As previously mentioned, because noninvasive methods for collecting urine must rely on a specimen that has passed through a contaminated milieu, quantitative cultures for the diagnosis of UTI are used to discriminate between contamination, colonization, and infection.

SPECIMEN COLLECTION

 Prevention of contamination by normal vaginal, perineal, and anterior urethral flora is the most important consideration for collection of a clinically relevant urine specimen.

Clean-Catch Midstream Urine

The least invasive procedure, the clean-catch midstream urine specimen collection, must be performed carefully for optimal results, especially in females. Good patient education is essential. Guidelines for proper specimen collection should be prepared on a printed card (bilingual, if necessary), with the procedure clearly described and preferably illustrated to help ensure patient compliance. The patient should be instructed to clean the peri urethral area well with a mild detergent to avoid contamination. Of importance, the patient should also be instructed to rinse well because the detergent may be bacteriostatic. Once cleansing is completed, the patient should retract the labial folds or glans penis, begin to void, and then collect a midstream urine sample. Studies showed that uncleansed, first-void specimens from males were as sensitive as (but less specific than) midstream urine specimens.

Straight Catheterized Urine

Although slightly more invasive, urinary catheterization provides a method for the collection of uncontaminated urine from the bladder. Either a physician or another trained health professional performs this procedure. Risk exists, however, that urethral organisms will be introduced into the bladder with the catheter. An example of a collection device to obtain “straight,” or “in and out,” catheterized urine is shown in Figure 1.

Fig1. Collection device to obtain urine by “in and out” or  “straight” catheterization. (Courtesy Tristate Hospital Supply Corp.,  Howell, Mich.)

Suprapubic Bladder Aspiration

With suprapubic bladder aspiration, urine is withdrawn directly into a syringe through a percutaneously inserted needle, thereby ensuring a contamination-free specimen. The bladder must be full before performing the procedure. This collection technique may be indicated in certain clinical situations, such as pediatric practice, when urine is difficult to obtain. In brief, the full bladder is punctured using a needle and syringe and sampled following proper skin preparation (antisepsis). If good aseptic techniques are used, this procedure can be performed with little risk in premature infants, infants, small children, and pregnant women and other adults with full bladders.

Indwelling Catheter

The number of patients in hospitals and nursing homes with long-term, indwelling urinary catheters continues to increase. These patients ultimately develop bacteriuria, which predisposes them to more severe infections. Specimen collection from patients with indwelling catheters requires scrupulous aseptic technique. Health care workers who manipulate a urinary catheter in any way should wear gloves. The catheter tubing should be clamped off above the port to allow the collection of freshly voided urine. The catheter port or wall of the tubing should then be cleaned vigorously with 70% ethanol, and urine aspirated via a needle and syringe; the integrity of the closed drainage system must be maintained to prevent the introduction of organisms into the bladder. Specimens obtained from the collection bag are inappropriate, because organisms can multiply there, obscuring the true relative numbers. Cultures should be obtained when patients are ill; routine monitoring does not yield clinically relevant data.

Specimen Transport

Because it is an excellent supportive medium for growth of most bacteria, urine must be immediately refrigerated or preserved. Bacterial counts in refrigerated (4° C) urine remain constant for as long as 24 hours. Urine transport tubes (BD Urine Culture Kit [Becton Dickin son Vacutainer Kits, Rutherford, New Jersey]) containing boric acid, glycerol, and sodium formate have been shown to preserve bacteria without refrigeration for as long as 24 hours when greater than 10⁵ CFU/mL (100,000 organisms per milliliter) were present in the initial urine specimen. The system may inhibit the growth of certain organisms, and it must be used with a minimum of 3 mL of urine. Another preservative system (Starplex Scientific, Inc., Etobicoke, Cleveland, TN) is also available. Both boric acid products preserve bacterial viability in urine for 24 hours in the absence of antibiotics. For patients from whom colony counts of organisms of less than 100,000/mL might be clinically significant, plating within 2 hours of collection is recommended. The kits provide a convenient method for preserving and trans porting urine from remote areas where refrigeration is not practical.

SCREENING PROCEDURES

As many as 60% to 80% of all urine specimens received for culture by the acute care medical center laboratory may contain no etiologic agents of infection or contain only contaminants. Procedures developed to identify quickly those urine specimens that will be negative on culture and circumvent excessive use of media, technologist time, and the overnight incubation period are dis cussed in this section. A reliable screening test for the presence or absence of bacteriuria provides physicians important same-day information that a conventional urine culture may take a day or longer to provide. Many screening methods have been advocated for use in detecting bacteriuria and/or pyuria. These include microscopic methods, colorimetric filtration, bioluminescence, electrical impedance, enzymatic methods, photometric detection of growth, and enzyme immunoassay. Because a discussion of all available urine-screening methods is beyond the scope of this chapter, only the more commonly used methods are highlighted.

