Cumitech 2B : Laboratory Diagnosis of Urinary Tract Infections

NOVEMBER

1998

Laboratory Diagnosis of Urinary Tract Infections JILL E. CLARRIDGE, JAMES AND MARIE T. PEZZLO COORDINATING

ALICE

R. JOHNSON,

EDITOR

S. WEISSFELD

Curmtech CUMULATIVE

TECHNIQUES

AND PROCEDURES

IN CLINICAL

MICROBIOLOGY

Cumitech Cumitech

1B 2B

Blood Cultures III l April 1997 Laboratory D~agnosls of Urinary

Cumitech Cumitech Cumitech Cumitech Cumitech

3A 4A 5A 6A 7A

Qualtty Control and Qualtty Assurance Practtceq tn Clm~cal Mtcrobtology l Mny 1990 Laboratory D~agnosts of Gonorrhea l Aprzl 1993 Practtcal Anaerobtc Bactertology l December 3 991 New Developments tn Antttntcrobtal Agent Suscepttbthty Tcsttng: a Practtcal Guide l l-ehrunry Laboratory Dtagnosts of Lower Kesptratory Tract Infecttons l September I987

Cumitech Cumitcch Cumitcch Cumitech

8 9 10 11

Detectton of Mtcrobtal Anttgens by Counterttnmunoelectrophorests l Deccwzbcr Collectton and Processtng of Bactertologtcal Spectmens l August I979 Laboratory Dtagnosts of Upper Rcsptratory Tract Infccttons l December 1979 Prxttcal

Methods

Aqpst

for Culture

Tract Infectmns

and Identtficatton

l

November

2 998

7 99 1

1978

of Fungi tn the Cltntcal Mtcrobtology

Laboratory

l

2 980

Cumitech Cumitech Cumitech

12A 13A 14A

Laboratory Laboratory Laboratory

Dtagnosts of Bactertal Diarrhea l Aprd I992 Dtagnoits of Ocular Infecttona l September 1994 Diagnosts of Central Nervous System Infecttons

Cumitech Cumitech Cumitech Cumitech

1.5A 16A 17A 18A

I.aboratory Laboratory Laboratory

Dtagnosts of Vtral Infecttons l August 1994 Dtagnosts of the Mycobactertoses l October 2 994 Dtagnosts of temale Genttal Tract Infecttons l June 2 993

Cumitech Cumitech Cumitech

19 20 21

Laboratory Laboratory Therapeutic 1,aboratory

Dtdgnosts of Ijcpatttts Vtruses l November I998 Dtagnosts of Chlamydtal and Mycoplasmal Infecttons l August Drug Monttortng: Anttmtcrobtal Agents l October I984 Dtagno\ts of Viral Respiratory Dtsease l March 1986

Cumitech Cumitech Cumitcch

22 23 24

Immunoserologv of Staphylococcal Disease l August 1987 Infecttons of the Sktn and Subcutaneous TISSLXX l /MMC 1988 Rapid Detectton of Vtruses by Immunofluorescence l August I988

Cumitech Cumitech Cumitech

25 26 27

Cumitech

28

Cumitech Cumitech

29 30

Current Concepts and Approaches to Anttmtcrobtal Agent Suscepttbtltty Testtng l Driember I988 Laboratory Dtagnosts of Viral Infecttona Productng Entcrttts l September 2 989 Laboratory Dtagnosts of Zoonottc Infections: Bacterial Infecttons Obtatned from Cotnpanton and Laboratory Animals l February 1996 Laboratory Diagnosis of Zoonottc Infections: Chlamydtal, I‘ungal, Vtral, and Parastttc Infecttons Obtatned from Companion and L,aboratorv Animals l February 7 996 Laboratory Safety tn Cltntcal Mtcrobtology l July 2 9% Selectton and U\e of Laboratory Trdct

Cumitech

Cumitechs of urinary

31

Editorial

Board

Jamson,

Karen

The

purpose

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0 1998

Washington,

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series

microbiology

American Avenue

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Allan

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as “standard” Society NW

1984

Procedures for Dtagnosts of Parastttc Infections of the Gastrotntesttnal

J. E., J. R. Johnson,

A. S. Welssfeld.

Frederick

McCurdy,

Cumitech

299 3

of Procedures m the Cltntcal Mtcrobtology

Clarridge.

ed.,

Cumitechs: Brenda

proposed

Massachusetts

as follows,

March

1996

and Vahdatton

Coordinating

Krisher,

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are not

Copyright

be cited infections.

of the

procedures given

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Septmzher

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Chamnan,

S. Nolte, Truant,

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Lorraine

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Laboratory

2B,

Washington,

Clarke,

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Fchruary

1997

Laboratory

diagnosis

D.C.

A Gleaves,

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Young. authors evaluating

as to appropriate roLltine

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procedures

Laboratory Diagnosis of Urinary Tract Infections Jill E. Clarridge Departments of Pathology and of Microbiology and Immunology, Baylor College of Medicine and Veterans Affairs Medical Center, Houston, Texas 77030

James R. Johnson Department

of Medicine, Division of Infectious Disease, University of Minnesota Medical School, and Medical Service, Veterans Affairs Medical Center, Minneapolis, Minnesota 55455

Marie T. Pezzlo Department

of Pathology,

University

of California

Irvine

Medical

Center, California

Orange, 92 668

COORDINATING EDITOR: Alice S. Weissfeld Microbiology

Specialists

Immunology, Introduction Clinical

Incorporated and Department of Microbiology and Baylor College of Medicine, Houston, Texas 77054

2

.........................................................................................

Considerations

..........................................................................

2

....... ... ....................... Significance of UTI ................................................................ Clinical Syndromes ..................................................................................................... Pathogenesis and Microbral Flora of UTI ...................................................................... Role of the Laboratory in the Management of UTI ........................................................

2 2 3 4

.............................................................................

4

........................................................ Suprapubic Aspiration and Straight Catheterization ............................................................................. Clean-Catch Midstream Specimens ............................................................... Collection Bags for Specimens from Children lndwellrng Catheters .................................................................................................. ............................................................................................................ lleal Conduits ....................................................................... Other Methods of Specimen Collection

4 4 5 5 5 5

Specimen Specimen

Transport Handling

5 6

Specimen

Workup

Specimen

Collection

............................................................................. .................................................... in the Laboratory ................................................................................

6

............................. Necessary Communication between the Clinician and the Laboratory .................................................................................. Approach to Laboratory Workup ............................................................... Selection of Media and Incubation Conditions ........................................................................................................ Routine Cultures Surverllance Cultures .................................................................................................. ......................................................................................................... Special Cultures ...................................... Guidelines for Specimen Workup and Interpretation of Results ................................... Recommended Supplementary Procedures for Special Situations .................................................................................................... Reporting Results

6 6 8 8 8 9 9 9 10

Rapid Urine

IO

Bacteriologic

Screens Culture

........................................................................... Systems

.............................................................

Direct Antimicrobial Susceptibility Testing of Urine Specimens .................................................................... Unacceptable Procedures References .........................................................................................

11 ..............

13 14 14

2

Clarridge

et al.

INTRODUCTION

U

rinary tract infections (UTIs) are among the most common infections in humans. For the major ity of cases, a limited number of bacterial species is the cause of the infection. The presence of microorganisms in urine is termed bacteriuria whether or not they cause infection. Since publication of the last Cumitech on this subject in 1987 (23), there has been increased emphasis on obtaining the information needed for treating patients in the most costeffective but accurate manner possible. Therefore, we have added several new sections in this updated edition. These include a discussion of the communication between the practitioner and the laboratory that is necessary to render cost-effective and accurate results of rapid urine specimen screening techniques and bacteriologic culture systems, which have been marketed to small hospital and physician office laboratories. Finally, because of space constraints due to the inclusion of so much new material, we have elected not to cover some procedural information, such as step-bystep collection instructions for clean voided midstream specimens for males or females and instructions on how to streak quantitatively, which can be found in other ASM Press publications (34, 89, 124).

