Current methods for antiviral susceptibility testing

Clinical Microbiology Newsletter March 15,1997

Vol. 19, No. 6

Current Methods for Antiviral Nell S. Lurain, Ph.D. Department of ImmunologylMicrobiology Rush University Chicago, IL 60612

Kenneth D. Thompson, Ph.D. Department of Pathology The University of Chicago Chicago, IL 60637

Over the past decade, the development of effective and less toxic antiviral drugs has led to substantial progress in the treatment of several viral diseases. There has been a significant increase in the use of these drugs for prophylaxis and long-term therapy of chronic infections. Most currently available compounds target viral nucleic acid synthesis, but compounds that inhibit other virus-encoded protein functions have been developed recently. In particular, several viral protease inhibitors have become available for treatment protocols. Whereas antiviral drugs can reduce the morbidity and mortality associated with some viral diseases, resistant virus strains frequently arise during treatment. The isolation of drug-resistant strains has led to a compelling need for antiviral susceptibility testing. The ease with which antiviral susceptibility testing can be accomplished is generally related to how efficiently a virus can be propagated in vitro. Viruses such as herpes simplex virus (I-ISV) and influenza virus, which replicate rapidly and produce high extracellular titers, are relatively easy to test for antiviral resistance at present. For viruses such as cytomegalovirus (CMV), varicellaCMNEEJ

19(6)4148.1997

Susceptibility

zoster virus (VZV), and human immunodeficiency virus (HIV), which replicate slowly and are cell associated, the most widely used assays are lengthy and labor intensive. The lack of an in vitro propagation system for other viruses such as hepatitis B presently precludes antiviral susceptibility testing. In general, there is a lack of standardization for the laboratory methods that are currently available for susceptibility testing of all of these viruses, although efforts are being made to address this problem (1,2). Recently, a new NCCLS subcommittee has been formed to attempt to standardize some aspects of virus susceptibility testing beginning with herpes simplex virus. The most frequently used method is usually a cell culture-based phenotypic assay such as a plaque reduction assay (IRA) or ELISA. For the rapidly growing viruses, these assays provide reliable, useful results. For the slower-growing viruses, however, these phenotypic assays often do not provide results within a practical time-frame to affect therapeutic decisions. The need for more rapid determination of drug resistance has led to efforts to develop better phenotypic assays (3-5) as well as genotypic assays (6,7). The genotypic assays, which usually are based on PCR, can detect the presence of drug resistance mutations even in the absence of virus replication; however, these assays detect only known mutations. Since new drugs inevitably lead to the selection of resistant virus strains, phenotypic assays are still required for the identification of new mutations Elsevier

Testing responsible for antiviral resistance.

Herpes Simplex Virus Acyclovir (ACV), a nucleoside analogue, has been approved for clinical use since 1982 and is still the drug of choice for treatment of serious HSV infections. ACV resistance is most commonly the result of mutations in the viral thymidine kinase (TK) gene (8). Other drugs that are available to treat HSV include the nucleoside analogues, penciclovir and vidarabine, and the pyrophosphate analogue, foscamet. Among the most frequently used susceptibility assays for HSV are the PRA, in situ ELISA, dye uptake, and DNA hybridization assays (9). The cells used in these susceptibility assays are usually human fibroblasts (I-IF), CV-1, or Vero cells. The PRA measures the ability of an antiviral compound to inhibit

In This Issue

Current Methods for Antiviral Susceptibility Testing . . . . . . . . . . . 41 A discussion of the methods available for susceptibility testing of important viral pathogens including herpes viruses and HIV

Nonusefulness of Quantitative Wound Cultures . . . . . . . . . . . . . . . 45 What information does the use of quantitative cultures of bum and other wounakprovide to the clinician?

