JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1994, p. 2755-27590095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Ciyptosporidium Antigen Detection in Human Feces byReverse Passive Hemagglutination Assay

M. FARRINGTON,l* S. WINTERS,1 C. WALKER,' R. MILLER,2 AND D. RUBENSTEIN3Clinical Microbiology and Public Health Laboratory' and Department of Medicine,3Addenbrooke's NHS Trust, Cambridge CB2 2QW, and Department of Medicine,

University College London Medical School, London W2N 8AA,2United Kingdom

Vol. 32, No. 11

Received 9 May 1994/Returned for modification 23 July 1994/Accepted 16 August 1994

A reverse passive hemagglutination (RPH) assay was developed for Cryptosporidium oocyst antigen with anantioocyst monoclonal antibody (MAb; MAb-Cl) coupled to stabilized sheep erythrocytes. RPH was comparedwith microscopy of auramine-phenol-stained smears of 56 oocyst-positive fecal samples, each of which wastested blindly by RPH with two oocyst-negative samples received on the same day (a total of 112 controls).Thirty-nine additional fecal samples from human immunodeficiency virus type 1 antibody-positive patientswith diarrhea (10 of which were positive in auramine-phenol-stained smears) were stored at -20°C beforetesting. Thirty specimens with a variety of other fecal pathogens (all negative for oocysts) were also tested. Ofthe 237 samples tested, 69 were positive by one or both methods: 65 by RPH and 66 by microscopy. The kappacoefficient of agreement between the methods was very high at 0.926. The sensitivity of RPH was 93.91%, thespecificity was 98.2%, the positive predictive value was 95.4%, and the negative predictive value was 97.7%.Visible oocyst numbers and RPH titers were measured after storage of fecal samples and oocyst concentratesfor 8 days at 4°C. Oocyst morphology was generally poor in specimens from the human immunodeficiency virustype 1 antibody-positive group, and it degenerated during the 8-day storage experiments. MAb-Cl-reactiveantigen eluted from oocysts to give progressively higher reciprocal titers during storage, and it was partiallyremoved from the oocysts by concentration. RPH is a promising technique for the detection of Cryptosporidiumantigen in human feces and may be useful when specimens are stored before testing. Studies of the sensitivityof Cryptosporidium immunoassays should take into account the possible release of antigen from oocysts.

Although long recognized as an animal pathogen, infectionof humans with Cryptosporidium spp. has been reported fre-quently only during the past decade. This followed apprecia-tion of its connection with intractable diarrhea in patients withAIDS (10) and of the public health implications of its frequentpresence in surface waters (13). Cryptosporidiosis has a world-wide distribution. It is detected in about 16% of patients withAIDS and diarrhea in hospitals in the United States, and thisproportion rises to about 50% in developing countries (10).

Laboratory diagnosis worldwide currently relies upon thewell-established but time-consuming method of microscopy ofZiehl-Neelsen- or auramine-phenol-stained fecal smears (3). Alow level of sensitivity is a problem with these methods, and itmay be increased only by technically demanding concentrationprocedures (17). Immunofluorescent stains are increasinglyused, but not all studies have shown their use to be straight-forward or always to improve sensitivity (2, 8, 9, 11). Experi-ence with several immunodiagnostic assays has been published(1, 4, 12, 14-16). However, few comparative studies of thesemethods have been performed outside the research laboratorysetting, none has shown significantly improved sensitivity, andnonspecific reactions have sometimes been observed (4). To-day, diagnostic microbiology laboratories in the United King-dom screen the majority of fecal specimens from patients withcommunity-acquired cases of diarrhea for Cryptosporidiumoocysts; in our laboratory about 7,000 microscopic examina-tions of auramine-phenol-stained material were performed in

* Corresponding author. Mailing address: Clinical Microbiology andPublic Health Laboratory, Box 236, Addenbrooke's NHS Trust, HillsRoad, Cambridge CB2 2QW, United Kingdom. Phone: (44) 223-242111. Fax: (44) 223-242775. Compuserve: 100115,3462.

1993. Elsewhere, many laboratories restrict the examination tofeces from human immunodeficiency virus type 1 (HIV-1)antibody-positive patients. A sensitive, specific, and cost-effec-tive diagnostic method would be a major advance.

