0095-1137/79/06-0681/07$02.00/0
Diagnosis of
Mycoplasma hominis Pyelonephritis
by
Demonstration
of Antibodies
inUrine
A. C.THOMSEN`* ANDH. O.LINDSKOV2
InstituteofMedical Microbiology, University ofAarhus,'andClinicalChemistry Departmentand Surgical University Clinic 1,2AarhusAmtssygehus, Denmark
Receivedforpublication22March1979
To evaluate thediagnostic significance of the demonstration in urine of anti-bodiestoMycoplasmahominis, 1,000samples of urine with morethan5 leuko-cytesperhigh-power fieldwereserologicallyinvestigatedby indirect
hemagglu-tination, usingglutaraldehyde-fixederythrocytescoated with M. hominis antigen. Thesampleswere collected from 702 patients. Antibodiesweredemonstrated in
theurine of ninepatients,allof whom had signs ofacuteattack of pyelonephritis. Insevenofthesepatients, characterized by mildormoderateclinical signs and
absence of lower urinary tract symptoms, bacterial causes were not observed,
whereasM.hominis organismswereisolated from theupperurinarytractinmost
casesand from the bladder urine inall cases.Intwopatients, characterized by
severe clinical signs and presence of lower urinary tract symptoms, both M. hominis and bacteria were isolated from the upper urinary tract and bladder urine. Thedemonstration of antibodiestoM.hominis in urine is of highdiagnostic valueastheywereonlyobserved in patients in whom M. hominis infection in the upperurinarytractwasevidentorlikely and only in thepresenceofclinical signs
ofacuteattacks ofpyelonephritis.
Mycoplasmahominis has been isolatedfrom the upper urinary tract of patients with acute pyelonephritis (16) and acute exacerbation of chronic pyelonephritis (15), in several cases without a bacterial cause of the disease being
demonstrated. M. hominis has not been culti-vated from the upperurinary tract ofpatients with noninfectious urinary tract diseases (14, 16). Theoccurrence of M. hominisin theupper urinary tractwasfrequentlyfollowed bya
sig-nificantserum antibody response and, further-more, by the appearance of urine antibodies,
whichhasbeenobservedonlyin cases of upper urinary tract infections by M. hominis (17).
Thus, it seemsreasonable to presume that M. hominis may be a cause of acute episodes of pyelonephritis in humans and that the demon-stration of antibodies inurine is of diagnostic significance.
Thepurposeof thepresentstudywas toverify
whetherpatientswith urine antibodies had M. hominisinfection in theupperurinarytractand to study the clinical signs and course and the diagnosticpossibilitiesofthe infection.
MATERIALS AND METHODS
Patients. A total of 1,000 urine specimens, each containing more than 5 leukocytes per high-power
field(x400 magnification)aftercentrifugationat1,500
rpmfor 5 min, were examined for antibodiestoM.
hominis. Thespecimensoriginated from702patients, viz., 250 men and 452 women whose ages averaged 66 (18to82)and 59 (16 to 78) years, respectively. Most of the cases were outpatients received for ordinary examinations, and the diagnosis and point in the courseof illnesscould not be established.
Only patients showing antibodies in the urine were studied further. Athoroughclinicalexamination was
performed;severalsamples of urine from the bladder
and, asfaraspossible, from the upper urinary tract werecollectedfor cultivation ofmycoplasmaand
bac-teria,andserumsampleswereobtained.
The diagnosis of acutepyelonephritiswasbasedon the simultaneous occurrence ofacute lumbar pain, elevatedbody temperature, andmore than five
leu-kocytesperhigh-power field incentrifuged urine, fol-lewedbycompleterecovery.
Acuteexacerbation ofchronic pyelonephritis was diagnosed by the presence ofacutelumbarpain and elevated body temperature inpatients withat least threeof thefollowing'signsof chronicpyelonephritis: repeated attacks ofurinary tract infections, pyuria, impairedrenal function, anddemonstrationof chronic
interstitial nephritis by histological examination or
irregularoutline ofthekidneyassociatedwithdilated calyces byradiography.
