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1151 Micro-Carrier-Test: Evaluating Disinfectants for HIV Yukiko SHIMAKOSHI Second Department of Internal Medicine, Department of Microbiology, Osaka Medical College, Osaka, Japan (Received:July 14, 1995) (Accepted: August 25, 1995) Key words: carrier test, HIV, disinfectant Abstract To of determine dry-fixed cells the in 5,ƒÊl of flat-bottomed was 10% to remain cells (1 •~ and the hypochlorite. discrepancies. are Micro-Carrier-Test to be cultured the same the useful The determined by reported previously, previous protocols of 96-well and Uninfected culture supernatant transcriptase cytotoxicity assay. was The those a 105 added PBS. disinfectants determined. (1 •~ of reverse glutaraldehyde screening well with 4 weeks. well-known and cells were times 0.01% as the each ethanol, present in of non-radioisotopic were 20% Micro-Carrier-Test Molt-4 Disinfectants three was several the 1-infected bottom for by disinfectants between promises of the washed disinfectant were almost differences the the contact were and I devised type temperature. activity of of of results The inoculated vitro, at room wells efficacy effects These the transcriptase virucidal 5 minutes in virus dry-fixed at and cytotoxicity method, after were minutes were reverse time-dependent concentrations 120 times cells/well) viruses immunodeficiency serum) for designated Residual new against Human bovine measure week. the disinfectants plate 104 to every evaluate Dose- fetal for harvested assay of cells. microtiter allowed Molt-4 effect virus-infected minimal effective and but 0.1% sodium there are To reevaluated. are some discussed. This disinfectants. Introduction Many (HIV) investigations have been or protocol activity activity was or infectivity. viral virus can To shorten the is sensitive Viral to were after 7 days Correspondence Department by the as and to: the have of dried Yukiko of Microbiology, 平 成7年10月20日 previously with short-term step been is is disinfection. SHIMAKOSHI, Medical to a cells In the types to these to rapid particles in of the Carrier tests tests could 5 days12,13). of plate. still be Hanson recovered et al.5) This which method the other on dried that 2-7 Daigaku-machi, Takatsuki-shi, Osaka, Japan small virus disinfect- M.D. College, is shorten- against stated a residual of particles from the with the method disinfectants virus of a disinfectant3). and other dry-fixed if the locus in disinfection The enzymes disinfection from a microtiter disinfectants. virucidal viral Micro-Suspension-Test8), target virucidal disinfectants according examine of of of virus studies activity effect virus the those HIV after two cells of indicator In virucidal the as test. Infectious the difficult uninfected carrier accepted separate used immunodeficiency disinfectants1). into It is efficacy reported9)11). HIV-infected Osaka divided introduced cells human components, of is needed co-cultivation washing targets from we of viral a carrier2)•`7). 1 (HIV-1)-infected viruses used on are for universally indicator assays or period major best ultracentrifugation determine simplifying disks infectivity no efficacy of is the disinfectants is virucidal antigenicity a suspension2,3) type pathogenic the of there the infectivity determined enough and by washing HIV efficacy Unfortunately because of infectivity ing in virucidal determining Viral cultured. tests suspension human for target suspension the reported. monitored be infectious of 569 1152 Yukiko ants effective against Therefore, a HIV-infected of the This step In well of the paper, method a microtiter results of the infectants described. or steel, of residual with the Cells, Virus Molt-4 1-infected and and cells, cell them fetal ml), bovine with for (RTA) in of the X-100) mixed and μl 0f culture hundred was added times the the p-nitrophenyl reaction was a measured in quadruplicate, Nacalai Alcohol Co. Ltd.) distilled were 96-well were fixed Five 50% FBS) as Drying of more The the to the the mean well must assay are be system. dry-fixed in the same several in well. a The well-known sodium dis- (10 after cells methanol at 107 cells/ml the The plate three stained bottom was with of the Daiso Ltd.), with was dried with Giemsa was for PBS. at 37℃. was solution. plate were from 2 •~ a the (99.9 counted blank % value. formalde- v/v %: Japan Pharmaceuticals saline