low positive predictive value (PPV).Objectives: To study the interpretation and diagnostic use of CMV-quantification by PCR in intestinaltissue biopsies to recognize CMV-ID. To develop cut-off intestinal CMV-loads attributing illness to CMV.Study design: CMV-genome copies in 163 biopsies from the lower intestinal tract of immunocompromisedpatients were determined by quantitative real-time PCR, normalized to the cell number, and retrospec-tively compared to histopathological analysis, clinical findings and occurrence of CMV-antigenemia. Twocut-off intestinal CMV-loads, cut-offhisto and cut-offclin, were defined using histopathological or clinicalcriteria as gold standard, respectively.Results: CMV was detected in 32.5% of biopsies with a more than six log range of CMV-concentrations(1 × 10−4–1.4 × 102 copies/cell). Notably, biopsies with histopathologically or clinically confirmed CMV-ID had a significantly higher CMV-load (p < 0.001). Cut-offhisto and cut-offclin were defined at the intestinal

CMV-load of 0.14 and 0.01 copies/cell, respectively, and improved the PPV. However, cut-offhisto showeda decreased sensitivity for clinically defined CMV-ID cases. Interestingly, many patients with CMV-IDshowed no concomitant CMV-antigenemia, suggesting a localized intestinal CMV-replication.Conclusions: Quantification of CMV in intestinal biopsies is a useful diagnostic tool allowing the definitionof cut-off values that can predict CMV-ID more accurate than qualitative PCR results. Further prospectivestudies have to clarify wether these cut-offs can improve diagnostics and treatment of CMV-ID in day- to-day clinical practice.

. Background

Cytomegalovirus (CMV) is a significant opportunistic pathogenor immunocompromised individuals causing CMV end-organ dis-

ase as a serious complication. One of its main manifestation sitess the gastrointestinal tract, with CMV intestinal disease (CMV-ID)eing most frequently observed.1,2

Abbreviations: CMV, cytomegalovirus; CMV-ID, cytomegalovirus intestinal dis-ase; IBD, inflammatory bowel disease; HIV, human immunodeficieny virus; PPV,ositive predictive value; NPV, negative predictive value; IHC, immunohistochem-

cal staining; ROC, receiver operating characteristic; AUROC, area under the ROCurve.∗ Corresponding author. Tel.: +49 511 5324328; fax: +49 511 5328736.

E-mail address: Ganzenmueller.Tina@mh-hannover.de (T. Ganzenmueller).

386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jcv.2009.08.008

© 2009 Elsevier B.V. All rights reserved.

Especially in recipients of stem cell or solid organ transplan-tations CMV-ID is a substantial cause of mortality and morbidity,despite improved antiviral prophylaxis and therapy.3–5 CMV-IDwas also very common in AIDS patients before introduction ofHAART.6,7 The role of CMV in inflammatory bowel disease (IBD)remains controversial, with CMV being most commonly reportedin patients with severe or refractory IBD receiving immunosup-pressant drugs.8,9 Furthermore, CMV-ID has been described inpatients with rheumatologic disorders, non-transplant malignan-cies, immunodefects and in elderly or critically ill.2,10,11

CMV-ID can be present with symptoms such as diarrhea,abdominal pain, weight loss, bleeding or fever, and can mimic

other entities such as ischemic colitis, toxic megacolon or evencolon carcinoma.12,13 Currently the diagnosis of CMV-ID is basedon combination of clinical symptoms from the lower intesti-nal tract consistent with CMV-ID, macroscopic findings uponendoscopy, histopathological findings, and demonstration of CMV

T. Ganzenmueller et al. / Journal of Clin

Table 1Patient demographics.

Characteristic Number of patients n = 163 (%)

Age in yearsa 46 (1–80)Sex, male 101 (62.0)

Underlying diseaseSCT 64 (39.3)SOTb 41 (25.2)HIV infection 13 (8.0)IBD 23 (14.1)Other diagnosisc 22 (13.5)

CMV: cytomegalovirus; SCT: allogenic stem cell transplantation, SOT: solid organtransplantation; HIV: human immunodeficieny virus; IBD: inflammatory boweldisease.

