Evaluation of the direct IDSX method on urinary tract samples: an accurate and inexpensive antimicrobial susceptibility test which

includes presumptive identification, providing a rapid diagnostic result for prudent antibiotic prescribing Running title: Evaluation of the direct IDSX kit

Adam R. Taintor+, Read R. Taintor, Elizabeth L. Frank* *Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
+

Corresponding author: Adam R. Taintor M.D.

Address: 9450 S. 1300 E. Sandy, Utah 84094 Telephone: 011-801-906-3186 E-mail address: adam.taintor@imail.org
Authors Note (August 2011): This paper was submitted in 2009 to Diagnostic Microbiology and Infectious Disease, however it was not accepted for publication for several reasons. Probably the most significant was the lack of a reference method. One reviewer remarked that: The Vitek 2 is a proprietary instrument and not a reference method. New AST methods need to be compared to existing reference methods published by CLSI (i.e., broth dilution, disk diffusion). In this study we used digital images to resolve discrepancies, but the reviewers did not consider this adequate. Another reviewer stated that: This manuscript describes an interesting approach to susceptibility testing of bacteria directly from urine. This approach may be useful and accurate. A new clinical study, incorporating reviewer recommendations, was not done because of constraints in time and funding. The IDSX kit is a manual antimicrobial susceptibility method. Current manual diagnostics are difficult to use, have long processing times, and are not designed for point-of-care use. Automated systems (such as the Vitek), while easier to use, are extremely expensive and require a substantial initial outlay that can take years to depreciate. Since 2009, several significant improvements to the IDSX kit have been patented. These involve the rapid placement of antimicrobial papers to the chambers using special applicators. Post incubation, the use of measuring transparencies provide a reading of Resistant, Intermediate or Susceptible directly from the chambers. This replaces the need to manually measure zones and consult standard interpretive charts to obtain results as with the Kirby-Bauer method for example.

Abstract We evaluated the IDSX kits novel direct antimicrobial susceptibility test using specimens from patients with urinary tract infection. The VITEK 2 was the reference method. The IDSX method performed: isolation with quantitation; presumptive identification with determination of predominating species; and antimicrobial susceptibilities, all at 12-18 hours from specimen receipt. Urine specimens evaluated were from 132 patients where antimicrobial agent treatment information was available. A total of 842 microorganism-antimicrobial agent combinations were evaluated. Comparison of susceptibility results from the IDSX method with those from the Vitek 2 method showed that 792 of 842 (94.1%) were in category agreement. In regards to antibiotic usage, 77 of 132 (58%) evaluated patients received an antimicrobial agent prescription on the day of their visit. Of those, 22% were negative for UTI and 16% received an antibiotic prescription later shown to be ineffective for treatment. IDSX kit usage would allow for much needed targeted prescribing.

1. Introduction Current guidelines for the management of uncomplicated urinary tract infection (UTI), such as those published by the Infectious Diseases Society of America (IDSA) (Warren et al., 1999) have supported empirical antibiotic treatment prescribed before definitive drug sensitivities have been performed. However, antimicrobial resistance among pathogenic agents of both cystitis and pyelonephritis is increasing (Gupta et al., 2001; Kahlmeter, 2001). A striking example is the increase in resistance to trimethoprim-sulfamethoxazole, the drug regimen of choice in the U.S. for empirical therapy of uncomplicated UTI's in women (Gupta et al., 2001). As a result, many clinicians prescribe more expensive fluoroquinolones for the treatment of acute uncomplicated cystitis (Taur et al., 2007). Although fluoroquinolones are effective antimicrobials and have an important role in treating cystitis, these drugs are not appropriate first line therapy (Hooton et al., 2004). Increasing resistance to broad-spectrum antibiotics, such as the fluoroquinolones, is a serious worldwide problem (Goettsch et al., 2000; Iqbal et al., 1997; Srinivasa et al., 1999; Thompson, 1999). It is essential that indiscriminate use of these broad-spectrum antibiotics is avoided so their efficacy may be preserved. Multidrug-resistant pathogens that are difficult and sometimes impossible to treat successfully are a major medical dilemma (Levy, 2002). Given the significant impact on morbidity and mortality, multidrug-resistant pathogens are considered a substantial threat to US public health and national security by the National Academy of Sciences Institute of Medicine (Smolinski et al., 2003), the federal Interagency Task Force on Antimicrobial Resistance (Interagency Task Force) (Antibiotic/antimicrobial resistance: action plan 1999), and the IDSA (Infectious Diseases Society of America 2004). In addition, a number of researchers have concluded that resistance is irreversible in many situations. Attempts to slow the pace at which resistance evolves may be the