residual an (PBS) into each time at well cells were image or room dry-fixed experiments with was cutoff v/v designated The 405 The as (37 5 nm inoculated The washed containing at Co.). Sumitomo FBS) 10 20. minutes phosphate-buffered 10% times 30 ethanol Hibitane(R): then buffer for formalin of Tween phosphatase was determined Ltd.), were addition absorbance InterMed 0.5% dTTP the reaction the by (rA)•E with O.02% well continued and S.D. % in the third Japan %: diluted %). washed and v/v (v/v plate. then was NaOH, (5 v/v being (2 •~ the Equipment gluconate and per a poly and by containing with measured of streptavidin-alkaline 37•Ž, of the was (diluted stopped ml transcriptase dUTP was per streptomycin well a sample buffer incubation times and each biotinylated at 1 N of of Reverse HIV in HIV- LAV-1BRU12) supplemented days. of reaction Persistently of 77 ƒÊg 4 brief, containing of three or 50 ƒÊl (NJ-2001, hypochlorite cabinet, cells and and 3 In cells. TCID RPMI-1640 indicator 1 hour Laboratories chlorhexidine in microliters 50 ƒÊl 2000 washing buffer plus TAAB the The for of uninfected containing fifty addition as with plate, electrophotometer %: with residual the was material the cells of penicillin assay14). with reaction microtiter tightly on tests. every as RT) 5 times hundred uninfected biosafety used continued added of medium overnight. concentrations flat-bottomed a and fixed disinfectant to performed maintained units buffer 37•Ž disinfectants of in duplicate. (LUZEX and designated microliters temperature in used Conditions a Inc.), to are used microtiter washed plate v/v Ltd.), water Five of Tesque Hanbai the infectious HIV-1-infected cells were the reaction second and (25 and concentrations were (158 was at One by microtiter Glutaraldehyde cells of reaction phosphate with All first the The stopped was transferred reported Molt-4 (non-RI was buffer. measured hyde: well washing was virus. Methods line, flat-bottomed the of well. dried particles3,5)•`7) material the and assay and antibiotics incubated microliters HIV tonometers, which effective cell supernatant Ail of the of infectivity, previously infecting transcriptase was and to with The 50 plate 5 MNaCl, One and 96-well and the T three-quarters reverse infectious pipetting, in of by 6 months. (FBS) (dT)-immobilized Triton 2. over serum non-radioisotopic human established replacement activity oligo were tips against of Disinfectants established lines culturing 10% Chemical an the infectivity minimal results effective tests biohazardous. Materials 1. the or and and compared on Micro-Carrier-Test disinfection equally such studies or and be many these complicated Micro-Carrier-Test were In scraping and to and sonication, the plate, assumed required determination by I describe be is glass the surface makes this of For the HIV been plates surface. from cannot for have individual on removed HIV test cells2,4)•`6) surface placed wet carrier SHIMAKOSHI done analyzer III). microliters were of inoculated infected into cells each (10-fold well of dilutions a 96-well flat-bottomed 107 to 2 •~ microtiter 104 cells/ml plate. 感 染症 学 雑 誌 in The 10, plate 第69巻 20 or was 第10号 Carrier dried for 120 cells were minutes inoculated replacement of measure 3. value of dry-fixed ,u l of PBS. 37•Ž 0D405 (Wako well of RT from an every wells all-exhausted cells/200 medium assay three in (1X104 the week type ,u 1/well) every was 1153 for HIV Disinfectant week. for and The All with the biosafety incubated culture 4 weeks. compared of cabinet. at 37•Ž in supernatant tests cutoff were Uninfected was done in 5% CO, with harvested to triplicate. The uninfected cells value. Assay microliters and 5% On react were the Chemical a disinfectant to cells CO2. Pure of allowed Uninfected in temperature each non-RI hundred were at into by Cytotoxicity One room three-quarters RTA mean at Test 7th for was 5 minutes. inoculated day of Industries inoculated The into culture, each the into well well viable each was (1 x cells then 104 were well in which washed cells/200 counted three times Jul/well) and with a Cell with 150 incubated Counting Kit Ltd.). Results 1. Residual Cells Residual air-dried well in after washing. the after Washing dry-fixed cells a safety washing. These after cabinet for After findings 30 and may washing 60 to were 120 180 minutes indicate counted minutes, of that with almost still the 100% air-drying, wet or image of almost the 25% completely dried analyzer (Fig. dry-fixed of cells cells the cells are 1) . After remained were easily being in removed removed the by from surface. 