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Median (range).b 15 kidney, 20 liver, 6 other transplantations.c Others included: immunodefects, non-transplant malignancies or rheumatic

isorders.

n an appropriate tissue specimen by culture, histopathologicalesting, immunohistochemistry or in situ hybridization.14,15 How-ver, according to these recommendations the detection of CMV inissue biopsies by PCR alone is not sufficient for the diagnosis ofMV-ID, as the positive predictive value (PPV) is too low due to theigh sensitivity of PCR, which may also detect latent CMV-DNA.his problem, which might lead to false positive results suggest-ng CMV-ID in cases of gastrointestinal disease of another etiology,

ight be solved by the development of quantitative PCR assays andnterpretive guidelines using suitable cut-off values.

. Objectives

Quantitative CMV-PCR in gut biopsies has been suggested1 tomprove the diagnostics of CMV-ID, but its interpretation and diag-ostic significance has not been studied in detail. Therefore weimed to evaluate the use of CMV-quantification by real-time PCR inissue biopsies from the lower intestinal tract for diagnosing CMV-D and tried to establish cut-off intestinal CMV-loads to attributentestinal disease to CMV.

. Study design

.1. Specimen

163 routine diagnostic biopsies from the lower intestinal tractor which CMV-PCR was requested to rule out CMV-ID were re-nalysed by CMV-quantification. Biopsies were obtained from 163atients with underlying diseases associated with immunosup-ression. Patient demographics are shown in Table 1.

.2. Histopathological criteria

Histopathological analysis of the biopsies consisted of morpho-ogic examination for enlarged nuclei and viral inclusions together

ith immunohistochemical staining (IHC) for CMV (monoclonalouse antibody, clone CCH2, DakoCytomation, Hamburg, Ger-any). IHC was performed on all CMV-PCR positive biopsies, and,

f CMV could not been excluded clearly by morphologic examina-ion, also on CMV-PCR negative biopsies. Patients were consideredo have histopathologically confirmed CMV-ID if either of these

ethods yielded a positive result.

.3. Clinical criteria

Besides classifying patients in groups with or without CMV-IDsing histopathology as gold standard we also used clinical criteria

ical Virology 46 (2009) 254–258 255

as gold standard to reclassify our patient collective: 26/163 patientswere considered to have clinically relevant CMV-ID, because com-patible symptoms or signs (typical endoscopic lesions) occurred inthe absence of another plausible etiology and/or the subsequentclinical course (i.e. improvement after antiviral treatment) con-firmed CMV as cause (“CMV-ID”). 82/163 patients with non-CMVetiology for intestinal illness, such as graft-versus-host disease,episodes of IBD or infectious pathogens (confirmed by endoscopic,histopathological or microbiologic findings) were considered ascontrol collective (“non-CMV”). For 47 patients the etiology ofintestinal symptoms was not clearly attributable (“unclear”) orinsufficient clinical data available (8 patients).

3.4. Real-time PCR

DNA was extracted from tissue biopsies using the Qiagen TissueKit (Qiagen, Hilden, Germany). The number of CMV-genomes wasdetermined by real-time PCR as previously described.16 A secondreal-time PCR for the human CRP-gene17 using the LightCyclerFast-Start Hybridization Kit (Roche Diagnostics, Mannheim, Germany)was performed to normalize the cell number and to exclude PCRinhibition. 1/10 of the extracted DNA was used for PCR reaction.The median cell number analyzed per PCR reaction was 7.2 × 104

(6.5 × 103–6.7 × 105).

3.5. CMV-antigenemia

CMV-antigenemia was determined by detection of CMV pp65,as previously described.18 High-level and low-level antigenemiawere defined as <10 or ≥10 CMV positive cells per applied 400 000leukocytes, respectively.

3.6. Statistical analysis

Statistical analysis was performed using SPSS version 16.0 (SPSSInc., Chicago). P-values <0.05 were considered significant. Pearson’sChi-square-test, Mann–Whitney U test or Kruskal–Wallis-test wereapplied to determine statistical significance of differences betweengroups.