CLINICAL

CONSIDERATIONS

Significance of UT1 UT1 is one of the most commonly encountered acute infectious diseases. It can occur from infancy (20,27, 53, 126, 138) through old age (1, 17, 95, 96) and in otherwise healthy persons, as well as in those who are compromised or debilitated (5, 65, 114). UT1 is responsible for substantial increased health care costs, morbidity, mortality, and lost productivity (104). Because UT1 is such a common problem and urine specimens are easy to obtain from patients, clinicians submit urine more frequently than almost any other type of specimen. As a result, urine cultures account for a major share of the workload in many clinical microbiology laboratories. Because of the large workload, it is important for microbiology laboratory workers to have some understanding of the clinical context from which these specimens originate, as clinical considerations often profoundly influence the laboratory evaluation of urine specimens (65). Clinical Syndromes Far from representing a single disease, the term “UTI” is actually an umbrella term that embraces a wide range of clinical syndromes, each with its own pathogenic mechanisms, host range, prognostic significance, and unique requirements for diagnosis and treatment (54, 65). A unifying theme among the di-

CUMITECH

2B

verse UT1 syndromes is the presence of microorganisms within the normally sterile urinary tract, usually accompanied by an acute inflammatory response. UT1 syndromes differ with respect to the specific site or the extent of infection within the urinary tract, the intensity of the inflammatory response, and the underlying status of the host. UT1 syndromes commonly encountered in clinical practice include asymptomatic bacteriuria, cystitis (as well as the overlapping entities of the frequency-dysuria syndrome and the urethral syndrome), and pyelonephritis (or UT1 with fever) (54,65). Any of these can occur in a host with an underlying urological abnormality that predisposes to UT1 or makes UT1 more difficult to treat effectively, in which case the infection is designated as “complicated” (114). Less commonly encountered UT1 syndromes include acute prostatitis ( 80, 8 7), intrarenal or perinephric abscess, and urosepsis (bacteremic UTI) (8, 54). Asymptomatic bacteriuria is defined by the presence of bacteria within the urinary tract in the absence of symptoms (54). Its clinical significance is controversial outside certain limited patient subsets, including children with vesicoureteral reflux (who may develop kidney damage from silent renal infection) (126, l49), pregnant women (because of the risks of progression to severe symptomatic UT1 and of possible harm to the fetus) (7, 42), and patients about to undergo an invasive procedure involving the urinary tract (who could develop urosepsis as a result of the instrumentation) (96). Because in asymptomatic patients bacteria isolated from the urine in low concentrations are more likely to represent colonization or contaminants than true urinary pathogens, more stringent colony count criteria are used to diagnose bacteriuria in this setting than in patients with clinically evident, symptomatic UT1 (65). Cystitis is the term applied to UT1 presumed to be confined to the bladder because of the presence of symptoms suggesting bladder involvement (e.g., dysuria [painful or burning urination], urgency, or frequency) and the absence of symptoms or physical signs suggesting inflammation at other sites within the urinary tract (54). However, clinical criteria are notoriously inaccurate in identifying the actual anatomical site of infection, as evidenced by the presence of occult infection of the upper urinary tract in a substantial subset of patients who appear clinically to have infection limited to the bladder (65). Fortunately, this has proven not to be a major problem for the management of UTI, because with contemporary antimicrobial agents treatment strategies based entirely on clinically categorized UT1 syndromes usually are highly successful (59, 65, 144). The term “frequency-dysuria syndrome” is applied to the syndrome manifested by patients (usually

CUMITECH

2B

women ) who have voiding symptoms suggestive of bladder irritation, irrespective of the underlying etiology. It includes patients who have ordinary acute bacterial cystitis (or urethritis), as well as those who have urethritis becau se of sexually tra nsmitted pathogens (such as herpes simplex virus or Chlamydia trachomatis) or who have a noninfectious urethral process (such as trauma or chemical irritation). The term “acute urethral syndrome” has been applied to the syndrome of the subset of these patients who have urine cultures showing either no growth or only low counts of uropathogenic bacteria, the assumption being that such patients do not have true bacterial bladder infections (133). It is now recognized that the colony count criteria traditionally used to separate bacterial cystitis from the urethral syndrome are artificial and that most dysuric women who would be assigned to the latter category on the basis of lowcount bacteriuria in fact require treatment for acute bacterial UT1 (6.5, 74, 133). Pyelonephritis and febrile UT1 are clinical diagnoses that connote more invasive infection than usually is thought to be present with superficial bladder infection alone (72). Inflammation of the kidney and renal pelvis (pyelonephritis) is assumed to be present when patients have pain or tenderness involving the flank, together with other clinical or laboratory evidence of UT1 (54). These local indicators of renal inflammation are often accompanied by evidence of a systemic inflammatory response, including fever, chills, malaise, headache, and nausea, which is absent in patients judged to have cystitis alone. In some patients these systemic manifestations occur without local signs or symptoms of renal invol vement, a situation sometimes termed febrile UTI. In such cases renal infection is often suspected, an.d treatment is given accordingly. Patients with the clinical syndrome of pyelonephritis or febrile UT1 may or may not have concomitant symptoms suggesting bladder infection (60) Patients with underlying anatomical or functional disorders that impair the normal defense mechanism of the urinary tract are at increased risk for UT1 (114). These patients also often develop serious sequelae from UT1 and usually do not respond to UT1 therapy. Intrarenal and perinephric abscesses, which may require surgical intervention, occur most commonly in such patients (5,54). The patient’s age and health status greatly influence the clinical manifestations that accompany UTI. Neonates up to 1 month of age with UT1 often have vague, nonspecific symptoms and signs, including bacteremia without fever, vomiting, poor feeding, and failure to thrive (20,27, 53, 126). After the neonatal period and until age two, children with UT1 commonly present with high fever but seldom have more

Urinary

Tract

Infections

3

specific symptoms of UTI, such as dysuria or frequency (53). From age two on, children with UT1 usually have more characteristic symptoms referable to the urinary tract. The syndromes of pyelonephritis and cystitis can be differentiated clinically, and girls with cystitis only can be spared urological investigation because of the low likelihood of underlying anatomic abnormalities or future renal problems, in contrast to children with other UT1 syndromes (53). In adolescents, hormonal changes and the onset of sexual activity lead to patterns of UT1 more similar to those of adults (53). The prevalence of bacteriuria among the elderly rises with increasing age .and degree of debility (1,17, 96,97,99), from about 20% (women) and 3% (men) for ages 65 to 75, to 25 to 50% (women) and 20% (men) for ages over 80. Higher prevalences occur among the bedridden or institutionalized elderly (95, 96). Acute pyelonephritis is more likely to give rise to bacteremia in elderly women than in younger women (3 8, 121). In the elderly, renal infection can precipitate acute renal failure (100, 153), sometimes in the absence of overt clinical symptoms of UT1 (153). Pathogenesis

and Microbial

Flora of UT1

UT1 most commonly is caused by bacteria from the patient’s own intestinal flora that enter the urinary tract in a retrograde fashion via the urethra (40), the so-called “ascending route” of infection ( 135). In children and adults without complicating factors, Escherichia coli is by far the most common organism to cause UTI, with Klebsiella and Proteus spp. running a distant second and third (128). This reflects not only the predominance of E. coli in the aerobic intestinal flora, but also the presence in certain E. coli strains of specific virulence factors that interact with the host to overcome host defenses in the urinary tract and to stimulate an inflammatory response (56). Sexual intercourse promotes UT1 in both sexes (11, 79, 98, 152), in part by facilitating the entry of intestinal (and vaginal) bacteria into the urinary tract. Females are at greater risk of UT1 throughout most of th .eir lives than are males, a difference often attributed both to the greater proximity of the female urethral orifice to the colonic bacterial reservoir and to the shorter female urethra (23). The vagina serves as an additional reservoir for uropathogenic bacteria in females. Vaginal colonization with E. coli is associated with the use of spermicide-based contraceptive methods (47), the fact that a woman is a nonsecretor of blood group substances (125), and the (hypoestrogenie) postmenopausal state (115). Although Staphylococcus saprophyticus accounts for a substantial minority of acute UTIs among young sexually active women and presumably also is acquired by the ascending route, the reservoir for this organism and its

4

Clarrldge

et al.