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the viral cytopathic effect (CPE) in the presence of varying concentrations of the drug compared with control tests without drug. The endpoint for determining the inhibitory concentration (I&I) is a 50% reduction in the number of plaques. The PRA requires 2 to 4 d post-inoculation to reach an endpoint, but the endpoint can be difficult to interpret. Variations in plaque size, especially at high drug concentrations, make the assay somewhat subjective. An important consideration with the PRA is the relatively small number of virions that can be tested. Approximately 50 plaque forming units (pfu) per well is the maximum. If the infection consists of a mixed population of susceptible and resistant strains, a minor resistant population may be missed with this assay. Another disadvantage is that the PRA is quite labor intensive. A modification of the PRA has recently been reported that uses a Vero cell line (VeroICP6LacZ#7) that is stably transformed with the E. coli 1ucZ gene that is under the control of an HSV-1 early promoter (10). These cells express pgalactosidase when infected with either HSV-1 or HSV-2. Susceptible strains of HSV are blocked in expression of pgalactosidase by antiviral drugs and this prevents the formation of blue plaques. The ELISA uses a 96-well microtiter plate format (llJ2). Cell monolayers are infected with the isolate at a multiplicity of infection between 0.01 and 0.1 and the virus is grown for 48 h in the presence of varying concentrations of drug. Viral antigens are detected by adding enzyme-labeled antibody to the wells followed by the addition of the enzyme substrate. The colored substrate product is read spectrophotometrically. The ICSOis the lowest concentration of drug that reduces the optical density of the colored substrate product by 50%

compared with the drug-free controls. The ELBA measures the level of a late viral protein product rather than CPE. The advantages of the ELISA are (i) an objective endpoint, (ii) rapid results, (iii) screening of a larger number of pfu, (iv) the ability to detect a resistant virus subpopulation in the presence of susceptible virus, and (v) good correlation with the standard PRA. The dye uptake assay is a colorimetric method based on the inhibition of the uptake of neutral red dye by virusinfected cells (13). The assay uses a %well microtiter plate format with control and drug dilution wells similar to that of the ELISA. Neutral red dye is added at 72 h post-infection. Dye incorporated by the viable cells is eluted into the microtiter wells, and the optical density of the eluted dye solution is determined spectrophotometrically. The endpoint is the drug concentration producing a 50% reduction in the viral inhibition of neutral red uptake. The advantages of the dye uptake assay are the objective endpoint and rapid time to obtain results. The main disadvantage is that the sensitivity of the assay makes it difficult to interpret the clinical significance of low-level resistance. In addition, the ICso results of the dye uptake assay tend to be high and do not always correlate with the PRA (13). The DNA hybridization assay (Hybriwix; Diagnostic Hybrids, Athens, OH) uses an ‘2SIodine-labeled probe to detect the presence of viral DNA in cultures of HSV in the presence and absence of drug (14). This assay is also performed in a %-well microtiter plate as described above for the FXISA and dye uptake assays. At 48 h post-infection, the cells are lysed and the lysate is absorbed onto membranes. The membranes are hybridized with the radiolabeled probe, and then counted in a

gamma counter. The IC50 is the lowest drug concentration that reduces the radioactive counts by 50%. This assay correlates well with the PRA and has the advantage of an objective endpoint. The major disadvantages of the DNA hybridization assay are the short half-life of the probe, the need for radioactive waste disposal, and the labor-intensive technical requirements. Genotypic assays for HSV drug resistance do not appear to be practical at present, because there is considerable heterogeneity in the TK mutations that confer resistance (15). The large number of mutation sites that can result in drug-resistance makes it necessary to sequence large portions of the TK gene.

Varicella-Zoster

Virus

ACV is the drug of choice for treatment of VZV. Resistance to ACV has been documented in patients with AIDS who have received prolonged ACV therapy (16). The majority of the ACV-resistant clinical isolates have mutations in the viral TK. Alternative drugs for treatment include famciclovir, vidarabine, and foscamet. The PRA using HP or CV-1 cells is probably the most common method for determining VZV drug susceptibility. Since VZV is a cell-associated virus, infected cells must be used as the inoculum, which is usually 40 to 60 infected cells per well in a 24-well plate. The plates are counted at 5 to 10 days postinfection, and the 50% endpoint is determined as described above for HSV. Other methods for determining the susceptibility of VZV include the DNA hybridization assay described for HSV (Hybriwix). As with HSV, there appears to be considerable heterogeneity in TK gene mutations conferring ACV resistance: therefore, genotypic assays are also not yet practical for VZV (17).

NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to peons or pmpcrty as a matter of prod&s liability, negligence or otherwise, or fmm any use or operation of any methods, products. instructions or ideas contained in the material herein. No suggested test or pmcedum should be carried out unless, in the reader’s judgment, its risk is just&d. Because of rapid advances in medical sciences, we recommend that the indcpmdcnt verification of diagnoses and drug dosages should be made. Discussions, views, and recommendations as to medical pmcedores, choice of drugs, and drug dosags are the responsibility of the authors. Chico1 Microbiology Nms&#er (ISSN 01964399) is issued twice monthly in one indexed volume per year by Elsevier Subwxiption price per year $222.00; for orders outside of the United States, Mexico nod Canada: $302.00. Second-class Postmaster:Sendsddrrss changrstoClinicalMicrobiologyNEwsle~er.ElsevierScimceInc.,655AvmueofthcAmericas,NewYorlc,NY TO&FREE for customers in the U.S.A and Canada: l-888-4ES-INFO (1-888-4374636) or fax: (212) 633-3680.

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Cytomegalovirus Ganciclovir, a nucleoside analogue, and foscamet are the two antiviral agents that have been approved for treatment of human CMV disease, which is prevalent among AIDS patients and organ transplant recipients. The long-term drug therapy required for these patients has led to the repeated isolation of resistant strains (3,6,18). The slow-growth of CMV in cell culture, however, has hindered the development of methods for the rapid determination of drug susceptibility of clinical strains. The conventional phenotypic assay is the PRA performed in HF cells (2). Because the virions are highly cell-associated, their numbers in clinical specimens are increased by passage in cell culture over a period of several weeks to obtain sufficient virus to perform the assay. Virus-infected cells are used as the inoculum for 24-well plates. Plaques are counted after 8 to 14 d of incubation. Advantages of this assay are that it is reproducible, variations in plaque morphology are evident, and all viable mutant viruses in the inoculum can be detected. The length of time and the amount of labor required to perform the assay, however, are deterrents for its general clinical use. In addition, a maximum of approximately 80 to 100 infectious units per well can be used in the inoculum in the 24-well plate format, which decreases the ability to detect low-level resistance. The Hybriwix DNA hybridization assay described for HSV and VZV is also available for CMV (19). The assay is reproducible and somewhat more rapid than the PRA. An even more rapid phenotypic assay has been described recently (3). Plaque reduction by antiviral agents is measured by reduction in CMV immediate early antigen production. Plaques are visualized by the addition of enzyme-tagged monoclonal antibodies followed by the addition of the enzyme substrate. Shell vials can be directly inoculated with peripheral blood leukocytes with plaque production evident in 4 to 6 d. Resistance is determined by comparison of plaque number in a control vial with plaque number in a vial containing the antiviral agent.

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Other phenotypic assays that am under development for cell-associated CMV clinical strains include an ELISA

(20) and flow cytometry(21). Both of these assays are based on determination of late CMV antigen production, which for drug-susceptible strains is reduced in the presence of drug. The major problem with the phenotypic assays for CMV is the need to expand the virus in cell culture, which, depending on the method used, can take up to several weeks. The delay reduces the clinical usefulness of the results; however, phenotypic methods are presently the only means of detecting drugresistant strains with previously unidentified mutations. Genotypic assays hold some promise for CMV, because most drug-resistance appears to result from a limited number of point mutations and short deletions in the UL97 phosphotransfemse gene (6,18). The most direct method is to use the PCR to amplify the areas of the gene known to contain the point mutations or short deletions. The PCR products can then be sequencedto demonstrate these changes. Sequencing is rapid and reliable especially with access to automated sequencers. An alternative to sequencing has been developed whereby the known mutations and deletions can be detected by restriction enzyme analysis (6,18). PCR products digested with specific restriction enzymes show loss or gain of sites in the presence of mutations conferring drug resistance. This method allows identification of resistance even in the absence of virus replication. The main disadvantage of the method is that it detects only known mutations in the UL97 phosphotransferase gene. Mutations in the DNA polymerase gene can also produce drug-resistance (22). The polymerase mutations that have been mapped so far are more widespread within the gene than are the mutations in the UL97 gene; therefore, rapid sequencing and restriction enzyme analysis may not be possible for mutations in the polymerase coding sequence.

