Most Numerous specimens are received in the Laboratory
30 to 40 % of specimens received in Microbiology laboratories are Urine specimens, to Identify the Infection.
Diagnostic information is important for the clinician.
Appropriate clinical information gives many clues for better diagnostic evaluations .
Specimen collection is the primary objective in getting an ideal sample.
Why women are at More Risk for Urinary Tract Infections
Women tend to have urinary tract infections more often than men do because the urethra is shorter in women than in men, so bacteria have a shorter distance to travel.
Who are at Risk with UTI
Urinary tract infection is much more common in adults than in children, but about 1-2% of children do get urinary tract infections. Urinary tract infections in children are more likely to be serious than those in adults and should not be ignored.
Most Important facts to establish Infection
Simple microscopic examination of wet films of unconcentrated urine for detection of Polymorphonuclear leucocytes - pus cells gives leading clues
Semiquantitative culture of urine to detemine wether urine contain potentially pathogenic bacteria in Numbers sufficent to identify it as causative agent causing infection.
Common Presenting Symptoms
Frequency of Micturation
Discomfort and pain in abodemen or on passing urine
Organs Involved in UTI
The following organs are infected
Donot include Urethra, Infections of Urethra is called as Urethritis, dealt under different clinical syndromes
Common site and Microbes
Patients in majority of cases present with infection of Urinary Bladder and called as Cystitis
Most common pathogen is Escherichia coli.
Common Pathogens causing UTI
Klebsiella pneumonia – var aerogenes or oxytoca
Proteus mirabilus, other coliforms
Candida being a fungus can cause infections in Diabetic or Immunocompromised patients.
Rare Microbes causing UTI
Serious Infections associated with UTI
May lead to Bacterimias detected by Blood culture
A prominent infection with Staphylococcus aureus can cause above manifestions.
UTI without Routine Bacterial isolates
Some times it puzzles the clinicians, patients present with symptoms of urinary tract infection but bacteria donot grow on Routine culture Media
May be associated with
Organisms that donot grow on selected media
Can be a Genito urinary tract Tuberculosis
Nationally exacting or anaerobic bacteria
Non Bacterial Urethritis,Cystitis and Urethral syndromes
One should be familiar with
Urethral or Bladder infections with
Simple tests to rule out Urinary tract infections
In les equipped laboratories routine testing for detection of
By rapid automated dipsticks can give basic information
But eliminates about ½ of culture negative specimens.
Collecting Urine for examination
Collect the Mid stream specimens of Urine
Donot collect spontaneously collected urine without instructions, which can lead to contamination with commensals bacteria Colonised on urethral orifice and perineum
The urine collected in a wide mouthed container from patients
A mid stream specimen is the most ideal for processing
Female patients passes urine with a labia separated and mid stream sample is collected
Specimen collected in young and Children
Non invasive methods are safe and ideal
Follow the Broomhall et al method
By tapping just above the pubis with two fingers place on suprapubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for aperiod of 1 minute, if not succesul tapping is repated once agin.
The child spontaneously pass the Urine and to be collected in a sterile container
Transport of Urine for Culturing Urine
All collected specimens of urine to be transported to laboratory with out delay
Delay of 1 – 2 hour deter the quality of diagnostic evaluations.
If the delay is anticipated the specimens are at preserved at 4 0 c
In field conditions Boric acid can be added at a concentration of 1.8 %
Proof of Urinary Tract Infection
Needs presence of potential pathogens in the freshly voided urine or scientifically preserved specimens in numbers greater than those likely to result from contamination from urethral meatus and the surrounding,
Kass suggested the Number to be about 10 5 or more ( 1,00,000 / ml of urine )
Diagnosis of Urinary Tract Infection
Microscopy of Urine for detection of Pyuria.
Leucocytes should be found in numbers of at least as great as 10 4 / ml before the pyuria is established
Wet Film examination of Urine
All wet films to be examined with high power ( x 40 ) objective.
Prepare the drop of urine after mixing the urine without centrifugation
Transfer 0.05 ml on the middle of the microscope slide and cover slip is applied.
The prepared specimen show a small excess of fluid along the edges of the cover slip.
A approximate finding of 1 leukocyte / 7 high power fields corresponds to presence of pyuria.
