Taibah University

Journal of Taibah University Medical Sciences (2014) 9(4), 263e267

Journal of Taibah University Medical Sciences

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Experimental Article

Comparison of Kinyoun’s acid-fast and immunofluorescent

methods detected an unprecedented occurrence of Cryptosporidium

in the Eastern Region of Saudi Arabia

Salah H. Elsafi, Ph.D. a,*, Somaya S. Al-Sheban, B.Sc. b, Khalid M. Al-Jubran, Ph.D. c,Mohamed M. Abu Hassan, M.Sc. c and Eidan M. Al Zahrani, Ph.D. c

aClinical Laboratory Science Department, Prince Sultan Military College of Heath Sciences, Dhahran,

Kingdom of Saudi ArabiabDepartment of Pathology and Laboratory Medicine, King Fahad Specialist Hospital, Dammam, Kingdom of Saudi ArabiacPrince Sultan Military College of Heath Sciences, Dhahran, Kingdom of Saudi Arabia

Received 12 January 2014; revised 1 March 2014; accepted 1 March 2014; Available online 26 September 2014

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ةعبتملاةيصيخشتلاةيعونلاوةيساسحلاةنراقملثحبلافدهي:ثحبلافادهأضمحللةمواقملاةلدعملانوينكةغبصيهو،ةيدوعسلاةيبرعلاةكلمملايفايلاح.مويديروبسوتبيركلادوجونعفشكللرشابملايعانملاقلأتلاةقيرطعم

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جئاتنتطعأو،ءادألايفلهسأتناكيعانملاقلأتلاةينقتنأنيبت:تاجاتنتسالاةنراقملابةيسيكلامويديروبسوتبيركلاتاضيوبفاشتكالةيعونوةيساسحرثكأ.ضمحللةمواقملاةلدعملانوينكهغبصمادختسابمتتيتلاةيديلقتلاةقيرطلاعم

Corresponding address: Head, Clinical Laboratory Science

partment, Prince Sultan Military College of Health Sciences,

. Box 33048, Dammam 31448, Kingdom of Saudi Arabia.

E-mail: salahelsafi@hotmail.com, salah@psmchs.edu.sa

.H. Elsafi)

r review under responsibility of Taibah University.

Production and hosting by Elsevier

8-3612 � 2014 Taibah University.

duction and hosting by Elsevier Ltd.

p://dx.doi.org/10.1016/j.jtumed.2014.03.008

Open access under CC BY-NC-ND lice

نمىلعأ%٦٦يهوةساردلاهذهيفاهيلإلصوتلامتيتلاةجيتنلانوكتكلذبونودضرملااذهىقبياليكلو.اقباستيرجأيتلاتاحوسملاوتاساردلاصحففاضينأبحصننلاهسإلاتالاحنمريثكدنعضيخشتنيباصملاىضرملللمعتيتلاةينيتورلاتاصوحفلانمضمويديروبسوتبيركلا.ةيصيخشتلاةيبطلاتاربتخملايفلاهسإلاب

صحفلا;ضمحللةمواقملاةلدعملانوينكةغبصبصحفلا:ةيحاتفملاتاملكلاةيدوعسلاةيبرعلاةكلمملا;مويديروبسوتبيرك;يعانملاقلأتلاةقيرطب

Abstract

Objectives: The aim of this study was to compare the

diagnostic sensitivity and specificity of the modified

Kinyoun’s acid-fast test used widely in Saudi Arabia

compared to the direct immunofluorscent assay (DFA)

for monitoring the occurrence of Cryptosporidium spp.

Methods: We compared the conventional, modified

Kinyoun’s acid-fast with the Merifluor direct immuno-

fluorscent assay for the detection of Cryptosporidium

oocysts in 100 stool samples, among patients who re-

ported to King Fahad Military Medical Complex during

AprileMay, 2012.

Results: The modified Kinyoun’s method and the DFA

revealed 49 and 66 Cryptosporidium oocyst positives,

respectively. Sensitivity and specificity of acid-fast when

compared to DFA, were 66.67% (95% CI: 53.99%

e77.79%) and 88.24% (95% CI: 72.53%e96.63%),

respectively, (Kappa ¼ 0.487 and the 95% confidence

interval was 0.328e0.645). The positive and negative

nse.

S.H. Elsafi et al.264

predictive values of the same method were 91.67% (95%

CI: 80.00%e97.63% and 57.69% (95% CI: 43.21%

e71.27%), respectively.

