APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2011, p. 7289–7295 Vol. 77, No. 200099-2240/11/$12.00 doi:10.1128/AEM.05553-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Microbial Inactivation for Safe and Rapid Diagnosticsof Infectious Samples�†

Jose-Luis Sagripanti,2 Birgit Hulseweh,1 Gudrun Grote,1 Luzie Voß,1Katrin Bohling,1 and Hans-Jurgen Marschall1*

Wehrwissenschafliches Institut fur Schutztechnologien (WIS), ABC-Schutz, Munster, Germany,1

and Edgewood Chemical Biological Center, U.S. Army, Aberdeen, Maryland2

Received 23 May 2011/Accepted 8 August 2011

The high risk associated with biological threat agents dictates that any suspicious sample be handled understrict surety and safety controls and processed under high-level containment in specialized laboratories. Thisstudy attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range ofpathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid frag-ments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogenperoxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or virusesafter 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliter-ated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses.High-level inactivation (more than 6 log10) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseu-domonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vacciniavirus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate withminimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide thebasis for quickly rendering field samples noninfectious for further analysis under lower-level containment andconsiderably lower cost.

The high risk associated with biological threats dictates thatany suspicious samples be handled under strict surety andsafety controls and processed under high-level containment inspecialized laboratories. These laboratories are complex, veryexpensive to operate, and need to be staffed by personnel froman extremely limited pool of experts. In addition, safe means oftransporting samples suspected of containing highly virulentagents to specialized high-level containment laboratories foranalysis also is expensive, requiring in many countries the cus-tody of armed personnel. It can be estimated that severalmillion dollars are spent annually worldwide to secure andsafely transport an ever-increasing stream of suspicious biolog-ical samples, which are collected in theaters of war as well as indomestic environments.

The objective of this study was to identify liquid chemicalreagents with high efficiency to inactivate microbial organismsand viruses that, at the same time, would preserve the highsensitivity of subsequent diagnostics performed at a lower levelof containment and cost.

Considerable knowledge has been accumulated regardingthe efficiency of microbicidal reagents and methods, partic-ularly on inactivating bacterial spores in liquids, on surfaces,or in aerosols (4, 19), but the subsequent effect of thesedisinfectants on the performance of either nucleic acid-

based or immunologically based diagnostics have receivedrelatively little attention. We selected for study two alde-hydes (formaldehyde and glutaraldehyde), a halogenatingagent (hypochlorite), two peroxides (hydrogen peroxide andperacetic acid), and a free radical-damaging agent (cupricascorbate), all of which previously have shown relativelyhigh sporicidal efficiency (3, 4, 18, 22). In addition, we ex-amined a chaotropic agent, guanidium thiocyanate, whichhas the well-established ability to dissociate biological struc-tures and liberate intact nucleic acids for analysis (26). Asmicrobial targets we selected spores of Bacillus atrophaeus,because these have been used frequently in sporicidal stud-ies and also because spores of B. atrophaeus show sensitivityto chemical germicides that is similar to that of virulentstrains of B. anthracis (24). Cells of Pseudomonas aeruginosawere selected for study because this bacterium was in thegroup with the highest resistance to disinfection among thevegetative cells of bacteria frequently causing human infec-tion in hospitals (23).

To assess the effect of disinfectants on viruses, mainly on thepotential hindrance of virus detection, we studied vacciniavirus (VacV), which is an orthopoxvirus (with a DNA genome)generally used as surrogate for smallpox virus, and Pixuna virus(PIXV), which is an alphavirus that has been used as simulantfor Venezuelan equine encephalitis virus and other highlypathogenic RNA viruses (1, 8, 9, 27). The inactivation of allthese microorganisms was monitored until a level of more than6 log10 was reached, since this is the standard assurance levelgenerally accepted for the safety of medical devices and con-taminated environments (ASTM standard E-2414-05 [2]). Theeffect of germicidal agents was studied on the performance ofPCR tests, as it is one of the most frequently used nucleic

* Corresponding author. Mailing address: WehrwissenschaftlichesInstitut fur Schutztechnologien ABC, Schutz Scientific Institute forProtection Technologies, NBC-Protection, German Federal Army,P.O. Box 1142, 29623 Munster, Germany. Phone: 49-5192-136377.E-mail: hansjuergenmarschall@bwb.org.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 19 August 2011.

7289

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

acid-based diagnostic methods, and on enzyme-linked immu-noassay (ELISA), which is one of the most widely employedimmunodetection methods (11, 13, 14, 17).

The results of this study should expand the knowledge of theeffects of germicidal agents on bacteria, bacterial spores, andviruses and provide new insights on the subsequent perfor-mance of detection and diagnostic methods on high-risk bio-logical samples.