Reverse passive hemagglutination (RPH) involves the ag-glutination by specific antigen of glutaraldehyde-stabilized,protease-sensitized sheep erythrocytes that have been coupledto immunoglobulin by chromic chloride (6). The sensitivity ofthe method exceeds that of enzyme-linked immunosorbentassay (ELISA) in some systems, and the technique has beensuccessfully used for antigen detection in several body fluids,including feces. Freeze-drying allows indefinite storage ofreagents, and the reactions are stable, easily read, and con-trolled (7).We compared RPH with semiquantitative microscopy of

auramine-phenol-stained smears of feces from patients withcommunity-acquired diarrhea, patients with AIDS and diar-rhea, and patients with a variety of other fecal pathogens.

MATERIALS AND METHODS

At the Clinical Microbiology and Public Health Laboratory,Addenbrooke's Hospital, Cambridge (CMPHL), stools werecultured for Salmonella, Shigella, and Campylobacter spp. byroutine methods. Clostridium difficile cytotoxin and Clostridiumperfringens enterotoxin were detected by Vero cell culture.Stools from patients with bloody diarrhea and hemolyticuremic syndrome and from children out of hospital wereinvestigated for Escherichia coli 0157. Fecal samples frompatients recently returned from areas where vibrios and Aero-monas spp. are endemic were also cultured for pathogenicvibrios, and samples containing blood were also cultured for

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2756 FARRINGTON ET AL.

Aeromonas spp. A wet preparation and a formol saline con-

centration for parasites were performed on patients recentlyreturned from outside the British Isles or those with bloodydiarrhea or AIDS or when requested clinically.Auramine-phenol staining for Cryptospondium spp. was per-

formed on all samples of feces from patients between the agesof 6 months and 44 years (3). A pea-sized portion was

emulsified in 3 ml of quarter-strength Ringer's solution to givea 20% solution, and a 10-,ul loopful was smeared in a well in a

monospot slide (code 011; Hendley, Loughton, United King-dom) and allowed to dry in air. A positive control was includedon each slide. Staining was performed with auramine-phenolfor 15 min, 3% acid-alcohol for 5 min, and potassium perman-ganate for 30 s, with washes after each step. After air drying,the slide was examined under a fluorescence microscope atX250 magnification, and the number of cysts per high-powerfield was recorded. Fecal samples found to be positive forCryptosporidium oocysts were each matched with two Crypto-sporidium-negative samples from other patients that had ar-

rived on the same day. Each set of three specimens wasrandomized and coded and submitted blind for RPH testing,usually on the day of arrival.

Fecal specimens from patients with AIDS and diarrhea werecollected from inpatients at the Middlesex Hospital, London.These were immediately frozen at -20°C and were trans-ported to CMPHL for routine fecal culture, parasite concen-tration and microscopy, and blind RPH testing of the results ofroutine investigations. As additional controls, fecal specimensfrom patients with diarrhea caused by other pathogens submit-ted to CMPHL and from patients with no causative diagnosiswere also tested by RPH.RPH technique. Anticryptosporidium oocyst monoclonal

antibody (MAb; MAB-Cl; batch 11), an immunoglobulin M(IgM) antibody of the kappa isotype, was generously donatedas ascites by the Central Public Health Laboratory, Colindale,London. Ascitic fluid was clarified by centrifugation and was

filtered through a 0.22-,um-pore-size Millipore filter, and thenIgM was precipitated at 4°C with 20 volumes of 2% boric acid.After centrifuging at 10,000 x g for 15 min, the precipitate wasdissolved in saline and was dialyzed overnight against physio-logical saline. A mouse kappa IgM (ABPC 22; no. M-7394;Sigma) was coupled to sheep erythrocytes as an immunoglob-ulin-erythrocyte control, and uncoupled erythrocytes were

used as the erythrocyte control.Antibody-coupled erythrocytes were prepared as follows (7).

Sheep erythrocytes washed in phosphate-buffered saline (PBS)were incubated for 20 min at 37°C with ac-chymotrypsin inacidified PBS-azide complement fixation test buffer (BR16;Oxoid, Basingstoke, United Kingdom). Stock chromic chloridecoupling solution was made by dissolving CrCl3 * 6H20 (BDH;Poole, United Kingdom) to a 1% concentration in pyrogen-free physiological saline and adjusting the pH to 5.0 with 5 Msodium hydroxide. Purified immunoglobulin was then coupledat a concentration of 0.5 mg ml-' to sheep erythrocytes inpiperazine buffer with an optimal 1:180 dilution of chromicchloride coupling solution (6, 7). Coupling was confirmed bytitration with sheep anti-mouse globulin (Serotec Ltd., Oxford,United Kingdom). Samples of 1 ml of coupled cells were

stabilized with 100 ,ul of glutaraldehyde (30 pI in 5 ml of PBS;BDS) and were then freeze-dried in PBS with 1.5% sucrose-

