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Evaluation of Enzyme Immunoassay Techniques for Detection of Some Intestinal Protozoa Antigens in Human Fecal Samples in Comparison to the Conventional Diagnostic Techniques 9+0A+6-. >?@ 9.:;<)= 8/ 4567- (",*123. ("/+0,-. ()*+#,-. %&' !""#$ (*:"7#<-. %&L-+@ (2K+#,-+@ (*&5J-. I.&J-. 9+0"/ GH (*F?,-. ("7E-. B:"CD M"E5<7-

:Thesis Proposal Submitted By Danah Abdulaziz S. AL-Turbak NOPOOQQQR

WI ISTTUDVCTIUS
Diarrheic disease is one of the greatest causes of morbidity and mortality worldwide. Gastrointestinal parasites, along with other infections, can be a significant problem for immunocompromised individuals, especially in resource-poor settings (Lewthwaite et. al, 2 !". #he ma$or parasitic causes of gastroenteritis are Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica, which will be discussed in this thesis, often have similar clinical presentations. #o complicate matters further, symptoms may also be similar to those caused by other gastrointestinal disease, such as bacterial and viral gastroenteritis. #he purpose of parasite detection is not limited to curing disease in an infected individual, but it is crucial in the prevention and spread of diseases. %or these reasons, detection methods must be held at the highest standard of both sensitivity and specificity, so that false negative and false positive results are avoided (&erwei$ et. al, 2 '".

Giardia lamblia is a flagellate proto(oan found in the small intestine of

humans and other animals (#hompson et. al, 1))*". +t is broad worldly distributed, being detected in both developing and developed countries. +t is the most commonly reported human intestinal parasite, with prevalence rates reaching 2 to ,-, in the developed countries
(.chant(, 1)) ".#he

prevalence rates may reach 2 to / - in some areas in the

developing countries (%arthing, 1)0)". #he overall prevalence rate in .audi 1rabia was 20.!-. %or children, adults, females, males, non-.audis2 .audis was ,/.0-, 1,.0-, '0.!-, 22.!-, 20.0- and 20.'- respectively. 3ationality was not a significant factor in the prevalence of giardiasis while age and se4 were significant factors (1L-#u5hi et.
al, 1))/".

.ince it is more fre6uent among eight months to ten years old children, it becomes a serious public health problem in this age group. #he symptoms of giardiasis in humans are e4tremely variable. .ome people may present the

asymptomatic form, others an acute or chronic diarrhea that can last for several months with mal-absorption syndrome and weight loss (7eyer and
8adulescu, 1),), Goldin et. al, 1)) " .

G. lamblia is usually pointed as one of the

causes of children9s retarded growth and development (Goldin et. al, 1)) ". Giardia infection can occur through ingestion of dormant cysts in contaminated water, food, or by the faecal-oral route (through poor hygiene practices". #he Giardia cyst can survive for wee5s to months in warm water. 1s well as waterborne sources, faecal-oral transmission can also occur so, 1utoinfection can also occur especially in children (:uang and ;hite, 2 /".

Cryptosporidium parvumX

an intestinal proto(oan parasite, is

considered as one of the ma$or diarrheagenic pathogens in humans throughout the world (3ime et.al, 1),/,<hai et.al, 1)) ,8hee et. al, 1))1,;ee et. al,
1))/".#he

gastrointestinal disturbances are self-limiting in immunocompetent

hosts, but chronic infections in immunocompromised sub$ects may become life-threatening (=iale5 et al., 2 2". 1mong patients with cryptosporidiosis, the ma$ority of immunocompetent individuals are initially symptomatic, with large number of oocysts present in their stools. :owever, as the acute infection resolves and the patient becomes asymptomatic, the number of oocysts decreases (;ee et. al, 1))/, =iale5 et al.
2 2".

1cute Cryptosporidium infection in children is usually associated with both acute and persistent diarrhea and various other gastrointestinal symptoms in immunocompetent persons and life-threatening illness in immunocompromised persons, e.g. in ac6uired immunodeficiency syndrome (1+D." patients (8amratnam and %lanigan, 1)),". 1symptomatic cryptosporidiosis, which represents potential reservoirs of unrecogni(ed, infected individuals who are capable of transmitting the infection to other individuals, was also documented (&uorio et. al, 1))1, >ettoello-7antovani et. al, 1))!, >alit et. al, 2 !". 1symptomatic individuals, livestoc5 such as cattle and drin5ing water are 5nown to be important vehicles in the epidemiology of the pathogen. 7ost cases of cryptosporidiosis occurred among children less than , years of age, and particularly in the first two years of life (>alit et. al, 2 !".