best course of action (Morell, 1997). Resistance to antibiotics frequently reduces the fitness of bacteria in the absence of antibiotics; this is referred to as the cost of resistance (Spratt, 1996). Decreased fitness due to a mutation or acquisition of resistance suggests that drug-resistant organisms might diminish if antibiotic use is decreased. However, increasing evidence indicates that compensatory mutations act to cause long-term persistence of resistant bacteria (Andersson, 2003; Campos et al., 1996; Schrag et al., 1997; Livermore, 2003). An example of this is the persistence of chloramphenicol-resistant Escherichia coli many years after the antibiotic ceased to be commonly used. Most suspected UTIs are confirmed by the quantitative culture of urine followed by conventional bacterial isolation, identification, and antimicrobial susceptibility testing (AST). During this process, which takes 48 to 72 hours or longer, the patient is often treated empirically with a broad-spectrum antimicrobial agent. Automated methods for identifying bacterial isolates and testing antimicrobial susceptibility have become the norm in most clinical laboratories and may shorten the process. However, an overnight pre-isolation step is still required in current methods. In contrast, the IDSX method is a direct antimicrobial susceptibility test (DAST) with results available within 12-18 hours following specimen collection. The DAST is validated by concurrently performing an isolation. This allows determination of a predominating species in a mixed culture or identification of a pure species as well as measurement of bacterial concentration in the specimen. A predominating species is defined as one that is present at greater or equal to ten times the colony count of any other species present. Species identifications are made by observation of colony morphology primarily. This manual method is directed toward a point-of-care, evidence-based, targeted approach to antibiotic treatment. The aim of

our study was to show that the IDSX method is a valid means whereby overnight antimicrobial susceptibility results could guide prudent therapy for urinary tract infections. 2. Materials and Methods 2.1 Setting The location for this Institution Review Board (IRB)-approved study was the laboratory of a university medical center located in an outpatient community clinic in Salt Lake City, Utah. 2.2 Study period and clinical samples From March to May of 2007 and from September through October of 2007, specimens were obtained from patients who presented with symptoms of lower urinary tract infection, as defined by their physicians. The initial criterion for inclusion in the study was submission of a urine specimen to the laboratory for culture. Following the observation that a substantial number of the specimens were negative for infection, we altered the criteria to collect samples that tested positive for nitrite on dipstick urinalysis and/or showed significant numbers of bacteria (2+ or greater ) on urine microscopic examination. IRB-approval to extract antimicrobial agent usage from the patient database for the period of our study was obtained and data analysis was limited to include only those patients for whom antimicrobial agent treatment information was available. 2.3 Microbiologic management of samples for direct IDSX All samples were midstream, clean-catch urine specimens. Specimens submitted for culture were aliquoted by laboratory staff into two Vacutainer Urine C& S Preservative Plus tubes (BD, Franklin Lakes, NJ). One tube was used for the IDSX method and the other was processed for the VITEK 2 Compact (bioMerieux, Inc., Durham, NC) method. Processing of a specimen was begun for both methods within the same workday. The de-identified IDSX-numbered tubes and corresponding urinalysis results were collected at the end of the workday and taken off-site for

