Table Of ContentWangetal.BMCInfectiousDiseases2013,13:36
http://www.biomedcentral.com/1471-2334/13/36
RESEARCH ARTICLE Open Access
Rapid screening for Chlamydia trachomatis
α
infection by detecting -mannosidase activity in
urogenital tract specimens
Ze-yu Wang1, Guang-yu Fu1, Shan-mei Wang2, Dong-chun Qin3, Zhong-quan Wang1 and Jing Cui1*
Abstract
Background: Chlamydiatrachomatis maycause multiple different urogenital tract disorders, but current
non-culture assays for rapidscreening of C. trachomatis typically use immunochromatography-based methods. We
established another new rapid non-culturemethod for detection of C. trachomatis based onthe measurement of
α-mannosidase enzymatic activity inurogenitaltractspecimens.
Method: Toevaluatetheperformanceofthismethod,α-mannosidaseactivitiesofC.trachomatisserotypeDstrain、
and29standardstrainsrelatedtoclinicalurogenitalpathogenswereinvestigated.Furthermore,553urogenitaltract
specimenswereusedforclinicalassaysviacellculturemethodandligasechainreactionmethod(LCR),adoptingan
expandedgoldstandard.
Results:OnlyC.trachomatiswaspositiveforα-mannosidaseactivityamongdifferenttypesofmicrobestestedinthe
research.Whenprostatefluidspecimens,whichhavesomeinterferingactivity,wereexcluded,thesensitivityand
specificityoftheenzymaticmethodwere91.8%(78/85)and98.3%(409/416),respectively.Therewerenosignificant
differences(P>0.05).
Conclusions:Theseresultsshowedthatα-mannosidaseactivitycouldbeutilisedasascreeningmarkerofC.
trachomatisinfection.
Keywords:Chlamydiatrachomatis,α-mannosidase,Activity,Goldstandard,Marker
Background of outpatient diagnosis, especially in terms of sensitivity,
C. trachomatis infection is the most common sexually specificity, time, and simplicity of operation. Currently,
transmitted disease (STD) in the United States [1]. non-culture assays for C. trachomatis screening typically
Mounting evidence has indicated that it not only evokes adopt immunochromatography-based methods. Tech-
nongonococcal urethritis (NGU), cervicitis, pelvic nologies based on chromogenic reactions of specific
inflammatory disease (PID), salpingitis, orchitis, and microbial enzymes have been widely applied in bacterial
epididymitis, but also increases risk of invasive cervical identification systems and chromogenic media [12-15].
cancer [2,3] and gives rise to serious reproductive However, no chromogenic assay for detecting C. tracho-
disorders such as infertility [4,5], miscarriage/premature matishasbeenmadeavailabletodate.
birth/missed miscarriage [6-9], and neonatal conjunctiv- Our previous findings [16] suggested that C. trachomatis
itis[10]. mighthaveveryhighα-mannosidaseactivity.Thepurpose
A large number of methods have been established for ofthestudywastoestablishanovelscreeningmethodfor
screening and diagnosis of C. trachomatis infection [11]. C. trachomatis infection without culture that would be
Nevertheless, few of these assays meet the requirements rapidandconvenientforuseinoutpatientclinics.
*Correspondence:[email protected]
1DepartmentofPathogenBiology,SchoolofBasicMedicine,Zhengzhou
University,Zhengzhou450001,China
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Wangetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Methods Specimens
Ethicsstatement This study evaluated 553 specimens from clinical patients
All patients were treated in accordance with the Helsinki attending the STD (257, 46.47%) and Gynaecology
Declarationontheparticipationofhumansubjectsinmed- (296, 53.53%) clinics at the First Affiliated Hospital of
ical research. Ethics approval for the study was obtained ZhengzhouUniversity(Zhengzhou,China)andtheHenan
from the First Affiliated Hospital Ethics Committee of Provincial People’s Hospital (Zhengzhou, China), respect-
Zhengzhou University (Approved No. 20100802) and ively.Forthe203malecases,threeurethraldischargespe-
Henan Provincial People’s Hospital Ethics Committee cimens (151 outpatients, 74.38%) or three prostate
(ApprovedNo.20100901). massage liquid specimens (52 outpatients, 25.62%) were
collected with sterile rayon swabs (Copan, Brescia, Italy).