Sampling.(i) Bladderurine.Urine from the blad-der was obtained before the onset of the attack in threecases(Table 1), andinallcasesitwasobtained at several times during the attack and at
approxi-mately monthly intervals during the3 monthsafter the attack. Theurine wascollected asclean-voided,
midstream urineorby catheterization. 681
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682 THOMSEN AND LINDSKOV
(ià) Ureteric urine. Ureteric urine wascollected from seven patients, in all cases during the attack (Table 1).After the introduction ofacystoscopeand washing of the bladder with water, urine from the upperurinarytract wascollectedthroughbilaterally placed ureteric catheters. The urine was separated intoinitial,middle,andterminalportions.
(iii) Serum. Serumsamples were collected inall cases atseveral times during the attack and at
ap-proximatelymonthly intervals duringa3-month pe-riodafter the attack.
Allspecimenswerestoredat4°C before examina-tion, whichwascarriedoutwithin24h ofcollection.
Storageat roomtemperatureduringcollection, trans-portation, and examination didnotexceed 2 h. After examination thespecimenswerestoredat-20°C.
Cultivation and identification. All urine
speci-mensfrompatientsshowingantibodiestoM.hominis werecultivated formycoplasmaand bacteria withina few hoursaftersampling.
Mycoplasmawerecultivatedon amodifiedHayflick
medium incandlejars and in 95%N2plus5%C02and identified bythe growthinhibition test and indirect immunofluorescenceaspreviouslydescribed (15).
Bacteriawerecultivatedonblood agar,lactose
bro-mothymolblue agar, and chocolate agar, in both
at-mosphericairand 100%H2.Thebacteriawere identi-fiedaccording tocolonymorphology,Gramstaining,
andbiochemicalproperties.
Demonstration of antibodies to M. hominids. Urinespecimenswereexamined within 24 h of sam-pling for antibodies to M. hominis by the use of indirecthemagglutinationperformedwith glutaralde-hyde-fixed sheeperythrocytes (1)andpooled antigen
(17). The titration was carried out in a microtiter system(5).
(i) Fixation oferythrocytes with glutaralde-hyde.Sheeperythrocytescollected in Alsever solution werewashed four times withphosphate-buffered
glu-cose,pH7.2, andresuspendedtoa20%suspensionin
phosphate-buffered glucose. Portions of this suspen-sionweremixed withanequalvolume of 0.2%
glutar-aldehyde inphosphate-buffered glucoseandincubated at37°C for15min ina waterbath. After thisfixation,
theerythrocyteswerewashed fivetimes, resuspended to a10%suspensioninnormal salinecontaining 0.1% sodiumazide,and storedat4°C.
(ii) Production ofantigen. Three strains of M.
hominis,allisolated from the upper urinarytractof patients withacutepyelonephritis (16),wereusedas antigen. Thestrainshaddifferenttiters when titrated against M. hominis (PG21) rabbit hyperimmune se-rum (17) by indirect immunofluorescence (10). Each strainwascultivated in600ml ofmodified Hayflick medium (2), harvested by centrifugation at 14,000 x g for1h,andresuspended in5mlof normal saline. This suspensioncontainedapproximately1010 colony-form-ing units of M. hominis per ml and was stored at
-20°C.
(iii)Coating with antigen. Glutaraldehyde-fixed
erythrocyteswerewashed twice anddiluted in
phos-phate-buffered saline, pH 7.0, to a 20% suspension. Primarily,erythrocyteswerecoated with each antigen
separatelyby mixing2mlofantigen suspension with 5ml of the 20%erythrocytesuspension. This mixture
wasincubated for18h at37°C,washed three times, andresuspendedto a 20%suspensionin phosphate-bufferedsalinecontaining50%glycerine.
(iv)Titration and poolingoftheantigen. Eryth-rocytescoated with twofold dilutions of eachantigen
werewashed threetimes,resuspendedin
phosphate-buffered saline to a 2% concentration, and titrated
separately by indirect hemagglutination against M.
hominis (PG21) rabbit hyperimmune serum in the microtiter systemasdescribed for fresherythrocytes
(5).Phosphate-bufferedsaline containing 1% normal rabbit serum wasused as diluent. According to the results of these titrations, the three antigens were
diluted in normal salineto the optimum concentra-tions.
Equal volumes of these antigen dilutions were mixed.Glutaraldehyde-fixed erythrocyteswerecoated with this antigen pool, titrated, and diluted to the optimum concentration of the pooledantigen as de-scribed above.