MATERIALS AND METHODS

Microbial species and sources. Bacillus atrophaeus (strain ATCC 9372) wasobtained from the American Type Culture Collection. Spores of B. atrophaeuswere prepared in accordance with DIN EN 14347 (5) and resuspended at aconcentration of 2 � 108 spores per ml. P. aeruginosa (strain DSM 1253) wasobtained from the German strain culture collection (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH [DSMZ], Braunschweig, Germany).On the day before each experiment, one colony of P. aeruginosa originallyisolated in tryptone soya agar (TSA; Oxoid, Wesel, Germany) was seeded into 50ml medium and grown for about 20 h at 37°C with agitation. On the day of theexperiment, cells near the end of their exponential phase of growth were washedthree times by centrifugation and resuspended in sterile distilled water. The titerof P. aeruginosa in the final working suspension ranged from 0.8 �109 to 2.4 �109 CFU/ml. Pixuna virus (strain BeAr 35645; Brazil 1961, ATCC VR-371) waspropagated in baby hamster kidney (BHK) cells (DSMZ-ACC 33; DMSZ,Braunschweig, Germany) at 37°C in Eagle’s minimum essential medium(EMEM), which was purchased from Biochrom (Berlin, Germany). It contained10% fetal calf serum (FCS) and 0.1% penicillin-streptomycin and was harvestedfrom infected cell monolayers at a concentration of 1.2 � 1010 50% tissue cultureinfective doses (TCID50)/ml.

Vaccinia virus, strain Elstree B5/Lister (WHO reference strain [7]), was grownin BHK cells at 37°C in EMEM containing 10% FCS and 0.1% penicillin-streptomycin and was used at a TCID50/ml ranging from 2 � 106 to 4 � 106.

Chemical disinfectants. Formaldehyde and guanidium thiocyanate were pur-chased from Roth GmbH (Karlsruhe, Germany), with the latter being dissolvedat a concentration of 7 M in sterile distilled water and its pH adjusted between7 to 8 at room temperature with Tris-HCl. Peracetic acid and sodium hypochlo-rite containing 6.7% active chlorine, as determined by iodometric determinationin our laboratory, were purchased from Sigma-Aldrich (Taufkirchen, Germany).Before use, the hypochlorite solution was adjusted to pH 7.5 with hydrochloricacid. Glutaraldehyde purchased from Sigma-Aldrich (Taufkirchen, Germany)was activated before each experiment by adjusting it to pH 9.3 with sodiumbicarbonate. Hydrogen peroxide at 30% (vol/vol) was obtained from Merck-VWR International, Darmstadt, Germany. Cupric chloride dihydrate (�99% per

analysis) was purchased from Roth GmbH (Karlsruhe, Germany). Cupric ascor-bate was generated directly before use from 0.5% cupric chloride dihydrate,0.1% L-ascorbic acid (�99% ACS reagent; Sigma-Aldrich, Taufkirchen, Ger-many), and 0.003% hydrogen peroxide; this last substance was added to ensureinstant liquid oxygenation for efficient free-radical generation (18, 23).

Cupric chloride and L-ascorbic acid were stored as solid powders at roomtemperature under vacuum. All dilutions were prepared by dissolving dry cupricchloride and ascorbic acid just before use as intended for disinfection in the field.We used peracetic acid as supplied in a 37.5 to 40.5% (vol/vol) stabilized solutioncontaining hydrogen peroxide and acetic acid for long shelf live (more than ayear) at room temperature. The chemicals peracetic acid and cupric ascorbate(selected for inactivation experiments) are sufficiently stable, even for field use,within a 6-month period, if not longer.

Microbial inactivation. A microbial suspension (typically 50 �l) containingeither 108 spores ml�1, 0.8 � 109 to 2.4 � 109 P. aeruginosa cells ml�1, 1.2 � 1010

Pixuna virus TCID50 ml�1, or 4 � 106 vaccinia virus TCID50 ml�1 was dispensedinto 1.5-ml Eppendorf-type tubes, carefully avoiding the microbial contamina-tion of the inside walls of the tube above the liquid level.

An equal volume of either phosphate-buffered saline (PBS) as a controlwithout inactivating effect or disinfectant at various concentrations wasadded, and the mixture with microbes and disinfectant was incubated at 21°Cfor 30 min with a 10-s initial mixing at 300 rpm (Eppendorf ThermomixerComfort; Eppendorf AG, Hamburg, Germany). Only cupric ascorbate wasprepared in situ, as previously described (20, 21, 23), with the exact concen-trations indicated in Table 1. After disinfectant treatment, nine volumes (900�l) of either ice-cold tryptone soya broth (TSB; Oxoid, Wesel, Germany) forbacteria or ice-cold EMEM with 10% fetal calf serum for viruses was addedto the treatment mixes to slow down any remaining inactivation process.Surviving bacteria were analyzed in aliquots by serial dilution and titrationonto TSA plates.

