Point-of-care urine Gram stain led to narrower-spectrum antimicrobial selection for febrile urinary tract infection in adolescents and adults - BMC Infectious Diseases - BMC Infectious Diseases

Study design and setting

This was a single hospital-based, retrospective observational study. The study setting was OCH, Japan. Approximately 39,000 patients visit the emergency room (ER) annually and nearly 7,000 patients are hospitalized after attending the ER each year [10]. Most patients with a suspected fUTI are initially examined in the ER. Adolescent and adult patients who are diagnosed with fUTI and who need to be hospitalized are admitted to the Division of Infectious Diseases. Patients with complicated pyelonephritis requiring surgical interventions such as double-J stent insertion, suprapubic cystostomy, or nephrostomy are admitted to the Department of Urology.

Case definitions and data collection

Febrile UTI was considered if a patient had symptoms of systemic inflammation (fever, chills, and malaise) and bladder symptoms (urinary frequency, urgency, and dysuria), which was supported by urinalysis (U/A) results that showed pyuria or bacteriuria (or both) and urine culture (U/C) results that showed substantial concentrations of a uropathogen [1]. Febrile UTI patients were divided into the following two groups: the uncomplicated group, which included patients with uncomplicated pyelonephritis; and the complicated group, which included patients with complicated pyelonephritis and prostatitis. Uncomplicated pyelonephritis was defined as acute pyelonephritis in women who did not have any underlying diseases and were not pregnant [11]. Complicated pyelonephritis was defined as acute pyelonephritis in patients who had underlying diseases such as neurogenic bladder, indwelling bladder catheter, diabetes mellitus, or prostate hypertrophy, or patients whose kidney had urolithiasis, abscess, or emphysematous pyelonephritis/cystitis. Undefined cases were categorized into the complicated group [11]. Prostatitis was defined as fUTI without flank pain, with prostate tenderness, or a significant increase in prostate-specific antigen (PSA) or an imaging diagnosis [9, 12].

In this study, all patient information was collected from medical charts between January 2013 and March 2015. The inclusion criteria were all patients aged 15 years or older who were admitted to the Division of Infectious Diseases and finally diagnosed with UTI. The exclusion criteria were as follows: (1) uncomplicated cystitis; (2) previous antibiotics exposure within 48 h; (3) PCGS not examined or U/C not submitted; (4) not diagnosed with a fUTI after admission; (5) patients with co-infection other than fUTI; (6) transferred to another hospital from the ER; and (7) data insufficient.

Urinalysis and urine culture

Clean-voided midstream urine or urine collected with a disposable catheter was used for PCGS in the ER. For patients with indwelling urinary catheters, urine was collected directly from the catheter rather than from the urine bag. The residue specimen was then delivered to the laboratory for U/A and U/C. If catheter-associated urinary tract infection was suspected, the catheter was replaced. U/A was performed after centrifugation without staining by a technician and using an automated urine analyzer (Aution Hybrid, Arkray, Kyoto, Japan). The pyuria result was considered to be positive when five or more leukocytes per 400 × magnification field (high power field: HPF) were observed, and the number of leukocytes was quantified. The bacteriuria result was considered to be positive if an average of one or more bacteria was identified in a HPF of view, and the number of bacteria was semi-quantified as follows: 1 + , an average of one or more in one visual field; 2 + , many in one visual field; and 3 + , a large number in one visual field.

U/C was performed by a technician using uncentrifuged urine. The number of bacteria was quantified using quantitative medium (Caldip Plus, Merck, Darmstadt, Germany). If the urine sample amount was insufficient, this quantification was omitted, but semi-quantification was confirmed using culture plates. If Gram-positive rods such as Bacillus or Corynebacterium, coagulase-negative staphylococci other than Staphylococcus lugdunensis or Staphylococcus saprophyticus, or other skin or vaginal normal flora grew in the culture, it was considered to be contaminated and non-pathogenic.

Enterobacteriaceae with ESBL-producing or AmpC-positive or carbapenem-resistant, carbapenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus were considered to indicate AMR.

Blood culture

At least two sets of blood cultures, which included aerobic and anaerobic bottles with Bactec Plus resin medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), were taken from the upper or lower limbs but not from the femoral vessels. All bottles were incubated for at least 5 days using the Bactec 9240 system, which is an automated blood culture system [13]. Coagulase-negative staphylococci, Bacillus, Propionibacterium, Micrococcus, Clostridium, and α-streptococci were considered to be potential skin contaminants [13]. With the exception of α-streptococci, if any of these grew in only one set of blood cultures, it was considered to be a contaminant.

PCGS and antimicrobial selection

PCGS was performed and interpreted by physicians in the ER soon after urine samples were obtained. Uncentrifuged urine specimens were placed on glass slides, fixed with a flame or hot air blower, and Gram-stained using Barmii M (Muto Pure Chemicals, Tokyo, Japan). We used a four-step dyeing procedure including crystal violet, 2% iodine sodium hydroxide, acetone ethyl alcohol, and 0.1% fuchsine. The slides were examined in the ER by in-house staff members including trained resident physicians (postgraduate years 1 and 2, called junior residents) to identify each etiologic agent and select an appropriate targeted antimicrobial therapy [7, 14]. Physicians examined the slides using conventional light microscopy at 100 × magnification and then at 1000 × magnification under immersion oil. If leukocytes were visible, the patient was considered to have pyuria, and the number of leukocytes was semi-quantified as follows: an average of < 1 in one per visual field (1000 ×) was ± ; an average of 1 to 9 was 1 + ; 10 to 99 was 2 + ; and > 99 was 3 + . Similarly, if bacteria were present, the patient was considered to have bacteriuria, and the number of bacteria was semi-quantified as follows: an average of < 1 per visual field (1000 ×) was ± ; an average of 1 to 9 was 1 + ; 10 to 99 was 2 + ; and > 99 was 3 + . All findings were confirmed by senior residents (postgraduate years 3 to 5) and attending physicians. If the junior residents' interpretation differed from that of senior residents or attending physicians, direct feedback to the junior residents was given from an educational viewpoint.