2. Survival The air-dried tions of was of Infectivity survival FBS of after infectivity determined in the (Table 1st and Drying of 2nd infected cells 1) . Infectivity weeks, and exposed of virus 1 x to 105 recovery various cells/well was concentrations was highest in detected 10% FBS of at in all the FBS and concentra1st week. Fig. 1 Residual Cells after Washing Uninfected cells (1 x 105 cells in 5 ,u1 of 10% FBS) were air-dried in a 96-well flat-bottomed microtiter plate for the designated times. After the drying the wells were washed three times with PBS. Residual cells were stained with Giemsa solution and analyzed with an image analyzer (LUZEX III). Table 1 Survived Concentrations *Virus recovery OD405 from 平成7年10月20日 three Infectivity of Air-dried Infected Cells in Various of FBS was monitored wells and by non -RI RT the cutoff value assay was . The 0.161. value was the mean Yukiko 1154 Table 2 Survived Infectivity SHIMAKOSHI of Air-dried Infected *Number of infected cells per well * *Virus recovery was monitored by reverse transcriptase (+) indicates positive RTA, and (—) negative Table 3 Effect Cells in 10% FBS activity (RTA) . RTA. of Disinfectants *Ethanol , glutaraldehyde, formalin and sodium hypochlorite were diluted with PBS and chlorhexidine gluconate with distilled water to the designated concentrations (v/v %). **The value was the mean 0D 405from three wells. The cutoff value was 0.170 at the 1st week and 0.192 at the 2nd week. ***The supernatant of the 3rd and 4th weeks was examined for RTA , and the residual infectivity was detected. 感 染 症 学雑 誌 第69巻 第10号 1155 CarrierTestforHIVDisinfectant To determine the detected, 10-fold Infectivity of rd week. detected 3. Construction cells (2 to remain for with triplicate. The was the when 1st 4. mean the 2nd was week, determined. week a well was survival 10% FBS and 1 that x the 1st of were of 103 1 or infectivity could examined x 104 lower, be (Table cells/well 2). in infectivity the could 3 not the week the the 3rd week. for and the each was was well 150 allowed ,u1 of at PBS. 37•Ž supernatant were cutoff to RTA If considered was be the ineffective, different the to in When was examined. in conducted value. considered the type and incubated tests a When weeks flat-bottomed culture All infected all-exhausted with The was disinfectant an and with effective. 4th in 4 weeks. of 96-well times compared be a in ,u 1/well) disinfectant to weeks, 5.0% exposure : 0.5% and was the and Effect of in RTA be according formalin; effect to the -•¢-: concentrations the 0.5 after in the effective. and 0.05% was used chlorhexidine of disinfectants 5 minutes of were 20% contact determined sodium 0.5% formalin after 2, 10, protocol. -•¡-: hypochlorite; ------: and 30 0.05% and 60 15% cutoff sodium minutes ethanol;- •~- contact 3). At gluconate, concentrations ethanol, of ethanol, (Table these Micro-Carrier-Test 0.05% of 5 minutes concentrations Disinfectants of 15% virucidal designated gluconate at 5.0, of after concentrations of chlorhexidine effects concentrations the effect performed formalin and Time-Dependent critical virucidal assay detected, hypochlorite, 平 成7年10月20日 was hypochlorite cytotoxicity effective 2 wells considered three every three the sodium Minimal medium of placed cells/200 week of was washed 104 every 2nd was the well microliters Micro-Carrier-Test was of the it then x Five temperature a disinfectant (1 devised. each room assay 2 consecutive glutaraldehyde, At and supernatant for the cytotoxicity Fig. from into at was well of Micro-Carrier-Test, and 1.0% well RT was minutes of each non-RI both the negative The 0D405 the in formalin, and 1st inoculated 120 three-quarters of both of evaluate glutaraldehyde, residual which in the in was for into by value in 2.0 in cells/well in microliters time. of negative determined, 102 cells Micro-Carrier-Test FBS) hundred RTA it was was x inoculated dried inoculated Characterization To 1 the 10% designated were positive and cells was One measure supernatant to detected results in plate replacement to and these The a cells CO2 105 was of cells/ml cabinet. harvested x infected Micro-Carrier-Test with 107 Uninfected 1 