Viruses were serially diluted in EMEM supplemented with 5% fetal calf serumand were plated in dilutions ranging from between 10�5 to 10�8 onto BHK cellmonolayers in 96-well cell culture microtest plates. Medium was removed fromcells, and virus in the sample (25 �l) was adsorbed for 1 h. The inoculum wasremoved and replaced with fresh EMEM containing 10% FCS (25 �l per well)before the plates were incubated overnight at 37°C in a 4% CO2 atmosphere. Theamount of surviving virus was determined by the TCID50 method on BHK cellmonolayers (9, 10, 28). Other aliquots of the same exposed sample were testedby PCR and ELISA as described in the sections below.

DNA and RNA preparation. Nucleic acids, DNA, or RNA from VacV orPIXV were extracted from a 100-�l aliquot of the sample inactivated with thestudied disinfectants. Purification was done by using either the QIAamp DNAMinikit or the QIAamp viral RNA Minikit (Qiagen, Hilden, Germany) accordingto the manufacturer’s instructions with minor modifications. Viral DNA andRNA were eluted with 100 �l of the cognate buffer. Generally, 1 �l of DNA was

TABLE 1. Screening of inactivating chemicals with Pixuna virus and Bacillus atrophaeus spores

Organism andchemical concna

Inactivating chemical and parameterg

Peracetic acid Na-hypochlorite Cupric ascorbate

Concn (%) Titerreduction

PCRdetectionb

ELISAsignalc Concn (%) Titer

reductionPCR

detectionbELISAsignalc Concnh (%) Titer

reductionPCR

detectionbELISAsignalc

Pixuna virusT1 0.00 1 log10 �� ��� 0.01 0 �� �� 0.1 �4 log10 �� �f

T2 0.03 �6 log10 �� � 0.05 3 log10 � � 0.5 �6 log10 �� �f

T3 0.30 �6 log10 � � 0.50 �6 log10 � � 2.5 �6 log10 � �f

Bacillus atrophaeus sporesT1 0.00 �0.1 log10 �� ��� 0.01 �3 log10 � �� 0.1 �1 log10 �� ���T2 0.03 �6 log10 �� ��� 0.05 �6 log10 � ��� 0.5 �6 log10 �� ���T3 0.30 �6 log10 �� ���/�� 0.50 �6 log10 � ��� 2.5 �6 log10 � ���

a Concentrations and titers given in italics generally apply to liquid sterilization (albeit sometimes for different incubation times and at other temperatures). T1,reduced concentration; T2, usual or standard concentration; T3, increased concentration.

b ���, �25 PCR cycles; ��, �30 PCR cycles; �, �35 PCR cycles; �, �35 PCR cycles.c ELISA performance, in units of optical density at 450 nm: ���, �75; ��, �50; �, �25; �, �25.d Guadinium-isothiocyanate.e ND, not determined.f After previous treatment of samples with 100 mM EDTA.g Reaction time of 30 min at room temperature.h Cupric ascorbate concentrations were the following: T1, 0.1 Cu, 0.02% AscH, and 0.0006% H2O2; T2, 0.5% Cu, 0.1% AscH, and 0.003% H2O2; T3, 2.5% Cu, 0.5%

AscH, and 0.015% H2O2.

7290 SAGRIPANTI ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

subjected to the VacV specific real-time PCR (rtPCR), and 5 �l RNA wassubjected to the PIXV specific real-time reverse transcriptase PCR (rtRT-PCR).

rtPCR and rtRT-PCR. For real-time PCR, the one-step RT-PCR, the Hot-StarTaq, or the TaqDNA polymerase system from Qiagen GmbH (Hilden, Ger-many) was used with either TaqMan or SYBR green I (TIB Molbiol GmbH,Berlin, Germany). The fluorescent reporter dye of the viral probe was 6-carboxy-fluorescein (FAM) and was located at the 5� end in all 5� nuclease assays. Thequencher, 6-carboxytetramethylrhodamine (TAMRA), was located at the 3�end.The 5� to 3� sequences of all primers and probes employed in this study aresummarized in Table S1 in the supplemental material. We used purified nucleicacids in viral PCR assays, and extensively washed and resuspended spores orvegetative cell suspensions were directly applied to the respective PCR tests.

rtPCR as well as rtRT-PCR assays were performed in microtest plates (BiozymScientific GmbH, Germany) in a final volume of either 25 or 20 �l on an Opticontype 1 device (Bio-Rad Laboratories, Inc.). The cycling parameters for each assayare given in Table S2 in the supplemental material. Primer, 5�-nuclease probe,SYBR green I, Mg2�, and deoxynucleoside triphosphate concentrations wereoptimized by titration and used as indicated in Table S2 in the supplementalmaterial.

Each real-time PCR and RT-PCR was performed in duplicate or triplicate,and individual experiments were repeated two to four times. Negative controlscontained water instead of a potential nucleic acid template. The impairment ofdisinfectants on either PCR or RT-PCR tests was correlated to the CT (cyclethreshold) value that corresponds to the first cycle number in which the fluores-cence signal significantly surpasses the background signal. The CT value is in-versely proportional to the amount of amplifiable target nucleic acid in thesample. Thus, a higher CT value corresponds to a smaller amount of amplifiabletarget nucleic acid in our samples that would result from increasing damage bydisinfectants. Nucleic acids that are not, or are only marginally, damaged bychemical inactivation result in CT values similar to those obtained with untreatedsamples. Those chemical agents that damage nucleic acids produce PCR testswith CT values higher than those obtained in the untreated controls or produceno measurable results at all.