1.0% bovine serum albumin (Calbiochem, La Jolla, Calif.) inan Edwards Modulyo freeze-dryer overnight. Before use, thecells were reconstituted with gentle mixing in PBS to theiroriginal volumes at 4°C for 24 h; shorter reconstitution timesgave a weaker distinction between positive and negative re-

sults.

Fecal samples were suspended in PBS at 25% (wt/vol). Thefecal suspensions were placed in a boiling water bath for 3 min,which we observed improved the quality of the agglutinationreactions. Following sedimentation and cooling for 15 min atroom temperature, the supernatants were diluted 1:2 and 1:20in PBS containing 0.1% normal mouse serum (Serotec). Serialtwofold dilutions of these supernatants were made along96-well microtiter plates (Serowell; Bibby-Sterilin, Stone,United Kingdom) with PBS containing 1% normal mouseserum, from 1:2 to 1:256 and from 1:20 to 1:1,280, respectively.

Plates were shaken to mix the contents of the wells, and theresult of hemagglutination was read by the sedimentationpattern after 30 min at room temperature. A negative result fora well was indicated by a button of unagglutinated erythrocytessettling at the bottom of the well, whereas a positive result wasindicated by a diffuse coating of agglutinated cells over thebottom of the well (7). An RPH result for a fecal specimen wasconsidered positive (i) if the titer in the MAb-Cl dilution seriesthat began at 1:20 was two or more dilutions higher than thehighest titer in either of the control dilution series that beganat 1:20 or, if the sample was negative by criterion (i), (ii) if thetiter in the MAb-Cl dilution series that began at 1:2 was threeor more dilutions higher than the highest titer in either of thecontrol dilution series that began at 1:2.

Effects of specimen storage and concentration on RPH titerand oocyst counts. Eight fecal samples that had been submit-ted to CMPHL for diagnostic purposes were chosen to studythe effects of storage and concentration procedures. They wereoocyst microscopy positive, large in volume, and gave highRPH reciprocal titers without nonspecific reactivities.

Twenty-five percent suspensions of each of the eight freshlyreceived samples were made in PBS, and aliquots of each ofthese were stored at 4°C. Their Cryptosporidium antigen recip-rocal titers and oocyst counts were determined on days 1, 3, 6,and 8. The RPH assays were performed in the same way as thediagnostic tests, with the exception that fecal suspensions werefiltered through 0.22-,um-pore-size filters (Millipore) afterremoval from the boiling water bath. This step was includedbecause preliminary experiments indicated that nonspecificreactivity increased with storage and that it could be removedby filtration without significantly altering the specific titer (datanot shown). Quantitation of the oocysts was performed by useof an improved Neubauer counting chamber, with calculationof the number of oocysts per milliliter in the original fecalsample.

Oocyst concentrates were made by mixing equal volumes ofthe eight freshly received fecal samples and dithiothreitol(Mucolyse PL. 701; Pro-Lab Diagnostics, Wirral, United King-dom) in 10-ml centrifuge tubes and holding the mixtures atroom temperature for 15 min. Ether (2 ml) was added to eachtube, and the contents were mixed by shaking before centri-fuging at 900 x g for 3 min. After the solid surface plug andsupernatant were discarded, the pellet was resuspended andwashed three times in PBS (pH 7.2), centrifuging at 900 x g for10 min after each wash, and was then resuspended in 1 ml ofPBS. One volume of each oocyst suspension was loaded to adiscontinuous step density gradient consisting of equal vol-umes of 0.5, 1.0, 2.0, 4.0, and 6.0% Ficoll (400) in PBScontaining 16% sodium diatrizoate. Each gradient was centri-fuged at 900 x g for 60 min at room temperature. The oocystswere harvested from a clearly defined band at the formerlocation of the 0.5% Ficoll layer. After washing three times inPBS, the oocysts were resuspended in 1 ml of PBS and theirconcentrations were determined by counting in an improvedNeubauer chamber. Oocyst concentrates were then added toeight aliquots of a Cryptosporidium-negative sample of feces to

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RPH FOR CRYPTOSPORIDIUM OOCYST ANTIGEN 2757

TABLE 1. Correlation between RPH assay" of Cryptosponidiumantigen and microscopy of auramine-phenol-stained

smears for oocysts in human stool specimens

No. of specimens with the followingRPH result microscopy result:

Positive Negative Total

Positive 62 3 65Negative 4 168 172

Total 66 171 237

a Sensitivity, 93.9%; specificity, 98.2%; positive predictive value, 95.4%; neg-ative predictive value, 97.7%; kappa coefficient of agreement, 0.926.