Culturing of urine for Isolation of Bacterial pathogens
Select the Media
For common isolates
Mac Conkey’s agar
helps in differentation of Lactose fermenting organisms from non lactose fermenting pathogens
Selection of Loop for Semiquantitative Method
Culture Media for isolations- CLED Medium
It is also an excellent universal culture medium owing to its wide spectrum of nutrients, lack of inhibitors and the fact that it allows a certain degree or differentiation between the colonies. It contains lactose as a reactive compound which, when degraded to acid, causes bromothymol blue to change its colour to yellow. Alkalinization produces a deep blue colouration. The lack of electrolytes suppresses the swarming of Proteus
Culture Media for Isolation
helps in isolation of fastidious, extracting strains
May extended incubation for isolation of pathogens for more than 48 hours with added atmosphere of 5 – 10 % co 2
All cultures processed by Semiquantitative method a loop of standard dimension of approximately known volume is inoculated into selected culture plate
In general a loop of SWG – 28 with a diameter of 3.26 mm internal diameter which can hold a drop of water or urine 0.004 ml.
After inoculation the culture plates are incubated at 37 0 c extending to > 18 hours are read
The colony counts are made, as each colony corropsdes to number of viable bacteria per ml of urine
Reading the Culture Plates
A true infection in the absence of prior antibiotic therpay the number of bacteria is likely to be at least 10 5 or more.
Contaminated specimens present with colony counts <10 4, however even less than 10 3
On several occasions the colonies are diverse species
Several studies prove counts >10 4 to be considered as presence of Urinary tract infection with the supporting clinical history
On some occasions more than one pathogen is isolated but should be processed for all practical purposes
eg E.coli along with Streptococcus fecalis
On few occasions even counts 10 3 are proved significant
Identification of Gram + organisms
All colonies identified morphologically as Staphylococcus to be characterized as
Enterococci - fecal group of organisms
Identification of Isolates Gram + isolates
The minimal tests to differentiate Gram + cocci include
2 Coagulase test
3 Bile esculin testing
4 Bacitricin in Streptococcus isolates
Biochemical tests in Gram - bacilli
Nitrite reduction test
Methyl red test
V P test
Lysine, ornithine, Arginine tests
Mac Conkey’s agar showing Proteus and E.coli
Blood agar showing Coagulase negative Staphylococcus
Yet many theories on Significant bactenuria are contraversioal.
The reporting of results should be maninly based on clinical history.
Even a true infection may contain only 10 3
In such circumstances tests to be reported as probably or possibly significant
A good coordination between a Microbiologist and Physcian is the best solution associated with best solutions to patients with Urinary tract infections.
Reporting of Contaminated specimens
Microscopy helps to detect pus cells and epithelial cells, an insignificant grwoth with few pus cells can ignored as contaminants
In females even the presence of leucocytes with Squamous epthelial cells without a defined significatn growth should be ignored.
Always use a pure grwoth of the isolates.
Perform testing for antibiotic sensitivity with inoculam which is comparable to defined Macfarland standards.
Most serious Infections presenting as Urinary tract Infections
May present with loin pain, fever,
Apart from Urine culture patients present with Bacteriaemia - can be detected by Blood culture.
Causative agent can be
Clinical Problems manifesting as Urinary tract Infections
An infection of the Genitourinary tract where the Microbes donot grow on selected media as in Mycobacterium tuberculosis
Nutritionally exacting or anaerobic bacteria
Non Specific Urethritis
Several conditions mimic Urinary tract infection which are associated with organism other than routinely isolated in simple and few selective media, they can be
Can be urethral or Bladder infections with
Dealing with UTI
The chemotherapy of proven infection may guided by in vitrosensitivity tests on the pathogen isolated by culture and outcome of therpay assessed by examination of urine at the conclusion of treatment.
Follow up examination of patients with UTI is important, if neglected can lead to chronic and permanent complications.
Antibiotic Sensitivity Testing
All the isolated bacteria identified as pathogenic to be tested for Antibiotic Sensitivity/Resistance pattern by disk diffusion methods
Always use a pure grwoth of the isolate.
Perform testing for antibiotic sensitivity with inoculam which is comparable to defined Macfarland standards
Rationalsim of Antibiotic Selection
If the patient is attending a General practice, outpatient clinic, drugs suitable by oral administration should be selected
The discks are with defined content to test the pathogens in the urine
Amoxycillin or Ampicillin 25
Cephelexin 30 µg
Nalidixic acid 30µg
ß Lactamase producing Gram negative strains
There is a grwoing incidence of ßlactamase producing strains in particular associated with Hospital acquired infections
The newly defined methods to be used as per the NCCL guidelines.
There is a growing need for testing for
There is a grwoing list organisms associated with Hospital Infections need better identification testing with new generation of drugs.
Created as Teaching Module for Medical Students in Devloping world Dr.T.V.Rao MD Email [email_address]