CUMITECH

pathogenetic mechanisms remain largely unknown (48, 78, 105). Complicating factors such as indwelling catheters, nephrostomy tu bes, urinary stones, surgical reconstructions of the urinary tract, and impaired bladder emptying not only help microorganisms enter and persist within the urinary tract, but also alter the spectrum of UT1 pathogens (65, 114). Although E. coli is the single most common organism in complicated and uncomplicated UTIs, other gram-negative bacilli such as Enterobacter, Prouidencia, Serratia, Acinetobacter, and Pseudomonas spp. figure more prominently, as do coagulase-negative staphylococci, enterococci, and yeasts, particularly among patients receiving antibacterial therapy and those with indwelling catheters (144). These non-E. coli organisms have a lesser intrinsic ability to invade the intact urinary tract than do uropathogenic strains of E. coli, but they sometimes can behave as formidable pathogens when host defenses are impaired (55). Polymicrobial UTI, which in other contexts is often (appropriately) interpreted as suggesting contamination of the urine specimen, is common in complicated UTI. The increased risk of UT1 in the elderly, particularly those who are functionally impaired, is probably multifactorial (95). Postmenopausal changes in the vaginal microflora ( 11 S), neurological alterations that interfere with normal voiding or required urinary tract instrumentation, atrophy of the periurethral tissues (in women), obstruction to urine flow from benign or malignant processes, diabetes mellitus (28), decreased urine flow due to decreased fluid intake, decreased urinary concentrating capacity, and fecal incontinence (with consequent gross contamination of the urethral meatus) all may contribute to the increased risk of UT1 in the elderly (9). Much less common than the ascending route of infection is infection of the urinary tract via the hematogenous route, in which bacteria or fungi seed the kidney through the bloodstream. The most common settings in which this occurs include staphylococcal bacteremia (such as in endocarditis); the occult bacillemia of pulmonary tuberculosis (which sets the stage for subsequent extrapulmonary tuberculosis, including of the genitourinary tract); and disseminated candidal infection (33,88). (It should be noted that most candidal UTIs are acquired by the ascending route, usually in patients with indwelling bladder catheters who are receiving antibacterial therapy [33, 881.) Role of the Laboratory

in the Management

of UT1

Clinicians require different types of assistance from the clinical laboratory in managing patients with suspected UTI, depending on the clinical circumstances. In healthy young women who have what appears clinically to be uncomplicated cystitis, the spectrum of

2B

pathogens and their likely susceptibility patterns are so predictable that short-course therapy can be started empirically (and perhaps even completed) before a urine culture result is available (65). A pretherapy urine culture usually is not necessary in this setting because it adds expense without improving outcomes (65,123). On the contrary, a screening test for pyuria (such as a dipstick test or urine microscopy) combined with the clinical impression (65), or even the clinical impression alone (l5l), can suffice to make the diagnosis of UTI. Posttherapy cultures also can be omitted safely in such patients (65, 123, 150). In contrast, in other patient groups, UT1 almost always should be documented by culture, and antimicrobial therapy (after a possible initial empiric phase) should be guided by the results of antimicrobial susceptibility testing (65). It can be helpful for the labowith clinicians regarding ratory to communicate which isolates from urine cultures are likely to be significant and thus warrant susceptibility testing and detailed identification efforts (58). Whereas routine aggressive workup of what often prove to be insignificant organisms can waste laboratory resources, postponing evaluation of urine isolates until specifically requested by the clinician can lead to unnecessary delays in providing results. It also is important for the laboratory to test antimicrobial agents that clinicians are likely to use to treat the patient’s infection, another reason for good clinician-laboratory communication.

SPECIMEN

COLLECTION

The quality of the laboratory evaluation of any clinical specimen is limited by the quality of the specimen. This certainly applies to urine specimens, where even with invasive collection methods there remains the possibility of contamination with bacteria from the skin, perineum, or urethral flora. Suprapubic

Aspiration

and Straight Catheterization

The “gold standard” method for ascertaining the presence of bladder bacteriuria is suprapubic aspiration (SPA), in which urine is aspirated by syringe directly from the bladder via a transcutaneously passed needle. Although SPA is used extensively by some (30), a more commonly used method for directly obtaining bladder urine is passage of a straight catheter. Although small numbers of organisms sometimes are introduced during catheter insertion (lO3), culture results from specimens obtained by this method are rarely confusing. Clean-Catch

Midstream

Specimens

Because both SPA and straight catheterization are invasive, the most frequently used approach to the

CUMITECH

2B

collection of urine specimens (from noncatheterized patients) is the clean-catch midstream method for voided urine (23, 103). To avoid contamination, any skin folds (i.e., the labia or prepuce) are carefully retracted from the path of the urine stream prior to voiding, the urethral area is methodically cleansed with a series of sponges and soap, and the first-void urine is passed into the toilet to clear the urethra before collection of the actual midstream specimen (23). However, readers should note that first-void and not midstream specimens are preferred for the molecular diagnosis of sexually transmitted disease (STD) and urethritis in men or women. Evidence has been presented that this elaborate process may not be truly necessary to obtain an uncontaminated voided urine specimen from men or even from women (19, 50, 75, 80, 91). On the other hand, some authorities insist that even when collected in a meticulous fashion with the assistance of a trained nurse, voided urine is often contaminated ( 110). It is likely that patient characteristics are important determinants of the suitability of voided urine specimens, with mobility-impaired or uncircumcised patients and those with poor hygiene probably less able to avoid contamination during specimen collection. A practical compromise may be to tailor the collection method and the interpretation of the result to the clinical situation (93). Collection

Bags for Specimens from Children

Collection of voided urine from children who lack voluntary bladder control requires the use of a collection bag. These devices are notorious for giving falsepositive results due to contamination from the perineal flora. Accordingly, cultures from collecting bags are meaningful only if negative, whereas they should be confirmed by a method such as straight catheterization or SPA if they are positive (20). Indwelling

Catheters

Urine specimens from patients with indwelling (Foley) bladder catheters can be collected using sterile technique by aspirating urine with a needle passed either through the catheter itself or through a special puncturable diaphragm incorporated into the collection tubing (63). It is important that the catheter not be disconnected from the collecting tubing to obtain the specimen because of the risk of introducing microorganisms into the system (113). Urine in the collecting bag can become contaminated via the drainage spout without infection of the catheter or the patient (63) and should not be sampled in place of urine specimens from the collecting tubing. Catheters that have been in place for an extended time often develop a microbial flora different from that present within the patient’s urinary tract, in which case urine specimens from the

