What is your diagnosis?
It could be a case of urinary tract infection. Some of the differential diagnoses include urethritis, PID, endometriosis, vaginitis and renal calculi.

Which are the bacteria that can cause urinary tract infection?
Common uropathogens of community acquire UTI include E.coli, Klebsiella pneumoniae, Proteus sps, and Enterobacter sps. In hospitalized patient, who have a urinary catheter,  Pseudomonas sps, and Enterococci are common pathogens. Staphylococcus saprophyticus is known to cause UTI in sexually active young women.

What is the common source of UTI?
In community acquired UTI, the uropathogens frequently are one's own enteric flora. UTI is more common in women than men due to proximity of anus to vagina and shorter urethra.

How are urinary tract infections classified?
UTI may be community acquired or hospital acquired, lower or upper, ascending  or descending, uncomplicated  or complicated.

How is the sample collected for laboratory diagnosis?
An early morning, freshly voided, clean-catch, mid-stream urine should be collected in a sterile, wide mouthed container after proper anogenital toilette. The external genitilia must be cleansed with mild antiseptic or soap before sample collection to avoid contamination of the urine by normal flora present in this region. In men, the prepuce is retracted and in women, the labia is spread apart and then the middle portion of the urine is collected in the container. The sample must be labeled and sent to the laboratory without delay.

Which are the other techniques to collect urine specimen?
In infants urine flow may be stimulated by tapping just above the pubis with two fingers at one hour after a feed. One tap per second is given for one minute and after an interval of one minute tapping is continued. Under certain conditions, suprapubic aspiration of urine directly from the bladder may be performed. Since this is an invasive technique, it must be performed only when absolutely necessary. Catheterization only for the purpose of collecting urine should be avoided as it may induce infection. In situations where the patient is already catheterized, the urine must not be collected from the bag, instead, it should be aspirated from catheter tube using needle and syringe.

How long can the urine be held before testing?
Ideally, urine must be processed as soon as possible since urine supports growth of bacteria. In case of delay of 1-2 hours the sample may be refrigerated or treated with boric acid at an concentration of 1.8%. Another way of preserving the sample in case of delay is by collecting urine in sterile vacutainer tubes containing boric acid-sodium formate transport medium. Samples that have been processed after a delay of five hours or more do not give reliable results.

Which investigations are performed on urine sample?
Urine wet mount and culture is commonly performed on urine specimen. Wet mount examination is performed to look for pus cells, RBCs and casts. A loopful of well mixed urine placed on the glass slide (without spreading) can be stained by Gram stain and observed.  Presence of single bacterium per oil immersion field in such a smear indicates significant bacteriuria. Screening test such as nitrate reduction, dipstick, tetrazolium reduction etc are not specific and are not routinely done. Leukocyte esterase dip test is helpful in detecting pyuria. Qualitative culture technique such as Miles and Misra are too cumbersome to perform for routine diagnosis, hence a semi-quantitative culture is performed by calibrated loop method. A loopful of well-mixed uncentrifuged urine is inoculated on to CLED agar/MacConkey agar and Blood agar without sterilizing the loop in between.

What is significant pyuria?
Presence of at least 1000 pus cells per ml of uncentrifuged urine is significant pyuria. Ordinarily, presence of ≥10 pus cells/HPF in centrifuged urine and ≥5 pus cells in uncentrifuged urine is considered significant. Some authors consider counts as low as 2-5 WBCs /HPF important in a centrifuged specimen in the female with appropriate symptoms. In women, contamination from vagina may introduce large numbers of pus cells into a sample of voided urine. The presence of squamous epithelial cells along with pus cells in the sample is evidence that contamination has occurred and the pus cell count is not significant.

What is significant bacteriuria?
Since normal voided urine tends to get contaminated with normal flora of the distal urethra, differentiation of contamination from urinary tract infection is made by quantifying the bacterial growth. Significant bacteriuria is a concept put forth by Kass EH, who stated that there should be at least 1,00,000 bacteria of single type per ml of urine. This count may not be applicable in all situations. Recent studies suggest that a count of 10² per ml in acutely symptomatic women and a count of 10³ per ml in symptomatic men may be significant. Any growth obtained from urine collected via suprapubic aspiration is significant. Lower counts may be significant when S. aureus is the pathogen.

How is semi-quantitative culture performed?
A loopful of well-mixed uncentrifuged urine is inoculated on the agar medium without sterilizing the loop and incubated at 37^oC overnight. Following incubation, the number of colonies of single type is counted. A bacteriological loop of 3 mm diameter approximately carries 0.001 ml of urine. If this amount of urine gives rise to at least 100 colonies then the numbers of bacteria present in 1 ml can be obtained by multiplying by 1000, i.e 1,00,000 per ml.

What is your observation?
Wet mount of urine shows plenty of pus cells and RBCs but few squamous epithelial cells. More than 100 colonies of pink coloured (lactose fermenting), smooth, low convex, circular colonies of a single type is seen on MacConkey's agar. Gram stained smear of the colony shows gram negative bacilli, hanging drop shows motile bacilli, and catalase test is positive. Results of biochemical reactions are positive indole test, negative urea hydrolysis, negative citrate utilization, positive MR test and negative VP test. TSI agar shows acid slant/acid butt with little gas but no H₂S. The isolate is identified as Escherichia coli was obtained in significant numbers.

What factors must be borne in mind while interpreting urine culture reports?
Urine in the bladder is sterile, small numbers of bacteria get into the urine from the distal part of urethra while voiding. In typical cystitis, most often urine culture are unimicrobial, recovery of more than one type of bacteria in urine indicates contamination. Bacterial counts in the range of 10²-10³ in the absence of pyuria and other symptoms usually indicates contamination. Rarely, mixed infection by more than one type can occur; in such situations a repeat culture with same results is reliable. Significant bacteriuria may not be applicable for suprapubic aspirated urine and when S. aureus is the pathogen. Bacterial counts can be lower if the patient is on antibiotics or has consumed large amount of water before voiding urine. Bacterial counts can be higher if there is a long delay between urine collection and culture.

What is sterile pyuria?
Presence of plenty of pus cells in urine but lack of growth on culture is sterile pyuria. The reasons for this condition include recent administration of antibiotics, UTI by a fastidious/auxotropic/anaerobic bacteria, urethritis due to gonococci,  non-gonococcal urethritis (due to Ureaoplasma, Chlamydia, Trichomonas, or viruses) or renal tuberculosis. 

What is baceriuria without pyuria?
It is the presence of large numbers of bacteria in the urine but lack of significant numbers of pus cells. This condition is usually seen in pregnant women where there is retention of urine or when voided urine is held for a long time before culture.

How is this condition treated?
Trimethoprim-sulfamethoxazole for 3 days is considered the current standard therapy for bacterial cystitis. Fluoroquinolones such as norfloxacin also works well. Antibiotics should be selected on the basis of susceptibility testing of the isolate.