#he prevalence rates may reach 0?1)- of cases of diarrheal disease. #he prevalence of cryptosporidiosis was highest among children 1*?2' months of age (!.2-" and least among those '0?/ months of age (2-". (%ayer et.al,
2 , #(ipori and ;ard 2 2".

+nfection can occur through contaminated material such as earth, water, uncoo5ed or cross-contaminated food that has been in contact with the feces of an infected individual or animal. <ontact must then be transferred to the mouth and swallowed. +t is especially prevalent amongst those in regular contact with bodies of fresh water including recreational water such as swimming pools. @ther potential sources include insufficiently treated water supplies, contaminated food, or e4posure to feces. 1lso autoinfection can occur especially in children (;ee et. al, 1))/, =iale5 et al. 2 2".

AmeYiasis

is an infection of human intestinal and e4traintestinal organs

by the proto(oan parasite Entamoeba histolytica. #he previously reported asymptomatic infections due to the non-pathogenic strains of E.histolytica currently are identified to be due to E.dispar (Diamond and <lar5, 1))*". +nfection is predominantly seen in the tropical and subtropical regions (#rol et.
al, 1)),".1ppro4imately

1 - of the worldAs population is infected by either E. ?1 , is due to E. histolytica,

histolytica or E. dispar of which ! million people have invasive disease due to E. histolytica. #he annual death of ' , place the amoebiasis as the second leading cause of death from parasitic disease worldwide (.tanley, 2 *". #he prevalence of amebiasis varies with the population of individuals affected, differing between countries and between areas with different socioeconomic conditions. +tAs the *rd ma$or parasite isolated from schoolchildren aged /?10 years in .audi 1rabia with percent of 2.!- (1L-.e5ait et. al, 1))*" #he highest recorded prevalence was found during household survey conducted in 8iyadh * .*(1l-.hammari et. al, 2 1"

;hile a former study conducted in 8iyadh reported #he prevalence rate among school children was

lower prevalence 1/.0*- among patientsA attending hospitals or health clinics

(=olbol and 7ahmoud, 1)0'"

!.2- in 1l-1siah Basim (1l-%aleh, 1)02"Z '.1- in 1bha (1sir" [@mar et. al, 1))1"Z 2.)- in 7a55ah (1l-:arthi, 2 '"Z finally, /.0- was detected among

'

asymptomatic school children2 while 1'- was reported among diarrheic school children in Ceddah (1l-=rai5en et. al, 2 *". #his parasite has an active tropho(oite stage e4ists only in the host and in fresh loose feces2 cysts survive outside the host in water, soils and on foods, especially under moist conditions on the latter. #he cysts are readily 5illed by heat and by free(ing temperatures, and survive for only a few months outside of the host (1l-:arthi, 2 '" ;hen cysts are swallowed they cause infections by e4cysting (releasing the tropho(oite stage" in the digestive tract. #he tropho(oite stage is readily 5illed in the environment and cannot survive passage through the acidic stomach to cause infection (.tanley, 2 *".

Diagnosis of parasitic infectionsD 1-\icroscopyW

+t is 5nown that fecal e4amination to detect Giardia lamblia and E. histolytica cysts or tropho(oites and cryptosporidium oocysts produces a high percentage of false-negative results. #raditionally, the diagnosis of these infections has relied upon microscopic e4amination of cysts or tropho(oites in fresh or fi4ed stool specimen in either single or multiple fecal specimens, by @E> (@va E >arasite" e4amination test. #he sensitivity of parasite identification has been reported to increase up to 0!- when three fecal samples obtained every other day or in ten days interval. +n some laboratories .edimentation #echni6ue 7ay be done routinely (1l-%aleh, 1)02, 1l-:arthi, 2 '". Cryptosporidium oocysts are not detectable by routine iodine staining of feces. 7odified acid-fast, .afranin F 7ethylene =lue and 7odified Giehl F 3eelsen staining techni6ues disclose intestinal coccidian (=a4by et al. 1)0'". :owever, these methods are laborious, re6uire s5ilful e4perience, time consuming, and have low sensitivity (the best only / - sensitive", this could be referred to the intermittently shedding of intestinal parasites in the stool as their e4cretion have cyclic basis, or due to small amount of stool used for

e4amination, which may not contain the parasite, due to scanty infection (>illai
and Hain, 2 *, #anyu5sel and >etri, 2 *".