Gram Stain

A Gram stain of urine is an easy, inexpensive means to provide immediate information as to the nature of the infecting organism (bacteria or yeast) to guide empiric therapy. After a drop of well-mixed urine is allowed to air-dry, the smear is fixed, stained, and examined under oil immersion (1000×) for the presence of 1 or 5 bacteria per oil immersion field (OIF). The performance characteristics of the urine Gram stain are not well defined in that different criteria have been used to define a positive result (1 or 5 bacteria per OIF). Using either 1 or 5 bacteria/OIF has a sensitivity of 96% and 95%, respectively, and a specificity of 91% when correlated with significant bacteriuria (>10⁵ CFU/mL). The Gram stain should not be relied on for detecting polymorphonuclear leukocytes in urine because leukocytes deteriorate quickly in urine that is not fresh or not adequately pre served. Many microbiologists have not adopted Gram stain examination of urine specimens because of its unreliability in detecting lower yet clinically significant numbers of organisms and because of its labor intensity. If employed, urine Gram stain should be limited to patients with acute pyelonephritis, patients with invasive UTIs, or other patients for whom immediate information is necessary for appropriate clinical management.

Pyuria

Pyuria is the hallmark of inflammation, and the presence of polymorphonuclear neutrophils (PMNs) can be detected and enumerated in uncentrifuged specimens. This method of screening urine correlates fairly well with the number of PMNs (neutrophils) excreted per hour, the best indicator of the host’s state. Patients with more than 400,000 PMNs excreted into the urine per hour are likely to be infected, and the presence of more than 8 PMNs/mm3 correlates well with this excretion rate and with infection. This test can be performed using a hemocytometer, but it is not easily incorporated into the work f low of most microbiology laboratories. The standard urinalysis (usually done in hematology or chemistry sections) includes an examination of the centrifuged sediment of urine for enumeration of PMNs, results of which do not correlate well with either the PMN excretion rate or the presence of infection. Pyuria also can be associated with other clinical diseases, such as vaginitis, and therefore is not specific for UTIs.

Indirect Indices

Frequently, screening tests detect bacteriuria or pyuria by examining for the presence of bacterial enzymes or PMN enzymes rather than the organisms or PMNs themselves.

Nitrate Reductase (Greiss) Test. This screening procedure looks for the presence of urinary nitrite, an indicator of UTI. Nitrate-reducing enzymes that are produced by the most common urinary tract pathogens reduce nitrate to nitrite. This test has been incorporated onto a paper strip that also tests for leukocyte esterase, an enzyme produced by PMNs.

Leukocyte Esterase Test. As previously mentioned, evidence of a host response to infection is the presence of PMNs in the urine. Because inflammatory cells produce leukocyte esterase, a simple, inexpensive, and rapid method that measures this enzyme has been developed. Studies have shown that leukocyte esterase activity correlates with hemocytometer chamber counts. The nitrate reductase and leukocyte esterase tests have been incorporated into a paper strip. Numerous manufacturers sell these strips commercially, and the strips are one of the most widely used enzymatic tests. Although the sensitivity of the combination strip is higher than either test alone, the sensitivity of this combination screening is not great enough to recommend its use as a stand-alone test in most circumstances. Of note, the leukocyte esterase test is not sensitive enough for determining pyuria in patients with acute urethral syndrome.

Catalase. The Accutest Uriscreen (JANT Pharmacal Crop., Encino, Calif.) is another rapid urine-screening system based on the detection of catalase present in somatic (pertaining to the body) cells and in most bacterial species commonly causing UTIs except for streptococci and enterococci. Approximately 1.5 to 2 mL of urine is added to a tube containing dehydrated substrate. Hydrogen peroxide is added to the urine, and the solution is mixed gently. The formation of bubbles above the liquid surface is interpreted as a positive test. Some studies have reported that this system does not offer significant advantages over the leukocyte esterase-nitrite strip.

Automated and Semiautomated Systems

Automated screening systems offer the promise of a large throughput with minimal labor and a rapid turnaround time compared with conventional cultures. However, these advantages may be offset by a substantial cost for the instrumentation. Often these costs can be justified only in laboratories that receive many specimens.

Various automated or semi-automated urine- screening systems are commercially available, such as the iRIcell Systems (IRIS International, Inc., Chatsworth, Calif.) and are capable of analyzing a urine or body fluid sample in one instrument. The instrument analyzes both the microscopic components and the urine chemistries by combining technology of both types of analyzers into one automated system. The Sysmex UF-100 (TOA Medical Electronics; Kobe, Japan) are able to recognize many cellular structures, including leukocytes and bacteria.