Urinary

Tract

Infections

5

collecting tubing may not reflect the microbiological status of the patient’s urinary tract (15). Because of this, it is preferable to collect urine specimens for culture after placement of a new catheter, rather than from the old catheter, when possible. Ileal Conduits Patients with surgically created ileal neobladders pose a challenge with respect both to the mechanical aspects of urine collection and to the interpretation of the resulting urine cultures. It can be difficult to obtain a sufficient volume of urine specimen from such conduits even with the use of a telescoping catheter, and the manipulations required increase the risk of introducing a contaminant. On the other hand, such conduits frequently are heavily colonized with a polymicrobial bacterial flora that includes potential uropathogens, making it sometimes impossible to distinguish by culture criteria between a contaminated specimen, a colonized conduit, and true polymicrobial UTI. Other Methods

of Specimen Collection

Occasionally, urine specimens may need to be collected from sites within the urinary tract by invasive means such as selective ureteral catheterization via a fiberoptic cystoscope passed through the bladder, or percutaneous nephrostomy, wherein a thin catheter is inserted through the patient’s flank into the renal pelvis to collect urine directly from the kidney. Such procedures, which are usually performed by urologists or invasive radiologists (37), may be used to determine where an infection is localized in the urinary tract (132) or to provide urinary drainage if there is an obstruction to flow. With such studies, and with the four-cup test for diagnosing prostatitis (86) and the bladder washout method for differentiating between infections of the upper and lower urinary tract (3 l), multiple urine specimens commonly are submitted for quantitative culture, in the hope of identifying an increase in the bacterial concentration nearest the site of infection. SPECIMEN

TRANSPORT

After the urine specimen has been collected, it should be sent to the laboratory as quickly as possible with the information necessary for complete processing. Cumitech 9 outlines specific details regarding requisition forms and required information (51). The method by which the urine sample was collected must be designated. In addition, the laboratory needs information on the clinical diagnosis, the precise time obtained, whether the patient has been receiving increased amounts of fluids, and whether any specific antimicrobial agents are being administered. Elec-

6

Clamdge

et al.

CUMITECH

tronic and handwritten laboratory requisitions should include a space for all of this information. Urine specimens should be cultured within 2 h after collection or should be refrigerated. Because this is not always possible, urine transport kits containing a preservative are available commercially. They have been designed to prevent microbial growth in urine specimens when processing is delayed, and to allow specimens to be held at room temperature. Ideally, they should also permit any microorganisms present in the specimen to remain viable without decreasing the concentration regardless of the volume of urine specimen in the container. Preservatives are available in both liquid and lyophilized forms. Although one urine transport kit with a liquid preservative has shown decreasing recovery of organisms at 24 and 48 h (41, 49), another with a lyophilized preservative gave results that support maintenance of organism concentration (147). To avoid an inhibitory or dilution effect, a urine volume of at least 3 ml is required. Urine specimens transported in a container with a preservative should be processed within 24 h of collection. For optimal results, regardless of transport system, urine specimens should be plated within 24 h after collection. Microbiology laboratories that close should make alternate arrangements for plating urine specimens.

SPECIMEN

HANDLING

IN THE LABORATORY

Specimens should be processed immediately on receipt; if this is not possible, they should be refrigerated at once. Most uropathogens grow well in urine held at room temperature (44,52,70,148). However, bacterial counts should remain stable for at least 24 h at 4OC. Specimens that are received more than 2 h after collection without evidence of refrigeration should be refrigerated, and a request for a repeat specimen should be telephoned. When information on collection time and method is not provided, similar procedures should be followed. No specimen should be discarded without contacting the requesting physician, because it may be irreplaceable. If improperly collected specimens must be plated, the final report should indicate that the specimen quality was compromised due to faulty collection, transport, or handling. As the time a specimen is left unrefrigerated increases, so does the percentage of mixed cultures. Less than 5% of urine specimens that are properly collected and transported contain multiple organisms (13). Exceptions to this rule are urine specimens from patients with neurogenic bladders or chronic indwelling catheters, in whom polymicrobic bacteriuria may be detected in 30 to 80% of cultures (46, 149, often without infection.

SPECIMEN

2B

WORKUP

Necessary Communication and the Laboratory

between the Clinician

The decision on how a specimen should be cultured depends on the mode of collection and the clinical signs and symptoms of the patient. This information should be conveyed to the laboratory by the clinician. The guidelines for specimen workup recommended in Cumitech 2A assumed that the laboratory receives all needed information, including method of urine collection, type of patient (for example, urologic or geriatric patient), clinical presentation (whether the patient was symptomatic), urinalysis results (the presence of inflammatory cells in the urine), and antecedent culture results. However, in actuality, the laboratory often receives incomplete 0 r inaccura te information, which results in its doing too much or too little work on a specimen. A possible way to communicate the necessary information might be for the clinician to designate a type of urine culture based on clinical criteria. In Table 1, we define three types -routine, surveillance, and special. The routine culture would be appropriate for outpatients and most patients with uncomplicated UTI. The surveillance culture would be appropriate for geriatric patients and individuals with indwelling catheters, where colonization or infection with multiple organisms is common. The special culture would be ordered if a previous culture failed to reveal a pathogen, if the patient was unresponsive to therapy, or if there was reason to suspect an unusual urinary tract pathogen. Specific protocols can be agreed on by the various members of the health care team, including physicians, administrators, and laboratorians. By ordering urine cultures based on type, the extent of the workup is directed by clinical necessity and the laboratory can choose the most appropriate plating media and quantitative inoculum (0.01 versus 0.001 ml). The distinction between this recommendation and previous protocols is that formerly the extent of workup was decided only after growth was assessed and was predicated largely on the quantity and number of different morphotypes growing at 24 h. Approach

to Laboratory

Workup

Although relatively few bacteria cause most UTIs, many different microorganisms are possible urinary tract pathogens. Some of these genera can be found normally colonizing the-skin in the genitourinary area and in the epithelial cells lining the urethra. The overall strategy for an effective urine culture protocol allows isolation of the most probable pathogens and distinguishes these from colonizing organisms. To decide the best culture strategy for an individual health care facility, we have broken down the various categories of pathogens below.

CUMITECH Table

1.

2B

Urinary

Appropriate

Type of urine culture ordered Routine

Surveillance

indications

and urine

Appropriate inoculum and plating media O.OOl-ml Mac

loop,

culture

One gram-negative a Abbreviations: b Mixed urethral

BA,

One isolate r104b

O.OOl-ml loop, BA, CNA, Mac

O.OOl-ml loop and O.Ol-ml loop, BA, Mac (chocolate, if needed); incubate plates for at least 2 days

Isolate

at IO5 with

for various

Growth

other(s)

Extent

at

types

of workup

Full ID and susceptibility

One or two gramnegattve isolates at r105 with other(s) at least 10X less Any other pattern

Full ID and susceptibility of GNR only

One isolate at >104b One gram-negative isolate at 21 O5 with other(s) at least 10X less Any other pattern

Full ID and susceptibility

Any/by

Special

methods

agreement

One or two isolates at rlO* to IO5

at least

Mac, MacConkey agar; CCMS, clean or skin flora at (IO4 can be Ignored.

10X catch

of urine

culture

Populations for which

Tract

Infections

7

ordersa and settings appropriate

Are urine screens helpful?

Ambulatory adults who are capable of collecting CCMS urine specrmens

Yes

Patients with neurogenlc bladder, catherized patients, geriatric patients

Probably

not

Selected urology and pedlatnc patients, urine specimen collected by in-and-out (straight) catheterization or suprapubic aspiration, four-specimen prostate massage, patients with continuing symptoms, selected females of child-bearing age for whom 1 O3 organrsms/mI may be significant as suggested In text

Probably

not

Presumpttve ID of Isolates at 404

Full ID and susceptibility of GNR only

Presumptive ID of isolates at 21 O4 Enumeration and presumptive ID only Full ID and susceptibility Full ID and susceptibility of GNR

less midstream;

ID, Identlflcatlon;

Organisms That Are Known Uropathogens and Grow Well on Routine Media at 24 h As previously discussed, some organisms are accepted uropathogens, although they can also be found as colonizing organisms or as contaminants introduced during collection. These include E. coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa, which generally comprise over 80% of the gram-negative rods isolated. Many other members of the family Enterobacteriaceae, especially Enterobatter spp. and Citrobacter spp., are also commonly isolated. Enterococcus spp., Staphylococcus saprophyticus (and other coagulase-negative staphylococci), Staphylococcus aureus, and Streptococcus agalactiae (group B) are the most frequently isolated grampositive organisms. In some laboratories, Candida spp. are frequently isolated, often as contaminants.