Conclusion: The DFA was found to be simple to perform

and has been demonstrated to have a higher sensitivity

than traditional staining procedures. The current posi-

tivity rate of 66% of Cryptosporidium in this study is

higher than indicated before by several studies. This

disease remains underdiagnosed in current routine labo-

ratory procedures. It is recommended that tests for

Cryptosporidium be done as part of a general diarrhea

screen during standard stool tests in diagnostic

laboratories.

Keywords: Cryptosporidium; Immunofluorescent; Kinyoun’s

acid-fast; Kingdom of Saudi Arabia

� 2014 Taibah University.

Production and hosting by Elsevier Ltd.

Open access under CC BY-NC-ND license.

Introduction

Cryptosporidium is a genus of coccidian parasites associ-

ated with gastrointestinal diseases in all vertebrates,including humans.1 Although many species ofCryptosporidium have been reported in humans, C. hominis

and C. parvum are responsible for the majority of humaninfections.2,3 C. parvum is found in humans as well as in anumber of other animals, whereas C. hominis apparently

infects only humans.4,5 In developing countriesCryptosporidium infections occur mostly in children underfive years of age,6e8 although outbreaks do occur

worldwide in all age groups.9 Immunocompromised peopleare most likely to be affected more seriously.10

Cryptosporidium is usually diagnosed by microscopicdetection of oocysts in stool specimens. Although various

methods have been described for the detection of oocysts, thediagnosis of C. parvum is usually made by using modifiedacid-fast,11 or immunofluorescence staining12,13 on

concentrated or unconcentrated fecal smears. The acid-faststain is not universally recommended for the detection ofC. parvum oocysts in fecal samples largely because of previ-

ous data that suggested a low specificity and sensitivity.14,15

Antigen detection by immunoassays has become widelyused for the diagnosis of cryptosporidiosis. Detection ofantigens on the oocyst wall of the organism in stool speci-

mens, using direct fluorescent-antibody for diagnosis ofcryptosporidiosis, has been reported several times. Goodsensitivities and specificities have been reported for some of

these tests in several comparative studies.16e21 Thesetechniques increase the sensitivity compared to routinelight microscopy and are easy to perform.13,22e24

The immunofluorescent technique has been demonstratedto have a higher sensitivity than traditional staining pro-cedures and is often considered as a gold standard for eval-

uating other diagnostic techniques.21,22,25,26

The Merifluor Cryptosporidium/Giardia Direct Immuno-fluorescence Assay (DFA) uses the principle of directimmunofluorescence. The combined fluorescein

isothiocyanate-labeled monoclonal antibodies are directed

against the cell wall antigens of G. lamblia cysts and Cryp-tosporidium parvum oocysts.

Cryptosporidium remains underdiagnosed under the cur-rent routine laboratory procedures, based mainly on thedetection of oocysts in fresh unconcentrated fecal smears

stained with modified ZiehleNeelsen (mZN). However, theuse of appropriate sensitive methods can improve casedetection of Cryptosporidium.

Most previous studies on the epidemiology of intestinalparasites in Saudi Arabia did not include Cryptospo-ridium27,28 and most of the available data on the occurrenceof the disease were hospital based. Cryptosporidium parvum

and C. hominis remain the two most frequent speciesdetected29 with various ranges of prevalence, mostly amongchildren below two years of age,30e32 and

immunocompromised patients.33 The Cryptosporidiumoocyst was detected in 20 (32%) of the symptomaticchildren, but only nine (4.7%) of the asymptomatic

children in Jeddah, Saudi Arabia.34

Typically, Cryptosporidium testing is currently notincluded in the routine examination of stool for ova andparasites. The aim of this study was to compare the diag-

nostic sensitivity and specificity of the mZN test used widelyin Saudi Arabia compared to the DFA for monitoring theoccurrence of Cryptosporidium spp.

Materials and Methods

Specimen collection

One hundred stool specimens were collected in Para-pak/Para-Pak 10% SAF (Sodium Acetate acetic acid) from in-dividuals of various ages who reported to the outpatient

clinic of King Fahad Military Medical Complex betweenApril-May, 2012 presenting with abdominal symptoms,mainly diarrhea. All specimens were anonymized and pro-

cessed separately.

Modified Kinyoun’s acid-fast technique for Cryptosporidium

The modified Kinyoun’s acid-fast stain was performed aspreviously described.35 In brief, fecal smears were dried on aslide warmer at 60 �C, before being fixed with absolute

methanol for 30 s, and stained with Kinyoun’s carbolfuchsin for one minute. The preparations were de-stainedwith acid alcohol for 2 min, then counterstained with Mal-

achite green for another 2 min. Slides were then rinsed withdistilled water, dried on a slide warmer at 60 �C for about5 min, mounted with a cover slip using Cytoseal 280

mounting medium, and examined under 100� oil objectivesfor the detection of Cryptosporidium oocyst.