(v)Titration ofspecimens.Abatch of
glutaral-dehyde-fixed erythrocytes coated with the final anti-genpool dilution was stored at 4°C and used for the
detection of antibodies in specimens from patients duringaperiod of6months. Titrations of the samples wereperformed as described above for antigen.
Thestability ofglutaraldehyde-fixedM. hominis-coatederythrocyteswaschecked bydailytitration of thesamehyperimmuneserum.The reproducibility of thetest wasstudiedby repeated titration of positive
specimens atmonthly intervals. The specificity was demonstrated by titration of sera containing antibod-iesto otherhuman mycoplasmas.The sensitivity of thetestusing glutaraldehyde-fixedand fresh erythro-cytes(5)wascompared.
RESULTS
Stability, specificity, and sensitivity of
glutaraldehyde-flxed,
M.hominu8-coated
sheep erythrocytes. Glutaraldehyde-fixed,M.
hominis-coated erythrocytes were stable for 6 months afterproduction,andduringthisperiod
the indirect hemagglutination test was repro-ducible as determined by repeated titration of serumspecimens. Urine antibodies were
gener-ally not preserved after storage at -20°C and were recovered from only 1 of12positive sam-plesby repeated titration.
Thespecificity and sensitivity of thetest were satisfactory. Glutaraldehyde-fixed erythrocytes
coated withM. hominis did not react with hu-man sera containing antibodies to seven other human Mycoplasma species, and theyshowed titersashigh as
those
of fresherythrocytes.
Patients with urine antibodiesto M. hom-inis. Nine patients, four menand five women, had urineantibody titers of 216 to M.hominis. All nine had acute attacksofpyelonephritis; six had acute pyelonephritis, and three had acute exacerbation of chronic pyelonephritis. Cases in which bacteria could not be isolated from the upperurinary tract andwith less than 104 of a
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mixed floraof bacteriaperml inbladder urine duringand after the attackwereconsiderednot tobecausedbybacteria.
Basedonclinicalsigns(pain, tenderness,lower
urinarytractsymptoms,bodytemperature,and number of leukocytes in the urine), the nine patientscould be divided into threegroups
(Ta-ble1).
(i) Mild acute attacks ofpyelonephritis. Twopatientshad tenderness in the
costoverte-bral angleonone side. Pain and lowerurinary tractsymptomswereabsent. Thetemperatures were37.9and38.0°C.The number ofleukocytes
inurinewasnothigherthan 30perhigh-power
field. Bacterial causes were not demonstrated. Inone patient (no. 634), ureteric urine from
theaffected side contained M. hominis and
an-tibodiestoM. hominis. Uretericurine from the
other side was normal. In bladder urine, M.
hominisand antibodiesweredemonstrated
con-stantly during the disease. Serum antibodies
were not demonstrated. After treatment with
tetracyclines, the symptoms, pyuria, and the yieldof M. hominis and antibodiesdisappeared, and relapse was not observed for the next 3
months.
Thesecondpatient (no. 611,showninFig. 1)
wasnotsubjectedtoureteric catheterization. In bladderurine, M.hominis and antibodies were
presentconstantly duringthe attack. Serum
an-tibodies were not detected. Without antibiotic
treatment, the symptoms, pyuria,and urine
an-tibodies disappeared, but M. hominiswas still
isolated from the urine. After 13 days without symptoms,the patientdiedfrom pulmonary
em-bolism. Atautopsy,theaffectedkidney revealed, close to the renal pelvis, asmall abscess from
which M. hominis, but no bacteria, could be
isolated. Otherpartsof the kidneywerenormal
macroscopically, and M. hominiswasnot culti-vated fromthesepartsor fromtherenal pelvis.
(il) Moderate acute attacks of pyelone-phritis. Five patients showed unilateral lumbar pain and tenderness, absence of lowerurinary
tract symptoms,temperaturesof 38.2to38.60C, and 20to40leukocytesperhigh-powerfield in
the urine. Bacterial causes were not demon-strated.