IDSX set-up, incubation and analysis. Briefly, specimens were applied directly to the IDSX kit or diluted and applied, antimicrobial-impregnated papers were then applied, and the plate was incubated at 35C overnight. The IDSX plate was divided into four quadrants. Two of the chambers contained media for bacterial isolation. The media were Columbia CNA agar with 5% sheep bred blood cells and MacConkey agar. The remaining chambers contained Mueller-Hinton agar and Mueller-Hinton agar with 5% sheep red blood cells. These chambers were used for antimicrobial susceptibility testing. Amikacin(AN), amoxicillin/clavulenate(AMC), ampicillin(AM), cefazolin(CZ), cefotetan(CTT), ciprofloxacin(CIP), gentamicin(GM), levofloxacin(LVX), nitrofurantoin(F/M), piperacillin/tazobactam(TZP), and trimethoprim/sulfamethoxazole(SXT) were the antimicrobial agents primarily evaluated. Other antimicrobial agents used include cephalothin(CF), doxycycline(D), ceftriaxone(CRO), tetracycline (TE) and cefdinir(CDR). The decision to perform a dilution or not was based on results from a urine dipstick. Leukocyte esterase, nitrite, blood or protein in the specimen may indicate bacteria in the urine. A positive nitrite generally indicated that a significantly large concentration of organisms was present. A 1:10 dilution of specimen in sterile water was made in this situation. The incubation time required for growth was 12 to 18 hours. Digital pictures were taken of the x-dish for documentation purposes after measurements of the zones of inhibition for each of the antimicrobial agents tested were made. The concentration of colony forming units per milliliter was determined by observing one of the isolation chambers and comparing it against a set of known concentration result images shown in Figure 1. The DAST is valid for all concentrations from 104 cfu/ml through 108 cfu/ml on the isolation chamber, and presumptive identification is determined on the isolated colonies using standard methods of colony morphology analysis along

with additional spot testing, such as the catalase test for gram-positive organisms and the cytochrome oxidase test for gram-negative organisms, when necessary. E. coli American Type Culture Collection (ATCC) 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853 were used as control strains. These are species with known zones of inhibition values used in control testing to assure that antimicrobial susceptibility systems are operating correctly. 2.4 Standard VITEK 2 quantitative cultures All samples were processed by the standard quantitative culture method using a calibrated 2.5 milliliter loop onto Columbia agar with 5% sheep blood and MacConkey agar plates. After 18 to 24 hours of incubation, the colonies were counted. Colony counts of greater than or equal to 104cfu/ml were considered significant. Identification (ID) and susceptibility testing of the microorganisms that were isolated were performed on the VITEK 2 system using cards ID-GNB (identification- Gram negative bacilli) and AST (antimicrobial susceptibility testing) N029 for gram-negative rods, and ID-GPC (identification Gram positive cocci) and AST-P524 for enterococci. Cards were inoculated with a bacterial suspension prepared in 0.45% saline equal to the turbidity of a 0.5 McFarland standard. The ID and interpretation of AST results were based on software version R 04.02 according to the manufacturers instructions. 2.5 Definitions and interpretation of results Comparison of the results of the test method (IDSX) with results from the reference method (VITEK 2) was made using qualitative susceptibility categories. The possible result for each microorganism-antimicrobial agent pair is (1) susceptible, (2) intermediate/moderately susceptible), or (3) resistant. An agreement or discrepancy category is assigned for each of the 9