Receiver operating characteristic (ROC) analysis was performedto calculate cut-off intestinal CMV-concentrations indicating CMVas cause of intestinal disease with histopathological or clinicalcriteria used as gold standard to attribute intestinal illness toCMV.19 The performance of the diagnostic variable “intestinalCMV-load” in relation to these reference methods was quantifiedby the area under the ROC curve (AUROC). The optimal cut-offvalues were identified at the maximum of Youden index (sensi-tivity + specifity − 1),20 and designated as cut-offhisto or cut-offclin,respectively. Positive and negative predictive value (PPV and NPV)were calculated for the selected cut-offs.

4. Results

4.1. CMV-detection in the biopsies by quantitative CMV-PCR

CMV was detected by real-time PCR in 32.5% (53/163) ofthe biopsies. Quantitative results showed a more than six logrange of CMV-DNA concentrations (1 × 10−4–1.4 × 102 copies/cell;median = 8 × 10−2) (Fig. 1).

4.2. Results of CMV-quantification in comparison tohistopathological diagnosis

None of the intestinal samples with negative CMV-PCR showeda positive histopathological result. In 12/53 (22.7%) of the CMV-PCR

256 T. Ganzenmueller et al. / Journal of Clinical Virology 46 (2009) 254–258

Fig. 1. Quantification of CMV-DNA levels in CMV-PCR positive gut tissue biopsies.Am

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Table 2Comparison of the diagnostic performance of qualitative CMV-PCR with cut-offhisto

using histopathology as gold standard.

Cut-off (CMV-copies/cell) Sensitivity Specifity PPV NPV

Qualitative PCR 1.0 0.73 0.23 1.0Cut-offhisto: 0.14 1.0 0.93 0.52 1.0

Cut-offhisto: cut-off adapted to histopathological results; PPV: positive pre-dictive value; NPV: negative predictive value. ROC analysis was calculatedusing histopathology as reference method and the Youden index (=sensitiv-ity + specifity − 1) was used to select an optimal cut-off intestinal CMV-load forattributing illness to CMV. The area under the ROC curve was 0.86 (confidenceinterval (CI) 0.80–0.93) for the qualitative approach and 0.98 (CI: 0.97–1.0)for the quantification. Subsequently the performance of this cut-off was com-

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wide range of CMV-DNA levels was observed in quantified gut biopsies with aedian CMV-load of 0.08 (0.0001–144.0) copies /cell.

ositive biopsies CMV-ID was detected by histopathological anal-sis. Notably, intestinal biopsies with a histopathological positiveesult showed a significantly (p < 0.001) higher median CMV-load13.3 copies/cell, range 0.14–144.0) than histopathologically nega-ive biopsies (0.0 CMV-copies/cell, range 0.0–4.0) (Fig. 2A).

.3. Results of CMV-quantification in comparison to clincal

iagnosis of CMV-ID

Clinical criteria were used to classify patients into a groupith clinically defined CMV-ID (26 patients) and a control groupith “non-CMV” etiology of intestinal disease (82 patients).

ig. 2. Intestinal CMV-load in histologically or clinically classified patient groups. (A)nalysis. The median CMV-load in samples with positive histopathology (13.3 copies/ceegative histopathological analysis (0.0 CMV-copies/cell; range 0.0–4.0). (B) Higher intesti

ntestinal CMV-concentration of the group with clinically defined CMV-ID (0.24 CMV-copMV (0.0 copies/cell; range 0.0–0.13) or unclear etiology (0.0 copies/cell; range 0.0–2.1) foroups “unclear” and “non-CMV” etiology.

pared to the qualitative approach for the histopathologically classified patientgroups.

The CMV-ID group had a significantly higher median intestinalCMV-concentration (0.24 CMV-copies/cell; range 0.0–144.0) com-pared to the groups with non-CMV (0.0; range 0.0–0.13) or unclearetiology (0.0; range 0.0–2.1) (p < 0.001) (Fig. 2B).

All biopsies from the control group were negative in histopathol-ogy. Notably, 14/26 biopsies from patients with clinically definedCMV-ID were also negative in histopathology, but were (withone exception) positive in CMV-PCR. Most of these had moder-ate intestinal CMV-loads of 0.01–0.5 copies/cell. Surprisingly, oneclinically defined case had a high intestinal CMV-concentration(4.0 copies/cell), but a negative histopathology.