CMPHL HIV+

l1280640

320

a 160

80

40

20

16

negative

IzzzjmIIIII

E

]

I

I

16 14 12 10 8 6 4 2 0 2 4Number

give a concentration of about 105 to 106/ml. These mixtureswere stored at 4°C. After 1, 3, 6, and 8 days the oocystconcentrations in each mixture were determined by lightmicroscopy with quantitation in an improved Neubauer cham-ber, and antigen concentrations were determined by perform-ing RPH assays.

RESULTSIn total, 237 specimens from patients were tested. These

included 56 from CMPHL with positive microscopy results andthe associated 112 controls with negative microscopy results.Thirty-nine stool samples came from episodes of diarrhea fromHIV-1 antibody-positive patients (10 microscopy positive), allof which were clinically compatible with Cryptosporidium in-fections and negative for other pathogens. Thirty samplescame from known Cryptosporidium-negative patients at CM-PHL (10 with giardiasis, 8 with C. difficile toxin, 2 with E. coli0157, 1 each with Endolimax sp. and Iodamoeba sp., and 8 withdiarrhea of unknown cause acquired in the United Kingdom).

Results from the dilution series starting at 1:20 usually gavemore easily interpretable reactions, stronger hemagglutinationpatterns, and less residual nonspecific reactivity than testsperformed in the dilution series that began at 1:2. Occasionalprozone phenomena were observed with the supernatantstested in the 1:2 series, which were diluted out in the 1:20series. In the 1:20 series, 62% of the tests showed no nonspe-cific reactions, 93% gave reactions of one or no wells, and notests showed nonspecific reactivity of greater than two wells.Only 36% of the nonspecific reactions were predominantlyagainst immunoglobulin-coupled erythrocytes (defined as atiter 2 or more dilutions greater against immunoglobulin-coupled erythrocytes than against erythrocytes alone). Nosamples reacted only or predominantly against uncouplederythrocytes.

Table 1 summarizes the results obtained from all 237samples from patients with diarrhea tested by microscopy andRPH, and Fig. 1 shows the RPH titers obtained from the 69specimens that were positive by one or both methods. Sixty-fivespecimens were positive by RPH; 63 of these were positive inthe MAb-Ci dilution series that began at 1:20 by criterion (i)above. The remaining two gave titers of 16 in the series thatbegan at 1:2. All 30 negative control samples from patientswith known causes of diarrhea and negative smears for Cryp-tosporidium oocysts were negative by RPH. Of the 66 speci-mens positive by microscopy, only 4 were negative by RPH, allof which were diagnostic samples submitted to CMPHL. Ofthe 171 specimens negative by microscopy, 3 were positive byRPH, all of which were from HIV-1 antibody-positive patients.Assuming microscopy of auramine-phenol-stained smears tobe a reference technique, the sensitivity of RPH was therefore

FIG. 1. RPH reciprocal titers obtained with the 69 stool specimensthat were positive by microscopy or RPH. CMPHL (open bars),samples referred to CMPHL for diagnostic purposes; HIV+ (shadedbars), samples from HIV-1 antibody-positive patients.

93.9%, the specificity was 98.2%, the positive predictive valuewas 95.4%, and the negative predictive value was 97.7%. Thekappa coefficient of agreement (5) between the methods wasvery high at 0.926.

Table 2 illustrates the results obtained from the 168 clinicaldiagnostic specimens referred to CMPHL combined with thoseobtained from the 30 negative control samples. The sensitivityof RPH for this group of specimens was 92.9%, the specificitywas 100%, the positive predictive value was 100%, and thenegative predictive value was 97.3%. The kappa coefficient ofagreement between the methods was very high at 0.949.