possible combinations. The categories are: (1) agreement, (2) minor error, (3) major error, or (4) very major error. These designations are displayed in Table 1. 2.6 Prescription information The medical record database was searched to obtain data concerning treatment during the period of the study. De-identified sample result information was linked to the patient record using HIPAA approved methods. The IRB protocol insured that appropriate measures were in place for the data extraction. 3. Results 3.1 Time to result During the study period, 132 comparable samples were processed concurrently by the IDSX and VITEK 2 methods. Eighty-one of the samples revealed a significant infection for which antibiotic treatment was indicated. Of these, the IDSX method produced a result within 1 day following receipt of specimen 78 of 81 times (96%). The remaining three specimens (4%) required an additional culture to speciate the predominant species; results were available on day 2. By contrast, the VITEK 2 method gave results 0 of 81 times (0%) on day 1 following receipt of specimen, 54 of 81 times (67%) on day 2, 11 of 81 times (14%) on day 3, 11 of 81 times (14%) on day 4, 3 of 81 times (4%) on day 5 and 2 of 81 times (2%) on greater or equal to day 6 (see Figure 2). 3.2 Comparison of antimicrobial susceptibility test results with associated discrepancies Among the 842 microorganism-antimicrobial agent pairs evaluated by the IDSX method, total agreement (category agreement) with the VITEK 2 was 94.1% (792/842). There were 37 (4.4%) minor-errors, 13 (1.5%) major-errors, and no (0%) very-major errors. 3.3 Patient therapy versus in vitro test results

There were 132 patients seen at the community clinic where therapy prescription information was available. Seventy-seven patients received empirical treatment (a prescription was written) on the day of their visit (see Table 2). However, microbiology culture results for 17 of the 77(22%) were negative for a urinary tract infection. Forty-eight of the 77(62%) tested culturepositive for a UTI and were treated with an antimicrobial agent that was subsequently bactericidal for the uropathogens. Twelve of the 77(16%) tested positive for a UTI but were treated with an agent that was not effective treatment for their uropathogens. Antimicrobial agent choices, diagnostic results, and treatment outcomes, for the day of visit empirical therapies, are shown in Table 2. Fifty-five patients did not receive empirical treatment on the day of the office visit. Microbiology results for 27 of the 55 were negative for a urinary tract infection and no treatment was given within a 30-day period. Five of the 55 were culture positive for a UTI but were not treated within the 30-day period. Four of the 55 were prescribed an antimicrobial agent by telephone from thirteen days to one day prior to their clinic visit. All 4 of these patients were infected with a pathogen that was resistant to the prescribed antibiotic and treatment failed. Effective antibiotic agents were prescribed based on VITEK 2 result. For example, one patient treated with ciprofloxacin for 11 days prior to the visit was switched to nitrofurantoin 3 days post visit. The remaining 19 of the 55 patients were treated with antimicrobial agents shown in the in vitro assay to be bactericidal, several days after their visit. The median time to treatment was 3.5 days post visit for these patients. 3.4 Five result examples (digitally illustrated) highlighting the need for evidence based therapy versus empirical therapy

Selected results comparing the IDSX and VITEK 2 are shown in Table 3 and Figures 3,4,5,6, and 7. Shown are the days to result (DTR), presumptive (IDSX) versus definitive (VITEK 2) identifications, AST results for each antimicrobial agent and the prescription medication history during the time period plus or minus 30 days from the day the patient was seen by the doctor and a specimen was collected and sent to the lab. Patient #48 (Figure 3, Table 3), was treated with SXT on the day of the medical visit. Category agreement was assigned as R-R for both SXT and AM. The patient waited 14 days for CIP therapy, which was effective against the infecting organism. AMC, F/M and TE would have been effective therapy also. Patient #91 (Figure 4, Table 3) was treated with CIP 11 days before an office visit. Results of AST showed category agreement (CA) of R-R for CIP, and the patient was prescribed with the effective agent F/M (CA S-S) 3 days post-visit. SXT would have worked equally well (CA SS). Patient #100 (Figure 5, Table 3,) was treated on the day of the visit with SXT. IDSX showed SXT and AM to be R by next morning. VITEK 2 however showed SXT and AM to be S (at day 3), which produced major errors between the two sets of results. One week later the isolated E. coli species was retested by VITEK 2. Results were reversed for both SXT and AM from a susceptible to resistant value. This brought these two antibiotics from major errors into category agreement. The SXT therapy failed and cefdinir was prescribed at 22 days post-visit (no susceptibility information was available). Patient #108 (Figure 6, Table 3) was treated for 23 days before the visit with AM. On the day of the visit the antimicrobial agent was switched to F/M. AST results from both methods showed