ELISA. All enzyme-linked immunosorbent assays (ELISAs) were performedin 96-microwell plates (Maxisorb; Thermo Fisher Scientific, Dreieich, Germany)that were coated with 3 to 4 �g antibody per well either by incubation overnightat 4°C or by incubation for 2 h at 37°C, while plates for viral ELISAs were washedand blocked with 1% FCS in PBS-T (PBS plus 0.01% Tween 20) for 1 h at roomtemperature. Plates for bacterial ELISAs were blocked with 1% low-fat milkpowder in PBS for 30 min at room temperature. In addition, plates for viralELISA were overlaid with liquid plate sealer (Candor Bioscience GmbH, Wan-gen, Germany) and used for ELISA studies within 3 to 4 weeks. Plates forbacterial ELISAs were freshly coated for each use.

For the detection of Pixuna virus, we used the species-specific monoclonalantibody (MAb) PixcT 6/2, kindly provided by V. Moennig (University of Vet-erinary Medicine, Hannover, Germany), as the capture antibody. For the detec-tion of vaccinia virus, we used an equimolar mixture of the MAbs 5B1 and 5B4(6, 7). P. aeruginosa and B. atrophaeus endospores were captured with specificrabbit polyclonal antibodies (PAbs) produced in our laboratory. Antigen incu-bation was performed for either 1 or 2 h at room temperature (21°C) or at 37°C.Bound viral and bacterial antigens were detected by using either biotinylated (b)species-specific MAbs or PAbs (PIXV bcT3/3/10, 1:10,000; VacV b5B1, 1:2,000;WIS pAb anti-B. atrophaeus spores, 1:400; WIS pAb anti-P. aeruginosa, 1:400).After extensive washing, the conjugate streptavidin-horseradish peroxidase

(PSA; GE Healthcare) was added to the wells and diluted 1:6,000 in PBS-FT(PBS plus 1% FCS plus 0.1% Tween 20).

Plates were incubated for 30 min at 21°C with agitation, and after three washeswith PBS-T staining was performed with the colorimetric substrate 3-3�,5,5�-tetramethylbenzidine (TMB; Serva, Heidelberg, Germany) for 10 min. Furthercolor development was stopped with 2 M sulfuric acid, and absorbance wasmeasured at 450 nm. The improvement of ELISA results obtained after cupricascorbate disinfection was evaluated by the addition of EDTA (pH 8; finalconcentration of 2, 10, 20, 40, and 100 mM). The enhancement of ELISA resultsafter peracetic acid treatment was attempted by the addition of either 1 M Tris,pH 8, or catalase at final concentrations ranging from 32 to 324 U. All threepotential ELISA enhancers (EDTA, Tris, and catalase) were purchased fromSigma-Aldrich and were added to samples after the 30-min inactivation withdisinfectants.

RESULTS

Screening for chemical methods that completely inactivatepathogens with minimal impairment of diagnostics. The con-centrations of inactivating reagents generally employed forchemical disinfection and sterilization of microbial pathogenscorrespond to hypochlorite at 0.05% (vol/vol), glutaraldehydeat 2% (vol/vol), peracetic acid at 0.03% (vol/vol), formalde-hyde at 8% (vol/vol), hydrogen peroxide at 10% (vol/vol), andcupric ascorbate at 0.5% (wt/vol) in cupric ions (20). There-fore, we exposed bacterial spores and viruses to the differentchemical germicides at the concentrations indicated above aswell as to one lower (generally one-tenth) and one higher(generally 10-fold) concentration. In addition to commonlyused germicides, we also studied the effect of guanidium thio-cyanate, which is known to disrupt cells and liberate nucleicacids without damaging them for further analysis (26). Ourmain requirement for the selection of disinfecting agents wasthe complete, fast, and reliable inactivation of spores at or nearroom temperature, since any method to be developed shouldbe rapid and performable in the field. The results of theseexploratory experiments with common disinfecting and inacti-vating chemicals are summarized in Table 1. We rated theefficiency of the reagents not only for their reduction of infec-tivity but also for their preservation of immunological reactivityand nucleic acid detection.

The sporicidal efficiency of the applied chemical germicidesthat we observed in dose-response experiments was similar topreviously reported results (23, 24). However, the comparisonof our data that of others was prevented by the limited datapreviously available about the virucidal efficiency of thesechemicals on RNA viruses (12, 15, 16).