Table 3 shows the results obtained with the 39 specimensfrom HIV-1 antibody-positive patients. For this group ofspecimens, the sensitivity was 100%, the specificity was 89.7%,the positive predictive value was 76.9%, and the negativepredictive value was 100%. The kappa coefficient of agreementbetween the methods was high at 0.816.One specimen from an HIV-1 antibody-positive patient that

was positive by RPH (reciprocal titer, 160) but that initiallyscreened negative by microscopy was found to contain veryscanty but characteristic oocysts (less than one per high-powerfield) on review of the film by senior staff. This sample wasscored as RPH positive and microscopy negative. Two otherspecimens from HIV-1 antibody-positive patients with diar-rhea were positive by RPH (reciprocal titers, 20 and 160) butnegative by microscopy even on repeat examination.

All four samples in which oocysts were seen by microscopybut which gave negative RPH results were from specimenssubmitted to CMPHL for routine diagnostic purposes. None of

TABLE 2. Correlation between RPH assay' of Cryptosporidiumantigen and microscopy of auramine-phenol-stained smears

for oocysts in stool specimens submitted toCMPHL for diagnostic reasons

No. of specimens with the followingRPH result microscopy result:

Positive Negative Total

Positive 52 0 52Negative 4 142 146

Total 56 142 198

" Sensitivity, 92.9%, specificity, 100%; positive predictive value, 100%; nega-tive predictive value. 97.3%; kappa coefficient of agreement. 0.949.

6 8 10 12 14 16

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2758 FARRINGTON ET AL.

TABLE 3. Correlation between RPH assay' of Cryjantigen and microscopy of auramine-phenol-stain(

for oocysts in stool specimens from HIV-antibody-positive patients

No. of specimens with the fRPH result microscopy result:

Positive Negative

Positive 10 3Negative 0 26

Total 10 29

a Sensitivity, 100%; specificity, 89.7%; positive predictive valtive predictive value, 100%; kappa coefficient of agreement, 0.

the samples gave high reciprocal titers in the coiseries that might have masked a true-positive ithree of the four samples had only low number,high-power field. Thirty-five characteristic oocypower field were seen in the fourth stool samptiters were identical on repeat testing. Unfortunaa very low volume specimen, and no additional sbe obtained from the patient, so further investigatperformed.

Figure 2 shows the effects of storage on theconcentrations (oocysts per milliliter) and the gecreciprocal RPH titers seen in the eight fecal sanafter 1, 3, 6, and 8 days of storage. Mean oocysteight clinical specimens of feces fell progressively106 (standard deviation [SD], 1.69 x 106) to 2.5:3.56 x 105) per ml, and in the fecal sample withconcentrates they fell from 5.80 x 105 (SD, 1.11 >x 104 (SD, 8.23 x 104) per ml, with many oocy:

degenerate at 6 and 8 days. Geometric mean rectiters rose progressively from 234 (range, 8 to,(range, 8 to 32,768) in the stored clinical specinand fell progressively from 144 (range, 2 to 1024)<2 to 8) in the samples to which oocyst conceadded.

Oocysts/mlx10o

1000

Ocysts (raw)

KCysts (conc.100 \ *-Tite (raw)

100

10

1 3 6 8

Days

FIG. 2. Effects of storage at 4°C on oocyst numlreciprocal titers. Each measurement is the mean ofBars, oocyst numbers per milliliter; lines, geometrreciprocal titers; open bars and triangles, eight fecal spatients with natural infections; shaded bars and s

negative fecal specimen to which oocyst concentrapatients with natural infections were added.

otosporidiumed smears

DISCUSSION

I1 In the preliminary assessment described here, RPH ap-peared to be a rapid and specific method for detecting

ollowing Cryptosporidium antigen in human feces, with a sensitivityequivalent to that of the most commonly used diagnostic

Total laboratory test, fluorescence microscopy with auramine-phenolstaining. The kappa coefficient of agreement between RPH

13 and microscopy was very high (0.926); this coefficient is the26 most appropriate measure, because no method for the routine

detection of Cryptosporidium oocysts can currently be consid-39 ered a "gold standard." Considering the results from all stool

ue, 76.9%; nega- specimens from patients with diarrhea, our experience of RPH816. compared with stool microscopy was similar to that of ELISA

in the hands of Newman et al. (12) (specificity, 93.9 versus96.4%; positive predictive value, 95.4 versus 97.6%). RPH,however, was more sensitive (93.9 versus 83.3%) and gave a