General Comments Regarding Screening Procedures

 In general, screening methods are insensitive at levels below 10⁵ CFU/mL. Therefore, they are not acceptable for urine specimens collected by suprapubic aspiration, catheterization, or cystoscopy. Screening methods may also fail to detect a significant number of infections in symptomatic patients with low colony counts (10² to 10³ CFU/mL) such as young, sexually active females with acute urethral syndrome. Further complicating the laboratory’s decision as to whether to adopt a screening method is whether screening results will be used to rule out infection in asymptomatic patients. Under these circumstances, testing for pyuria is essential.

Therefore, given the importance of the 102 CFU/mL count and the PMN count, no screening test should be used indiscriminantly. Selecting a screening method largely depends on the laboratory and the patient population being served by the laboratory. For example, there will be a cost advantage in screening urine in laboratories that receive many culture-negative specimens. On the other hand, urine from patients with symptoms of UTI plus a selected group expected to have asymptomatic bacteriuria should be cultured. For example, patients in their first trimester of pregnancy should be cultured because these women might appear asymptomatic but have a covert infection and become symptomatic later; UTIs in pregnant women may lead to pyelonephritis and the likelihood of a premature birth. Other situations in which patients with no symptoms of UTI might be cultured include the following:

• Bacteremia of unknown source

 • Urinary tract obstruction

 • Follow-up after removal of an indwelling catheter

• Follow-up of previous therapy

Other factors that must be considered when selecting a rapid urine screen include accuracy, ease of test performance, reproducibility, turnaround time, and whether bacteriuria or pyuria is detected.

URINE CULTURE

Inoculation and Incubation of Urine Cultures

Once it has been determined that a urine specimen should be cultured for isolation of the common agents of UTI, a measured amount of urine is inoculated to each of the appropriate media. The urine should be mixed thoroughly before plating. The plates can be inoculated using disposable sterile plastic tips with a displacement pipetting device calibrated to deliver a constant amount, but this method is somewhat cumbersome. Most often, microbiologists use a calibrated loop designed to deliver a known volume, either 0.01 or 0.001 mL of urine. These loops, made of platinum, plastic, or other material, can be obtained from laboratory supply companies.

The calibrated loop that delivers the larger volume of urine (0.01 mL) is recommended to detect lower numbers of organisms in certain specimens. For example, urine collected from catheterization, nephrostomies, ileal conduits, and suprapubic aspirates should be plated with the larger calibrated loop. The communication of pertinent clinical history to the laboratory is essential so that appropriate processing can be performed.

The choice of media to inoculate depends on the patient population served and the microbiologist’s preference. The use of a 5% sheep blood agar plate and a MacConkey agar plate allows detection of most gram negative bacilli, staphylococci, streptococci, and enterococci. To save cost and somewhat streamline culture processing, many laboratories use an agar plate split in half (biplate); one side contains 5% sheep blood agar and the other half contains MacConkey agar.

In some circumstances, enterococci and other streptococci may be obscured by heavy growth of Enterobacteriaceae. Because of this possibility, some laboratories add a selective plate for gram-positive organisms, such as Columbia colistin-nalidixic acid agar (CNA) or phenyl ethyl alcohol agar. Although some discriminatory capability may be added, cost is also added to the procedure. In addition to increased cost, inclusion of plated media selective for gram-positive organisms generally provides no or limited additional information. Many European laboratories use cystine-lactose electrolyte deficient (CLED) agar. In recent years, chromogenic media have been introduced and become commercially available from a number of manufacturers, allowing for more specific direct detection and differentiation of urinary tract pathogens on primary plates, such as BD CHROMagar (Becton Dickison, Heidelberg, Germany). This medium uses enzymatic reactions to identify E. coli and Enterococcus without additional confirmatory testing from urine specimens as well as providing presumptive identification of S. saprophyticus, Streptococcus agalactiae, Klebsiella-Enterobacter-Serratia and the Proteus-Morganella-Providencia groups.

Before inoculation, urine is mixed thoroughly and the top of the container is then removed. The calibrated loop is inserted vertically into the urine in a cup. Otherwise, more than the desired volume of urine will be taken up, potentially affecting the quantitative culture result (Figure 2). If the urine is in a small-diameter tube, the surface tension will alter the amount of specimen picked up by the loop. A quantitative pipette should be considered if the urine cannot be transferred to a larger container. Once inoculated, the plates are streaked to obtain isolated colonies (Figure 3).

Fig2. Method for inserting a calibrated loop into urine to  ensure that the proper amount of specimen adheres to the loop.

Fig3. Method for streaking with calibrated urine loop to  produce isolated colonies and countable colony-forming units. 

