Adenovirus sampling and transport

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Graefes Arch Clin Exp OphthalmolDOI 10.1007/s00417-006-0283-9 LABORATORY INVESTIGATION

Roman WlfelMartin PfefferSandra EssbauerSylke Nerkelun

Gerhard Dobler

Received: 23 August 2005Revised: 23 December 2005Accepted: 20 January 2006# Springer-Verlag 2006

Evaluation of sampling technique and transport

media for the diagnostics of adenoviral

eye infectionsAdenovirus sampling and transport

Abstract Background: Human ade-noviruses (HAdV) may cause pha-ryngoconjunctival fever, follicularconjunctivitis or epidemic keratocon-

junctivitis (EKC). Especially, out-breaks of the latter may lead to severe

economic losses when preventivemeasures are implemented too late.Thus, a safe sampling method, properspecimen transport conditions and afast and sensitive diagnostic techniqueis mandatory. Methods: Two com-mercially available virus transportsystems (VTS) were compared withtwo NaCl-moisturised sampling de-vices, one of which comprises Da-cron-tipped plastic-shafted swabs andthe other a cotton-tipped wood-shafted swab, available in most

ophthalmologists

offices. Down-stream methods for specific detectionof HAdV included directimmunofluorescence assay (IFA) ofconjunctival swabs, virus isolation bycell culture and quantitative real-time

polymerase chain reaction (qPCR).Furthermore, the influence of appli-cation of local anaesthetics prior toswabbing on subsequent detection ofHAdV was investigated. Results:Application of local anaesthetics hada positive influence on the amount of

swabbed cells, thus increasing thechance of obtaining positive results

by IFA. Neither isolation of HAdV bycell culture nor by qPCR was nega-tively influenced by this pretreatment.Surprisingly, both commerciallyavailable VTS performed signifi-cantly worse than the NaCl-mois-

turised swabs. This was shown withregard to virus recovery rates in cellculture as well as viral genome copynumbers in the qPCR. Conclusions:Based on our results, the followingrecommendations are provided toimprove sampling, transport and di-agnostic techniques regarding con-

junctival swabs for diagnosis ofhuman adenovirus infection:(1) application of local anaesthetics,(2) NaCl-moisturised VTS for ship-ment of specimens, and (3) detection

of HAdV by qPCR. The latter methodproved to be superior to virus isola-tion by cell culture, including subse-quent identification by IFA, because itis faster, more sensitive and allowssimultaneous handling of a number ofsamples. Hence, countermeasures to

prevent further virus spread in anoutbreak situation can be implemen-ted earlier, thus reducing the numberof subsequent adenoviral infections.

Keywords Viral transport . Clinical

samples.

Adenoviruskeratoconjunctivitis

Introduction

Human adenoviruses (HAdV) are a leading cause of acuteconjunctivitis in clinical practice [7, 8, 15]. HAdVinfections of the eye may lead to pharyngoconjunctivalfever, follicular conjunctivitis or epidemic keratoconjunc-

tivitis (EKC). The latter is commonly caused by speciesHAdV-D, particularly by serotypes 8, 19 and 37, whereasfollicular conjunctivitis and pharyngoconjunctival fever aremost frequently caused by HAdV-B serotypes 3 or 7 andHAdV-E4 [1, 12, 18]. Most EKC outbreaks are commu-nity-based and transmitted from person to person by

R. Wlfel (*) . M. Pfeffer .

S. Essbauer . G. DoblerBundeswehr Institute of Microbiology,Neuherbergstrasse 11,80937 Munich, Germanye-mail: [email protected].: +49-89-31682637Fax: +49-89-31683292

S. NerkelunBundeswehr Medical Specialist Service

Section of Ophthalmology,Munich, Germany


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fingers or have been reported to occur due to contaminatedophthalmologic solutions and instruments [5, 8, 23].Because of crowded living conditions, military personnelare at particularly high risk for ocular adenoviral infections.For example, in spring 2004, an outbreak of conjunctivitisappeared at different locations of the German armed forces,causing disruption of military training and even closure of

some military facilities. Finally, a total of 940 eye swabsamples of symptomatic patients were analysed, but only12 adenovirus-positive samples were identified. In conclu-sion, a majority of patients who where clinically diagnosedas EKC contracted conjunctivitis of another, yet unidenti-fied, aetiology [24]. This incidence stresses that during anepidemic outbreak of conjunctivitis, rapid laboratorydiagnosis is required to adopt appropriate preventivemeasures and to limit spread of the virus if necessary[21]. Collection and preparation of patients specimens isof fundamental importance for diagnosis of adenoviruses.Because HAdV are cell associated, specimens have to becollected in such a way that they comprise as many intact

cells as possible [9]. During transport and shipment of thespecimens for further investigation, samples should be keptunder conditions allowing later identification of HAdVeither by virological or molecular methods.