10

that the infecting organism was S to F/M. (It is instructive to note that CIP and SXT were both Resistant. Therefore empirically F/M was a good guess.) Patient #115 (Figure 7, Table 3) was treated for 13 days before the office visit with a combination of AM and F/M. Both methods showed these agents to be ineffective in treating the organism. No treatment was prescribed on the day of the visit. CIP was used as therapy two days post visit. The uropathogen was a very resistant P. aeruginosa with few effective oral treatment options. LVX, another fluoroquinolone, would have been effective as well. 4. Discussion The IDSXs DAST method and the VITEK 2s AST method were considered to be in total agreement when both methods presented the same susceptibility category (susceptible, intermediate, or resistant) by both methods. When the results from the methods were not in agreement the severity of the discrepancy is ranked (from least severe to most severe): Minorerror (the IDSX method indicated intermediate and the VITEK 2 method indicated susceptible or resistant; the IDSX indicated resistant and the VITEK 2 indicated intermediate; or IDSX indicated susceptible and the VITEK 2 indicated intermediate); Major-error (the IDSX indicated resistant and the VITEK 2 indicated susceptible); and Verymajor-error(the IDSX indicated susceptible and the VITEK 2 indicated resistant) Nine of the discrepancies from the study involved microorganism-pipericillin/tazobactam (TZP) couples. In several recent studies with the VITEK 2, unacceptable levels of error (minor, major, and very major) were detected. In some cases systematic biases toward false susceptibility for (piperacillin-tazobactam and imipenem) were noted: "More serious very major errors (false-susceptible) were detected at rates several fold greater than the acceptable limit

11

(15%)for piperacillin-tazobactam when using the VITEK (15.0%) and VITEK 2 (20.0 to 21.7%) systems (Juretschko et al., 2007). Four of the discrepancies concerned the microorganism-SXT couple. With trimethoprim and the sulfonamides, antagonists in the medium allowed some slight growth which was not disregarded although CLSI guidelines recommend disregarding slight growth (20% or less of the lawn growth) and measurement of the more obvious margin to determine the zone diameter (CLSI M2-A9, 2006). Several other observations concerning the discrepancies were: 1) Seventeen of the errors were within 0.5 mm of being in category agreement. 2) Twenty-five of the 37 minor errors were either intermediate for IDSX and susceptible for VITEK 2 or resistant for IDSX and intermediate for VITEK 2. These errors fall on the side of a safer (less chance for treatment failure) therapeutic approach to the infection. 3) Discrepancy resolution if validated by digital image could have reduced the major errors from 13 to 2. See for example the IDSX TZP result for patient #91 in Table 3, and Figure 4, TZP disk-quarter for a visual: the result was E.coliTZP-resistant versus the VITEK 2 result of E.coli-TZP-susceptible which creates a major error combination (ME). This would be a very major error for VITEK 2 if the IDSX method was the reference method. For future studies, the Kirby-Bauer Disk Diffusion method could serve as the reference for resolving discordant AST results. A major advantage of the IDSX kit would be to direct therapy with the most prudent antimicrobial agent within 12 to 20 hours from the office visit. As it was stated in the introduction, fluoroquinolones are effective antimicrobials and have an important role in treating cystitis but these drugs are not appropriate first line therapy (Hooton et al., 2004). It was instructive that for 21 patients treated with ciprofloxacin empirically on the day of their visit,

