TRACING THE PATHOGEN
Part twelve:

 Cooperation at investigation or
Clinical Microbiology III
Institute for Microbiology shows
L

List of topics
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Infections of urinary tract – survey
Urine sampling
Possibilities for urine specimen processing
Result interpretation, prevention and treatment of UTI
Infections of genital tract – introduction
Sampling and examination of genital infections

Urinary tract infections – survey



Importance of urinary tract infections (UTI)
lBesides respiratory infections, this is another very important group of infections with economic
losses and inconvenience for patients
lDangerous is risk of complications — for example, from cystitis and pyelonephritis may become the
bearing may become the emergence of urosepsis, i. e. infection of the bloodstream
lIMC are very common, especially in women
lCausative agents are usually bacterial, antibiotic therapy is therefore often (though not always)
indicated

Urinary tract of a healthy person
•Kidney – without microbes normally
•Renal pelvis – without microbes normally
•Ureters – without microbes normally
•Bladder of young and middle aged people – without microbes normally
•Bladder of seniors – even under normal circumstances it can be settled in microbes, which does not
make problems and becomes a "normal flora"
•Urethra – normally without the microbes with the exception of the final part (bacteria from the
skin + partly specific flora: viridans Streptococcus, aerococci, etc.)

Anatomic classification of UTI – 1
lUrethritis cases are rather connected with genital infections and are discussed in the context of
this issue
lCystitis are the most common UTIs, especially for women (they have a shorter urethra). They are
often associated with situations where a stream of urine as a natural protection system is weaker,
for example:
lthe pelvic floor disorders (typically in women after childbirth)
lfor hyperplasia of prostate (that is, by contrast, for women rare;-))
lComplications of cystitis may be pyelonephritis
l

Clinical description of cystitis and pyelonephritis
lBurning during urination
lFrequent urination, small amounts of urine
lSometimes urine is bloody and turbid
lOrientation strip test may show leucocytes and eventually also pH elevation
lIn case of back pain and fever, it is usually not only cystitis, but pyelonephritis
lIn case of pyelonephritis, the strip test would show also presence of proteins in the urine

Attention – cystitis symptoms are not specific
•Diseased urination (frequent urination, incontinence, burning) may have another cause than
cystitis, which should be revealed or excluded
•It can be a sexually transmitted disease (Chlamydia or Mycoplasma infection, gonorrhoea)
•It can be a non-infectious inflammation (mechanical irritation of catheterization, etc.) or other
non-infectious cause (even incipient tumour!)
•It could also be an inflammation of the walls of the bladder (e.g., infectious,
parasitic/Schistosoma, as well as non-infectious)
•In all these cases is finding in the urine culture is negative

Pyelonephritis
lPyelonephritis is an inflammation of the renal pelvis, unlike glomerulonephritis.
lGlomerulonephritis affects the glomeruli and is usually non-infectious; however, it may be an
autoimmune origin after a streptococcal infection.
lIt is more serious than cystitis, it does not only affect the lumen of the urinary tract, but
kidney tissue, so antibiotic therapy should respect it
lUsually arises as a complication of cystitis, but the origin can also be haematogenous
lRecurrent pyelonefritis may also be complicated by urolithiasis (bladder stones)
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Urine sampling



UTI diagnostics
lAnamnesis, that might also include sexual life (gonorrhoea and other urethritis)
lClinical examination
lOrientation diagnostic strip examination  (the presence of bacteria in urine)
lBiochemical examination – the presence of bacteria, proteins, etc.
lMicrobiological testing is recommended for uncomplicated and necessary for complicated one (not
speaking about pyelonephritis)

Sampling and transport of urine
lThe most reliable urine is obtained by suprapubic puncture. In practice, however, rarely used
lQuite good is also catheterized urine (catheterization performed due to sampling)
lCommonly collected urine sample may not be bad, if it is properly sampled and transported
lUrine from permanent catheter is the worst of possible samples, sometimes, of course, there is
nothing for us. Sometimes also urine from nephrostomy is used.
•There is no reason to use cathethrized urine globally. It only makes sense when the outcome is
repeatedly doubtful.