Meanwhile, for the 350 female cases, three cervical secre-
Organisms tion specimens (232 outpatients, 66.29%) or three vaginal
Reference strains and cell lines were obtained from the secretion specimens (118 outpatients, 33.71%) were col-
organisationsshowninTable1. lected with sterile rayon swabs using vaginal forceps.
Table1Referencestrainsandcellline
Strainsorcellline Accessionnumbers Organizations
Acinetobacterbaumannii ATCC19606 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Candidaalbicans ATCC10231 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Candidaglabrata ATCC15126 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Candidaguilliermondii ATCC6260 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Candidakrusei ATCC14243 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Cryptococcusneoformans CMCC(F)D2q ChinaMedicalMicrobiologicalCultureCollectionCenter(fungi)(Nanjing,China)
Candidaparapsilosis CGMCC2.1846 ChinaGeneralMicrobiologicalCultureCollectionCenter(Beijing,China)
Candidatropicalis ATCC750 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
ChlamydiatrachomatisSerovarD VR-885 AmericanTypeCultureCollection(Manassas,USA)
Enterococcusfaecalis ATCC29212 HuankaiMicrobialSci&Tech.Co.,Ltd(Guangzhou,China)
Enterococcusfaecium ATCC700221 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Escherichiacoli ATCC25922 HenanProvincialInstituteofFoodandDrugControl(Zhengzhou,China)
Gardnerellavaginalis ATCC14018 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Haemophilusinfluenzae ATCC10211 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Klebsiellapneumoniae CMCC46117 TianheMicroorganismReagentCo.,Ltd(Hangzhou,China)
McCony CRL-1696 AmericanTypeCultureCollection(Manassas,USA)
Mycoplasmahominis ATCC15488 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Neisseriagonorrhoeae ATCC19424 HenanProvincialCenterforDiseaseControlandPrevention(Zhengzhou,China)
Pseudomonasaeruginosa ATCC25619 LandBridgeBiotechnologyCo.,Ltd(Beijing,China)
Proteusmirabilis CMCC(B)49005 HuankaiMicrobialSci&Tech.Co.,Ltd(Guangzhou,China)
Salmonellaenteritidis ATCC13076 LandBridgeBiotechnologyCo.,Ltd(Beijing,China)
Staphylococusaureus ATCC25923 HenanProvincialInstituteofFoodandDrugControl(Zhengzhou,China)
Staphylococcusaureus ATCC29213 CapitalInstituteofPediatrics(Beijing,China)
Staphylococcusepidermidis ATCC12228 HuankaiMicrobialSci&Tech.Co.,Ltd(Guangzhou,China)
Staphylococussaprophyticus ATCC49453 LandBridgeBiotechnologyCo.,Ltd(Beijing,China)
Stenotrophomonasmaltophilia ATCC17666 LandBridgeBiotechnologyCo.,Ltd(Beijing,China)
Streptococusagalactiae ATCC13813 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Streptococuspneumoniae ATCC49619 NationalCenterforClinicalLaboratory(Beijing,China)
Streptococuspyogenes ATCC19615 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Trichomonasvaginalis ATCC30001 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
Ureaplasmaurealyticum ATCC15531 HarmonyBiotechnologyCo.,Ltd(Shanghai,China)
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Three swabs collected for each specimen, wereno signifi- salt (J&K, Shanghai, China). Aliquots(50μl)ofsubstrate
cant differences in sampling link and randomly used with solutionandchromogenicreagentwereaddedsequentially
three methods. Noneofthe patients received anyantibio- into sample solutions (extracted from every swab sample
tics one week before sample collection, when samples with500μlsamplediluent)ortheaforementionedmicro-
weretakenbeforediagnosis. bial suspensions as well as the chlamydial suspension
mentioned in the section called limit of detection (LOD)
Media,cultureandinoculation oftheenzymaticmethod.Theresultwasconsideredtobe
Liquid media A (LMA) and liquid media B (LMB) were positive (OD ≧ 0.150) if the colour changed from
512
prepared for mycoplasma culture. The components of colourless to red or brown after 15 min of incubation at
LMA were shown in Table 2, but LMB consisted of 37°C; otherwise, the result was considered to be negative
50mg/lphenolredbesidesLMAcomponents.Themedia (OD < 0.150). The suspensions containing bacteria or
512
were inoculated with both Ureaplasma urealyticum and cells was centrifuged at 5,000 rpm for 5 min, and then
Mycoplasma hominis and analyzed by using the colour- OD valueofthesupernatantsweremeasured.