4.4. Definition of a cut-off intestinal CMV-load using ROCanalysis with histopathology as gold standard

Using histopathology as gold standard the cut-offhisto was deter-mined at 0.14 CMV-copies/cell by ROC analysis (AUROC = 0.98, CI:0.97–1.0). Applying cut-offhisto to the groups classified by his-tolopathology into patients with or without CMV-ID increased

specifity (=0.93) and PPV (=0.52) compared to qualitative PCRresults (specifity = 0.73; PPV = 0.23). Sensitivity and NPV remained1.0 (Table 2).

Higher intestinal CMV-load in biopsies with a positive result in histopathologicll, range 0.14–144.0) is significantly higher (p < 0.001) compared to samples withnal CMV-load in biopsies from patients with clinically defined CMV-ID. The medianies/cell; range 0.0–144.0) is significantly higher compared to the groups with non-r intestinal symptoms (p < 0.001). There was no significant difference between the

T. Ganzenmueller et al. / Journal of Clin

Table 3Comparison of the diagnostic performance of qualitative CMV-PCR with cut-offhisto

and cut-offclin using clinical criteria as gold standard.

Cut-off (CMV-copies/cell) Sensitivity Specifity PPV NPV

Qualitative PCR 0.96 0.85 0.68 0.99Cut-offclin: 0.01 0.96 0.91 0.78 0.99Cut-offhisto: 0.14 0.69 1.0 1.0 0.91

Cut-offhisto: cut-off adapted to histopathological results; Cut-offclin: cut-off adaptedto clinical criteria; PPV: positive predictive value; NPV: negative predictive value.ROC analysis was calculated using clinical criteria as reference method and theYouden index (=sensitivity + specifity − 1) was used to select an optimal cut-offintestinal CMV-load for attributing illness to CMV. The area under the ROC curvewas 0.91 (confidence interval (CI) 0.84–0.97) for the qualitative approach and 0.96(tc

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CI: 0.92–1.0) for the quantification. Subsequently the diagnostic performance ofhe cut-offclin was compared to the qualitative approach and the cut-offhisto for thelinically classified patient groups.

.5. Definition of a cut-off intestinal CMV-load using ROCnalysis with clinical criteria as gold standard

Using the clinical classification as gold standard a secondut-off, cut-offclin, was determined at 0.01 CMV-copies/cell byOC analysis (AUROC = 0.96; CI: 0.92–1.0). Application of cut-ffclin to the clinically defined groups led to further improvedPV (=0.78) and specifity (=0.91), with unchanged sensitivity=0.96) and NPV (=0.99) compared to the qualitative PCR approachTable 3).

.6. CMV-antigenemia

Data concerning CMV-antigenemia within 10 days before orfter biopsy sampling was available for 108 patients. 24.1%26/108) showed CMV-antigenemia (15 of these high-level, 11ow-level antigenemia). 68.2% of the individuals with clinicallyefined CMV-ID showed no (n = 11/22) or low-level (n = 4/22)MV-antigenemia. 40% of patients with histopathologically con-rmed CMV-ID showed no (n = 3/10) or low-level (n = 1/10)MV-antigenemia.

. Discussion

Due to its low PPV qualitative CMV-PCR from tissue samples isonsidered to be inappropriate for recognizing CMV-ID,14,15 a com-on and serious complication in immunosuppressed patients.2

ighly sensitive PCR assays, which are used in an increasing num-er of laboratories, might produce positive results without clinicalorrelate and lead to unnecessary treatment with adverse sideffects. Thus, it is crucial to develop interpretative guidelines.21 Wevaluated CMV-quantification by real-time PCR in 163 gut biop-ies and determined cut-off intestinal CMV-loads to improve thebjective diagnostics of CMV-ID.