ntrol dilution higher negative predictive value (97.7 versus 77.1%). Beforereaction, and firm conclusions are drawn, these methods should be com-s of cysts per pared with the same samples of feces, but RPH appears to bests per high- a screening test that is at least as promising as ELISA. RPHle, but RPH should also be compared with immunofluorescence micro-ttely, this was scopic methods. It is possible that bias toward the increased,amples could sensitivity of RPH was introduced into the present study:ions were not because one of the three fecal samples submitted for testing

was known to be positive. However, preliminary experimentsmean oocyte showed that storage of fecal samples altered RPH titers, and)metric mean only about one positive sample was submitted to CMPHL pernples studied week; hence, practicability dictated that the study be designedcounts in the in this way.(from 1.05 x Diagnostic assays for Cryptosporidium antigens may have3 x 105 (SD, different sensitivities and specificities for samples from HIV-1added oocyst antibody-positive and healthy patient groups because of theK106) to 5.48 differences in the prevalence of Cryptosporidium spp. and othersts appearing causes of diarrhea between the groups. Oocyst morphology in-iprocal RPH most specimens from the HIV-1 antibody-positive patient2,048) to 861 group was poor, and inexperienced staff found it difficult tonens of feces, assess this group of smears. One sample judged to be micros-to 2.5 (range, copy negative routinely was found to contain very scantymntrates were typical oocysts on careful review by senior staff. We suspected

that the negative microscopy results in this and the other twospecimens from HIV-1 antibody-positive patients that alsogave positive RPH reactions may have been artifacts as a resultof oocyst degeneration during the storage period. There was

Geometric mean no evidence of cross-reactivity of these samples with eitherlt*e control reagent, and we found that MAb-Cl did not react with

fecal specimens containing Giardia cysts, C. difficile toxin, E.coli 0157, Endolimax sp. or Iodamoeba sp. Others have alsofound that MAb-C1 does not react with Campylobacterjejuni,

100 Salmonella enteritidis, or rotavirus (4). We therefore performedJ the 8-day storage experiments to investigate the effects of time

and concentration procedures on oocyst morphology andantigen release. Chapman et al. (4) used an enzyme immuno-

10 assay with MAb-Cl and found similar titers in fecal suspen-sions and supernatants, suggesting that the antigen may besoluble or finely particulate. We found the antigen to elutefrom fresh oocysts, reaching progressively higher titers in thesoluble phase, while the oocyst structure deteriorated duringthe same period. RPH with MAb-Cl therefore may be espe-

,bers and RPH cially useful for the detection of Cryptosporidium antigen inbeightrsampeR specimens that have been stored or delayed in transit. Much

-ic gmteaSn RPlH detectable and elutable antigen was removed if oocysts werespecimens from subjected to a vigorous concentration procedure, and the,quares, oocyst- initially low RPH titers fell progressively during the 8-day study,tes from eight period. Presumably, this was caused by degradation of residual

soluble or particulate antigen, with little antigen remaining on

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RPH FOR CRYPTOSPORIDIUM OOCYST ANTIGEN 2759

the oocysts and being eluted. Comparison of the sensitivities ofmicroscopic and RPH-based methods is therefore complicatedby the progressive elution of antigen from and the loss of thecharacteristic morphologies of fresh oocysts in clinical speci-mens. Similar assessments of the roles of free and oocyst-bound antigen and of the way in which they change withstorage have not been reported for other Cryptosporidiumimmunoassays with MAb-Cl or other MAbs, but measure-ments of the sensitivities of other methods should take thisobservation into account.None of the specimens positive in auramine-phenol-stained

smears but negative by RPH showed signs of nonspecificreactivity that might have masked significant reactions. Threeof the four specimens contained only low numbers of morpho-logically typical oocysts and might have been at the limits ofsensitivity of the RPH assay. One sample contained 35 char-acteristic oocysts per high-power field, but was repeatedlynegative by RPH. These oocysts might have belonged to aCryptosporidium species that did not react with MAb-Cl, butfurther investigations were not possible because of a limitedsample volume.Two RPH tests were performed on each microtiter tray, with