TABLE 1—Continued

Inactivating chemical and parameterg

Glutaraldehyde Formaldehyde Hydrogen peroxide Guadinium-ISCNd

Concn (%) Titerreduction

PCRdetectionb

ELISAsignalc

Concn(%)

Titerreduction

PCRdetectionb

ELISAsignalc

Concn(%)

Titerreduction

PCRdetectionb

ELISAsignalc

Concn(M)

Titerreduction

PCRdetectionb

ELISAsignalc

0.40 �6 log10 � � 4 �6 log10 � �� 1 �1 log10 �� ��� 0.035 0 �� ���2 NDe � � 8 toxic � � 10 3 log10 � � 0.35 0 �� ��4 NDe � � 16 toxic � � 15 �6 log10 � � 3.5 �5 log10 �� �

0.40 �2 log10 �� ��� 4 �0.1 log10 �� �� 1 �0.1 log10 �� ��� 0.035 �0.5 log10 �� ���2 �2 log10 �� ��� 8 �0.1 log10 � �� 10 �0.1 log10 �� ��� 0.35 �0.5 log10 �� ���4 �6 log10 �� ��� 16 �1 log10 � � 15 1 log10 �� ��� 3.5 �0.5 log10 �� �

VOL. 77, 2011 MICROBIAL INACTIVATION FOR DIAGNOSTICS 7291

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

As demonstrated by the data in Table 1, some chemicalgermicides, like hydrogen peroxide, formaldehyde, and glutar-aldehyde, did not kill completely all the spores challenged inour tests. These same chemicals, at their concentrations of use,also impaired the performance of PCR and ELISAs to detectspores and viruses, as illustrated in Table 1. This impairingeffect on PCR and ELISA was more pronounced on virusesthan on spores (Fig. 1). Eight percent formaldehyde decreasedthe relative fluorescence of PCR testing of spores but had onlya minimal effect on their CT values (Fig. 1 C). In contrast,sodium hypochlorite, at the concentration generally used inliquid sterilization (0.05%, vol/vol), completely inhibited PCRassays of spores and viruses as well as the ELISA of PIXV (Fig.1B and D). Guanidium thiocyanate at 3.5 M, close to its max-imal aqueous solubility, did not hinder PCR or ELISA but hadlittle inactivating effect on spores and Pixuna virus (Table 1).Incubation at room temperature (21°C) with either cupricascorbate at 0.5% in cupric ions or with 0.03% peracetic acidreduced spores and virus titers by at least 6 log10, hindering thediagnostic performance of PCR and ELISA slightly, exceptthat for Pixuna virus the preliminary ELISA results were neg-ative.

Selected chemical inactivation methods suitable for subse-quent diagnostics. The results from the agent screening shownin Table 1 clearly indicated that peracetic acid and cupricascorbate are the most promising two chemical inactivatingreagents for the development of a field-ready inactivatingmethod that would not impair subsequent diagnostics. Forfurther and more detailed studies, we included vaccinia virus asa surrogate for smallpox virus and vegetative cells from P.aeruginosa as a bacterium with relatively high resistance todisinfection (23). Peracetic acid, which at 0.03% (vol/vol) killedspores (�6 log10) and Pixuna virus (�7 log10) beyond detec-tion levels, affected the corresponding PCR and ELISAs dif-ferently, as shown in Fig. 2. Whereas for both spores andviruses PCR sensitivity was only marginally affected (Fig. 2A),the immunoassay of viruses was completely inhibited at theinactivating concentrations (Fig. 2B). The ELISA signal resultsobtained for spores (Fig. 2B) were affected only by peraceticacid at a concentration 10 times higher than both the concen-tration providing high microbicidal efficacy (0.03%) (Table 1)and the concentration generally used.

The effect of cupric ascorbate at a concentration of 0.5% inCu2� ions (0.5% cupric, 0.1% ascorbate, and 0.003% hydrogen

FIG. 1. Deleterious effect of selected germicidal agents on PCR assays. The diagram depicts the deleterious effects on PCR analysis of Pixunavirus (PIXV) and of B. atrophaeus spores after incubation with different concentrations of formaldehyde or sodium hypochloride at 21°C for 30min compared to effects on untreated controls. Microbes were treated with either water as a nonmicrobicidal control (T0), with formaldehyde(FA), or with sodium hypochlorite (NaHClO) at the concentrations indicated. PCR was done as described in Materials and Methods, and therelative fluorescence as a function of the number of amplification cycles is shown for PIXV (A and B) or for spores of B. atrophaeus (C and D).A complete inactivation (�6 log10) of PIXV and spores was achieved with 0.05% Na-hypochlorite after 30 min of incubation at 21°C; additionally,complete inactivation with formaldehyde was attained for PIXV after 30 min of incubation at 21°C but was not achieved with spores. Symbols inpanel A: black squares, no FA (T0); open squares, 4% FA (T1), 8% FA (T2), and 16% FA (T3). Symbols in panel B: black squares, no NaHClO(T0); open squares, 0.0005% NaHClO (T1); crosses, 0.005% NaHClO (T2); black triangles, 0.05% NaHClO (T3). Symbols in panel C: black circles,no FA (T0); black triangles, 4% FA (T1); open triangles, 8% FA (T2); open circles, 16% FA (T3). Symbols in panel D: black circles, no NaHClO(T0); black triangles, 0.005% NaHClO (T1); open circles, 0.05% NaHClO (T2).