In our study a negative CMV-PCR result in gut biopsies wasighly predictive for absence of CMV-ID, and consequently canule out CMV end-organ manifestation, as already proposedy others.22,23 A very dynamic range of CMV-concentrationsas observed in CMV-PCR positive gut biopsies, indicating

ocal CMV replication in gut tissue during active infection oreactivation, but possibly also standing for low-level detec-ion of latent CMV-DNA. However, not all CMV-PCR positiveiopsies showed histopathological signs for CMV-ID, which onhe one hand is a known phenomenon,24 but on the other

and could indicate that histopathology alone is not sensitivenough as suitable gold standard. Therefore, we also used clinicalriteria, considering samples with negative histology as clini-ally significant, if compatible symptoms occurred, and couldeveal several additional cases with clinically defined CMV-ID.

ical Virology 46 (2009) 254–258 257

Interestingly, most of these cases showed moderate intestinalCMV-loads. One of the clinically defined cases had an unex-pected high intestinal CMV-load, but could not be confirmedby histopathology, which might have been caused by samplingerrors.

Remarkably, both, samples with a positive result in histopatho-logical analysis and samples with clinically defined CMV-ID showeda significantly higher intestinal CMV-load than the respectivecontrol groups. These results support the application of quanti-tative PCR and the definition of cut-off intestinal CMV-loads todistinguish between CMV-ID and CMV-detection without clinicalillness.

Using histopathology or clinical criteria as gold standard weestablished cut-off intestinal CMV-loads of 0.14 copies/cell (cut-offhisto) and 0.01 copies/cell (cut-offclin), respectively, leading toimproved specificity and PPV compared to qualitative PCR results,with maintained sensitivity for both cut-offs. However, if cut-offhistowas applied to the clinically classified groups, specifity (1.0) andPPV (1.0) were highly increased, but nearly one third of cases clin-ically suspicious for CMV-ID were overlooked, as the sensitivitydecreased (0.69). This highlights the usefulness of the more sen-sitive clinically defined cut-offclin in addition to the highly specifichistopathologically determined cut-offhisto.

In our study a high percentage of patients with clinically orhistopathologically defined CMV-ID showed no or only low-levelCMV-antigenemia, indicating a localized CMV replication in thegut and stressing the limited value of CMV blood tests for predic-tion and diagnosis of CMV-ID. Similar results have been reportedby others: Mori et al. showed that only 21% of patients had a posi-tive CMV-antigenemia test before developing CMV-ID, and, thoughall patients subsequently developed antigenemia, in 47% the val-ues remained at low-level.25 The phenomenon that CMV diseasedoes not always correlate with peripheral blood viral load was alsodescribed for other CMV end-organ manifestations.26 Furthermore,the CMV load in the end-organ sample bronchoalveolar lavage hasbeen reported to be more predictive for CMV-pneumonitis thanblood viral load.27 This underlines the importance of examiningappropriate tissue samples from the diseased organ to recog-nize CMV-ID.15 Therefore, and since an early presentation of CMVcolitis can be mild and nonspecific especially in immunocompro-mised, early colonoscopy is considered to be indispensable fordiagnosis.28,29

In summary these data show that quantification of CMV-DNAin gut biopsies by real-time PCR is a useful diagnostic tool, allow-ing the definition of cut-off intestinal CMV-loads, which are morepredictive for CMV-ID than qualitative PCR results. However, anabsolute, universally acceptable value cannot be given. In thisstudy cut-offhisto gave similar results to the reference methodhistopathology, and due to its high PPV is useful to confirm thediagnosis CMV-ID. The clinically defined cut-offclin seems to bemore suitable for screening, as it provides higher sensitivity thanhistopathologic analysis. Therefore, results above cut-offclin shouldlead to further diagnostic efforts to confirm or exclude CMV-ID.Despite its subjective character and dependence on the exam-iner’s experience,30 histopathologic testing cannot be replaced, butefficiently complemented by quantitative PCR on tissue samples,which is an objective, fast and highly standardized method. Futurestudies will reveal wether the cut-offs described here can improvediagnostics and treatment of CMV-ID in the day-to-day clinicalpractice.

Conflict of interest

Funding: none.Competing interests: none declared.Ethical approval: not required.

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58 T. Ganzenmueller et al. / Journal

cknowledgements

The authors thank G. Harste, E. Holthoff-Steinkopf, R. vonietrowski, and K. Sendler for excellent technical assistance.

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