Influenza The clinically useful antiviral drugs for treatment of influenza A viruses are the adamantane derivatives, amantadine

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and rimantadine. These drugs inhibit the hydrophobic transmembrane domain of the M2 protein of influenza A but not other influenza viruses (23). Sequence data from drug-resistant strains indicate that resistance is determined by a single amino acid change in one of four residues of the M.2 protein (23). Although the PRA has been used for drug susceptibility testing of influenza viruses, an ELISA has been reported (24). The ELISA uses MDCK cell monolayers in 96-well plates. Various dilutions of the virus are grown in the presence and absence of 1.0 pgJm1 of drug. The virus antigen is detected by an enzyme-conjugated antibody and the colored substrate product is detected spectrophotometrically. No genotypic assay for influenza A has yet been developed.

Human Immunodeficiency Virus There arethree generalclassesof antiviral agents available for treatment of HIV: (i) nucleoside analog inhibitors of the reverse transcriptase (RT), such as AZT, ddI, and 3TC; (ii) non-nucleoside analog inhibitors of the RT, such as Nevirapine; and (iii) protease inhibitors, including Indinavir, Saquinavir, and Ritonavir. In spite of the availability of a variety of drugs with different inhibitory mechanisms, resistance develops in response to treatment with each type of drug (25-27). This is a result of both the high rate of viral replication and the frequency of nucleotide misincorporation by the RT. A PRA was developed for testing AZT resistance (28). The assay uses the adherent cell line HT4-6C derived from HeLa cells by introducing the human CD4 gene for stable CD4 expression. Syncytium-forming strains of HIV produce multinucleated cell plaques, which are inhibited by drugs to which the virus is susceptible. Although the assay is reliable for determination of resistance, it is useful only for strains that form syncytia. A second phenotypic assay developed by the AIDS Clinical Trials Group (ACTG) is based on peripheral blood mononuclear cell (PBMC) culture (1). The original assay involves two steps. First a stock of virus is obtained by cocultivating the patient’s PBMC with

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HIV-negative donor PBMC that have been stimulated with phytohemagglutinin. The stock is titered in donor PBMC to determine the 50% tissue culture infectious dose (TCIDso). For the second step, a standardized inoculum is used to infect donor PBMC in micro&r plates. A range of antiviral drug concentrations is tested in replicate wells to determine resistance. The inhibitory effect of the drug is measured by quantitation of viral ~24 antigen in the supematant fluid of each well using commercially available ELISA kits. The advantage of this method is that the majority of HIV strains can be tested. A significant disadvantage is the length of time (4 to 6 wk) and the labor required to perform the steps of coculture, titering the virus, and p24 assay. In addition prolonged culture of the virus may alter the virus population such that susceptibility testing does not reflect the level of resistance of the original virus isolate. The procedure is undergoing modification by the ACTG working group to eliminate steps to reduce both the time and work needed to obtain results (29). Two newer phenotypic assays have been reported. The recombinant virus assay involves PCR amplification of a patientderived pool of RT sequences, which are introduced into cells carrying an RT-deleted proviral clone (4). Recombinant virus containing potentially drug-resistant RT sequences can then be analyzed for the production of syncytia by the HT4 HeLa cell PRA. An advantage of this assay is that there is no co-cultivation that might select out subpopulations of virus. An RT expression assay is being developed to detect RT activity in bacterial cells (5). The RT gene from patient isolates is introduced into a bacterial expression vector. RT activity resulting in incorporation of a radiolabeled nucleotide into a primed template substrate is detected in situ in membrane-lysed bacterial colonies by autoradiography. Resistance can potentially be measured by reduction in RT activity in the presence of antiviral drugs. Specific mutations conferring resistance to all approved drugs have been identified. Often more than one mutation or a group of mutations is associ-

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ated with resistance. Combination therapy also adds to the number of mutations that potentially can occur in the virus isolated from a patient (30). The number and wide distribution of these mutations within the affected genes makes development of genotypic assays difficult. At present, genotypic analysis of resistance is limited to primer-specific amplification or direct sequencing of PCR products.

to detect human immunodeficiency virus reverse transcriptase activity expressed in bacteria. J. Biol. Chem. 264:1668!&16693. 6.