7292 SAGRIPANTI ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

peroxide) on PCR and ELISAs is presented in Fig. 3, showingthat the effect of disinfection on the PCR assay of spores orviruses was negligible. The CT values (means � standard devia-tions) obtained for the untreated controls were 28.5 � 0.58 forspores, 16.5 � 0.58 for P. aeruginosa, 24.9 � 0.9 for Pixuna virus,and 25.6 � 0.4 for vaccinia virus. The PCR signal was lost forPixuna and vaccinia viruses at concentrations 5-fold higher thanthose generally recommended for liquid sterilization, but theserelatively high concentrations did not impair the PCR of spores orP. aeruginosa.

The data presented in Fig. 2 and 3 indicate that peracetic acidand cupric ascorbate increase the number of PCR cycles (relativeto the number of cycles in the controls not exposed to germicidalagent) to detect Pixuna and vaccinia viruses by about 4 and 0cycles, respectively. The same figures demonstrate that no changewas observed in the number of PCR cycles to detect spores orvegetative bacteria (between 0 and 1 cycle, respectively) afterinactivation with either peracetic acid or cupric ascorbate.

The limit of detection of ELISAs to determine spores wasmarginally reduced after treatment with peracetic acid or with

cupric ascorbate. In contrast, ELISA to detect viruses was sensi-tive to disinfection (Fig. 2B). Several substances were included inthe experiments in an attempt to minimize the impairing effect ofperacetic acid and cupric ascorbate on viral ELISA. The additionof catalase (32 to 320 U) to degrade the remaining peroxide or ofTris-EDTA, pH 8, to neutralize acid could not restore the Pixunavirus ELISA signal after disinfection with peracetic acid. Amongall substances studied, EDTA best prevented the impairment ofviral ELISAs by disinfection with cupric ascorbate. The resultsshown in Fig. 3B indicate that cupric ascorbate without posttreat-ment with EDTA completely inhibited immunological reactionswith Pixuna and vaccinia viruses. However, the addition ofEDTA, pH 8, up to a final concentration of 100 mM after thedisinfection with cupric ascorbate protected the signal from vac-cinia virus ELISA to about 90% of the level of the untreatedsample. Immunoassays to detect Pixuna virus were more sensitiveto cupric ascorbate, since EDTA retained only about 20% of thesignal seen for the untreated antigen. These results suggest thatantigens of Pixuna virus are more sensitive to oxidation and in-activation by cupric ascorbate than antigens of vaccinia virus.

FIG. 2. Effect of peracetic acid on PCR and ELISA. All microorganisms were treated with phosphonoacetic acid (PAA) at concentrations of0.003, 0.03, and 0.3%. The range of inactivation of �6 log10 is indicated as a gray area in both graphs. The standard deviations from the meansare indicated by lines extending beyond the symbols. (A) Effect of PAA treatment on the CT values of PCR analysis of Bacillus atrophaeus spores(black circles), Pseudomonas aeruginosa (open circles), PIXV (black squares), and vaccinia virus (VacV; open squares) relative to its effect onuntreated controls. The CT values of untreated controls were 27.5 � 0.58 for B. atrophaeus, 15.8 � 1.16 for P. aeruginosa, 24.7 � 2.0 for PIXV,and 23.6 � 2.7 for VacV. (B) Effect of the same treatment with peracetic acid on the corresponding ELISA of B. atrophaeus spores (black circles),P. aeruginosa (open circles), PixV (black squares), and VacV (open squares) relative to its effect on untreated controls. OD, optical density.

VOL. 77, 2011 MICROBIAL INACTIVATION FOR DIAGNOSTICS 7293

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

Peracetic acid reduced the sensitivity of spore immunoassays tonearly 20% of the results obtained with untreated controls, whilecupric ascorbate had no impact. It is important to note that theinactivation of vegetative bacteria, bacterial spores, or viruses didnot lead to false-positive signals in subsequent PCR or ELISAtesting.

DISCUSSION

This study attempted to find a rapid, reliable, and simplemethod for the complete inactivation of a wide range of patho-gens, including spores, vegetative bacteria, and viruses, while pre-serving microbial nucleic acid fragments suitable for PCRs andproteinaceous epitopes for detection by immunoassays. Sevencommonly used liquid disinfectants were included in the experi-ments. The majority of these substances either partially inacti-vated the microbial load or severely impaired subsequent PCRand ELISAs. Our data demonstrate that high-level inactivation(more than 6 log10) of vegetative bacteria, bacterial spores, andDNA or RNA viruses can be attained within 30 min at 21°C with

either peracetic acid (0.03%) or cupric ascorbate (0.5% in Cu2�

ions) treatment with only minimal hindrance in the subsequentperformance of PCR. Concentrations higher than those generallyrecommended for liquid sterilization (i.e., 5-fold) did not impairthe PCR amplification of P. aeruginosa cells or B. subtilis spores.These findings were unexpected, considering that cupric ascor-bate is well known to produce reactive oxidative species (25).Since even relatively long sequences, such as the 1.3-kb fragmentof P. aeruginosa, was amplified at the concentration used, we canconclude that the inactivation by cupric ascorbate and by per-acetic acid preserves enough of the molecular structure of nucleicacids to allow the subsequent amplification by PCR.