@E> I4aminationD
Direct wet fecal smear is done either by saline or iodine, and it is prepared by mi4ing in a slide a small amount of stool (about 2 mg" with a drop of reagent2 which is eitherD saline ( .0!- 3a<l" or iodine, the slide is covered and e4amined at low-power ob$ective (1 J" under low light intensity2 any suspicious ob$ects may then be e4amined with the high dry ob$ective (' J"
(Garcia, 1)))".

7odified GiehlF3eelsen 1cid %ast stainD

:ere we use carbol fuchsin stain for staining then use a milder decolori(er which allows the organisms to retain more of their pin5-red color (Garcia, 1)))".

2-+mmunological methodsD
#he diagnosis of parasitic infection is usually based on demonstration of antibodies (1bs", however, high percentage of people have 1bs without active infection. 1lso, such tests used for 1b detection are still sub$ected to false positive and negative reactions, as a result of lac5 of specificity of the antisera
(%achado et al2 1)) ".

%urthermore, serological methods for detection of 1bs may

be inconclusive or unreliable in many instances as in patients receiving immunosuppressive therapy (3ath and .inai2 2 1". #hus, facing these serodiagnostic problems, a rapid method for the direct detection of antigenic components (1g" of the parasites in body fluids or tissues might offer a valuable aid for rapid and specific diagnosis of acute infection (:ammouda et al., 2 /". Ifforts to improve the diagnostic testing have been developed in recent years. +ntestinal parasites are intermittently shedded in the stool as their e4cretion have cyclic basis, this problem can potentially be overcome if antigen detection assays are developed (>illai and Hain, 2 *". /

1ntigen detection assays in sera have proved to be very useful in the diagnosis of some parasitic infections. 1ntigen based IL+.1 has many significant advantages for the diagnosis of giardiasis, cryptosporidiosis and amoebiasis (7ichel et al.2 2
".

.ome of the assays are able to differentiate between E.histolytica and E.dispar, and have e4cellent sensitivity and specificity. #hey can be performed by none e4pert laboratory technicians and outperform microscopy in their potential as large-scale screening tools in epidemiological studies, such as waterborne outbrea5s situations. #hese assays range in sensitivity from //.*- to 1 )2./- to 1 - (<aballero-.alcedo et al.2 1))'". - and in specificity from

#he #riage parasite panel en(yme immunoassay (#riage" (=+@.+#I Diagnostics" is a 6ualitative en(yme immunoassay panel for the detection of Entamoeba histolyticaFE. dispar, Giardia lamblia and Cryptosporidium parvum in fresh or fro(en fecal samples. #he single immunochromatographic strip is coated with monoclonal antibodies specific for E. histolytica/E. dispar antigen 2) 5Da and for antigens of Giardia lamblia and Cryptosporidium parvum. 1ntigens from clinical fecal samples that are specific for these three parasites are isolated and immobili(ed on a membrane using specific antibodies. 1n antibody-en(yme con$ugate then binds to specific sites on these antigens. #he antigens are detected after the addition of substrate by the formation of colour bars in different areas depending on the parasite present and show on the test device as blue-blac5 lines (.harp et al. 2 1, #anyu5sel and >etri, 2 *".

*-7olecular methodsD
During the last decade, the molecular diagnostic tools has been developed for detection of parasite D31 in stool samples, it is started as a research for differentiation of Entamoeba histolytica and Entamoeba dispar and still not routinely done. 3ow they have developed a range of molecular tests, ,

including several multiple4 real time ><8As for the detection of Giardia, Cryptosporidium, Entamoeba histolytica, Microsporidia, hookworm species and Strongyloides (%ra(ar and @rlandi2 2 ,". .ensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing ma5e ><8 a useful tool for future diagnosis and studies on the molecular epidemiology of parasitology. .o far, molecular techni6ues have been mainly used for small epidemiological surveys and in reference laboratories. #he problem of these techni6ues is that they are of high cost, need e4perience, and the ris5 of contamination, that is the reason why it is not routinely applicable [7organ et al. 1))0X Frazar an] Urlan]iZ OQQ^_.