Permanent catheter urine sample
lIf it is possible to wait a few days e. g. for the exchange of the catheter, it is better – the
result of the newly exchanged a catheter will be far better (but it is advisable to wait after
replacing some time until the colonizing bacteria from the old catheter are washed away)
lIf we cannot wait, we sample the urine, but we must reckon with the fact that the interpretation
is ambiguous (inflammation and not just the colonization is likely in case of leucocytes present in
the urine)
lWe must think whether we are considering treatment with antibiotics at all; If not (asymptomatic
bacteriuria) the examination looks to be useless
lMicrobiological examination of the catheter itself, sent to a laboratory, is not recommended
(nearly impossible interpretation), although some laboratories perform it

Sampling of spontaneously urinated urine
lIt is the sampling from the middle stream of urine spontaneously urinated (routine type with
secondary risk of contamination during sampling)
lTechnique: The container for the collection of urine must be sterile, wide-neck (e.g. a beaker*),
knowledgeable patient thoroughly washed prior to the collection's external genitals with soap and
water and (possibly) wipe off the outer estuary of the urethra's swab moistened with disinfectant
in solution (especially in children, however, the use of disinfectant is not recommended)
*this is as it is written in the official recommendations, in practice, it depends on the
situation; If the patient urinates directly into a test tube, it is better

Sampling in males and females
lMales by one circulating movement
lFemales should be in wide position over the toilet, they stretch out the labia by one hand and
they wash the genital by a tampon using the other hand from front to back.
lAfter that, the patient urinates the first part of urine and the midstream is taken into sterile
vessel without breaking the urination. Sampled urine is placed into a sterile container for
transport.
•(Czech Medical Society of John Evangelist Purkyně, RECOMMENDED GUIDLINES FOR GENERAL
PRACTICIONERS, Project of Healthcare Ministry of Czechia by help of a grant IGA MZ ČR 5390-3)

Urine sampling in a female – technique
moczeny
http://www.lab-turnov.ic.cz/schema_1.php
Wash your genital by water and soap
Stretch your labia out
Wash your hands please

Urine sampling in a female – technique
moczeny
http://www.lab-turnov.ic.cz/schema_1.php
Let the first portion flow into the toilet
Catch approx. 50 ml of urine to the vessel without interrupting urination. Do not touch the inner
part of the vessel.
Place the container with the urine according to  directions
Let the remaining part of urine flow into the toilet

Urine sampling in a male – technique
http://www.lab-turnov.ic.cz/schema_2.php
mocmuzi
Wash your hands please
Pull down your foreskin please
Wash the end of your penis

Urine sampling in a female – technique
http://www.lab-turnov.ic.cz/schema_2.php
mocmuzi
Let the first portion flow into the toilet
Catch approx. 50 ml of urine to the vessel without interrupting urination. Do not touch the inner
part of the vessel.
Let the remaining part of urine flow into the toilet
Place the container with the urine according to  directions

Exceptions of urine sampling rules
lIn suspicion for urethritis we take first portion of urine (we wash out the microbes from
urethra).
lIn prostatitis we rather use last portion of urine
lFor schistosomosis we collect last portions of urine several time, at least 20 ml needed. (In the
laboratory, the urine will let settle and then we look for the eggs of the parasite in the sediment
on the bottom). Transport should be quick.