The gold standard for laboratory diagnosis of HAdVconjunctivitis is virus isolation by cell culture, usuallyrequiring 12 weeks for completion [16]. More rapidassays have been developed, including enzyme immuno-assay, direct immunofluorescence, DNA hybridisation andelectron microscopy [4, 16, 17, 19, 22]. These techniquescan be complex, laborious and often inappropriate forroutine situations. In contrast, real-time polymerase chainreaction (real-time PCR) assays are now widely used for

diagnosis of viruses in most microbiological laboratories.Protocols have been developed for HAdV detection inocular swab samples, offering significant improvement inspeed and sensitivity [10].

In cooperation with the German National ReferenceLaboratory for Adenoviruses, Hanover Medical School, wewere involved in the investigation of a suspected outbreakof EKC in the German Armed Forces, as mentioned above.We found that in the past, recommendations for samplingtechniques were partially based on popular or traditionalassumptions [9]. Several studies investigating the ability ofdifferent viral transport systems (VTS) to maintain viabilityof HAdV were carried out [2, 6, 11, 13, 14]. However, it is

difficult to compare these results since they were carriedout under different conditions, employing a variety ofmethods for viral detection. However, none of these studiescompared rapid virus isolation and quantitative real-timePCR (qPCR). The purpose of our study was to address thefollowing questions:

Is application of local anaesthetics to the eye prior toswabbing necessary, and does it affect the detection ofHAdV by cell culture or quantitative real-time PCR?

Does the material of the swabs or the swab shaftinfluence the results of qPCR?

Are there significant differences in the performance ofcommercially available VTS compared with a simple

NaCl-moisturised transport method regarding detec-tion of viruses by cell culture and/or qPCR?

Based on our experiments and results, we providerecommendations on adequate VTS for efficient investiga-tion of EKC outbreaks.

Materials and methods

Conjunctival samples

A total of 73 conjunctival swabs were collected from adult patients with suspected viral conjunctival infections byphysicians at the Ophthalmology Specialist Service Sec-tion, Munich. The eyes of 51 patients were anaesthetised

topically with one drop of oxybuprocaine (4 mg/ml,Conjuncain single dose, Mann-Pharma, Berlin, Germany)1 min prior to sampling; controls were taken from 22

patients without topical anaesthesia. Using a Dacron-tippedswab, both upper and lower conjunctival surfaces werevigourously wiped, rotating the swab during the sampling

process to ensure that the entire conjunctival surface wassampled. The specimen swab was rolled, using slight

pressure, onto the 6-mm well area of a microscope slide. Itwas ensured that the whole swab tip was used to preparethe slide. The smear was air-dried at room temperature andthen fixed in ice-cold acetone for 10 min. After immediatetransfer to the laboratory, the number of conjunctival cells

on the slide was counted using a phase-contrast microscopeprior to diagnostic treatment of the specimens.

Cells and media

Culture cells employed in this study were A549 cells(human lung cell line) kindly provided by Frank Hufert,University of Freiburg, Germany. Cell cultures were grownin minimum essential medium (MEM; Invitrogen, Karls-ruhe, Germany) containing 3% foetal calf serum (FCS;Biochrom, Berlin, Germany), sodium bicarbonate, penicil-lin, streptomycin and amphotericin B (all from Invitrogen,

Karlsruhe, Germany).

Viruses

HAdV type 8 from a clinical isolate was kindly provided byHermann Schtzl, Technical University of Munich,Germany. A viral stock was obtained by inoculation ofA549 cells. Supernatant was harvested when a nearlycomplete cytopathic effect was observed and was briefly


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centrifuged (10 min, 1,200 g) to pellet cellular debris. Thetitre of the viral stock was determined by immunofluores-cence (IFA), as described below. Stock aliquots containing6105 focus-forming units (FFU)/ml were stored at80Cuntil use. The HAdV stock was serially diluted in MEM totest for different initial inoculum concentrations.

Topical anaesthetics

Oxybuprocaine-containing eye drops (50 l, 25 l and12.5 l) (novesine, 4 mg/ml oxybuprocaine, chlorhexidinediacetate 90 g/ml; OmniVision, Puchheim, Germany)were mixed in a reaction tube with 50 l of the followingHAdV solutions: FFU 2105/ml, 2104/ml, 2103/ml.MEM was added up to a final volume of 1,000 l. Reactiontubes were incubated for 60 min until further cell-cultureand qPCR processing.