Although the sensitivity of immunoassays depends on the af-finity and concentration of available antibodies, the numerousELISAs that we performed with diverse liquid disinfectants andvarious microbes provide support to our finding that in general,PCR assays withstand treatment with a variety of disinfectantsbetter than immunoassays. Peracetic acid disinfection maintainedELISA sensitivity from 84 to 90% of that of untreated controlsduring the detection of spores and vegetative bacteria and nearly

FIG. 3. Effect of cupric ascorbate (CuAscH) on PCR and ELISA. All microbes were treated with CuAscH at one of three concentrations (0.1,0.5, or 2.5%). The range of inactivation of �6 log10 is indicated as the gray area, and the standard deviations from the means are indicated by linesextending beyond the symbols. (A) Effect on PCR and RT-PCR of treatment with different levels of CuAscH. Indicated are the CT values obtainedby the PCR analysis of B. atrophaeus spores (black circles), P. aeruginosa (open circles), PIXV (black squares), and VacV (open squares). (B) Effecton ELISA after treatment with the three levels of CuAscH on bacteria or viruses without chelation or after the chelation of Cu2� ions with 100mM EDTA (black circles, B. atrophaeus; open circles, P. aeruginosa; open squares, VacV with EDTA; open diamonds, VacV without EDTA; blacksquares, PIXV with EDTA; black diamonds, PIXV without EDTA).

7294 SAGRIPANTI ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.

50% for the detection of vaccinia virus, and it completely inhib-ited the ELISA detection of Pixuna virus.

Disinfection with cupric ascorbate preserved the sensitivity ofELISA for vegetative bacteria and bacterial spores (within 80 to90% of untreated controls), but without further treatment it im-paired the results of viral immunoassays. The addition of EDTAafter incubation with cupric ascorbate preserved the signal inELISA to detect vaccinia virus (a DNA virus) at nearly 90% ofthe level of that in untreated controls and at 20% of the signalobtained with Pixuna virus (an RNA virus). It is important to notethat inactivation did not lead to false-positive signals.

The presented results were obtained with pure cultures of thecorresponding bacteria and viruses, and therefore the perfor-mance of peracetic acid and cupric ascorbate on a variety ofenvironmental samples containing soil, foliage, or other organicmaterial remains to be established. However, the relatively rapidinactivation of the high microbial loads in our samples at roomtemperature by cupric ascorbate or peracetic acid used as de-scribed herein could provide an effective means of quickly ren-dering field samples suspected of containing infectious agents safefor further analysis under lower-level containment and consider-ably lower costs. Sample disinfection with peracetic acid is simpleand could be used before shipping suspected samples to thoselaboratories relying exclusively on PCR methods for the rapiddetection of hazardous infectious agents. However, decontamina-tion with peracetic acid should be selected only if one is certainthat immunoassays will never be performed on the disinfectedsamples, since the inhibition of immunoassays by peracetic acidwas considerable.

Although an additional step involving the addition of EDTAwas needed, the relatively rapid and complete inactivation of alltested microbes at room temperature by cupric ascorbate ap-peared to be the most promising method to render field samplesnoninfectious and thus easily and safely transportable for subse-quent analysis and diagnostics by PCR and/or immunodiagnos-tics. Some loss in immune reactivity for viruses should be ex-pected during the disinfection of samples with either cupricascorbate or peracetic acid, but this disadvantage should be morethan compensated for by the concomitant gains in surety, safety,and economy resulting from handling samples as noninfectiousand, thus, at a much lower containment level.

ACKNOWLEDGMENTS

We gratefully acknowledge the kind and excellent technical assis-tance provided by Claudia Langermann. We thank Barbel Nieder-wohrmeier and the team in the bacteriological laboratory at WIS fortheir substantial support in providing the bacterial/spore suspensionsand their technical assistance in developing and performing the im-munoassays for bacteria and spores.

This study was done at the WIS while Jose-Luis Sagripanti was aparticipant in the U.S.-German Technical Officers Exchange program.

REFERENCES

1. Aguilar, P. V., et al. 2004. Endemic Venezuelan equine encephalitis innorthern Peru. Emerg. Infect. Dis. 10:880–888.

2. ASTM International. 2005. Standard test method E-2414-05. Quantitativesporicidal three-step method (TSM) to determine sporicidal efficacy of liq-uids. Liquid Sprays, and vapor or gases on contaminated carrier surfaces.American Society for Testing and Materials, West Conshohocken, PA.