12

cultures from 13 of the 21 patients were susceptible to the oral antimicrobial agent ampicillin, cultures from 18 of the 21 patients were susceptible to amoxicillin/clavuanic acid, cultures from 19 of the 21 patients were susceptible to nitrofurantoin, and cultures from 16 of the 21 patients were susceptible to trimethoprim/sulfamethoxizole. Simple DAST of urine specimens by a disc diffusion assay have been shown in the past to offer a means of rapidly and inexpensively guiding antimicrobial therapy for patients with urinary tract infections when used selectively and interpreted carefully (Johnson et al., 1995; Bronnestam 1999; Blue et al., 1991; Gillenwater et al., 1996). However, the DAST of these former studies did not have the benefit of a concurrent isolation for presumptive identification or a means to quantitate the concentration of pathogens in the specimen as the IDSX kit does. The DAST portion of the IDSX method operates on the principle of a disc diffusion assay. It is important to note that the DAST portion of the IDSX method produces results that are equivalent to a standardized disc diffusion assay such as the Kirby-Bauer assay, only when the antibiotic papers are placed so that they are against the walls or into the corners of the test chambers. Also, the IDSX kit gives valid results over a broad concentration range (4 logs) on the plate from 104 cfu/ml to 108 cfu/ml. Ability to determine a predominating species in a mixed culture is another advantage The results of this study show that patients with lower urinary tract

infections can benefit from reliable antimicrobial susceptibility data from IDSX results in less than 24 hours. The results were comparable with those obtained with the VITEK 2 method (overall correlation of 94.1% without discrepancy resolution) that was available in no less than 48-72 hours. The IDSX kit is a novel direct antimicrobial susceptibility test (DAST). The kit allows presumptive identification based on isolated colonies, determination of a predominating species

13

in mixed culture, and yields bacterial cell concentration, all concurrently performed with the DAST. Since the IDSX kit provides overnight results, it is prudent for the healthcare provider to delay prescription of antimicrobial therapy for 12-18 hours when results of DAST are available to guide patient care. Patients can be given an analgesic such as phenazopyridine to minimize symptoms and provided with literature explaining antibiotic resistance. A prescription can be telephoned to the pharmacy the next morning. This would allow the healthcare provider to prescribe the most appropriate antibiotic instead of using empirical treatment. Antimicrobial therapy that is evidence-based is prudent use of our antibiotic armamentarium. Acknowledgements The authors thank the microbiology staff at Central Laboratory, the Utah Health Research Network (study sponsor), and Atoosa Kourosh MD, MPH.

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References Andersson DI (2003) Persistence of antibiotic resistant bacteria. Curr Opin Microbiol 6:452-456 Antibiotic/antimicrobial resistance: action plan. Atlanta (1999): US Department of Health and Human Services Centers for Disease Control and Prevention. Available at http://www.cdc.gov/drug- resistance/action plan Blue AP, Gordon DL (1991) Is primary testing on urine samples valid? Pathology 23:149-152. Bronnestam R (1999) Direct antimicrobial susceptibility testing in bacteriuria. APMIS 107(4):437-444. Campos J, Roman F, Georgiou M, Garcia C, Gomez-Lus R, Canton R, Escobar H, Baquero F (1996) Long-term persistence of ciprofloxacin-resistant Haemophilus influenzae in patients with cystic fibrosis. J Infect Dis 174(6):1345-1347 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Ninth Edition. Clinical and Laboratory Standards Institute document M2-A9. Clinical and Laboratory Standards Institute (CLSI). Wayne, PA, 2006. Gillenwater JY, Clark MM (1996) Tentative direct antimicrobial susceptibility testing in urine. J Urology 156(1):149-153. Goettsch W, VanPelt W, Nagelkerke N, Hendrix MG, Buiting AG, Petit PL, Sabbe LJ, Van Griethuysen AJ, de Neeling AJ (2000) Increasing resistance to fluoroquinolones in Escherichia coli from urinary tract infections in the Netherlands. J Antimicrob Chemoth 46:223-228.