Ureteric urine obtained from four patients contained, onthe affected side, M. hominis in
threecases(no. 10, 136,and683)andantibodies
in allcases. One patient (no. 154) wasnot sub-jectedtouretericcatheterization. Bladder urine fromonepatient(no. 136), examined beforethe appearance ofsymptoms, did notcontain anti-bodiestoM.hominis(Fig. 2). Duringtheattack, M. hominis and antibodies were demonstrated
in bladderurine of all five patients; intwocases
(no. 10 and 683), the antibodies were not ob-served constantly (no. 10, shown in Fig. 2). A significantrise inserumantibodieswas
demon-strated in four patients (no. 136, [Fig. 2], 154, 683, and 701), whereas in one patient (no. 10)
antibodies, althoughpresent,didnotchange
sig-TABLE 1. Clinicalcharacteristics, occurrence of M. hominis in urine, and occurrence of antibodies to M. hominis in urine and serum in nine patients with acute attacks ofpyelonephritis
Clinicalcharacteristics Occurrence ofM.hominis Antibodies to M. hominis (highest titer)
Group Patient Lower Uretericurine
Urine'
Serumno. Lum- Ten- u Body Leuko-
Blad-bar der- urinary temp cytes inderur-
Conva-pain ness syp-((C) urine' ine Right Left Before During After Acute descent
toms aide aide
phasephs
I 634 - + - 37.9 30 + + - NTc 32 <2 <2 <2
611 - + - 38.0 20 + +d NT NT 512 <2 <2 <2
Il 10 + + - 38.4 40 + - + NT 1,024 4 128 256
136 + + - 38.2 20 + _ + <2 128 <2 <2 512
683 + + - 38.2 40 + + - NT 128 4 32 256
701 + + - 38.6 40 + - - NT 128 <2 <2 128
154 + + - 38.5 20 + NT NT NT 256 2 64 512
III 251 + + + 39.3 200 + + - <2 64 NT 16 256
414' + + + 39.8 300 + - + <2 64 2 32 512
aInrelationtothe attack.
bNumber perhigh-power fieldincentrifugedurine.
'NT,Nottested.
dCultivated
from
thekidneyatautopsy.'Thispatient hadtwoattacks(seeFig. 4).
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684 THOMSEN AND LINDSKOV
MYCO-LEU -PLAS-KO -MAS CYTES
10' >40-10'
40-10' 30-10' 20-10'>
10-2 3 L WEEKS
5 6 7 8 9 10 Il 12
FIG. 1. Apatient (no. 611) witha mildacuteattackofpyelonephritis. Symbols: Occurrence of symptoms (xxxxx); leukocytes incentrifuged urine per high-power field (vertical bars); M. hominis, colony-forming units permilliliterofurine(-);M. hominisantibody titers in urine(*)andserum (A).
WEEKS
FIG. 2. Two patients (no. 10 and 136) withmoderate acuteattacks ofpyelonephritis. Symbolsasin Fig. 1.
nificantly. In two cases (no. 136 and 683), urine antibodies were demonstrated before serum an-tibodies (no. 136, shown in Fig. 2). After
treat-mentwith tetracyclines, symptoms, pyuria, M. hominis, and urine antibodies disappeared in all five cases, and relapse did not occur in the following 3 months (no. 10 and 136, shown in Fig. 2).
(iii)Severe acute attacks of pyelonephri-tis. Two patients suffered from intense unilat-eral lumbarpain, tenderness, and lower urinary
tract symptoms, and temperatures of 39.3 and 39.8°Cand200 to 300leukocytes per high-power field in the urinewererecorded. Mixed infections
with M. hominis and bacteria were demon-strated.
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ANTIBODIES TO M. HOMINIS IN URINE 685 In one patient (no. 251, shown in Fig. 3),
uretericurine from theaffectedsideyieldedM.
hominisandantibodies. Bladderurine examined before the onset of the disease did notharbor antibodies, but urine sampled duringthe attack contained M. hominis and>10i Klebsiella pneu-moniae per ml, and antibodies to M. hominis occurredperiodically. Asignificantserum anti-body response to M. hominis was demonstrated. Aftertreatmentwith ampicillin, thepatient im-proved, but symptoms were still present; the numberofleukocytesin the urinedecreasedto 20 per high-power field. K.pneumoniae could notbedetected,but M. hominis and antibodies werestillpresentinthe urine. At this time, the examinations were stopped as the patient suf-fered from cancer of the colon in the terminal stage.
The secondpatient (no.414,shown in Fig. 4) hadtwo acuteepisodes of pyelonephritis during a 2-monthinterval. During the first episode, a bacterial causecouldnotbe demonstrated. Ur-eteric catheterizationwas notperformed, butM.