air-dried 4 weeks. plate. biosafety of of cells/well number of x of RTA 105 accordance microtiter 5% x the in number dilutions 1 When be In optimum ethanol, were not 0.01% 1156 YukikoSHIMAKOSHI glutaraldehyde and 0.1% sodium hypochlorite. Five minutes of contact with 0.5 % formalin or 0.005% chiorhexidine gluconate did not achieve complete disinfection. Their virucidal effect of the critical concentrations of 15% ethanol, 0.5 % formalin and 0.05 % sodium hypochlorite was determined at 2, 10, 30 and 60 minutes (Fig. 2) . All these disinfectants showed time-dependent effects in 1 week. Discussion In the determination of the effect of chemical disinfectants against HIV, carrier tests with cell-free viruses3'5)-7) and virus-infected cells2'4)^-6)as targets of disinfection have been described. Hanson et al.5) reported that virus-infected cells were more resistant to disiufectants than cell-free viruses. When disinfectants are examined only against cell-free viruses, their efficacy for practical use may be overestimated. Moreover, virus-infected cells are easier to handle than cell-free viruses. Therefore, virus-infected cells were used as targets of disinfection in the present study. In previous studies2'-7), the surface of the fixed target was treated with a disinfectant and later it was removed from the surface by sonication, scraping or pipetting and transferred to a culture system to measure residual infectivity. This was a complicated and hazardous method. Also some disinfectants, such as alcohols and aldehydes, may cause the infectious materials to be so tightly attached to the surface that the target cannot be collected totally and the residual infectivity cannot be measured accurately. In the present Micro-Carrier-Test the target is fixed to the bottom of a well in a microtiter plate, and the treatment with disinfectants and the measurement of residual infectivity can be performed simply in the same well. With this simple procedure large numbers of samples can be examined safely. Since the infectivity assay is performed in the same well, residual disinfectant might affect the results of the assay. The cytotoxicity of the residual disinfectant did indeed affect the monitored cells in the present test. Aranda-Anzaldo et a1.15)stated that the residual cytotoxicity of the chemical might mask or mimic the presence of true virucidal activity and lead to erroneous conclusions. Therefore, a cytotoxicity assay must be performed during the test, and the virucidal effect is not determined at the concentration of disinfectant which causes residual cytotoxicity. In order to evaluate the Micro-Carrier-Test, well-known disinfectants were tested again. Minimal effective concentrations of ethanol and glutaraldehyde in the Micro-Carrier-Test were the same as in the Micro-Suspension-Test. The minimal effective concentration of sodium hypochlorite in the Micro-Carrier-Test was slightly higher than in the Micro-Suspension-Test, probably because of the greater resistance of dried material. Lloyd-Evans et a1.9) noted that tests conducted on suspensions often overestimate the ability of a product to disinfect contaminated surfaces. Hanson et a1.5) also stated that disinfectants effective against wet HIV cannot be assumed to be equally effective against dried virus. To avoid overestimation of virucidal efficacy, carrier tests should be used for the evaluation of disinfectants. In carrier tests, 1.1 % glutaraldehyde for 10 minutes') and 1% for 15 minutes') was found not to be effective, but the minimal effective concentration in the present test was 0.01% for 5 minutes of contact. Sattar and Springthorpen noted that virus protected by body fluids may be equally or more stable. Prince et al.'), Hanson et al.5) and I used 20, 50 and 10% FBS, respectively. So the differences were probably due to the different concentrations of FBS. The virucidal effect of chiorhexidine gluconate could not be examined by the Micro-SuspensionTest previously reported8), but could be by the Micro-Carrier-Test. Since the cells are destroyed by low osmotic pressure and low pH, it is difficult to collect the cells by centrifugation in the Micro-Suspension-Test. The present carrier test does not require centrifugation, so chemicals such as chlorhexidine gluconate can be tested. 