WII UY`ectives a Strategies

1- #o evaluate the clinical utility of an antigen capture en(yme immunoassay in detecting the ma$or causes of the proto(oal diarrhea in humans stools in comparison to the conventional diagnostic techni6ues.

2- #o differentiate E. histolytica (pathogenic" from E. dispar (commensal" to avoid the unnecessarily treatment with amoebicidal drugs.

WIII \aterials an] \etho]s

bc stool specimensW

%ecal samples will be collected from symptomatic patients attending Hing %ahad 3ational Guard hospital in 8iyadh city and complaining from gastrointestinal problems.

Oc Toutine UaP edaminationW

+mmediately after collection, each stool specimen will be divided into three parts, the first will be fresh, second part will be fro(en in an empty stool container, while the third part will be fi4ed in container containing 1 formalin. 1ll specimens fi4ed in 1 - formalin will be microscopically e4amined for the e4pected parasites by @E> e4aminationD Direct saline eet mount preparations Direct io]ine eet mount preparations .0!- 3a<l2 or iodine solutions. #hese mi4tures will

#he direct wet smear is prepared by mi4ing a small amount of stool (about 2 mg" with a drop of provide a uniform suspension under a coverslip.. #he entire coverslip should be systematically e4amined with the low-power ob$ective (1 J" under low light intensity2 any suspicious ob$ects may then be e4amined with the high dry ob$ective (' J". #he use of an oil immersion ob$ective (1 slide (Garcia, 1))), 1l-=rai5en, 2 *". J" on mounts of this 5ind is not routinely recommended unless the coverslip is sealed to the

The formol ether concentration techniqueW

=y centrifugation, the concentration procedure leads to the recovery of all proto(oa, eggs, and larvae present2 however, the preparation contains more debris than is found with the floatation procedure. Ither is used to e4tract debris and fat from the feces and leave the parasites at the bottom of the suspension. #his test is recommended as being the easiest to perform, allowing recovery of the broadest range of organisms, and being the least sub$ect to technical error (Garcia, 1))), 1l-:arthi, 2 '".

Staining Yy Trichrome stainW

#his stain is a modification of GomoriAs original staining procedure for tissue. +t is a rapid, simple procedure which produces uniformly wellstained smears of intestinal proto(oa, human cells, yeast cells, and artifact material in about '! min or less. +t will be used for staining of Giardia lamblia and Amoeba (Garcia, 1))), .tanley, 2 *". Staining Yy aci]cfast stainingW

@ocysts of <ryptosporidium in clinical samples may be difficult to detect without special staining. 7odified acid-fast stain (7odified Giehl 3eelsen stain" is recommended to demonstrate this organism. <oncentrated sediment of fresh or formalin-preserved stool may be used (=a4by et al2 1)0', Garcia, 1)))".

;hatever the samples will be positive or negative or uncertain for parasites, they will be retested by the en(yme immunoassay techni6ues. fc Enzyme Immunoassay [EIA_ metho]sW
1ll I+1 5its will be used with fresh, freshly fro(en and formalini(ed stool specimens.

Triage parasite panelW

#he immunoassay diagnostic 5it will be used according to the manufacturerKs directions. 1ntigens from clinical specimens that are specific for these three parasites are isolated and immobili(ed on a membrane using specific antibodies. 1n antibody-en(yme con$ugate then binds to specific sites on these antigens. #he antigens are detected after the addition of substrate by the formation of color bars in different areas depending on the parasite present and show on the test device as blue-blac5 lines (1l-:arthi, and Cam$oom2 2 ,". 11

#he tubes, pipettes, devices, and all reagents are provided with the 5it. >ositive and negative controls are included in the device, and the total time is appro4imately 1! min (.harp et al. 2 1, 1l-:arthi, and Cam$oom2 2 ,".