one tray for each of the dilution series beginning at a dilution1:2 and one beginning at a dilution of 1:20. A diagnostic systembased on RPH could involve a more limited series of dilutionsand use a single immunoglobulin-coupled erythrocyte control;hence, more tests could be accommodated per plate. Alterna-tively, a pair of screening dilutions for test and controlspecimens could be used, with further dilutions set up only ifnonspecific agglutination was seen. Testing of additional sam-ples will be necessary to resolve this issue. After reconstitutionof the reagents, RPH results are available within 40 min, whichincludes only about 15 min of hands-on time. This is consid-erably faster than microscopy if more than a few tests are to beperformed, and quantitative results are obtained. As with thecurrently available ELISA-based methods (12), batch testingof large numbers of samples by RPH is possible, and RPHneeds little capital expenditure because the results are assessedby eye and expensive reading equipment is not required.We found RPH with MAb-Cl to be a promising technique

for the detection of Cryptosporidium antigen in human feces,and it may be especially useful when specimens are storedbefore testing and when batch testing of large numbers ofsamples is required. Studies of the sensitivity of Cryptospo-ridium immunoassays should take into account the possiblegradual release of antigen from oocysts during storage of fecalspecimens.

ACKNOWLEDGMENTS

We are grateful to R. R. A. Coombs (emeritus professor, Universityof Cambridge, Cambridge, United Kingdom) and J. McLauchlin(Central Public Health Laboratory, Colindale, London) for many

helpful discussions and to the Addenbrooke's Hospital Trust FundsCommittee, the East Anglian Regional Health Authority ResearchCommittee, and Serotec Ltd. for support.

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2. Arrowood, M. J., and C. R. Sterling. 1989. Comparison of conven-tional staining methods and monoclonal antibody-based methodsfor Cryptosporidium oocyst detection. J. Clin. Microbiol. 27:1490-1495.

3. Casemore, D. P., M. Armstrong, and R. L. Sands. 1985. Labora-tory diagnosis of cryptosporidiosis. J. Clin. Pathol. 38:1337-1341.

4. Chapman, P. A., B. A. Rush, and J. McLauchlin. 1990. An enzymeimmunoassay for detecting Cryptosporidium in faecal and environ-mental samples. J. Med. Microbiol. 32:233-237.

5. Cohen, J. 1960. A coefficient of agreement for nominal scales.Educ. Psychol. Measurement 20:37-46.

6. Coombs, R. R. A., M. L. Scott, and M. P. Cranage. 1987. Assaysusing red cell-labelled antibodies. J. Immunol. Methods 101:1-14.

7. Farrington, M., S. Winters, D. Rubenstein, J. Greatorex, and R.Whetstone. 1991. Streptococci from primary isolation platesgrouped by reverse passive haemagglutination. J. Clin. Pathol.44:670-675.

8. Garcia, L. S., T. C. Brewer, and D. A. Bruckner. 1987. Fluores-cence detection of Cryptosporidium oocysts in human fecal speci-mens by using monoclonal antibodies. J. Clin. Microbiol. 25:119-121.

9. Garcia, L. S., A. C. Shum, and D. A. Bruckner. 1992. Evaluation ofa new monoclonal antibody combination reagent for direct fluo-rescence detection of Giardia cysts and Cryptosporidium oocysts inhuman fecal specimens. J. Clin. Microbiol. 30:3255-3257.

10. Gellin, B. G., and R. Soave. 1992. Coccidian infections in AIDS.Med. Clin. N. Am. 76:205-234.

11. MacPherson, D. W., and R. McQueen. 1993. Cryptosporidiosis:multiattribute evaluation of six diagnostic methods. J. Clin. Mi-crobiol. 31:198-202.

12. Newman, R. D., K. L. Jaeger, T. Wuhib, A. A. M. Lima, R. L.Guerrant, and C. L. Sears. 1993. Evaluation of an antigen captureenzyme-linked immunosorbent assay for detection of Cryptospo-ridium oocysts. J. Clin. Microbiol. 31:2080-2084.

13. Report of a Group of Experts on Cryptosporidium in WaterSupplies. 1990. Department of Health, Department of the Envi-ronment. Her Majesty's Stationery Office, London.

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15. Siddons, C. A., P. A. Chapman, and B. A. Rush. 1992. Evaluationof an enzyme immunoassay kit for detecting cryptosporidium infaeces and environmental samples. J. Clin. Pathol. 45:479-482.

16. Ungar, B. L. P. 1990. Enzyme-linked immunoassay for detection ofCryptosporidium antigens in fecal specimens. J. Clin. Microbiol.28:2491-2495.

17. Weber, R., R. T. Bryan, and D. D. Juranek. 1992. Improved stoolconcentration procedure for detection of Cryptosporidium oocystsin fecal specimens. J. Clin. Microbiol. 30:2869-2873.

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