Direct immunofluorescence assay

The MeriFluor Cryptosporidium/Giardia direct immuno-fluorescence detection kit procedure was used (Meridian

Diagnostics, Inc., Cincinnati, Ohio) according to the man-ufacturer’s directions. In brief, a drop of fecal material wassmeared over the entire slide well and dried at room tem-perature. Positive and negative controls were applied when-

ever the procedure was performed. One drop of a detection

Kinyoun’s acid-fast and immunofluorescent methods detected an unprecedented occurrence of Cryptosporidium 265

reagent (FITC labeled anti-Cryptosporidium and anti-Giar-dia monoclonal antibodies in a buffered solution containing

a protein stabilizer and 0.1% sodium azide) was added toeach well. A drop of counterstain (eriochrome black solu-tion) was added and mixed in each well. Slides were incu-

bated in a humidified chamber in a dark room, for 30 min.Slides were then washed and a coverslip was affixed withmounting medium, Buffered glycerol containing formalin, an

antiquencher and 0.05% sodium azide, before it was scannedthoroughly, using an X40 Olympus with (FITC filter set(excitation 490 nm; emission 510 nm). The presence ofCryptosporidium oocysts as confirmed at higher magnifica-

tion apple green color and characteristic morphology ofCryptosporidium oocysts.

Data analysis

We used Meriflour as a reference test, and accordingly,the sensitivity and specificity of the Kinyoun’s acid-fasttechnique were calculated. Cohen’s kappa coefficient of

agreement was calculated using SPSS version 20.0 StatisticalPackage for the Social Sciences (SPSS, Inc., Chicago, IL).

Results

All 100 fecal samples were tested for Cryptosporidium bythe Kinyoun’s acid-fast technique and DFA. The ModifiedKinyoun’s method and DFA revealed 48 and 66 Crypto-

sporidium positive samples respectively (Table 1). Forty-fourspecimens tested positive using both methods, whereas 30tested negative using both methods. Twenty two samples

which tested positive by DFA were negative by theKinyoun’s acid-fast technique, while 4 samples were positiveby the latter, but negative by the former with Cohen’s Kappaindex equal to 0.4866.

The sensitivity, specificity, positive predictive value andnegative predictive value of the Modified Kinyoun’s methodwere 66.67%, 88.24%, 91.67%, and 57.69%, respectively

(Table 2).

Discussion

We compared the conventional microscopy of Kinyoun’sacid-fast technique with DFA for the detection of Crypto-sporidium and found the former to be considerably less sen-sitive and less specific than the latter test. In another study

the sensitivity of the Kinyoun’s acid-fast technique was75.4%, when compared against real-time PCR, while that ofimmunofluorescence microscopy was 97.4%.36 The

Table 1: Correlation between DFA and Kinyoun’s acid-fast

stain for the detection of Cryptosporidium oocyst.

Kinyoun’s acid-fast Meriflour (DFA) Total

Positive Negative

Positive 44 (91.7) 4 (8.3) 48

Negative 22 (42.3) 30 (57.7) 52

Total 66 34 100

Cohen’s Kappa index ¼ 0.4866 SE ¼ 0.0935.

Chi square test ¼ 27.10; P-value ¼ 0.000.

diagnostic sensitivity of the acid-fast method is moderate,at 66.67% CI ¼ 53.99e77.79%) with a specificity of 88.24%

(CI ¼ 72.53e96.63%) which needs to differentiate stainingartifacts and other organisms. The modified Kinyoun’s acid-fast technique requires a high degree of interpretation and

continued practice. Although the speed of turnaround time isgood, it is highly labor intensive, despite the low cost per test,as has been reported before.37 Kinyoun’s acid-fast technique

was unable to detect 22 positive cases. These false negativeresults were probably obtained from specimens with lowparasite numbers. The Kinyoun’s acid-fast technique testalso detected four false positives. The acid-fast nature of the

oocyst makes it difficult to differentiate them from othernoncryptosporidial acid-fast organisms of comparable size.11

The lower sensitivity of the modified Kinyoun’s acid-fast

technique in this study may be in part due to the use ofunconcentrated fecal smears. Concentrating oocysts in thesample can increase the sensitivity of Kinyoun’s acid-fast

technique for its detection.23

The accuracy of the acid-fast stain has not been wellestablished, and depends, in part, upon the number of stoolspecimens examined, since the number of oocysts shed in

feces is not constant and multiple samples are required toimprove its diagnostic yield.38 In one report the examinationof a single stool specimen identified only 30 percent intestinal