Viral transport systems (VTS)

Two different VTS were evaluated. One was the Copan New Virus Transport System (Copan VTS, Copan,Bovezzo, Italy) comprising a liquid virus transport mediumin a sponge carrier located at the bottom of the tube. TheDacron swab tip is placed in direct contact with themedium-soaked sponge. About 900 l transport mediumcould be easily squeezed out of the tube for further sample

processing. The second was the Viral Culturette (BD VTS,Becton-Dickinson, Heidelberg, Germany), a swab-tubesystem containing Hanks balanced salt solutions. Mediumis sealed in a reagent cradle, which has to be broken after

insertion of the rayon-tipped swab. There is no instructiongiven by the manufacturer as to how to recover the samplefrom the tube: We added 900 l MEM, inserted the swabagain and vortexed the tube. Thus, about 900 l mediumcould be extracted. For comparative purposes, we took 15-ml reaction tubes (VWR, Darmstadt, Germany) comprising900 l sterile 0.9% NaCl (Braun, Melsungen, Germany)

plus either Dacron-tipped plastic-shafted swabs or cotton-tipped wood-shafted swabs (all from VWR, Darmstadt,Germany). All experiments were performed at least twice

by different investigators.

Inoculation of transport systems

Concentration-dependent stability Both commercial VTS,Dacron-tipped plastic-shafted swabs and cotton-tippedwood-shafted swabs stored in NaCl, were evaluatedusing the following virus inocula: FFU 6105/ml,6104/ml, 6103/ml, 6102/ml, 6101/ml and 6100/ml.All swabs were inoculated with 50 l of the specified viralstock concentration and stored for 24 h in the dark at

21C. The diluted stock controls were stored at 80Cuntil final analysis.

Time-dependent stability The swab of each transport typewas inoculated with 50 l of a virus dilution (6104

FFU/ml) and immediately placed into the respectivetransport medium. Dacron-tipped plastic-shafted swabs

stored in NaCl were used for comparison. Tubes weremaintained in the dark at 21C for 0 h, 24 h, 48 h, 72 h,96 h and 120 h. Subsequent to the respective storage

periods, tubes were frozen at80C and finally analysedby IFA and qPCR. As controls, 50 l-aliquots of swab-freevirus dilutions stored at 80C for the correspondinginoculation period were used.

Centrifugation culture and detection of viruses by IFA

Titres of viral samples and stocks were determined using96-well plates containing 80% confluent A549 cells.

Samples were vortexed again prior inoculation. Followingaspiration of the medium, each well was inoculated with100 l sample. The plates were sealed with an adhesivefilm and centrifuged at 2,000 g for 30 min at 25C. After anadditional incubation period of 30 min, the inoculate wasremoved. To each well was added 200 l MEM containing3% FCS, and plates were further incubated at 37C and 5%CO2. After 48 h, the cell culture supernatant was carefullyremoved; plates were air dried and fixed with freshmethanol-acetone (1:1 v/v) at room temperature for 30minutes. IFA staining was done using the Imagen adeno-virus kit (DakoCytomation, Cambridgeshire, UK): Themonoclonal antibody solution was diluted 1:3 with phos-

phate-buffered saline (PBS) without any remarkable loss ofsensitivity (data not shown). To each well, 40 l of dilutedantibody solution was added. Plates were then incubatedwithin a moist chamber for 30 min at 37C. Excess reagentwas washed out with 100 l PBS. PBS was then drainedoff, and the plates were again air dried at room temperature.Mounting fluid (DakoCytomation, Cambridgeshire, UK)was added, and wells were examined using an invertedfluorescence microscope at x100 magnification. Cells withan intracellular, nuclear or cytoplasmic granular apple-green fluorescence were considered HAdV-positive. Theaverage turn-around time for clinical samples examined bythis technique is 5660 h.

Quantitative real-time PCR (qPCR)

DNA was extracted from samples using the Qiagen DNAkit (Qiagen, Hilden, Germany), according to the manu-facturers instructions. qPCR was performed using aLightCycler (Roche, Mannheim, Germany) with generic

primers, as described previously [10]. Cycling conditionswere modified slightly by reducing the extension period at


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65C from 60 s to 15 s. The average turn-around time forclinical sample preparation and qPCR analysis was 48 h.The positive control standard, a HAdV-2 PCR amplicon(nt. 1885619137 of the HAdV-2 sequence) cloned into a

pGEM-T easy plasmid vector, was kindly provided byAlbert Heim, Medical University of Hanover, Germany.Concentration of the plasmid was determined by photom-

etry and calculated as genome equivalents (DNA copies)per millilitre. Serial dilution of the HAdV plasmid was usedto create a standard curve. DNA concentrations werecalculated considering the sample dilution factor using theLightCycler software. The standard was stored at20C inaliquots until use.