TechgaY panelW
#his test will be used to differentiate between E.histolytica and E.dispar, the detection will be carried out as suggested by the manufacturer. 1ll stool specimens diagnosed of being either Entamoeba histolytica or Entamoeba dispar by the #riage >arasite >anel assay will be tested by techlab panel (=uss et al. 2 0". #he microtiter wells will be coated with immobili(ed polyclonal antibody that binds adhesin of E.histolytica/ dispar. #he con$ugate is a monoclonal antibody-pero4idase con$ugate specific for E.histolytica adhesin. +f adhesin is present in the specimen, it will bind to the con$ugate and immobili(ed polyclonal antibody. #he well strips will be read in a microtiter plate reader at '! nm. #he E. histolytica ++ test detects appro4imately adhesion (Geehaida et al. 2 0". .2 to .' ng per well of

Statistical AnalysisW
Sample sizeW Depending on the references (.harp et al. 2 1, 1l-:arthi, and Cam$oom2 2 ," showing that the sensitivity of conventional diagnostic techni6ue used for stool e4amination was ranging from ,/.)- to 0 - and the sensitivity of antigen capture IL+.1 techni6ues was almost 1 increased to 1 -, the sample si(e was calculated to be '! cases (Goldsmith, 1),0". :owever, the number of samples will be in this thesis to get more accurate results.

Data entry and data analysis will be done using .tatistical >ac5age for .ocial .cience (.>..", software version 1/. Descriptive analysis will be performed for demographic findings and categorical variables.

12

Happa statistics and %isherKs I4act #est will be applied to determine the agreement of In(yme +mmunoassay techni6ues which will be used in this thesis with the traditional microscopic techni6ues. 1 value less than
(Goldsmith, 1),0".

. ! will be considered to be statistically significant

WIh Teferences
1- 1l-=rai5en, %.1., =eeching,1.3.C., :ommel, 7. and :art, <.1. Detection of Cryptosporidium amongst diarrhoeic and asymptomatic children in Ceddah, .audi 1rabia. 1nn. #rop. 7ed. >arasitol., 2 *, ),D ! !-!1 . 2- 1l-%aleh, %.G. #he prevalence of Entamoeba histolytica and other parasite in school children.<omm. :ealth in .audi 1rabia, 1)02, *2-*'. 1*

*- 1l-:arthi, ..1. >revalence of intestinal parasites in schoolchildren in 7a55ah, .audi 1rabia.#he 3ew Igypt. C. 7ed., 2 ', *1D *,-'*. '- 1l-:arthi, ..1. and Cam$oom, 7.=. Diagnosis and differentiation of Entamoeba infection in 7a55ah 1l 7u5arramah using microscopy and stool antigen detection 5its. ;orld C.7ed..ci., 2 ,, 2(1"D 1!-2 . !- 1l-.hammari, .., Hho$a, #., Il-Hhwas5y, %. and Gad, 1. +ntestinal parasitic diseases in 8iyadh, .audi 1rabiaD prevalence, sociodemographic and environmental associates. #rop. 7ed. +nt. :ealth, 2 1, /D 10'-10). /- 1L-.e5ait, 7.1, 1L-1nsary, L.1, and 1L-Gamel, %.1. >revalence of pathogenic intestinal parasites among .audi rural schoolchildren2 #he Iuropean Cournal of >ublic :ealth, 1))*, *('"D2*2-2*/. ,- 1L-#u5hi, 7.:.2 1L-1hdal, 7.3.2 1c5ers, C. >., and >eters, ;. >revalence of Giardia lamblia infection in the city of 8iyadh, .audi 1rabia. .audi 7ed. C., 1))/, 1, ('"D '02-'0/. 0- =a4by, D., =lundell, 3. :art, <.1. #he development and performance of a simple, sensitive method for the detection of Cryptosporidium oocysts in faeces. !yg., 1)0', )*D *1,-*2*. )- =iale5, 8., =inder, 3., Diet(, H., Coachim, 1., Hnobloch, C., and Gelc5, L.I. <omparison of fluorescence, antigen and ><8 assays to detect Cryptosporidium parvum in fecal specimens. Diag. 7icrobiol. +nfect. Dis., 2 2, '*D 20*-200. 1 -=olbol, .., and 7ahmoud, 1. Laboratory and clinical study of intestinal pathogenic parasites among the 8iyadh population. .audi 7ed. C., 1)0', !D 1!)-1//. 11-=uss, .., Habir, 7., >etri, ;.1., and :a6ue, 8. <omparison of #wo +mmunoassays for Detection of Entamoeba histolytica. C <lin 7icrobiol., 2 0, '/(0"D 2,,0?2,,).