感染 症 学 雑 誌 第69巻 第10号 CarrierTestforHIVDisinfectant 1157 The present Micro-Carrier-Test is a simple procedure which shortens the period of contact between the infectious target and the disinfectant. There is still the problem that residual disinfectant can affect subsequent co-cultivation. However, the use of the cytotoxicity assay solves this problem. Accordingly, both the previous Micro-Suspension-Test and the present Micro-Carrier-Test are required in the screening of disinfectants. Acknowledgments Grateful acknowledgments are made to Prof. Masuyo Nakai, to Prof. Ken-ichi Katsu, Second Department of Internal Medicine wish to express my thanks to Dr. Kouichi Sano for helpful support Ms. Akie Hanada for their expert technical assistance. Part of this work was supported by a grant for AIDS Research Public Health. Department of Microbiology and for their guidance in this study. I and to Mr. Yoshihiko Fujioka and from The Osaka Association for References 1) Sattar, S.A. and Springthorpe, V.S.: Survival and disinfectant inactivation of the human immunodeficiency virus: A critical review. Rev. Infect. Dis. 13: 430-447, 1991. 2) Resnick, L., Veren, K., Salahuddin, S.Z., Tondreau, S. and Markham, P.D.: Stability and inactivation of HTLV-III/ LAV under clinical and laboratory environments. JAMA 255(14): 1887-1891, 1986. 3) Tj cktta, E., Hungnes, 0. and Grinde, B.: Survival of HIV-1 activity after disinfection, temperature and pH changes, or drying. J. Med. Virol. 35: 223-227, 1991. 4) Fauvel, M. and Ozanne, G.: Immunofluorescence assay for human immunodeficiency virus antibody: Investigation of cell fixation for virus inactivation and antigen preservation. J. Clin. Microbiol. 27(8): 1810-1813, 1989. 5) Hanson, P.J.V., Gor, D., Jeffries, D.J. and Collins, J.V.: Chemical inactivation of HIV on surfaces. BMJ 298: 862-864, 1989. 6) Pepose, J.S., Linette, G., Lee, S.F. and MacRae, S.: Disinfection of Goldmann tonometers against human immunodeficiency virus type 1. Arch. Ophthalmol. 107: 983-985, 1989. 7) Prince, D.L., Prince, R.N. and Prince, H.N.: Inactivation of human immunodeficiency virus type 1 and herpes simplex virus type 2 by commercial hospital disinfectants. Chemical TIMES & TRENDS 13: 13-16, 1990. 8) Shimakoshi, Y., Sano, K., Nakano, T., Nakamura, T., Ohshiba, S., Katsu, K. and Nakai, M.: A micro-suspensiontest for evaluation of disinfectants against human immunodeficiency virus. J. Jpn. Assoc. Infect. Dis. 69(5): 532-538, 1995. 9) Lloyd-Evans, N., Springthorpe, V.S. and Sattar, S.A.: Chemical disinfection of human rotavirus-contaminated inanimate surfaces. J. Hyg. 97: 163-173, 1986. 10) Tyler, R. and Ayliffe, G.A.J.: A surface test for virucidal activity of disinfectants: preliminary study with herpes virus. J. Hosp. Infect. 9: 22-29, 1987. 11) Sattar, S.A., Springthorpe, V.S., Karim, Y. and Loro, P.: Chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses. Epidem. Inf. 102: 493-505, 1989. 12) Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L.: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868-871, 1983. 13) Bueren, J., Simpson, R.A., Jacobs, P. and Cookson, B.D.: Survival of human immunodeficiency virus in suspension and dried onto surfaces. J. Clin. Microbiol. 32(2):571-574, 1994. 14) Nakano, T., Sano, K., Odawara, F., Saitoh, Y., Otake, T., Nakamura, T., Hayashi, K., Misaki, H. and Nakai, M.: An improved non-radioisotopic reverse transcriptase assay and its evaluation. J. Jpn. Assoc. Infect. Dis. 68(7): 923-931, 1994. 15) Aranda-Anzaldo, A., Viza, D. and Busnel, R.G.: Chemical inactivation of human immunodeficiency virus in vitro. J. Virol. Methods 37: 71-82, 1992. 平成7年10月20日 1158 Yukiko Micro-Carrier-Test: SHIMAKOSHI HIVに 対 す る消 毒 薬 の 評 価 方 法 大阪医科大学 第2内 科学教室,微 生物学教室 島 要 越 assayで 残 存 す る消 毒 薬 の細 胞 毒 性 を検 討 した. 旨 ウイル ス に対 す る消 毒薬 の効 果判 定 方 法 とし て,乾 燥 固 定 した ウ イ ル ス 感 染 細 胞 を用 い た Micro-Carrier-Testを イ ル ス1型 考 案 し た.ヒ ト免 疫 不 全 ウ に 感 染 し たMolt44を96穴 平 底 マ イ ク ロ タ イ タ ー プ レ ー トの ウ エ ル 底 に 室 温 で120分 乾 燥 固 定 し 消 毒 薬 を 作 用 さ せ た 後,PBSで し,非 感 染 のMolt-4を 加 え4週 由紀 子 間 洗浄 RT assayで 転 写 酵 素 活 性 を 測 定 し た.ま たcytotoxicity 果 を再 検 討 した.濃 度,時 られ,5分 ル,0.01%グ 間依存性 の効果 が認 め 間 で の最 低 有効 濃 度 は,20%エ ル タル ア ル デ ヒ ド,0.1%次 タ ノー 亜 塩素酸 ナ トリウ ム で あ っ た.こ れ は,こ れ ま で の 報 告 と 部 違 い が あ っ た の で,今 回 と これ ま で の 方 法 に 一 つ い て そ の 違 い を検 討 し た.こ のMicro-Carrier- 間 培 養 し た.1週 間 毎 に 培 養 上 清 を 回 収 しnon-RI この 新 しい 方 法 を評 価 す るた め既 存 の 消 毒 薬 の 効 逆 Testは 消 毒 薬 の効 果 判 定 の 一 つ の ス ク リー ニ ン グ法 と して 有 効 で あ る と考 え る. 感 染症 学 雑誌 第69巻 第10号