Statistical analysis

The Wilcoxon signed rank test was used to determinesignificant differences between the different samplegroups. Statistical analyses were performed by using

GraphPad prism 4.00 for Windows (GraphPad Software,San Diego, CA, USA). Results are given as mean standard error of the mean (SEM). Pvalues are given whenappropriate. Significance was accepted at P


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cant (P>0.9). We did not see any comparable cell damagein the diagnostic process of specimens from locallyanaesthetised patients. Likewise, by using DNA extractionand qPCR protocols described above, we did not find anysignificant influence of oxyprocain on real-time detectionof HAdV-DNA (data not shown), indicating that neither thelocal anaesthetic nor the conservation material inhibited

detection of HAdV by qPCR.

Influences of VTS on cell culture and qPCR results

We compared the performance of NaCl-moisturised cotton-tipped wood-shafted swabs and Dacron-tipped plastic-shafted swabs to the Copan VTS and BD VTS: Titration ofseveral solutions with different virus concentrationsemploying the culture centrifugation technique providedno significant difference between controls and cotton-tipped wood-shafted swabs. In strong contrast to theseresults, almost 90% of the infectious virus was lost by

storage in Copan or BD VTS. (Table 2, Fig. 5). The numberof DNA copies of samples stored in Copan or BD VTS wassignificantly lower compared with controls. The corre-sponding qPCR results of the remaining two swab systemsrevealed no sign of inhibition by cotton- and/or wood-containing swabs after storage for 2 days (Fig. 3).

Stability of adenovirus storage in different VTS

Clinical HAdV specimens are commonly sent by postalservice for further virological investigation. Therefore, wetested the stability of a 6104 FFU/ml virus solution stored

in the dark at room temperature. At day 0, similar amountsof HAdV were detected in all transport media when tested

by immunofluorescence (Table 3) However, the HAdVtitre decreased in the Copan VTS as well as the BD VTS asearly as 24 h in the storage period. After 3 days, no viruscount was detected for either commercial systems employ-ing culture centrifugation technique or IFA. In the

corresponding samples stored with NaCl-moisturisedDacron-tipped plastic-shafted swabs, viable virus wasdetected for up to 5 days using cell culture. With qPCR,differences between the tested transport media were lessdistinct (Fig. 4). Inoculated virus was detected over 5 daysin commercial VTS as well as Dacron/NaCl swabs.

Discussion

Specimen collection and transport is an important issue invirological diagnostics. In case of a suspected outbreak ofEKC, reliable microbiological diagnosis is needed toconfirm clinical diagnosis or to avoid inappropriatetreatment and overreactions. The first objective of this

Table 2 Concentration-dependent stability of human adenoviruses (HAdV) after 24-h storage

Log-focus-forming units per millilitreInoculum

Control Copan VTS BD VTS Cotton/wood/NaCllog FFU/ml

Mean SEM Mean SEM Mean SEM Mean SEM

5.8 a 4.6 0.08 4.5 0.03 a

4.8 4.7 0.07 3.4 0.03 3.3 0.03 4.6 0.02

3.8 3.8 0.05 2.4 0.06 2.4 0.10 3.6 0.09

2.8 2.7 0.07 b b 2.5 0.16

1.8 b b b b

0.8 b b b b

FFU focus-forming units, VTS viral transport system, SEM standard error of the meanaMore than 300 focus-forming units per wellbLess than one focus-forming unit per well

Fig. 3 Concentration-dependent stability of human adenovirus(HAdV) DNA in different transport media: The amount of viralDNA that can be recovered from commercial virus transport systems(VTS) is up to 2 logs lower when compared with a NaCl-

moisturised transport system (P=0.03). The quantitative real-time polymerase chain reaction (qPCR) results of cotton- and wood-containing swabs did not significantly differ from the Dacron-madeswabs


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study was to determine if local anaesthesia of the eyeshould be recommended for HAdV specimen collection.Because HAdV replicates mainly within the nucleus [3], itis most important to collect as many conjunctival cells as

possible. We showed that topical application of an

anaesthetic is an appropriate way of collecting materialfor diagnostics, which does not interfere with downstreamtechniques such as qPCR, an important screening method,or virus isolation by cell culture. The cytotoxic-like effectsthat we observed in cell culture at the highest concentrationof local anaesthetics are most likely not relevant for theinvestigation of clinical samples. The appropriate amountof anaesthetic eye drops (50 l) corresponds to thecomplete dose normally administered to a patients eye.Therefore, the amount of local anaesthetics in the specimenwill always be much lower.