512
changingunit(CCU) method,as previouslyreported[17].
Once the concentrations of mycoplasma reached 106
CCU/mlinLMB,theculturesofLMAshouldbeimmedi- LODoftheenzymaticmethod
atelystoredat4°C. McCoy cells infected with C. trachomatis serovar D were
Trichomonas vaginalis was obtained after 24 h stored at −80°C, and frozen-thawed twice to obtain chla-
incubation at 37°C in Trichomonas medium (Oxoid, mydialsuspensionbeforeuse.Serial10-folddilutionsofthe
Basingstoke, UK) supplemented with 8% horse blood chlamydialsuspensionwereinoculatedinsixduplicateinto
and 1,000 units/ml penicillin sodium and 500 mg/ml wells (100 μl/well) of cycloheximide-treated McCoy cell
streptomycin.The collectionwasstored at 4°C. monolayers that had been incubated with MEM medium
All other micro-organisms exceptC. trachomatis used in (Gibco, Grand Island, NY, USA) in a 96-well flat-bottom
the study were inoculated and cultured as described in microtiter plate (Nunc Inc., Roskilde, Denmark) at 37°C
Table 3. The collections described in Table 3 were resus- under 5% CO for 48 h. Inclusion body titers of the chla-
2
pended to 0.5 McFarland standards in a sterile solution mydialsupernatantwerequantifiedbytitratingthenumber
of0.9%NaClandthenstoredat4°C. ofinclusion-formingunits(IFU).Thecontentsofeachwell
were stained with a C. trachomatis direct fluorescent anti-
Enzymaticmethod body kit (Academy of Military Medical Science, Beijing,
The enzymatic method was based on the substrate of China) and examined by microscopy for IFU counts. The
α-D-mannosidase. The substrate solution contained averageIFUofeachdilutioncultureofthreereplicatewells
1.5 mg/ml 6-chloro-3-indolyl-α-D-mannoside (J&K, was taken as the concentration of C. trachomatis in the
Shanghai, China), 100 mM citrate buffer (pH 4.0), and corresponding dilution of the chlamydia suspension. Each
1% Triton X-100. The sample diluent was 0.9% NaCl. dilution culture of the other three replicate wells was
The chromogenic reagent contained 0.08% fast violet B examined via the enzymatic method after collection to de-
termine the LOD for C. trachomatis of the enzymatic
Table2CompositionofLMAforMycoplasma(perliter)* method. 10-1 u/ml α-D-mannosidase (EC3.2.1.24; Sigma,
USA) solution was 5-fold serially diluted to 10-4 u/ml.
Ingredient Concentration Ingredient Concentration
These enzyme solutions fold-diluted were detected via the
NaCl 6.4g Beefheartextract 7.2g
power enzymatic method to determine the LOD for α-D-
mannosidase.
CaCl 112mg Yeastextract 2.72g
2
power
Referencemethod
MgCl·6HO 80mg Peptone 8.0g
2 2
Cell culture and LCR method were used to evaluate the
MgSO·7HO 80mg Horseserum 200ml
4 2 clinical performance of the enzymatic method. The
KCl 320mg Penicillinsodium 1,000,000units
swabsusedforculture weredippeddirectlyintoinciden-
Na2HPO4·12H2O 122mg Ampicillinsodium 375mg tal transport medium (Copan, Brescia, Italy) and cul-
KHPO 48mg Vancomycin 40mg tured according to the aforementioned method. The
2 4
Cysteine 0.8g PolymyxinB 40,000units cultures were tested using a C. trachomatis direct
hydrochloride fluorescent antibody kit. LCR was carried out using the
Arginine 4.0g Nystatin 15,000USP LCx C. trachomatis assay (Abbott Laboratories, Abbott
hydrochloride units Park, Israel) according to the manufacturer’s instruc-
Urea 4.0g tions. Although culture method has a good specificity,
*AdjustedtopH6.25. its sensitivity may be influenced by various factors.
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Table3Themediaandculturemethodsforbacteriaand 97.6%) and 98.3% (95% CI, 97.1% to 99.5%), in the
candida meantime the sensitivity and specificity of the LCR were
Strains Media Temp. Time 95.3% (95% CI, 90.8% to 99.8%) and 100% (95% CI,
Bacteria 100% to 100%) (Table 6), respectively. Therewerenosig-
A.baumannii,E.faecalis, Bloodagarbasea 37°C 24h nificantdifferencesinperformancebetweentheenzymatic
methodandtheexpandedgoldstandard(P>0.05).