Sampling in small children and catheterized patients
lIn children
lUrine is obtained by collecting the bags tightly
lMethod is burdened with a relatively high risk of secondary contamination
lThe bag should not be attached more than 30 minutes
lIt should be removed immediately after the pee
lIn catheterized patients we should reckon with the fact that any result is indicative for the
colonization of the catheter, rather than for the infection. The sampling must be carried out so as
to minimize the risk of further contamination

Urine transport
lFor evaluation of an UTI, the quantity is important – see further. The quantity can only be
evaluate if the microbes would not multiply in the urine during the transport. If they do, the
quantity is changed
lTherefore, it is essential to return to the urine laboratory within two hours after sampling (or
even faster)
l If, exceptionally, this cannot be fully met, storage in a refrigerator should be used (unlike for
other specimens)

URICULT type devices
lThe purpose of these units is to totally eliminate the time lag between the collection of urine
and the beginning of cultivation. The urine is sampled and a special tool with cultivation media is
placed in it. Then the urine is removed and the media start to be cultured (sometimes without
sending to the lab, if a small incubator is available for it)
lOn these plates, however, the microbes are difficult to diagnose. This method is therefore doesn't
apply, as it was originally expected. In its use of large regional differences.
lIf it is used, it is necessary to strictly follow the correct procedure

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How to use an URICULT
lThe cap with culture media should be screwed out carefully (to let the cap in the air during
sampling)
lUrine midstream is used for filling in the Uricult container to 3/4 (directly or from a sterile
container).
lThe device with cultivation media is placed into the urine in the container
lAfter several seconds the device is removed
lExcess urine is left to drain on the bottom of the plates, then sucked out with filter paper (do
not touch the media)
lThe urine is removed from the vessel including the remaining drops
lExceptionally it is possible to perform the sampling by placing the both sides of the media in the
stream of urine

Urine specimen processing



Qualitative and semiquantitative urine examination
•When a quantitative examination of the urine is diluted and given on a few of the culture media.
•At semiquantitative examination, urine is not diluted, but a calibrated single use loop on is
used. The examination is less laborious, but also less accurate.
•Of course, not only quantity is assessed, but also the normal way to diagnose the microbe is used.

Semiquantitative processing 1
•A calibrated plastic loop for 1 µl is used
•That means that when it is placed into the water or a liquid with similar surface tension just one
microlitre is kept in the „eye“ of the loop •This microlitre is inoculated to one half of blood
agar plate (in practical session: on a total plate)
•After that it is normally incubated (24 h, 37 °C)
•The other day colonies are counted. According to the number of colonies the result is interpreted
•In our laboratory we use now chromogenic medium instead of formerly used Endo or McConkey agar

Bacteria on a chromogenic medium
P1010007u
Foto O. Z.

Semiquantitative processing I
lNumber of colonies after incubation corresponds to the number of CFUs in 1 µl of original urine
–CFU = colony forming unit: one microbe, a pair, a short chain, a small group. In practice we
neglect the difference between a microbe and a CFU, so we say that we count microbes when we really
count CFUs
lIf the number of colonies approximately corresponds the number of microbes in 1 µl of original
urine, then the number of colonies × 1000 corresponds the number of microbes in 1 ml of original
urine. 10 colonies – 104 microbes in one millilitre, 100 colonies – 105 microbes/ml

Automated culture systems
lSome companies now offer automated culture systems, which detect positivity already after four
hours and they even refer the antibiotic sensitivity (Italian system UroQuick). Some, especially
the private laboratory systems welcome it and base their microbiological examination of urine on
this system.
lHowever, this approach is very risky, because the determination of the antibiotic susceptibility
testing without specifying the type of bacteria is very treacherous. If there is such a system
combined with the classic diagnosis, the damage is not necessary. However, it is unacceptable to
use such a system without its results being interpreted the microbiologist (e.g. location of the
instrument into biochemical laboratories).