Results of this study on HAdV infectivity in centrifu-gation indicate a significant difference between the testedVTS for virus preservation. In contrast to specific virus

Fig. 5a, b Representative details of immunofluorescence-stainedhuman adenovirus (HAdV)- infected cells in 96-well cell culture(100-fold magnification). a Virus stored on an NaCl-moisturisedswab for 2 days. b Same virus sample stored in Copan VTS

Fig. 4 Time-dependent stability of human adenovirus (HAdV)DNA in the investigated transport media stored at room temperature.Curve progressions for Copan virus transport system (VTS) and BDVTS differ significantly from controls (P=0.03). However, differ-ences between control and Dacron/NaCl are nonsignificant (P=0.13)

Table 3 Time-dependent stability of human adenovirus (HAdV) in virus transport systems (VTS) over 5 days

Log-focus-forming units per millimetre

Day Control Copan VTS BD VTS Dacron/NaCl

Mean SEM Mean SEM Mean SEM Mean SEM

0 4.7 0.13 4.3 0.04 4.2 0.06 4.7 0.01

1 4.7 0.08 3.7 0.05 3.5 0.06 4.7 0.042 4.7 0.13 2.8 0.06 2.6 0.00 4.7 0.02

3 4.7 0.03 2.3 0.00 a 4.6 0.01

4 4.7 0.03 a a 4.3 0.03

5 4.6 0.01 a a 3.8 0.01

aLess than one focus-forming unit per well


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transport media, sterile cotton-tipped wood-shafted swabsare available in most general practitioners and ophthal-mologists offices. However, no investigation is availableon inhibition of PCR or interference with cell culture by thenatural materials these swabs are made off [20]. In bothcommercial systems (Copan VTS and BD VTS), HAdVsurvival was reduced when compared with NaCl-mois-

turised Dacron-tipped plastic-shafted swabs. These resultsare in contrast to previous studies [6, 13], which foundadenovirus to be rather stable in commercial VTS. Thisdiscrepancy may partly be explained by differences in thetest protocols, such as incubation time (up to 17 days) orvirus quantification (TCID50). For example, high concen-trations of HAdV type 2 were used to inoculate differentVTS, and this may be a further reason why no decrease invirus titre was detected at 22C over a period of 10 days[13]. Nevertheless, our data demonstrate a loss of HAdVviability depending on the VTS if a routine diagnostic test(rapid cell culture detection method) is used as the read-outsystem.

The surprising results of the well-preserving nature ofNaCl-moisturised swabs without any additives are also noteasy to interpret. One suggestion we can come up with isthat at least some of the ingredients of the commercial VTSinterfere with virus viability. This might be due to thenonenveloped morphology of HAdV naturally having ahigh tenacity. But we have no explanation as to why acommercially available VTS causes more harm to a virusthan physiological NaCl moisture does. Similar experi-ments with other nonenveloped viruses, e.g. enteroviruses,may show whether this HAdV-specific phenomenon doesapply to other nonenveloped viruses as well.

Concerning recommendations for adenovirus viraltransport medium, HAdV DNA was fairly stable in allmedia tested, but both commercially available systemsfrom Copan and Becton-Dickinson did not perform as wellas the NaCl-moisturised swabs in preserving HAdVinfectivity for rapid cell culture detection. Hence, neithersystem can be recommended for specimen transport of

ophthalmologic samples for HAdV diagnosis, regardless ofthe downstream methods used. Furthermore, both com-mercial VTS were up to three times more expensivecompared with the swab/tube combinations, which wereused in this study. In conclusion, our recommendation foran adenovirus transport medium is an NaCl-moisturisedDacron-tipped plastic-shafted or cotton-tipped wood-shafted swab stored and shipped in a standard reactiontube. Although virus titres maintained the initial level foralmost 4 days, fast transport and shipment is crucial toachieve diagnosis in due course. Because of its much lowerturn-around time and wider availability compared withrapid cell culture detection, we strongly recommend qPCR

for a fast and sensitive screening of ophthalmologicallyrelevant HAdV.

Acknowledgements We acknowledge the excellent technicalsupport of Heike Prabel, Aileen Lorber, Peter Klein, RahimeTerzioglu and Gudrun Zller during the course of this study. Wethank Dr. Albert Heim, German National Reference Laboratory forAdenoviruses, Hanover Medical School, Germany, for his helpfulsupport in setting up the quantitative HAdV real-time PCR. Theviews expressed in this article are those of the authors and do notnecessarily reflect the official policy or position of the GermanMinistry of Defence or the German Government.

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