Chou, S., et al. 1995. Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. J. Jnfeet. Dis. 171:576-583.

7.

Shafer, R.W., et al. 1996. Jnterlaboratory comparison of sequence-specific PCR and ligase detection reaction to detect a human immunodeficiency virus type 1 drug resistance mutation. J. Clin. Microbial. 34:184%1853.

8.

Hill, E.L., G.A. Hunter, andM.N. Ellis. 1991. In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 35:2322-2328.

9.

Leahy, B.J., K.J. Christiansen. and G. Shellam. 1994. Standardisation of a microplate in situ ELISA (MJSE-test) for the susceptibility testing of herpes simplex virus to acyclovir. J. Virol. Methods 48:93-108.

Future Outlook for Virus Susceptibility Testing Antiviral susceptibility testing for rapidly growing viruses is moving toward standardization of the phenotypic assays. Susceptibility testing for slowgrowing viruses is beginning to change from phenotypic assay to molecularbased techniques that may make susceptibility testing mote practical. These changes are a result of recent research elucidating the mechanisms of drug resistance. Both functional assays of protein targets of the drugs and sequence analysis of known mutations are currently at the forefront of assay development. References 1. Japour. A.J., et al. 1993. Standardized peripheral blood mononuclear celJ culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 37: 1095-1101. 2. Crumpaker, C.M., et al. 1996. Development of a consensus assay to determine susceptibility of clinical isolates of CMV to antiviral therapy. Abstract 164. Third Conference on Retroviruses and Opportunistic Infections, Washington, D.C. 3. Gema. G., et al. 1995. Rapid screening for resistance to ganciclovir and foscarnet of primary isolates of human cytomegalovints from culture-positive blood samples. J. Clin. Microbial. 33:738-741. 4. Kellam, P., and B.A. Larder. 1994. Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeticiency virus type 1 isolates. Antimicrab. Agents Chemother. 38:23-30. 5.

Prasad, V.R., and S.P. Goff. 1989. A novel in situ colony screening method

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10. Tebas, P., E.C. &bell, and P.D. Olivo. Antiviral susceptibility testing with a cell lime which expresses B-galactosidase after infection with herpes simplex virus. Antimicrob. Agents Chemother. 39:1287-1291. 11. Rabalais, G.P.,M.J. Levin, andF.E. Berkowitz. 1987. Rapid herpes simplex virus susceptibility testing using an enzyme-linked immunosorbent assay performed in situ on fixed virus-infected monolayers. Antimicrob. Agents Chemother. 31:94&948. 12. Safrin, S., E. Palacios, and B.J. Leahy. 1996. Comparative evaluation of microplate enzyme-linked immunosorbent assay versus plaque reduction assay for antiviral susceptibility testing of herpes simplex virus isolates. Antimicrob. Agents Chemother. 40:1017-1019. 13. McLaren, C., M.N. Ellis, and G.A. Hunter. 1983. A calorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res. 3:223-234. 14. Swierkosz, E.M., et. al. 1987. Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus. Antimicrob. Agents Chemother. 31:1465-1469. 15. Kimberlin,

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D.W., et. al. 1995. Assays

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for antiviral drug resistance. Antiviral Res. 26:40-13. 16. Talarico, C.L.. W.C. Phelps, and K.K. Biron. 1993. Analysis of the thymidine kinase gene from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS. J. Virol. 67:102+1033. 17. Boivin, G., et. al. 1994. Phenotypic and genotypic characterization of acyclovirresistant varicella-roster viruses isolated from persons with AIDS. J. Infect. Dis. 170:68-75. 18. Hanson, M.N., et al. 1995. Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir. Antimicrob. Agents Chemother. 39:1204-1205. 19. Dankner, W.M., et al. 1990. Rapid antiviral DNA-DNA hybridization assay for human cytomegalovirus. J. Virol. Methods 28:293-298. 20. Tatarowicz, W.A., N.S. Lurain, and K.D. Thompson. 1991. In situ ELISA for the evaluation of antiviral compounds effective against human cytomegalovirus. J. Virol. Methods 35:207-215.