GNR,

gram-negative

rod.

Because most of these organisms grow well in urine, an increase in concentration during transit can be a problem and specimens should be held at 4OC until inoculation. Organisms That Are Known Uropathogens (or Genitourinary Pathogens) and Do Not Grow on Routine Media Mycobacterium tuberculosis is an infrequent uropathogen that is associated with a high leukocyte count. It requires special media and is discussed elsewhere (21). Chlamydia trachomatis grows only in cell culture and is associated with sexually transmitted urethritis and salpingitis rather than UT1 but is mentioned here because first-voided urine is one of the preferred diagnostic specimens ( 14). Ureaplasma urealyticum does not grow on routine media. Al-

8

Clarridge

et al.

CUMITECH

though it may be a genitourinary tract pathogen, it is often isolated along with other pathogens (30). Neisseria gonorrhoeae can be grown on chocolate agar or modified Thayer Martin (MTM) medium if overgrowth by other organisms is expected. Investigations have shown that culture of first-voided urine is 92 to 99% sensitive in detecting gonorrhea in males, although it is not nearly as useful in females (2, 122). Organisms That Can Be Uropatbogens but May Require Special Media or Longer Than 24 h To Grow There are other, less pathogenic organisms that are found as normal periurethral or vaginal flora but when isolated from urine in high numbers have been associated with urinary tract disease. These include some species of Corynebacterium, Gardnerella uaginalis, Haemopbilus influenzae, and Haemophilus parain fluenzae. Corynebacterium urealyticum can be an important but rare cause of UTIs (22). Rates for significant isolations may vary from essentially 0 to 3.5% for UTIs in nonselected populations (35). Soriano and colleagues have documented the causative role of C. urealyticum in alkaline-encrusted cystitis, an ulcerative inflammation of the bladder in which localized ulcers are covered with deposits of struvite (MgNHJO, 6H,O) crystals (129). C. urealyticum bacteriuria occurs in patients hospitalized for a long period who are severely immunocompromised, urologically manipulated, or elderly (83, 130). It has also been associated with pyelonephritis (3) and can spontaneously disappear from urine in the absence of antibiotic therapy (24). Corynebacterium glucuronolyticum (also called Corynebacterium seminale) has been isolated from semen and the male genitourinary tract and may cause prostatitis or urethritis (35, 116). It is a strong urease producer. CDC group Fl, synonymous with some strains of Corynebacterium pseudogenitalium and also a urease producer, has been implicated as a urinary tract pathogen. C. urealyticum and other corynebacteria grow well on 5% sheep blood agar (BA) or Columbia colistin-nalidixic acid (CNA) agar but are more reliably detected after a 48-hour incubation. There are multiple studies linking H. infhenzae and H. parainfluenxae to UTI, although their frequency of occurrence and significance have not been confirmed (4, 16). When Boyd et al. (18) used chocolate agar as a primary plate for 200 specimens, they isolated Haemophilus spp. from three patients; one of the isolates was found to be significant, but the authors concluded that using chocolate media was not cost-effective. Some investigators have suggested the inclusion of a chocolate plate for urine cultures for children (36, 137) because of the association of H. infiuenzae and l

2B

H. parainfluenxae with other infections in this age group. The significance of G. vaginalis in urine is problematic. The main pathogenic association for G. vaginalis is with vaginosis in the female (22). Although low concentrations in urine seem to reflect normal flora from the urethra in both males and fern ales (6), concentrations above lo4 are reported to be associated with symptomatic bacteriuria, recurrent UTI, cystitis in women, and urethritis and prostatitis in men (67, 76, 127, 139, 154). Coisolation of U. urealyticum is common (29). However, even when found in high numbers, there is often no leukocyte response (6,67). The organism is found more often in females and patients with predisposing factors, including renal disease and instrumentation (6, 67, 76). Colonies of G. vaginalis are barely visible on BA at 24 h. CNA or HBT (human blood bilayer Tween) plates and incubation for 48 h in CO2 (76, 154) are recommended. Organisms That Are Probably Not Uropatbogens but May Be Isolated from the Urethra and Urine A variety of organisms colonize the genitourinary area but are not recognized as causes of UTI. These include anaerobes, Actinomyces spp., Lactobacillus, alphahemolytic streptococci, coagulase-negative staphylococci, corynebacteria other than those previously mentioned, and small numbers of gram-negative rods. Selection of Media and Incubation

Conditions

Unfortunately, there is not one “best way” of performing all urine cultures. Ideally, testing should be performed and plating media should be selected on the basis of patient population and clinical diagnosis. Guidelines for the appropriate inoculum and plating media for routine, surveillance, and special cultures are presented in Table 1. Routine Cultures Routine urine cultures should be plated onto 5% sheep blood agar and either MacConkey or a similar selective medium (such as eosin-methylene blue agar). For routine urine cultures, plates are incubated overnight (at least 16 h) at 35 to 37OC in ambient air; alternatively, the blood agar plate may be incubated in elevated (3 to 8%) C02. An additional 8 to 10% increase in isolates can be achieved with further incubation; however, most of these are yeast or coryneform organisms (66, 120). Surveillance

Cultures

For populations in which mixed organisms are common, a CNA agar plate should be added to demonstrate the presence of enterococci, other gram-positive organisms, or yeasts in mixed culture. However, this agar should never be substituted for blood agar. Phenylethyl alcohol agar does not seem as effective as

CUMITECH

2B

CNA agar, since it does not allow as luxuriant growth of streptococci, corynebacteria, or Gardnerella uaginalis and is less inhibitory to some gram-negative rods. Cultures should be incubated for 48 h at 35OC. Special Cultures Special cultures are those designed for specific diagnoses, fastidious organisms, or particular patient groups. For fastidious organisms, chocolate agar may be added to the CNA and MacConkey agar plates. The CNA and chocolate plates should be incubated in 5% CO2 and held for 48 h. For specific patient populations such as those with chronic UT1 or anatomic abnormalities, it may be appropriate to culture for anaerobes and slower-growing organisms. However, it has generally not been cost-effective to routinely screen cultures for gram-positive organisms or fastidious organisms (10, 18). Special cultures are also appropriate if there is a discrepancy between the clinical presentation or the direct Gram stain and previous culture results. Guidelines for Specimen Workup and Interpretation of Results The principles for the interpretation of the quantitative bacteriology of urine specimens were carefully defined by E. H. Kass (69, 70). He demonstrated a high correlation between patients with three consecutive clean-catch morning urine specimens containing greater than 100,000 CFU/ml and UTI. Goldberg et al. (39) initially emphasized the difficulty in applying the lOO,OOO-CFU/ml rule to all patients. They demonstrated that the time of collection was an important variable that may significantly influence the number of CFU per milliliter of urine. All women had counts of >lOO,OOO CFU/ml in their first-voided morning specimen. By the time they appeared in the clinic, four of the six patients studied had counts for voided urine specimens between 100 and 10,000 CFU/ml. The fact that these low counts represented significant bacteriuria was confirmed by the demonstration of similar counts of the same organism in urine obtained by suprapubic needle aspiration of the bladder. Stamm et al. (133) have confirmed these conclusions. Similarly, urine voided after prostatic massage may contain the organism(s) responsible for prostatitis. Infecting organisms are usually present at a density of 10,000 CFU/ml, but a density of 100 CFU/ml can be clinically important (71, 131). Commonly used guidelines for routine specimens state that for a single isolate a density of > 100,000 CFU/ml indicates infection, < 10,000 CFU/ml indicates urethral or vaginal contamination, and between 10,000 and 100,000 CFU/ml needs to be evaluated based on clinical information (12, 70, 112). Most cases of pyelonephritis and cystitis can be correctly