MYCO- LEU
-PLAS- KO-MAS CYTES
10' >40
10' 40
10' 30 10' 20 10'>
10-w
FIG. 3. Apatient (no.251)withasevereacutec
* i1 * T I
2 3 4 5 6 7 8 9
hominis and antibodies were demonstrated in the bladderurine, which contained25leukocytes perhigh-power field. A significant serum anti-body response toM. hominis developed. This episodewas nottreated, butsymptoms,pyuria, and antibodiesdisappeared; however, M. hom-iniswasstillpresentin the urine. Two months later, the patient hadasecondattack, this time the attack was accompanied by severe symp-toms. In ureteric urine from the affected side and in bladder urine, M. hominis, antibodies, and Escherichia coli were recovered. After treatmentwithtetracyclines,thesymptoms dis-appeared, and the urine returnedtonormal.
DISCUSSION
The resultsof thepresentandprevious studies oncultivation (15, 16) andantibody demonstra-tion (17) indicate that M. hominis isa cause of some acuteepisodes ofpyelonephritis.
In the presentstudy, urine antibodies to M. hominisweredemonstrated in ninepatients,all
SOLATED
ANTI
-ETERIC CATHETERIZATION BOY
4096 2048 1024 512 256 128 64 32 16 <16
2 3 4 5 6 7 8 9 10 Il 12
FEEKS
attackofpyelonephritis. Symbols as in Fig. 1.
URETERIC CATHETERIZATION
t E. COLI ISOLATED
ANTI-tEtRACYCLINE
BsODY
4096 2048
- 1024
_,~~~~~~~---
- 512256
,v«Ft - ~~~~~~~~128
16 <16
WEEKS
FIG. 4. Apatient (no. 414) with amoderate and a severe acute exacerbation ofchronicpyelonephritis. SymbolsasinFig.1.
PATIENT
NO 251 971
YEARS
f+
K.PISPEUMONIAE
I' Ç AMPICILLINf URI VOL. 9,1979
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686 THOMSEN AND LINDSKOV
of whom showedsigns ofacute attacks of pye-lonephritis.In sevenofthepatients, abacterial causeof the diseasewas notobserved, whereas M.hominisandantibodies toM. hominiswere demonstratedintheupperurinarytractof five andinbladder urine ofallseven cases. Further-more, in four of thesepatients a significant
se-rumantibody responsetoM. hominisdeveloped. Intheremainingtwopatients,amixed infec-tion of M.hominis and bacteria occurred in the upperurinary tract,and antibodies toM. hom-inisweredemonstratedinureteric urine. Inone of thesecases,antibioticeradication of the bac-teriawasfollowedbyimprovement, butnot dis-appearance, of the clinical signs.The other pa-tient hadtwoattacks,the first of whichseemed to be caused by M. hominis and was cured
spontaneously butwas followed by continuous yielding of M. hominis in the urine.Duringthe secondattack, amixed infection of M. hominis and bacteriawasdemonstrated. Whetheran in-teraction between themicroorganismsoccurred isnotknown,but it has been demonstrated that rats experimentally colonized in the upper uri-nary tract with Mycoplasma arthritidis were moresusceptibletoE. coliinfection of the kid-ney (18).
Insome casesof pure M. hominisinfections,
theclinicalsignsweremild andserumantibody responses were notelicited, and in othercases
the clinical signs were moderate andregularly
followed byserumantibodyresponses. In exper-imental M. arthritidispyelonephritis(11), it has been demonstrated thatratswithlesions limited totherenalpelvis and associatedareasproduced
only urine antibodies, whereas rats with more
extensive lesionsproducedbothurine andserum antibodies. Inexperimentalupperurinarytract infection causedby E. coli (3), itwas observed that rats with pyelitis did not develop serum antibodies, whereasratswithpyelonephritis did. Allpatients withpureM. hominisinfections werecharacterizedby absence of lowerurinary tractsymptoms,whichisin accordancewith the commonopinion that M. hominis does not cause urethritis or cystitis. In contrast to pure M. hominis infections, mixed infections including bacteriawerefollowedby severe clinical signs as
weil
aslowerurinary tractsymptoms.Withouttreatment, the course of M. hominis infections seems to be self-limiting. However, the two untreated patients continued to harbor M.hominis in the urinary tract, in one case in the kidney. In the other case a relapse with mixed infection ofM. hominis and bacteria in the upper urinary tract occurred. In patients with bacterial pyelonephritiswho receive no or insufficient treatment the bacteria may remain inthekidney andcause relapse (19).