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Gupta K, Hooton TM, Stamm WE (2001) Increasing antimicrobial resistance and the management of uncomplicated community-acquired urinary tract infections. Ann Intern Med 135:41-50 Gupta K, Sahm DF, Mayfield D, Stamm WE (2001) Antimicrobial resistance among uropathogens causing community-acquired UTI in women: a nationwide analysis. Clin Infect Dis 33:89-94. Hooton TM, Besser R, Foxman B, Fritsche TR, Nicolle LE (2004) Acute uncomplicated cystitis in an era of increasing antibiotic resistance: a proposed approach to empirical therapy. Clin Infect Dis 39:75-80 Infectious Diseases Society of America (2004) Bad bugs, no drugs: as antibiotic discovery stagnates, a public health crisis brews. Alexandria, VA: Infectious Diseases Society of America Iqbal J, Rahman M, Kabir MS, Rahman M (1997) Increasing ciprofloxacin resistance among prevalent urinary tract bacterial isolates in Bangladesh. Jpn J Med Sci Biol 50(6):241-250 Johnson JR, Tiu FS (1995) Direct antimicrobial susceptibility testing of acute urinary tract infections in women. J Clin Micro 33(9):2316-2323. Juretschko S, LaBombardi V, Lerner SA, Schreckenberger PC (2007) Accuracies of -lactic susceptibility test results for Pseudomonas aeruginosa with four automated systems. J Clin Micro 45(4):1339-1342 Kahlmeter G (2001) An international survey of the antimicrobial susceptibility of pathogens from uncomplicated urinary tract infections: the ECO-SENS Project. J Antimicrob Chemo 51:69-76.

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Levy S (2002) Factors impacting on the problem of antibiotic resistance. J Antimicrob Chemoth 49:25-30. Livermore DM (2003) Bacterial resistance: origins, epidemiology, and impact. Clin Infect Dis 36(Suppl 1): S11-S23. Morell V (1997) Antibiotic resistance: road of no return. Science 278(5338):575-576. Schrag SJ, Perrot V, Levin BR (1977) Adaptation to the fitness costs of antibiotic resistance in Escherichia coli. Proc R Soc Lond B Biol Sci 264(1386): 1287-1291 Smolinski MS, Hamburg MA, Lederberg J (2003) Microbial threats to health: emergence, detection, and response. Washington, DC: Institute of Medicine Spratt BG (1996) Antibiotic resistance: counting the cost. Curr Biol 6:1219-1221 Srinivasa H, Parija SC, Bhattacharya S, Sehgal R (1999) A high incidence of ciprofloxacin resistance in urinary isolates in eastern Nepal. J Commun Dis 31(1):45-47 Taur Y, Smith MA (2007) Adherence to the Infectious Diseases Society of America guidelines in the treatment of uncomplicated urinary tract infection. Clin Infect Dis 44:769-774 Thomson CJ (1999) The global epidemiology of resistance to ciprofloxacin and t he changing nature of antibiotic resistance: a 10 year perspective. J Antimicrob Chemother 43(Suppl A):31-40. Warren JW, Abrutn E, Hebel JR, Johnson JR, Schaeffer AJ, Stamm WE (1999) Guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women. Clin Infect Dis 29:745-758

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Table 1. Possible combinations of category results with agreement category(agreeement or discrepancy) Reference method (VITEK-2) Resistant Intermediate Susceptible Resistant Intermediate Intermediate Susceptible Susceptible Resistant Test Method (IDSX) Resistant Intermediate Susceptible Intermediate Resistant Susceptible Intermediate Resistant Susceptible Agreement category Agreement Agreement Agreement Minor error Minor error Minor error Minor error Major error Very major error

18

Table 2. Empirical treatment on the day of visit. 77 of 132 (58%

Empirical antibiotic SXT CIP F/M CF AMC AM D LVX CRO+LVX CDR

Number of patients Rx with the antibiotic 19 27 15 4 4 2 1 3 1 1 77 total

Eventual diagnosis plus pathogen susceptibility No UTI UTI-UTI--antibiotic (% of antibiotic not total) susceptible susceptible(%) (%) 5(26%) 6(22%) 2(13%) 2(50%) 1(50%) 1(33%) 17(22%) 9(47%) 19(70%) 11(73%) 2(50%) 3(75%) 2(66%) 1 1
a b

5(26%) 2(7%) 2(13%) 1(25%) 1(50%) 1(100%) 12(16%)