Urinary

Tract

Infections

9

assessed by using these parameters. However, these guidelines fail to offer suggestions regarding the handling of urine specimens from some symptomatic patients, whereas as few as 100 CFU/ml can be clinically important. Special consideration also has to be given to specimens from children, for whom collection can be more di .fficult, Gram sta in ca.n be more reliable than urinalysis, and infections can be manifest at lower numbers (lo3 to lo4 CFU) (82). Infection is more often seen in patients with anatomic abnormalities and premature infants (82,90). Special guidelines are also needed for those with neurogenic bladders and for the elderly in nursing home care. For these patients, screening tests are not as accurate and the prevalence of bacteriuria is high in the asymptomatic (30%) and the symptomatic (60%) (102). Typical guidelines for specimen workup are given in Table 1. Guiding principles are that too much workup is a waste of resources and can lead to prescribing unnecessary antibiotics and that abbreviated workups can lead to the etiological agent of the UT1 not being discovered. However, it must be stressed that protocols for individual laboratories must be established based on close cooperation between the medical and laboratory staff. Recommended Supplementary Special Situations

Procedures

for

How best to determine whether a UT1 is confined to the bladder or involves the upper tract as well has stimulated considerable discussion and exploration of various testing methods over the years (64). However, precise knowledge of the actual anatomical site of infection is rarely critical to the management of patients with UT1 because syndrome-based treatment algorithms usually lead to acceptable clinical outcomes (57, 73, 134). Furthermore, the accuracy of many of these localizing methods is questionable (103). Similarly, there is little evidence that the multiple-sample culture method involving prostatic massage that has been recommended for determining whether or not a man with UT1 has prostatitis (86) is accurate or leads to improved outcomes compared with empiric treatment based strictly on clinical impression ( 8 1). A variety of specialized tests are available for determining whether different isolates of the same species represent the same or different strains. This question arises in the context of UT1 when an individual patient has multiple episodes of UT1 because of what appears to be the same organism (such as a woman with recurrent UTI) ( 119), or when multiple individuals are infected with what may be the same strain (such as in a suspected nosocomial or community-wide outbreak) (111,141, 142). Traditionally, 0 lipopolysaccharide, K capsular, and H flagellar antigens have

10 Table

Clarndge 2.

Microscoprc

et al.

Rapid

urine

Urine

screen

method

CUMITECH

2B

screens

(Gram

Pnncrple stain)

Comments

Method based on the staining reaction mrcroorganrsm, reaction IS dependent on cell wall composition

of

Enzyme drpstrck (Chemstnp LN; Bra-Dynamics, DIV. Boehnnger Mannherm Dragnostrcs, IndIanapolls, Ind )

Detects nitrate reductase present most gram-negative bacrllr and leukocyte esterase, a leukocyte enzyme

Enzyme tube test (Unscreen; Bard Patient Care Drvrsron, C. R. Bard, Inc., Murray Hill, N.J )

Detects catalase present In bacteria, leukocytes, and erythrocytes by the addition of H,O;, to urine

Formation of foam above the lrqurd surface IS positive; requires 1.5 to 2 ml of urine

Colonmetnc frltratron (FrltraCheckUTI, Meridian Dragnostrcs, Inc., Crncrnnatr, Ohio)

Bacteria and leukocytes are trapped In a filter and detected by a residual pink color remaining from safranrn 0 dye

High concentratron of cells may clog the filter; filtration correlates wrth infection (92)

been used to define serotypes for comparing different isolates (61), but these tests are not generally available. Today, strain typing is done more conveniently by using one of a variety of genome-based typing methods. These include pulsed-field gel electrophoresis, ribotyping, conventional restriction digest DNA fingerprinting, multilocus enzyme electrophoresis, and either arbitrarily primed or repeat-element PCR methods (62, 84, 119, 143). Biotypes, antimicrobial susceptibility patterns, and plasmid profiles are often insufficiently stable or discriminating to be useful in this application (62). Reporting

Results

The laboratory report should accurately reflect the specimen workup so that the physician understands the limits of the procedure. Reports of “no growth” are misleading; reports should read “no growth ( 10’ CFU/ml and >8 to 10 leukocytes/pi. A sample (0.01 m 1) o f we 11-mixed, uncentrifuged urine should be placed on a glass slide and allowed to air dry. The slide should be methanol or heat fixed and Gram stained. Using this method, the sensitivity of the Gram stain for the presence of 2 1 bacterial cell/oil immersion field is at least 85% when correlated with a colony count of X0’ CFU/m1(107,108,109,146). It has also been reported to be the least expensive method when compared with other screens (107, 108). One report recommended that if an uncentrifuged urine specimen from an asymptomatic patient was negative by microscopy for bacteria and leukocytes, a culture could be eliminated (64). However, because of its decreased sensitivity at < 10’ CFU/ml, most investigators have recommended the Gram stain as a screen only for those patients with predictably high colony counts, mainly asymptomatic patients and those with presumed pyelonephritis. Although the Gram-stained smear of uncentrifuged urine specimen is accurate at >105 CFU/ml and inexpensive, it is not the routine screening method in most laboratories because it is time-consuming and relatively insensitive. To improve the sensitivity at levels of 2 lo4 CFU/ml, the use of the cytocentrifuge technique with the Gram stain has been suggested (117). Although the Gram stain has limitations as a urine specimen screen, it should be available for use in selected cases. When positive, it has the advantage of

CUMITECH

2B

correlating with significant bacteri uria and providing specific information such as Gram stain cha racteristic and organism morphology, which may be used to select initial antimicrobial therapy. The Chemstrip LN is a plastic strip with reagent paper attached for detecting the presence of leukocyte esterase and nitrite in urine. The sensitivity of this simple, rapid screen ranges from 79 to 95% (106, 107, 108). It has the advantage of being easy to perform and interpret. It can be easily used as a single test or in batches and can be readily incorporated into the laboratory’s routine work flow. The Uriscreen test is an enzyme test detecting catalase that is present in most bacteria causing UTIs as well as in leukocytes and erythrocytes. When urine specimens containing bacteria are mixed with hydrogen peroxide, foam from the release of oxygen indicates the presence of catalase originating from bacterial or somatic cells in the urine specimen. This method also has the advantage of being easy to perform and interpret. It can be used as a single test or in batches and can be readily incorporated into the laboratory’s routine work flow. The Uriscreen has a sensitivity of 93% for specimens with ~10~ CFU/ml and 95 % for specimens with 1 lo2 CFU/ml and leukocytes (108). The FiltraCheck-UT1 is a calorimetric filtration device that detects both bacterial and somatic cells that have been trapped in the filter and subsequently stained with safranin dye. The residual pink color is compared to a color guide. Results may be interpreted immediately or within 10 minutes. The preceding methods have been compared for accuracy and performance. A collaborative study of the Chemstrip LN and the Uriscreen found that the two methods had similar sensitivities for specimens with both ~10~ CFU/ml and pyuria, or for specimens with ~10~ CFU/ml with or without pyuria (108). Another evaluation compared the FiltraCheck-UT1 and the Gram stain cytospin (117). For the 425 urine specimens tested, the sensitivities of the two methods were similar (~93%) for specimens with ~10~ CFU/ ml; however, the sensitivity of the FiltraCheck-UT1 decreased to 89% for specimens with 21 O4 CFU/ml, while the sensitivity of the Gram stain cytospin remained essentially unchanged. Murray et al. reported that the filtration method was a better predictor of infection than either the culture at 2 lo5 CFU/ml, the Gram stain, or the Chemstrip LN (92). The purpose of urine specimen screening should be to rapidly eliminate those specimens that do not warrant culture without eliminating any that do. In addition, to have a clinical impact on patient care, the test must have the ability to detect 2 lo4 CFU/ml, results should be available within 1 h of specimen collection for outpatients and -~2 h for inpatients, and results of

Urinary

pyuria should not be met, a ing. Although duced within screening has crobiologists. tories struggle

Tract

Infections

11

be included (136). If these criteria canlaboratory should not consider screena number of screens have been introthe last two decades, urine specimen not been well accepted by clinical miThis may change, however, as laborato remain fiscally competitive.