12-<aballero-.alcedo, 1.7., &iveros-8ogel, =., .alvatierra, 8., #apia<onyer, C., .epMlveda-1mor, G., and @rti(-@rti(, L. .eroepidemiology of amebiasis in 7e4ico. 1m. C. #rop. 7ed. :yg., 1))', ! D '12-'1). 1*-<hai, C.N., .hin, ..7., Nun, <.H., Nu, C.8., and Lee, ..:. I4perimental activation of cryptosporidiosis in mice by immunosuppression. Horean C >arasitol2 1)) , 20D *1-,. 1'-Diamond, L..., and <lar5, <.G. 1 redescription for of Entamoeba histolytica .haudin, 1) * (amended ;al5er, 1)11" separating it from

1'

Entamoeba histolytica/dispar =rumpt 1)2!. C. Iu5. 7icrobiol., 1))*, ' D *' -*''. 1!-%achado, 1., %onte, L., 8o$as, L., 1lberti, I., and 7achin, 8. #echni6ue for the detection of "o#oplasma gondii antigens in mouse urine. 7em.+nst.@swaldo <ru(, 1)) , 0!(1"D /!-/0. 1/-%ara(, <.D. and @rlandi, >.1. Ivaluation of two D31 template preparation methods for post-immunomagnetic separation detection of Cryptosporidium parvum in foods and beverages by ><8. 1ppl. Invir. 7icrobiol., 2 ,, ,*(22"D ,','-,',/. 1,-%arthing,7.C.G. :ost parasite interactions in human giardiasis. Buart. C. 7ed., 1)0), , D 1)1-2 '. 10-%ayer 8., 7organ, L., and Lpton, ..C. Ipidemiology of CryptosporidiumD transmission, detection and identification. +nt. C. >arasitol2 2 ,* D 1* !?1*22. 1)-Garcia, L... >ractical guide to diagnostic parasitology. 1.7 press, ;ashington D<. 1))), >D /*,1 2. 2 -Goldin, 1.C.2 ;erner, 1.>.#.2 1guilera, 1.I. Ifficient diagnosis of giardiasis among nursery and primary school children in .antiago, <hile by capture IL+.1 for the detection of fecal Giardia antigens. 1mer. C. trop. 7ed. :yg., 1)) , '2D !*0-!'!. 21-Goldsmith, <.:. .ample si(e in clinical and laboratory research, 1),0. 22-:ammouda, 3.1., 1min, ..1., Hhalifa, 1.7., 1bou-Il 3aga, +.%., Gaafar, 7.8. and 3asr, 7.1. #he use of IL+.1 and immunohistochemistry techni6ues for detection of "o#oplasma gondii antigen in tissues of e4perimentally infected mice. C.Igypt..oc.>arasitol.,2 /. */(*"D )2!)*!. 2*-:uang, D.=., and ;hite, 1.<. 1n updated review on Cryptosporidium and Giardia. Gastroenterol. <lin. 3orth 1m.2 2 /, *! (2"D 2)1?*1'. 2'-Lewthwaite, >., Geoffrey, G., and :art, 1. Gastrointestinal parasites in the immunocompromised. <urr @pin +nfect Dis2 2 !, 10(!"D '2,-'*!. 2!-7eyer, I.1., and 8adulescu, .. - Giardia and giardiasis. 1dvanc. >arasit., 1),), 1,D 1-',. 2/- 7ichel, 7.N., Hhalifa, 1.7., and +brahim, +.8. Detection of Cryptosporidium parvum antigen by co-agglutination test and IL+.1. Iast.7editerr.:ealth C., 2 , /(!"D 0)0-) ,. 2,-7organX V \X PallantX g X DeyerX i j X ForYesX D A X TichX k an] ThompsonX 8.<.1. <omparison of ><8 and 7icroscopy for Detection of

1!