PCGS has been used for a practical and useful diagnostic tool since 1976 at OCH [7]. All junior residents in the first year rotate to the division of infectious diseases for 2 weeks and receive basic education on infectious diseases. In addition, junior residents of internal medicine or primary care in the second year rotate to the division for over a month, as do senior residents in their elective months. All attending physicians in the division also received education on infectious diseases as alumni of the internal medicine residency program of OCH.

Annual cumulative antimicrobial susceptibility test data were reported and distributed to medical staff by the bacteriology laboratory. The organisms that were estimated using PCGS led physicians to select antibiotics along with the cumulative antibiogram. For example, 1415 cultures were positive for Escherichia coli in 2015, the sensitivity of which to ampicillin was 62%, ampicillin/sulbactam 69%, cefotiam (a second-generation cephalosporin, alternative to cefuroxime in Japan) 82%, cefmetazole (a cephamycin, alternative to cefoxitin in Japan) 99%, cefotaxime 84%, tobramycin 94%, and ciprofloxacin 79%. ESBL strains accounted for 16% of the bacteria cultured. In another example, 404 cultures were positive for P. aeruginosa, and the sensitivity to piperacillin was 95%, while that of ceftazidime was 96%, aztreonam 91%, imipenem/cilastatin 95%, tobramycin 99%, and ciprofloxacin 93%. During this study, the cumulative antibiogram was updated yearly from 2013 to 2015.

In urine PCGS, medium- or large-sized Gram-negative rods suggested E. coli, Proteus mirabilis, or Klebsiella pneumoniae. In these cases, cefotiam 1 g every 6 to 8 h (q6–8 h) intravenously (iv) was mostly administered. A third-generation cephalosporin (cefotaxime 1 g q6–8 h iv or ceftriaxone 1–2 g q12–24 h iv) was selected when Citrobacter, Enterobacter, or other Enterobacteriaceae was suspected based on previous culture findings. If an ESBL-producing bacterial strain was suspected based on a living place or previous culture results, cefmetazole 1 g q6–8 h iv was selected. If a patient's condition was unstable due to septic shock, a carbapenem (meropenem 1 g q8h iv or imipenem/cilastatin 0.5 g q6h iv) was used. Small-sized Gram-negative rods suggested P. aeruginosa, ceftazidime 1 g q6–8 h iv, aztreonam 1 g q6–8 h iv, tobramycin 120–240 mg q24h iv, or a carbapenem (meropenem or imipenem/cilastatin) was chosen. Gram-positive cocci in chains suggested Enterococcus or Streptococcus, and mainly ampicillin 1 g q6–8 h iv or rarely vancomycin 1 g q12h was selected. Gram-positive cocci in clusters suggested Staphylococcus or Aerococcus, and cefazolin 1 g q6–8 h iv or rarely vancomycin was chosen. If Gram-negative rods and Gram-positive cocci in chains were observed, ampicillin/sulbactam 1.5 g q6–8 h iv was chosen to cover both Enterobacteriaceae and Enterococcus. When more than two types of bacteria were confirmed, a polymicrobial infection including AMR was suspected, and a cephalosporin, a cephamycin, or a carbapenem was selected. These polymicrobial infection cases were excluded from the analysis in Table 3, because they were unevaluable on PCGS. If Gram-positive rods, small amounts of Gram-positive cocci or mixed organisms were observed, they were considered to be non-pathogenic.

Penicillins and first- or second-generation cephalosporins were defined as narrow-spectrum antibiotics; fourth-generation cephalosporin, carbapenems, and vancomycin as broad-spectrum antibiotics; and all other antibiotics as intermediate-spectrum antibiotics [7].

Outcome measures

The primary outcome was the susceptibility of targeted therapies such as initial antimicrobial selection based on PCGS according to urine and/or blood culture susceptibility test results in the uncomplicated and complicated groups. Secondary outcomes were pyuria and bacteriuria detection by PCGS compared to U/A, and bacterial type estimation by PCGS compared to U/C.

Statistical analysis

To ensure an adequate sample size to calculate the susceptibility of the initial antimicrobial choice, we hypothesized that there would be a difference between the uncomplicated and complicated groups. We assumed that the rate of susceptibility in the uncomplicated group would be more than 0.9 because the patients were young and the rate of AMR was still low. In the complicated group, however, we expected that the susceptibility could be lower than 0.9 because they were older, and repeated antimicrobial exposure increases AMR colonization. Therefore, assuming that the rate of susceptibility would be 0.95 in the uncomplicated group and 0.8 in the complicated group, and assuming that the uncomplicated group-to-complicated group ratio was 1:4, 80% power, and a two-sided alpha level of 0.05, we calculated that 260 patients would be required.

The Chi-squared or Fisher's exact test was used for categorical variables, and the Student's t-test was used for numerical variables. Cuzick's test for trend was used to compare PCGS and U/A results. Kappa statistics were used to evaluate the agreement between PCGS and U/C results. Additionally, p < 0.05 was considered to be significant. The statistical analysis was performed using Stata software (version 16.1; StataCorp, College Station, TX, USA).