BACTERIOLOGIC

CULTURE

SYSTEMS

Another approach to the detection of microorganisms in urine specimens is the use of self-contained bacteriologic culture systems. These systems initially were designed to be used in physician offices, in ambulatory care clinics, and at other patient care sites where transport to a diagnostic laboratory may be delayed and refrigeration may not be available. The last decade has seen the introduction of a number of similar systems that can be incorporated in the larger diagnostic laboratory to increase efficiency and decrease turnaround time and cost. Most of these primary culture systems permit quantitation and the identification of commonly isolated urinary tract microorganisms using a single agar plate or agar/dipslide. Some of the more commonly used systems are summarized in Table 3. The Bactercult Urine Culture Tube is a urine collection device that contains a medium for the isolation and identification of commonly isolated urinary tract pathogens. Urine is collected in a sterile di sposable tube whose inner surface is coated with a nutrient indicator culture medium that contains peptone, lactose, urea, phenol red, agar, and para-nitrophenyl to prevent the swarming of Proteus spp. The urine is poured out of the tube, and the container is incubated at 35OC overnight or at room temperature for 48 h. If the culture is positive, discrete colonies appear on the medium. A phenol red indicator in the medium provides presumptive differentiation of the common organisms causing urinary tract infections. Individual colonies may be selected for further identification. The Bullseye Urine Plate is a five-chambered plate containing media designed to assist in the isolation and presumptive identification of common bacterial urinary tract pathogens and the determination of their antimicrobial susceptibility patterns. The media incorporated in the system include an enriched generalpurpose growth medium, Trypticase soy agar (TSA) with 5% sheep blood, and selective and differential agars including Levine EMB, citrate urinary agar, modified XLD agar, and Mueller-Hinton (MH) agar for antimicrobial susceptibility testing. The combination of growth patterns and typical colonial morphologies on these media when compared to a color chart provides a colony count and presumptive identification of the bacterial pathogen(s). A disposable inoculating loop, provided by the manufacturer, is used to inoculate all the media except for the MH agar, which

12

Clarridge

Table

3.

et al

Summary

CUMITECH

of bacteriologic

Culture Cranbury,

Bullseye Urine Dlagnostlcs,

Prtncrple

N .J .)

Plate (HealthLInk Jacksonvrlle, Fla.)

CPS ID 2 (bfoM&eux Hazelwood, MO.)

Diaslide (Diatech Boston, Mass

Vltek,

Diagnostics 1

Inc.,

Inc ,

DIP N COUNT (Starplex Sclentlflc, Etoblcoke, Ontario, Canada) Rainbow Calif.)

(Biolog,

U R I-CH ECK (Troy Troy, Mlch.)

Inc , Hayward,

Biologicals,

Un-Klt/Urr-Three (Culture Norwich, N.Y.)

Uricult Trio (Onon Espoo, Finland)

B Abbrevlatms:

systemsa

system

Bactercult (Carter-Wallace/Wampole Division,

culture

Inc.,

Kits Inc.,

DIagnostIca,

AST, antmcroblal

susceptlblhty

26

Sterile, disposable tube coated with culture medium; contains phenol red Indicator for presumptive ID of common uropathogens Five-chambered plate contalnlng media for lsolatlon and Identlflcatlon of common uropathogens and their AST patterns Enumeration and presumptrve ID of common urinary pathogens

Transparent hinged plastic casing contains CLED and MacConkey agars for detection and presumpttve rdentlficatron of common UTI pathogens Paddle contains CLED and MacConkey or EM5 agar Rainbow agar CP-8 used with 8 confirmation spot tests for ID of 8 mlcroorganisms causing UTI; medium contains chromogenic substrates which color microorganisms Dipslide culture for the enumeration and ID of uropathogens; dlpsllde contarns CLED and MacConkey or EM5 agar; slmllar to DIP N COUNT Agar plate systems used to detect common uropathogens; Urt-Kit IS plastic hinged case containing CLED medium; Uri-Three triplate contains 5% BA and MacConkey and CLED agars Three-medium dipsllde containing CLED, MacConkey, and a /3-glucuronldase substrate for enumeration of microorganisms and presumptive Identlflcatlon of E. CO/I testmg,

Comment(s) Colonies

counted

Colonies

counted;

direct

AST

Identifies E- cob, P mirabdis, Enterococcus spp , and Indolepositive Proteus; presumptive ID of K. pneumoniae, P. aeruginosa, S. aureus, S. agalactiae, and Cad/da spp. Growth IS compared to reference photographs for quantitatron; morphology and color determine presumptive ID Colonies are compared to a color density chart; color chart is used for ID Interpretation may occur within 3 to 6 h for rapidly growing organisms

Growth chart

density

IS compared

Growth density is compared colony density chart

E. CO/I appears

as brown

to

to a

colonres

ID, ldentrflcation

is inoculated with a cotton swab. Antimicrobial susceptibility disks are applied to the MH agar, and the plate is then incubated at 33 to 37OC for 18 to 24 h. Interpretation of colony count and organism identification are similar to standard methods. However, direct antimicrobial susceptibility testing (DST) is not a standardized National Committee for Clinical Laboratory Standards (NCCLS) (94) method, a limitation which will be further discussed in the next section (“Direct Antimicrobial Susceptibility Testing of Urine Specimens” ). CPS ID 2 is a culture medium containing substrates for P-D-glucuronidase and P-D-glucosidasefor use in the enumeration and presumptive identification of microorganisms causing urinary tract infections. This medium provides a rapid, reliable method for the detection and identification of the common urinary pathogens and, becauseof the reported accuracy, can replace the standard primary plating media, that is, 5% sheepblood agar and MacConkey agar, currently

usedin most laboratories. Mazoyer et al. reported a 97 to 100% sensitivity for identification of microorganisms when cultures were monomicrobial; this decreasedto 70 to 97% when they were polymicrobial(85). The Diaslide Urine Culture system is a semiquantitative culture device for detecting and presumptively identifying bacteria in urine specimens.Each Diaslide consists of a transparent hinged plastic casing containing face-to-face plates of cystine-lactose electrolyte-deficient (CLED) and MacConkey agars. A special plastic sampler is designed to inoculate a measured quantity of the urine specimen to the agar surfaces so that after incubation, colonies can be quantitated and identified by standard methods. The sensitivity of the Diaslide was >98% in comparison to culture (32, 118). The DIP N COUNT dipslide is a semiquantitative culture system for the detection of common microorganisms causing UTI. The paddle contains either MacConkey or Levine eosin-methylene blue (EMB)