3. Block, S. S. (ed.) 1991. Disinfection, sterilization, and preservation, 4th ed.Lea & Febiger, Philadelphia, PA.

4. Block, S. S. (ed.) 2001. Disinfection, sterilization, and preservation, 5th ed.,Lippincott Williams & Wilkins, Philadelphia, PA.

5. British Standards Institution, London. 2005. European standard: chemicaldisinfectants and antiseptics–basic sporicidal activity–test method and re-quirements (phase 1). Beuth Verlag GmbH, Berlin, Germany.

6. Czerny, C. P., H. Meyer, and H. Mahnel. 1989. Establishment of an ELISAfor the detection of orthopox viruses based on neutralizing monoclonal andpolyclonal antibodies. J. Vet. Med. B 36:537–546.

7. Czerny, C. P., and H. Mahnel. 1990. Structural and functional analysis oforthopoxvirus epitopes with neutralizing monoclonal antibodies. J. Gen.Virol. 71:2341–2352.

8. Fitzgibbon, J. E., and J. L. Sagripanti. 2008. Analysis of the survival ofVenezuelan equine encephalomyelitis virus and possible viral simulants inliquid suspensions. J. Appl. Microbiol. 105:1477–1483.

9. Greiser-Wilke, I. M., V. Moennig, O. R. Kaaden, and R. E. Shope. 1991.Detection of alphaviruses in a genus-specific antigen capture enzyme immu-noassay using monoclonal antibodies. J. Clin. Microbiol. 29:131–137.

10. Kaerber, G. 1931. Beitrag zur kollektiven Behandlung pharmakologischerReihenversuche. Arch. Exp. Pathol. Pharmakol. 162:480–487.

11. Kinney, R. M., D. W. Trent, and J. K. France. 1983. Comparative immuno-logical and biochemical analyses of viruses in the Venezuelan equine en-cephalitis complex. J. Gen. Virol. 64:135–147.

12. Maillard, J. Y. 2001. Virus susceptibility to biocides: an understanding. Rev.Med. Microbiol. Virol. 12:63–74.

13. Morinet, F. 1992. Polymerase chain reaction (PCR) in microbiology. Valueand practical applications. Presse Med. 21:1377–1380.

14. Peterson, E. M. 1981. ELISA: a tool for the clinical microbiologist. Am. J.Med. Technol. 47:905–908.

15. Poschetto, L. F., et al. 2007. Comparison of the sensitivities of norovirusesand feline calicivirus to chemical disinfection under field-like conditions.Appl. Environ. Microbiol. 73:5494–5500.

16. Rice, W. E., et al. 2007. Chlorine inactivation of highly pathogenic avianinfluenza virus (H5N1). Emerg. Infect. Dis. 13:1568–1570.

17. Rodriguez, J. M. 1997. Detection of animal pathogens by using the polymer-ase chain reaction (PCR). Vet. J. 153:287–305.

18. Sagripanti, J. L. 1992. Metal-based formulations with high microbicidalactivity. Appl. Environ. Microbiol. 58:3157–3162.

19. Sagripanti, J. L. 2009. A knack for survival. CBRNe World 4:60–66.20. Sagripanti, J. L., and A. Bonifacino. 1996. Comparative sporicidal effect of

liquid agents. Appl. Environ. Microbiol. 62:545–551.21. Sagripanti, J. L., and A. Bonifacino. 1997. Effect of salt and serum on the

sporicidal activity of liquid disinfectants. AOAC Int. 80:1198–1207.22. Sagripanti, J. L., L. B. Routson, A. Bonifacino, and C. D. Lytle. 1997.

Mechanism of copper-mediated inactivation of herpes simplex virus. Anti-microb. Agents Chemother. 41:812–817.

23. Sagripanti, J. L., et al. 1997. Comparative sensitivity of thirteen species ofpathogenic bacteria to seven chemical germicides. Am. J. Infect. Control25:335–339.

24. Sagripanti, J. L., et al. 2007. Virulent spores of Bacillus anthracis and otherBacillus species deposited on solid surfaces have similar sensitivity to chem-ical decontaminants. J. Appl. Microbiol. 102:11–21.

25. Sagripanti, J. L. 1999. DNA damage mediated by metal ions, p. 179–209. InA. Sigel and H. Sigel (ed.), Interrelations between free radicals and metalions in life processes. Metal ions in biological systems series, vol. 36. MarcelDekker, New York, NY.

26. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY.

27. Shope, R. E., O. R. Causey, A. H. P. Andrade, and M. Theiler. 1964. TheVenezuelan equine encephalomyelitis complex of group A arthropod-borneviruses, including Mucambo and Pixuna from the Amazon region of Brazil.Am. J. Trop. Med. Hyg. 13:723–727.

28. Spearman, C. 1908. The method of right and wrong cases (constant stimuli)without Gauss formulae. Br. J. Psychol. 2:227–242.

VOL. 77, 2011 MICROBIAL INACTIVATION FOR DIAGNOSTICS 7295

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 24

Jan

uary

202

2 by

189

.90.

223.

52.