Cryptosporidium parvum in :uman %ecal .pecimensD <linical #rial. C. <lin. 7icrobiol., 1))0, */('"D ))!-))0. 20-3ath, 1 and .nai, 1. <erebral to4oplasmosis. <ur. #reat. @pt. +nf. Dis., 2 1, *D ',1-'0 . 2)-3ime %.1., =ure5, C.D., >age, D.L., :olscher, 3.1., and Nardley, C.:. 1cute enterocolitis in a human being infected with the proto(oon Cryptosporidium. Gastroenterology2 1),/, , D !)2-0. * -@mar, 7..., 1bu-Geid, :.1. and 7ahfou(, 1.1. +ntestinal parasitic infections in school children of 1bha (1sir", .audi 1rabia. 1cta C. of #ropica, 1))1, *'D 1)!-2 2. *1->alit, 1., .ur, D., 7itraDhar, H., and .aha, 7.8. 1symptomatic cryptosporidiosis in periurban slum setting in Hol5ata, +ndia ? a pilot study. C. +nfect. Dis.2 2 !, !0D 11 -1. *2->ettoello-7antovani, 7., Di, 7.L., Dettori, G., &a$ro, >., .cotti, .., and Ditullio, 7.#. 1symptomatic carriage of intestinal Cryptosporidium in immunocompetent and immunodeficient childrenD a prospective study. >ediatr. +nfect. Dis. C.2 1))!, 1'D 1 '2-,. **->illai, D.8. and Hain, H.<. +mmunochromatographic strip-based detection of Entamoeba histolytica$E. dispar and Giardia lamblia coproantigen. C. <lin. 7icrobiol., 2 *, *,D * 1,-* 1). *'-8amratnam, =., %lanigan, #.>. <ryptosporidiosis in persons with :+& infection. >ostgrad. 7ed. C.2 1)),, ,*D ,1*?/. *!-8hee, C.H., .eu, N..., and >ar5, =.H. +solation and identification of Cryptosporidium from various animals in Horea. Horean C. >arasitol.2 1))1, 2)D 1*)-1'0. */-.chant(, >.7. >arasitic (oonoses in perspective. +nt. C. >arasit., 1)) , 21D 1/1-1, . *,-.harp, ..I., .uare(, <.1., Duran, N., and >oppiti, 8.C. Ivaluation of the triage microparasite panel for detection of Giardia lamblia, Intamoeba histolyticaF Intamoeba dispar, and <ryptosporidium parvum in patient stool specimens. C. <lin. 7icrobiol., 2 1, *)(1"D **2?**'. *0-.tanley, ..L,. 1moebiasis. #he Lancet, 2 *, */1D 1 2!-1 *'.

*)-#anyu5sel, 7., and >etri, ;.1. Laboratory Diagnosis of 1mebiasis. <lin. 7icrobiol. 8ev., 2 *, ,1*-,2). ' -#hompson, 8.<.1.2 8eynoldson, C.1., and 7endis, 1.:.;. Giardia and giardiasis. 1dvanc. >arasit., 1))*, *2D 1-1/ .

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'1-#rol, :., 7arti, :., and ;eiss, 3. .imple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and ><8. C.<lin. 7icrobiol., 1)),, *!D of 1, 1-1, !. '2-#(ipori ., ;ard :. <ryptosporidiosisD biology, pathogenesis and disease. 7icrobes +nfect2 2 2, 'D 1 ',?1 !0. '*-&erwei$, C.C., =langO, 8.1., and #emplton, I.D., .imultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in %ecal .amples by Lsing 7ultiple4 8eal-#ime ><8. C.<lin.7icrobiol.2 2 ', '2(*"D122 -122*. ''-&uorio, 1.%., Co5ipii, 1.7., and Co5ipii, L. Cryptosporidium in asymptomatic children. 8ev. +nfect. Dis.2 1))1, 1*D 2/1-2/'. '!-;ee, ..:., Coo, :.D., and Hang, N.=. Ivaluation for detection of Cryptosporidium oocysts in diarrheal feces of calves. Horean C. >arasitol.2 1))/, *'D 121-/. '/-Geehaida, 7., ;an 3or 1milah, ;.1.;., 1mry, 1.8., :assan, .., .arimah, 1. and 8ahmah, 3. 1. study on the usefulness of #echlab Entamoeba histolytica ++ antigen detection IL+.1 in the diagnosis of amoebic liver abscess (1L1" at :ospital Lniversiti .ains 7alaysia (:L.7", Helantan, 7alaysia. #ropical =iomedicine2 2 0, 2!(*"D 2 )? 21/.

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