CUMITECH

2B

agar on one side, and CLED agar with or without polymyxin B sulfate on the other side. The paddle is dipped into the urine specimen and returned to the unit, which is closed tightly and incubated at 37°C for 16 to 24 h, For interpretation, the number of colonies is compared to a color density chart. Presumptive identification is based on colonial morphology and color. Confirmatory identification and antimicrobial susceptibility testing can be performed on colonies picked from the paddle surface. The Rainbow UT1 System is a multicolor diagnostic system for the simultaneous enumeration and identification of common urinary pathogens on a single medium, Rainbow agar Q-8. This medium, when combined with eight confirmatory spot tests, can identify eight of the common gram-positive and gramnegative urinary pathogens. Rainbow agar Q-8 contains chromogenic substrates that color the microbial colonies. For example, E. co/i appears as red colonies and can be confirmed by the indole spot test, while K. pneumoniae appears as blue colonies and P. mirabilis is orange. In some cases, the rapid growth of the microorganism (within 3 to 6 hours) and the appearance of a distinctive color permits same-day identification. URI-CHECK is a semiquantitative dipslide culture test, using MacConkey or Levine EMB agar for one side and CLED agar with or without polymyxin B sulfate for the other side. The test is performed by dipping the slide into a urine specimen followed by overnight incubation. Growth density is compared to a chart provided by the manufacturer. This test is similar to the DIP N COUNT dipslide previously described, Uri-Kit and Uri-Three are two agar plate systems used to detect common urinary tract pathogens. UriKit is a small clear plastic hinged case containing CLED medium. Uri-Three is a tri-plate system that contains three media, TSA with 5% sheep blood agar, MacConkey agar, and CLED agar. A special swab is used to inoculate the media in both systems; the presumption is made that this swab can deliver 0.01 ml and allow su bsequent quantitation. After overnight incubation, the density of the colonies may be compared to a chart or actual colony counts can be made. Presumptive identification is attempted based on colonial moiphology and color. Subsequent positive identification and antimicrobial susceptibility testing can be performed on colonies picked from the agar surface. Usicult Trio is a three-media dipslide including CLED agar, MacConkey agar, and a medium containing hydroxyquinoline-/3-D-glucuronide to detect P-glucuronidase-producing colonies (E. coEi). This system was compared with other methods for the rapid identification of E. coli on primary media (77). Overall, 92% of the 324 E, coli isolates were identified by this dipslide, compared to 84% by a methylumbelliferyl$-D-glucuronide-containing medium.

Urinary

Tract

Infections

13

In summary, the use of bacteriological primary culture systems can extend beyond the physician office into the diagnostic laboratory. These systems have the advantage of incorporating colony counts, presumptive identification, and, in some cases, definitive identification of common urinary tract pathogens. Preliminary evaluations of multiple-agar single-unit systems report results comparable to those of the conventional culture methods, and the data suggest that these primary culture systems can replace the multiple plates that are widely used in diagnostic laboratories. The ease of use and interpretation, the increased efficiency, and the potential cost savings warrant consideration of this approach to urine cultures in the diagnostic laboratory. However, some of these kits have not been cleared by the Food and Drug Administration (FDA) for diagnostic use; therefore, for liability and accreditation reasons, users should be cautious about which ones they use. DIRECT ANTIMICROBIAL SUSCEPTIBILITY TESTING OF URINE SPECIMENS For at least 2 decades, investigators have been evaluating the accuracy of DST of urine specimens compared to standard methods (25,26,43,45,59,68,94, 101, 136). There are clinical conditions and circumstances that warrant DST, although the issue remains controversial. Urine specimen systems that include DST are available and have been used in physicians’ offices, but, in most cases, they are not FDA cleared. The concern is that the results obtained from DST will not be equivalent to those obtained using the standardized disk diffusion testing method described in NCCLS standard M2-A5 (94). Polymicrobic cultures and those with < lo5 CFU/ml are also of concern. The use of the Gram stain to screen urine specimens for the purpose of detecting positive specimens (2 lo5 CFU/ ml) and to eliminate mixed cultures has been suggested (43). The overall concern is that DST of urine specimens, or any clinical material, will yield grossly misleading results because it is not a standardized procedure. On the other hand, physicians would prefer to have results as soon as possible. The usefulness of rapid data depen .ds on , compl .eting the total laboratory test in a short time period (136). Proponents of the DST assert that i .t has great value in the management and treatment of UT1 (106); however, in most situations, treatment is begun empirically without the need for any susceptibility testing as outlined in the section above entitled “Role of the Laboratory in the Management of UTI. ” Urine specimen systems that include the option of DST are the Bullseye Urine Plate previously described and the Urine Duo (Culture Kits, Inc., Norwich, N.Y .), The Urine Duo includes two separate devices: a culture kit with a small plastic hinged case containing

14

Clamdge

et al.

modified CLED medium and a loo-mm plate of MH agar. In the Bullseye Urine Plate, the MH agar is incorporated into the center of a five-chambered plate aspreviously described. The procedure includes DST; however, the manufacturer states in the product information sheetthat DST is not a standardized procedure as outlined by NCCLS and suggeststhat the standard method should be used as a fullow-up procedure. Dornbusch and colleagues evaluated the Sensicult (Orion Diagnostica, Espoo, Finland) dipslide method, a glass slide coated with MH agar (25, 26). They concluded that DST, performed by personnel trained in bacteriological analysis, seemedto be useful as a screening test to differentiate strains likely to respond to therapy from resistant strains (26). The major advantage was the decreased time for obtaining results. In another study, these investigators confirmed that for DST the results must be evaluated by experienced bacteriologists because the results displayed unacceptable disparities when interpreted by others (26). They also concluded that DST of mixed cultures is clearly inadvisable. Kallenius et al. reported a 96% agreement between DST and the standardized disk diffusion test, which included testing 700 outpatient urine specimensand 2,800 observations (68). Timesaving is an advantage of DST, and specimensgiving indistinct zones of inhibition and mixed culture should be excluded. There is a high degree of correlation (>95 %) between DST using infected urine as the inoculum and the standardized disk diffusion method (59, 102, 106). These studies suggestthat DST is equivalent to the standard method when infected urine with ~10~ CFU/ml of a pure culture is tested. The major reported advantages of DST are the earlier reporting of results and possibly lower costs related to shorter hospitalization and earlier treatment with the appropriate antimicrobial agent (106). The procedure has been recommended fur urine specimens alone with the conditions that only a pure culture at ~10~ CFU/ml should be reported and that all other findings be repeated (140). In summary, DST is of value in selected caseswith ~10~ CFU/ml of a pure culture; however, its use on a routine basis is not recommended (45). If DST is performed, results from polymicrobial cultures must be repeated, as must cultures with fewer than lo5 CFU/ml, The challenge is to determine when DST should be performed, with the goal of maximizing clinical utility while minimizing errors and laboratory workload (59). We suggestthat laboratories only set up direct susceptibility tests on urine specimenslikely to be from monomicrobic infections. Inclusion criteria should include a positive Gram stain fur white blood cells and a single bacterial morphotype. Exclusion criteria should include specimensfrom bag urine

CUMITECH

28

specimensand Gram stains that show multiple bacterial morphotypes or no bacteria or white blood cells. Becausemany direct susceptibility testing results will need to be repeated using a standardized method (59), the ultimate decision will involve consideration of whether the patient is ill enough to require more immediate results in the face of multiple susceptibility test charges. UNACCEPTABLE

PROCEDURES

Several procedures sometimes used for the diagnosis of urinary tract infections are considered unacceptable. These include the following. 1. Do not perform cultures of:

a+ Foley catheter tips b . urine specimensin broth medium C. urine specimen sediment d voided or catheterized urine specimen anaerobically anaerobes as a routine procedure f . urine specimensdelayed longer than 2 h without refrigeration or preservative l

e.

2. Do not perform direct antimicrobial susceptibility testing: a. as a routine procedure b. of mixed cultures c. of specimenswith