E.faecium,E.coli,K.
The result of the chlamydial suspension quantified
pneumoniae,
with 617 IFU/ml was light pink, and might be consid-
P.mirabilis,P.aeruginosa,
ered positive (OD = 0.162). Meanwhile, the result of
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S.enteritidis,S.aureus,
the chlamydial suspension was colourless if quantified
S.epidermidis,S.saprophyticus, with 126 IFU/ml, and might be considered negative
S.maltophilia,S.agalactiae, (OD = 0.098). Therefore, the LOD was 617 IFU/ml
512
S.pyogenes,S.pneumoniae for C. trachomatis. In addition, the LOD was 10-3 u/ml
(OD =0.155) for α-D-mannosidase.
G.vaginalis* Bloodagarbase 37°C 48h 512
H.influenzae* ThayerMartinmediab 24h
Discussion
N.gonorrhoeae* 48h
There are various well-known methods for detecting
Candida C. trachomatis, including cell culture-, immunology-,
C.albicans,C.tropicalis, Sabourauddextrose 37°C 24h molecular biology-, and biochemistry-based methods.
agarc
C.glabrata,C.parapsilosis, Cell culture is complicated to perform and requires ex-
perience to produce accurate results, and it also has
C.guilliermondii,C.krusei,
more stringent requirements for the sampling swabs and
C.neoformans 30°C 48h
transporting before inoculation [21,22]. Therefore, cell
a,b,cOxoid,Basingstoke,UK;asupplementedwith5%sheepblood;b
culture is rarely used in clinics. Among the available
supplementedwith5%horseblood;*culturedinacandlejar.
immunological methods, serological tests for the
Therefore, the research adopted an “expanded gold C. trachomatis antibody have significant limitations [23],
standard” [18-20] described as follows: any positive by but methods for C. trachomatis antigen detection
either culture or LCR was classified as a true positive, (mainly referring to the lipopolysaccharide, LPS), espe-
whatevertheresultoftheenzymaticmethod. cially immunochromatography-based methods, have
been widely used due to their simplicity of operation.
Results There are two ways to extract the LPS antigen for
In our developed assay, only C. trachomatis samples C. trachomatis immunochromatographic assays: heat ex-
tested was positive for α-D-mannosidase activity; but traction and acid extraction. Neither of these methods
OD of both other organisms and cell cultures which guarantees the full extraction of LPS as an intact anti-
512
were not inoculated with C. trachomatis used in the gen, which influences the sensitivity of this method. The
study was all below 0.100, which fell into the range of biochemical detection of glycogen in C. trachomatis
negative results negative with 0.150 (OD ) as the cut- inclusions [24] is greatly affected by Candida spp. that
512
off value, even if the reactions were allowed to proceed often exist in these specimens and are especially com-
for 1 h at 37°C. Of the 553 clinical samples, 132 samples mon during the female menstrual cycle and pregnancy.
were positive with OD ranging from 0.161 to 1.955, With the increasing glycogen in vaginal epithelial cells,
512
and 421 samples were negative with OD ranging from thismethodmayalsocausefalsepositives.Thedetection
512
0.013 to 0.142. C. trachomatis detection results of 553 of C. trachomatis in the United States and Europe has
cases with culture, LCR and enzymatic method used an mainly focused on molecular biology methods [25-30].
expanded gold standard as the reference standard were Although these methods are both high sensitivity and
showed in Table 4.Theenzymatic method wasleastreli- high specificity, it can be challenging for molecular biol-
able when prostate specimens were used. The sensitivity ogy methods to meet the requirements of the actual ap-
of the enzymatic method was 91.5% (95% confidence plicationinclinical screening.
interval [CI], 85.9% to 97.1%), and the specificity of this Enzymatic studies of C. trachomatis, especially for
method was 90.0% (95% CI, 87.3% to 92.7%) (Table 5). enzymes with diagnostic significance, have not been
The sensitivity and specificity of the LCR were 94.7% reported in the literature. Previous studies [31] and our
(95% CI, 90.2% to 99.2%) and 100% (95% CI, 100.0% to research have shown that C. trachomatis secretes extra-
100.0%), respectively. However, in those specimens other cellular enzymes with high α-D-mannosidase activity.