Non-susceptible antibiotic switched with susceptible antibiotic listed below (days from visit) CIP(14), CIP(4), LVX(4), CDR(22), CIP(2) F/M(23), F/M(2) SXT(3), No replacement

SXT(13) F/M(6) No replacement

48(62%)

Antibiotics listed: amoxicillin/clavulenate(AMC), ampicillin(AM), ciprofloxicin(CIP), levofloxicin(LVX), nitrofurantoin(F/M), trimethoprim/sulfamethoxazole(SXT), cephalothin(CF), doxycycline(D), ceftriaxone(CRO), cefdinir(CDR),
a

CRO was not tested but LVX showed susceptible;

CDR was

not tested but assumed to be susceptible because all cephalosporins showed susceptible.

19

TABLE 3. Selected results of AST, ID for IDSX and VITEK-2 method. Also includes time to result and antimicrobial agent intervention history. See Figures 3-7.

PRIOR
FIG. # TEST AMC DTR SXT

DAY 0 RX SXT

POST RX(D) CIP(+14)

ID 1:10dil.,10-6 col/ml gm(-), LF > 10-5 col/ml Escherichia coli 1:10dil.,10-7 col/ml gm(-) LF,w minor gm(+) > 10-5 col/ml Escherichia coli 1:10dil.,5x10-7 col/ml,gm(-),LF > 10-5 col/ml Escherichia coli repeated 1 week later: > 10-5 col/ml E. coli

CTT

TZP

LVX

F/M

CIP

GM

AM

CC

AN

VA

CZ

TE

RX(D)

IDSX VITEK IDSX VITEK IDSX

1 2 1 4 1 3

R R R R R S R R R R R

S S R R S S S I I R R

S S I I S S S S S R R

R N R N

R N R N

S S R R S S S R R S S

R N R N R N N R N R N

S S S S S S S S S S N

S S S S S S S R R S N

S S R R S S S R R S S

S S S S S S S S S R N

S S R S S S S S S S S

S N S N R N N R N R N

R R S S R S R R R R R S S S S S R N CIP(-11)

NONE

F/M (+3)

VITEK VITEK IDSX VITEK IDSX VITEK

SXT

CDR(+22)

1 2 1 2

1:10dil.,5x10-6 col/ml,gm(-),non-LF,w minor gm(+) > 10-5 cfu/ml Escherichia coli 1:10dil.,10-6 col/ml,gm(-),grn fluorMH,non-LF, ox(+) pseudomonas aeruginosa, >10-5 col/ml

AM(-23)

F/M

AM(-13) F/M(-13)

NONE

CIP(+2)

DTR=days to result; ID=identification;1:10 dil=1:10 dilution of specimen; gm(-)=gram negative organism; gm(+)=gram positive organism; LF=lactose fermentor; w minor=minor species present; col=colony,sometimes refered to as colony forming unit or cfu; grn fluorMH=green fluorescence on Mueller-Hinton agar; ox(+)=cytochrome oxidase positive colonies; R=resistant,I=intermediate,S=susceptible; See text for the list of antimicrobial agents; CDR=the antibiotic Cefdinir

104 cfu/ml

105 cfu/ml

106 cfu/ml

107 cfu/ml

108 cfu/ml

Fig.1. Set of incubated IDSX plate images of known bacterial concentration. Test sample results are matched for a determination of concentration.

90 n u 80 m b 70 e r 60 o f p a t i e n t s
50

40 30 20
10

IDSX result

VITEK 2 result

0 1 2 3 4 5 >=6 days post receipt of specimen in lab

Fig.2. The IDSX kit antimicrobial susceptibility test result compared to the VITEK 2 result in regards to time from receipt of specimen to result.

Fig.3. Cultured IDSX plate of specimen from patient #48

Placement key

Fig.4. Cultured IDSX plate of specimen from patient #91

Placement key

Fig.5. Cultured IDSX plate of specimen from patient #100

Placement key

Fig.6. Cultured IDSX plate of specimen from patient #108

Placement key

Fig.7. Cultured IDSX plate of specimen from patient #115

Placement key