After treatment withtetracyclines, cessation ofsymptoms anddisappearance of M. hominis andantibodies occurred within2or3daysinall of theseventreatedpatients,and fora3-month periodnorelapsewasobserved.
Fordiagnosticpurposes,it haspreviouslybeen established that M. hominis may be cultivated frombladderurine withoutbeingpresentin the upper urinarytract (16). Cultivation from ure-teric urinerevealing quantitative equal growth
of M. hominis in three subsequently collected
samplesindicatesinfection,but theprocedureis laborious.
The demonstration ofserumantibodies isof value in those cases in which a significant
re-sponse occurs, but, as shown in the present study,thisusuallydidnotdevelopuntil2weeks after theonset of disease. Furthermore, itwas
demonstrated in the present and in previous
studies(17) thatpatientsmayhave M.hominis infection of theupperurinary tract not associ-ated witha serumantibodyresponse,and it has also been established (17) that patients may developserumantibodiestoM.hominisduring
acutepyelonephritis without M. hominisbeing
isolated from theurinarytract atall.
Thedemonstration ofurine antibodies seems tobeofgreatvalue. All patientsshowing urine antibodies to M. hominis had signs of acute episodes ofpyelonephritis andone or more signs indicating infection of theupperurinarytract by M. hominis alone or combined with bacteria. Urineantibodiesseemonlytobepresentduring infections associated with symptoms. In cases where the urinewas examined before the onset of the disease (no. 136, 251,and414), antibodies couldnotbe demonstrated, and theurine anti-bodies disappeared with the clinical signs of disease.Duringanattack, thepresence of urine antibodies may fluctuate and periodically dis-appear (no. 10 and 251). This fluctuation may possiblybe duetoopeningand closing of lesions or toblockingofthe antibodiesbymycoplasmas.
The urine antibodies were not stable when storedat-20°C. Theinfluence oftemperature,
pH, and content ofalbumin on the stability of theseantibodies have to be studied.
In aprevious study(17),urine antibodieswere demonstratedinonly4of 10patientswith acute episodesofpyelonephritis andM.hominis infec-tion in the upperurinary tract.However, only one sample of urine from eachpatient was ex-amined, and thespecimens had beenstored at -20°C. Thus, it has not been established whetherM.hominis infection of the upper uri-nary tract may be present without shedding of urineantibodies, but the results of the present study emphasizethe needfor immediate exam-ination ofthe urine and the needinsome cases
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forcollectionofseveralurinesamples during the
attackin order todemonstrateantibodies. Theantibody level of diagnostic significance isdifficult to establish even when the examina-tions are performed with one reproducible method. In this study, the demonstrated levels varied greatly, and they were not correlated to the severity of the infection (Table 1). However, a urine antibody titer of -32 in one or more sampleswasdemonstrated toimply M. hominis infectionof the upper urinary tract.
The urine antibodies seem to originate from the kidneys. They were detected in ureteric urine, but only from the infected side, and in
severalcasesthey were present in urine but not in simultaneously collected serum. They oc-curred only during the acute attack and pre-ceded the development of serum antibodies. This is inaccordance with theobservations of
experimentalM. arthritidispyelonephritis (11). Inbacterialpyelonephritis, urine antibodies oc-cur both in natural (20) and in experimental
infections (4, 7). Under theexperimental condi-tions,they seemed tooriginatefrom thekidneys and atleast in some cases to be secretory im-munoglobulin A antibodies (12). Moreover, the occurrencein urine of bacteria coated with an-tibodies issupposed to imply an upper urinary tractinfection (13).
Forserological diagnosis of M. hominis infec-tion, some recent studies (8) have indicated that indirect hemagglutination is more reliable than the tests for metabolism inhibition, immunoflu-orescence, and growth inhibition. The use of
glutaraldehyde-fixed sheep erythrocytes is rec-ommended for indirect hemagglutination test-ing.Antigen-coatederythrocyteswerestable for 6 months, and reproducible results were ob-tained during this period. The specificity and
sensitivity were satisfactory, as has also been shown in other studies on M. hominis (6), M.
pneumoniae (9), and bovinemycoplasmas (1).
ACKNOWLEDGMENT
Wethank H.Ern0forexcellent adviceduringthisstudy. LITERATURE CITED
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