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Urine
Basic diagnostic schedule
•Day 0: start of culture only
•Day 1: result of primary culture of specimen on BA, EA/URI, expedition of all negative results,
pathogen testing •Day 2: expedition of positive results, if bacterial susceptibility is sufficient
(if not, à more tests)
•Day 3: expedition of remaining results

Result interpretation, prevention and treatment of UTIs



Urine – result interpretation
•There is no common flora, nevertheless, in elderly often asymptomatic bacteriuria (ABU), it is not
necessary to treat it
•Differentiation of contamination, but also colonisation (especially in cathethrized patients) is
often very difficult, often possible only based on clinical situation (the microbiology finding
itself is not sufficient)
•Among pathogens, the most common is Escherichia coli, more Enterobacteriaceae, yeasts,
enterococci, Streptococcus agalactiae, Staphylococcus saprophyticus etc

Interpretation of urine examination I
lWhen we find one microbe, it is valid that
lQuantity over 105 microbes in 1 ml is considered likely uroinfection. In elderly it nevertheless
might be a colonization
lQuantity 104–105 is borderline. It is not sure whether the specimen was taken properly (e. g. in
babies) it is rather considered a contamination. It is rather important in men and children.
Antibiotic susceptibility is nevertheless tested
lQuantity < 104 is considered a contamination
lNot valid for punctured and cathethrized urine

Semiquantitative urine evaluation at finding one microbe
Number of colonies
Number of CFU
(bacteria) in 1 µl of urine
Number of CFU
(bacteria) in 1 ml of urine
Evaluation (valid for 1 bacterium)
Less than 10
Less than 10
Less than 104
Contamination
10–100
10–100
104–105
Borderline
More than 100
More than 100
More than 105
Infection

Interpretation of urine examination II
lIn case of finding two microbial species approx.:
lQuantity < 105 is evident contamination
lQuantity > 105 is borderline (unsure)
lIn case of finding three microbial species:
lNearly always we take it as contamination
lException: one microbe in quantity > 105, two other microbes < 104 à first microbe is considered
pathogen, the other two contamination
lIn practice we also take into account what species of microbes do we have (staphylococci use to be
taken less seriously)

Asymptomatic bacteriuria (ABU)
lOnly real actual infection that is causing the problem should be treated – not the mere presence
of bacteria in urine (particularly in older people),
lHowever, there may be exceptions:
lpregnant women – we treat even ABU because urinary infection can become a focus of a vaginal
infection à infection during delivery
lor some other risk situations, e. g. a person with immunodeficiency; here also that bacteria might
be the source of infection of other organs

Treatment of UTI
lFor uncomplicated community (= not hospital) cystitis sometimes plant extracts (cranberries) are
sufficient.
lAs to antibiotics, for cystitis suitable nitrofurantoin is suitable (does not concentrate in the
blood, but in the urine). Another option is to co-trimoxazole, amoxicillin, second generation
cephalosporins, doxycycline, and more
lFor hospital cystitis treatment should be chosen according to the susceptibility (but this is
useful even for outpatients)
lFor pyelonefritis (inflammation of the renal pelvis) the antibiotics must penetrate not only into
the urine, but also into the renal tissue. Nitrofurantoin or norfloxacin is therefore not
applicable. Targeted treatment for the causative agent is used.

Prevention of UTIs
lVery effective preventive techniques:
–To urinate after sexual intercourse (especially in women)
–To prefer hormonal contraception to barrier methods
–Change frequently menstrual devices
–Do not use spermicide gels, creams, gels and perfumed napkins
lCompletely wrong and dangerous techniques:
–Excessive hygiene
–Overuse of so called disinfection gels and soaps
–Frequent bathing in bathing foams
•According to the „Recommended guideline for antibiotic treatment of community  infections of
kidneys and urinary tract infections in the primary care“
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Infection of reproductive organs – introduction



Importance of this group of infections
lInfections of sexual organs are also quite frequent infections
lThe problem is that it is difficult to assess how frequent they really are. Ill people often try
self-treatment and remain hidden to the healthcare, because they are ashamed and they have shame to
speak about it (including with a doctor)
lAnother problem is difficult implementation of effective measures for treatment and prevention.
Also in diseases where sexual intercourse does not play the main role (e. g. vaginal mycoses) it is
useful to treat both (all) partners

Normal situation of genital organs
lIn normal situation there are no microbes:
–In females in uterus, tubas, ovarias
–In males in prostate, ducts, testes
lSpecific normal flora is in vagina (lactobacilli, some more aerobic and anaerobic microbes). Also
microflora of distal part of urethra is partially specific
lVulva is the borderline between vaginal and skin flora
lIn males also prepuce bag is specific is specific, besides skin flora there are also e. g.
non-pathogenic mycobacteria etc.