Once plated, urine cultures are incubated overnight at 35° C. For the most part, incubation for a minimum of 24 hours is necessary to detect uropathogens. Thus, some specimens inoculated late in the day cannot be read accurately the next morning. These cultures should either be reincubated until the next day or interpreted later in the day when a full 24-hour incubation has been completed.

Interpretation of Urine Cultures

As previously mentioned, UTIs may be completely asymptomatic, produce mild symptoms, or cause life-threatening infections. Of importance, the criteria most useful for microbiologic assessment of urine specimens is dependent not only on the type of urine submitted (e.g., voided, straight catheterization) but the clinical history of the patient (e.g., age, sex, symptoms, antibiotic therapy).

One major problem in interpreting urine cultures arises because urine cultures collected by the voided technique may be contaminated with normal flora, including Enterobacteriaceae. Determining what colony count represents true infection from contamination is of utmost importance and is related to the patient’s clinical presentation. A number of studies have proposed the use of different cutoffs in colony counts based on clinical presentation; an example of one such set of guidelines is given in Table 1.

Table1.  Criteria for Classification of Urinary Tract Infections  by Clinical Syndrome

Ideally, the clinician caring for the patient should provide the laboratory with enough clinical information to allow specimens from different patient populations to be identified. These specimens could then be selectively processed using the guidelines in Table 1. However, because microbiology laboratories frequently receive little or no clinical information about patients, questions have been raised as to whether these cutoffs are practical and realistic for routine laboratory use. Further complicating urine culture interpretation is the increasing difficulty in distinguishing between infection and contamination as the criterion for a positive culture is lowered from 105 CFU/mL to 10² CFU/mL. Because of these issues, many laboratories establish their own interpretative criteria for urine cultures based on the type of urine submitted (e.g., clean-catch midstream, catheterized, and surgically obtained specimens such as supra pubic aspirates). Variations in interpretative guidelines occur from one laboratory to another but some generalities can be made; these are listed in Table 2. Some examples of urine culture results are shown in Figure 4 to illustrate some of these interpretations.

Table2.  General Interpretative Guidelines for Urine Cultures

Fig4. Culture results illustrating some of the various interpretative guidelines. A, Growth of ≥10⁵ CFU/mL of a lactose-fermenting  gram-negative rod in a clean-catch midstream (CCMS) urine from a patient with pyelonephritis; complete workup would be done. B, Growth  of ≥10⁵ CFU/mL of a lactose-fermenting gram-negative rod (arrow A) and 104 to 105 CFU/mL would be worked up completely. 

اخر الاخبار

اخبار العتبة العباسية المقدسة

العتبة العباسية المقدسة تشهد تشييع جثمان المحقق الشيخ محمد رضا المامقاني

قسم الشؤون الفكرية يختتم برنامج (فتية سيد الماء) لرعاية الأيتام بنسخته الثانية

لتطوير المهارات الإدارية.. العتبة العباسية المقدسة تطلق الدورة التعليمية الأولى لمسؤولي وحداتها

قسم التطوير يقيم دورة تدريبية عن الظهور الإعلامي لملاكاته

المزيد

الآخبار الصحية

دراسة تكشف "السبب الخفي" وراء ترك التمارين الرياضية

كشف التوقيت "المثالي" لتغيير فرشاة الأسنان

بلاستيك في أنسجة بشرية.. دراسة تكشف مفاجأة طبية

دراسة تحذر من الإفراط في ممارسة الرياضة.. كيف يضر دماءك؟

المزيد

الآخبار التكنلوجية

"ديب سيك" تحجب أحدث نماذجها عن شركات الرقائق الأميركية

"سامسونغ" تطلق سلسلة هواتف "غالاكسي إس 26"

حملات سرية ورسائل تشويه.. كيف يتم استغلال "تشات جي بي تي"؟

أثر نفسي غير متوقع للرياضة.. مرتبط بالغضب

المزيد

مواضيع ذات صلة

Identification of Mycoplasma Infections

Laboratory Diagnosis of Infections of the Eyes

Laboratory Diagnosis of Central Nervous System Infections

Diagnosis and Differential Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

Differential Diagnosis of Pancytopenia

Diagnosis of Aplastic anemia

Diagnosis of Upper Respiratory Tract Infections

tuberculosis testing (TB testing, Interferon gamma release assay [IGRA, T-Spot.TB], QuantiFERON-TB Gold [QFT, QFT-G, TB Gold test, TB blood test], Nucleic acid amplification for TB [NAAT], TB antibody)

thyrotropin-releasing hormone stimulation test (TRH stimulation test, Thyrotropin-releasing factor stimulation test [TRF stimulation test])

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Processing

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Collection and Transport

thyroid scanning (Thyroid scintiscan)