21. McSharry, J., et al. 1996. Standardized flow cytometry based assay for HCMV antiviral susceptibility. Abstract 555. Third Conference on Retroviruses and Opportunistic Infections, Washington, D.C. 22. Lurain, N.S., et al. 1992. Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents. J. Virol. 66:7146-7152. 23. Klimov, A.I., et. al. 1995. Prolonged shedding of amantadine-resistant influenza A viruses by immunodeficient patients: detection by polymerase chain reaction-restriction analysis. J. Infect. Dis. 1721352-1355. 24. Belshe, R.B.. et al. 1988. Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection. J. Virol. 62:1508-1512. 25. D’Aquila, R.T., et al. 1995. Zidovudine resistance and HIV-l disease progression during antiretroviral therapy. AM. Intern. Med. 122:401+08. 26. Havlir, D., et al., 1995. High-dose nevirapine: safety, pharmacokinetics, and antiviral effect in patients with human

immunodeficiency virus infection. J. Infect. Dis. 171:537-545. 27. Ho, D.A., et al. 1994. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor. J. Virol. 68:201&2020. 28. Larder, B.S., B. Chesebro, and D.D. Richman. 1990. Susceptibilities of zidovudine-susceptible and -resistant human immunodeficiency virus isolates to antiviral agents determined by using a quantitative plaque reduction assay. Antin&rob. Agents Chemother. 34:436441. 29. Brice. A., et al. 1995. Zidovudme (ZDV) susceptibility screening assay for clinical HIV isolates. Abstract I285. ‘IX&y-fifth International Conference on Antimicrobial Agents and Chemotherapy, San Francisco. 30. Iverson, A.K.N., et al. 1996. Multidrugresistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy. J. Viral. 70:1086-1090

Editorial

Nonusefulness of Quantitative Wound Cultures Ian Alan Holder, Ph.D. Director, Department of Microbiology Shriners Hospitals for Crippled Children Bums Institute-Cincinnati Unit Cincinnati, OH 45229-3095

By using a scalded rat Pseudomonas infection model, the elegant studies of Teplitz et al. (1,2) demonstrated that bum wound sepsis results from progressive bacterial colonization of the wound and subsequent invasion from nonviable tissue to healthy tissue of numbers more than lo5 colony forming units (CFU) of the bacterium/g of wet weight tissue. Krizek et al. (3) reported that skin graft “take” usually does not occur in experimental or clinical wounds that

Clinical

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contain more than ld CFU/g tissue whereas Robson and Krizek (4) suggested that cutaneous allograft “take” does not occur when bacterial densities in wounds exceed 10’ CFU/g. Studies such as these prompted the development of a wide variety of methods to quantify the number of organisms associated with patient wounds.

Early meth-

ods involved quantifying organisms obtained from the surface of the wound by “absorption” of the bacteria into wet gauze pads by capillarity or by swabbing the wound surface with wet swabs(5-7) followed by extraction of the bacteria horn the gauze pads or swabsand quantifying CFUs by dilution and plate counts.

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To reduce the time needed to get quantitative results without waiting for the microorganisms to grow on culture plates, attempts were made to use quantitative microscopic techniques. One such method involved the use of surface swab cultures in which the numbers of bacteria washed off one surface swab, which had been rubbed back and forth over a prescribed surface area of burn wound, were quantified and compared with a Gram-stained smear of a second swab similarly treated (8). Visualization of bacteria on the smear was reported to indicate that lo6 CFU or more bacteria were present per swab. Because quantitative surface culture, even with Gram-

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