than prostate fluid samples, the sensitivity andspecificity Although some organisms used in the study such as
of the enzymatic method were 91.8% (95% CI, 86.0% to C. albicans have genes encoding α-1,2-mannosidase
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Table4C.trachomatisdetectionresultsof553caseswithculture,LCRandenzymaticmethodusedanexpandedgold
standardasthereferencestandard
Method No.oftruepositives/positives No.oftruenegatives/negatives
Enzymaticmethod 86/132 413/421
Urethraldischarge22/24 Urethraldischarge126/127
Prostatemassageliquid8/47 Prostatemassageliquid4/5
Cervicalsecretion31/33 Cervicalsecretion196/199
Vaginalsecretion25/28 Vaginalsecretion87/90
Cellculture 60/60 459/493
Urethraldischarge18/18 Urethraldischarge128/133
Prostatemassageliquid7/7 Prostatemassageliquid43/45
Cervicalsecretion21/21 Cervicalsecretion198/211
Vaginalsecretion14/14 Vaginalsecretion90/104
LCR 85/89 459/464
Urethraldischarge22/22 Urethraldischarge128/133
Prostatemassageliquid8/8 Prostatemassageliquid43/45
Cervicalsecretion32/32 Cervicalsecretion198/211
Vaginalsecretion27/27 Vaginalsecretion90/104
Expandedgoldstandard 94 459
Urethraldischarge23 Urethraldischarge128
Prostatemassageliquid9 Prostatemassageliquid43
Cervicalsecretion34 Cervicalsecretion198
Vaginalsecretion28 Vaginalsecretion90
[32], α-D-mannosidase activity was invisible by naked false positive results. Some human sperm surface pro-
eyes with 6-chloro-3-indolyl-α-D-mannoside as sub- teins possess α-D-mannosidase activity [36], which may
strate. Thismaybebecausetheextracellularα-D-mannosi- be the reason that prostate specimens produce less reli-
dase from these organisms is much less or the enzyme able results. Serotype D was only one of the most preva-
activity is relatively low. Previous records [31,33-35] on lent (11.1%), and no serovar L2 was found in China [37].
substratesofα-D-mannosidasemainlyinvolvedp-nitrophe- Although the results of this study suggested that other
nol-α-D-mannoside and 4-methylumbelliferyl-α-D-manno- serotypes, such as serotypes E, F, G, K, H, J, I, and Ba, at
side. However, 6-chloro-3-indolyl-α-D-mannoside is a least most of them, might have α-D-mannosidase
novel chromogenic substrate, and colour reaction of its activity, there seems to be some limitation of tests on
chromogenismuchmoresensitiveespeciallyinthecaseof C. trachomatisculturesbased ononlyone strain ofsero-
thepresenceofanazoreagentsuchasfastvioletBsalt. type D. Further studies and more comprehensive clinical
Our results showed that clinical specimens such as evaluations should be conducted due to little research
urethral discharge, cervical secretions, and vaginal secre- on α-D-mannosidase activity of C. trachomatis. In
tions did not interfere with the chromogenic detection addition, our studies did not evaluate C. pneumoniae or
of α-D-mannosidase activity to screen for C. trachoma- C. psittaci; the α-D-mannosidase activities of these spe-
tis, although prostate massage liquid produced more cies shouldbestudied aswell.
Table5ClinicalperformancesofthreeassaysforC. Table6ClinicalperformancesofthreeassaysforC.
trachomatisusingspecimensincludedprostatemassage trachomatisusingspecimensexcludedprostatemassage
liquid liquid
Methods %Sensitivity(95%CI) %Specificity(95%CI) Methods %Sensitivity(95%CI) %Specificity(95%CI)
Enzymaticmethoda 91.5(85.9,97.1) 90.0(87.3,92.7) Enzymaticmethoda 91.8(86.0,97.6) 98.3(97.1,99.5)
Cellcultureb 63.8(54.1,73.5) 100.0(100.0,100.0) Cellcultureb 64.6(54.3,74.9) 100.0(100.0,100.0)
LCRc 94.7(90.2,99.2) 100.0(100.0,100.0) LCRc 95.3(90.8,99.8) 100.0(100.0,100.0)
aΧ2=8.030,P=0.005,P<0.05;bΧ2=8.721,P=0.003,P<0.05;cΧ2=0.164, aΧ2=0,P=1,P>0.05;bΧ2=8.605,P=0.003,P<0.05;cΧ2=0.116,
P=0.685,P>0.05. P=0.734,P>0.05.
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