Cryptosporidium infections.10

On the other hand, DFA, which detected more cases, withmoderate degree of interpretation, moderately labor intensive,

and very good turnaround time and simplicity to perform wasconsidered a gold standard method in this study because of itspreviously reported sensitivity and specificity.13,16,20,25,39,40 It

is also used to detect Crysptosporidium oocysts in non-fecalsamples.41 Thus, DFA, with its higher sensitivity thantraditional staining procedure methods, identifying oocystsin concentrated or unconcentrated fecal samples, is the

current test of choice for diagnosis of cryptosporidiosis.22

Cryptosporidium is often thought to be underdiagnosedin current routine laboratory procedures. Since all infected

persons are not symptomatic, the actual number of infectedpeople remains undetected and could be much higher thanthe reported number. The true extent of underdiagnosed

cryptosporidosis in Saudi Arabia is not known. A likelyreason for the underdiagnosis of the disease is due to the lackof routine examination of stool specimens with appropriate

sensitive technique.42,43 Most clinical laboratories do notroutinely include any stool assay for Cryptosporidiumunless requested specifically by the ordering physicians.

The use of an immunoassay technique when examining

stool specimens for Cryptosporidium would be the preferredprocedure, given the moderate insensitivity of acid-fast stain

Table 2: Sensitivity, specificity, and positive and negative pre-

dictive values together with the upper and lower 95% confi-

dence interval (in parenthesis) of Kinyoun’s acid-fast technique

for detecting C. parvum.

Kinyoun’s acid-fast techniques

Sensitivity% 66.67 (53.99e77.79)

Specificity% 88.24 (72.53e96.63)

Positive predictive value% 91.67 (80.00e97.63)

Negative predictive value% 57.69 (43.21e71.27)

S.H. Elsafi et al.266

microscopy, the technical time involved, and the skillsrequired for such testing.

Because of the low prevalence of cryptosporidosis inmany areas, and the difficulty of testing for the presence ofthe parasites, examination for these particular parasites is

often not routinely performed.42,43

Out of the 100 samples tested, the DFA, reference methodin this study, detected a 66% positivity rate, while the

Kinyoun’s acid-fast technique detected 44% true positives.The detected positivity rate is considered to be higher thanever detected before in similar settings. It is worth mentioningthat most of the specimens examined in this study were not

diarrheic and belonged to different age groups.A study to investigate the prevalence of cryptosporidiosis

in Dammam and Alkhobar, Saudi Arabia, when testing 209

stool specimens from children and 112 from adults withdiarrhea, only two (1%) of the children, were positive forCryptosporidium spp., both aged 2 years.30 A one year

hospital based study in Riyadh, Saudi Arabia,cryptosporidiosis was detected in two out 174 children (0e120 months of age) with diarrhea using a safranin-methylene blue stain.31 The Cryptosporidium oocyst was

detected in 20 (32%) of the symptomatic preschoolchildren under 5 years but only nine (4.7%) of theasymptomatic ones in Jeddah, Saudi Arabia.34 A higher

prevalence of Cryptosporidium of 69.7% and 64.2%infection rates were also detected, but amongimmunocompromised Saudi patients of various ages.33 A

cross-sectional hospital based study in Jeddah, Saudi Ara-bia, detected cryptosporidiosis among 8.3% inpatients and2.3% outpatients using concentrated fecal smears, examined

with iodine and the Ziehl Neelsen staining methods.44

Another hospital based study, with a modified ZiehleNeelsen technique on concentrated mostly diarrheic stoolrevealed 8.1% Cryptosporidium parvum.45 Concentrated

fecal smears of 812 children (2e12 years) were examinedusing the modified Ziehl Neelsen in Tabuk, Saudi Arabia,and revealed 0.6% prevalence of cryptosporidium.46

The current positivity rate of Cryptosporidium stronglyindicated that this disease remains underdiagnosed in currentroutine laboratory procedures. It is recommended that tests

for Cryptosporidium be done as part of a general diarrheascreen during standard stool tests in diagnostic laboratories.

Fluorescent detection procedures offer both increased

sensitivity and specificity, compared to conventional stainingmethods and may be particularly helpful in screening largenumbers of patients in an outbreak situation, or whenscreening patients with minimal symptoms.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Prince Sultan MilitaryCollege of Health Sciences. We thank Mr. Mohi Hussein forhis technical assistance. Thanks are due to Mr. Ali Gazwany

for translating the Arabic abstract and Mr. Mazin Elsafi fortyping the manuscript.

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