Classification of sexual infections
lThe classic sexual diseases are transmitted almost exclusively by sexual way. They are a subject
of registration and reporting under the special laws. For us, this includes primarily gonorrhoea
and syphilis
lOther infections of genital organs are those that affect the sexual organs, but sexual
transmission is not the only or even the most important
lThere also exist infections transmitted sexually, but not affecting directly sexual organs
(hepatitis B, AIDS, etc.)
•There exists the term "sexually transmitted infections" – STI (formerly STD – sexually transmitted
diseases). The content of the term is rather changeable by its user.
uuseuser.vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv

Classical sexual diseases
Gonorrhoea
Neisseria gonorrhoeae („gonococcus“)
Common also in Europe
Syphilis (lues)
Treponema pallidum
Lymfogranuloma venereum
Chlamydia trachomatis serotypes L1, L2, L3
Exceptionally also in Europe
Chancroid (ulcus molle)
Haemophilus ducreyi
In Europe rare, from other countries
Granuloma inguinale
Klebsiella (ex: Calymmatobacterium) granulomatis

Other agents of sexual infections – 1
•Human papillomavirus (related to cervical carcinoma – almost types 16 and 18, other types –
causing condyllomata acuminata etc.)
•Herpes simplex virus type 2, eventually also type 1
•Virus of molluscum contagiosum
•Chlamydia trachomatis – serotypes D to K
•Ureaplasma urealyticum, Mycoplasma hominis and more urogenital mycoplasms •Gardnerella vaginalis,
Mobilluncus mulieris, anaerobic bacteria (bacterial vaginosis – more later)

Other agents of sexual infections– 2
•Enterobacteriaceae, streptococci, enterococci, staphylococci and more agents of so called areobic
vaginitis
•Yeasts especially of genus Candida
•Trichomonas vaginalis
•Pubic lice also maybe classified here, although directly reproductive organs are not attacked

Interpretation of „positive“ findings
•Like the other places with normal microflora (intestine, the oral cavity) vagina can also be
considered an ecosystem. Its stability is influenced both by microbes and the host-side factors •In
many cases the culture positivity itself is no reason to treatment, what's important is the
clinical context. This concerns in particular the anaerobic bacteria, gardnerel, urogenital species
of Mycoplasma and Chlamydia •Microscopy is often useful for the interpretation. Unlike the culture
we see ratios of bacteria

Bacterial vaginosis (BV)
lBacterial vaginosis is a condition where the normal vaginal flora in the vagina is diseased and
vagina contents larger amounts of Gardnerella, Mobiluncus, and anaerobic bacteria. All of them may
be in the vagina also normally, but usually not so many
lWe can't determine a unique causative agent
lAlmost no leucocytes are present. Some bacteria blocate their migration to the inflammation site.
On the other hand, In microscopy  we see epithelial cells covered with bacteria – clue cells
lTreatment: metronidazole, probiotics

Nugent scoring system
lSome laboratories use microscopy of vaginal smear for counting of so called Nugent score. Score
over 7 is nearly sure for vaginosis
lNugent score is based on „morphotypes“ = „bacteria that look like…“ – there exist three typical
morphotypes that are counted:
lGardnerella/Bacteroides morphotype (slender, rather Gram-negative or Gram-variable straight rods)
lMobilluncus morphotype (slender curved rods)
lLactobacillus morphotype (long, robust, rather Gram-positive rods) – unlike the previous two, the
presence of this morphotype speaks against and not for the diagnosis of bacterial vaginosis
•Note: Nugent scoring systém can only show us cases of classical bacterial vaginosis. Nevertheless,
there also exist cases of a simillar disorder, but just different clones of lactobacilli (and not
Gardnerella etc.) present. Obviously, in these cases the Nugent score is low, and such a case
cannot be distinguished by microscopy.

Nugent score counting in the original article
Nugent original.gif


Simplified counting “+“ to “++++“
•+ = not in every field
•++ = in every field, but not frequent
•+++ = in every field, frequent
•++++ = very frequent, first object you see when looking to the microscope

Nugent score counting
•Due to the fact that it is a microscopic and not the culture proof, we work with so-called
morphotypes. For example, bacteria belonging to the "morphotype Lactobacillus" may no
Lactobacillus, but it is very likely
•Morphotype Gardnerella/Bacteroides: not present = zero point, + = one point, ++ = two points, +++
= three points, ++++ = four points
•Morphotype Lactobacillus: the contrary: not present = four points, positivity ++++ = zero points
•Curvet Gram-labile rods: none = 0 points, + or ++ = one point, +++ or ++++ = two points

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Aerobic vaginitis (AV)
lBesides bacterial vaginosis there also exists classical (i.e., containing leucocytes) bacterial
vaginitis (colpitis; however, the concept of vaginitis, an incorrect combination of Latin and
Greek, unfortunately took and used)
lHowever, it is very difficult to distinguish the agent of the inflammation from the accidental
discovery or colonization of the vagina
lMost commonly we find Enterobacteriaceae, enterococci, Streptococcus agalactiae, Staphylococcus
aureus
lTreatment depends on the presence of symptoms, with the exception of Streptococcus agalactiae
(outside of pregnancy is recommended rather woman healing due to the transfer to the newborn; in
pregnancy itself we do not treat, but the delivery is protected)

Sampling and examination in genital infections



Possible specimens in genital infections – anatomic classification
•Vaginal swab – usually from the rear side of vagina, using gynaecologic mirrors, must not be
contaminated by the microbial flora of the vulva
•Cervical swab also using gynaecological mirrors
•Urethral swabs in both genders
•Swab from penis, prepuce, glans in men
•Ejaculate (or swab from ejaculate)
•Swab from labia in females
•Invasive specimens (content of cyst etc.)

Possible specimens in genital infections – according to the causative agents
•Amies swab – for aerobic bacteria, Gardnerella, anaerobic bacteria, event. also urogenital
mycoplasms (some laboratories use special media for mycoplasms) •Dry swab is almost used for
non-cultivation detection of antigens and DNA, i. e. in chlamydias, papillomaviruses etc.; if we
wish to have a specimen from deeper layers of the mucosa, we would use a brush •E-swab may
eventually replace both previous swabs (as the producer says, it enables both culture and PCR)
•C. A. T. swab is for yeasts and Trichomonas
•Smears may be sometimes very useful
•Clotted blood is used for antibody detections (e. g. syphilis)

Smears from vagina or urethra
•It is a situation where the clinician directly makes a smear of secretions on the slide. Caution –
If the slide is not sterile, the swab should not be used for culture
•We evaluate both quantity of individual formations, and also final appearance of the preparation.
According to communication, it is eventually possible to coung Nugent score of BV
•In gonorrhoea we rather send urethral and cervical swabs, an usually only one slide to Gram stain.
More in the material to Neisseria

Classical „mikrobial appearance of vaginal microflora“ (MAVM)
•A classic, recently already less commonly used variant of microscopy in gynaecology used to be
microbial appearance of vaginal microflora
–They used to send two slides with vaginal smears
–On was Gram stained for bacteria, but also epithelial cells, WBC-s, yeast cells etc., the other
one was Giemsa stained (mainly for Trichomonas)
–Indivitual formations were evaluated (+ to ++++) and finally all smear was graded as a Roman
number I to VI (sometimes with sub-types, e. g. IIa2)
•Following pictures show some possibilities of vaginal smear results
•

027a.JPG [USEMAP]
End