Performance evaluation of direct saline stool microscopy,Formol ether concentration and Kato Katz diagnostic methods for intestinal parasitosis in the absence of gold standard methods

Abstract

Background

The parasite load within the sample and the amount of sample taken during examination greatly compromise the sensitivity of direct saline stool microscopy.

Methods

A cross-sectional study was conducted in March 2011 in Bahir Dar city among 778 fresh single stool samples to evaluate the performance of direct saline (DS), Kato Katz (KK) and Formol ether concentration (FEC) methods against the ‘Gold’ standard.

Result

Among 778 stool samples from school age children, the highest prevalence of intestinal parasites was recorded by FEC (55.1%). The sensitivity of DS, FEC and KK were 61.1%, 92.3% and 58.7%, respectively.

Conclusion

FEC is more sensitive than DS and KK. Hence, use of the latter is preferred.

Keywords

Direct stool microscopy sensitivity NPV Kato Katz Formol ether concentration

Introduction

Intestinal parasites are among the most common human infections throughout the world especially in tropical and subtropical countries.¹ The diagnosis of parasitic infections commonly relies on the detection of helminth eggs or larvae and protozoal cysts or trophozoites. The commonly used diagnostic techniques are direct saline (DS), Formol ether concentration (FEC)² and Kato Katz (KK).³

The sensitivity of DS is low as compared to KK and FEC. DS is the most commonly used method for the diagnosis of intestinal parasitic infections generally in Africa, and Ethiopia in particular.^4,5

Moreover, a low sensitivity of DS has been noted in the situation of low intensity infections.⁶ On the other hand, the detection performance of FEC is higher than DS^7,8 and KK with regard to helminthes diagnosis.⁹ Consequently, the use of DS as a routine diagnostic method may lead to under-diagnosis of intestinal parasitosis, which generally requires a more rapid, easy, reliable and sensitive diagnostic method.

DS is simple to perform, of low cost and takes little time but its sensitivity may be compromised by a single stool examination, and the amount of stool sample. Moreover, the available information with respect to KK and FEC in Ethiopia in general and in the study area in particular is limited. This study was therefore carried out to evaluate the performance of DS, FEC and KK techniques.

Materials and Methods

A cross-sectional study was conducted in Bahir Dar city, the capital city of the Amhara National Regional State, situated in north-western Ethiopia, 560 km from Addis Ababa, among school-age children in March 2011. Based on the 2007 Census, the city has a total population of 221,991 (108,456 boys/men).¹⁰

A total of 778 school age children were selected as study participants from 12 primary schools among 50 primary schools. Eligible children were randomly selected in a class. Those students taking anti-parasitic drugs and those who refused to participate were excluded from the study. Training of laboratory staff and explanation about the study were given before samples were collected.

Three stool slides, labelled with the students’ ID numbers, were processed for DS, KK and FEC techniques and examined microscopically. Examination was performed as soon as the stool specimens arrived in the laboratory. KK and FEC techniques were followed according to test protocol.

The performances of the three techniques were compared. Since there is no ‘gold’ standard method, the sensitivity and negative predictive value (NPV) of the diagnostic tests were estimated using the combined results of all three tests.¹¹

Tests were carried out as follows:

DS: A fresh stool sample (approximately 2 mg) was put on a slide with a wooden applicator, and emulsified with saline. Depending on the stool consistency, iodine was used. Parasites were identified under microscopy first using a 10× and then a 40× objective.

FEC: 0.5 g of faeces in 10 mL of normal saline was mixed thoroughly in a glass. Two layers of gauze were placed in a funnel and the contents strained into a 15 mL centrifuge tube. A total of 2.5 mL of 10% formaldehyde and 1 mL of ether were then added. The solution was mixed well and centrifuged at 1000 revolution/min for 3 min. Two slides (one for saline and the other for iodine) were prepared from the sediment, covered with cover slide and finally examined microscopically.

KK: 41.7 mg of sieved stool was transferred to the templates according to the manufacturer’s instructions (Mahidol University, Thailand).¹² The stool was covered with cellophane which was previously immersed with malachite green. Identification of the ova or cysts of the parasites and quantification of the ova of S. mansoni and geo-helminths were performed.¹³

To eliminate observer bias, two laboratory personnel performed the microscopic examination independently of each other, being blinded to the other’s findings, and the slides were then checked by the principal investigator. The results of their observation were recorded for later comparison on separate sheets. A quality control was done by repeating all discordant results.

Data were entered and analysed using SPSS version 20. Sensitivity, specificity, positive predictive value (PPV), negative predictive values and Kappa value of DS, KK and FEC techniques were calculated against the notional ‘gold’ standard.

Ethical clearance was obtained from College of Medicine and Health Science ethical review committee, Bahir Dar University and from Bahir Dar city zone educational office. Written informed consent was obtained from every study participant including the parents and the guardians of the children. Intestinal parasite positive cases were treated with anti-helmenthic or anti-protozoal medication depending on the types of species identification.

Results

From a total of 778 samples, the overall prevalence of intestinal parasitosis was high (55.1%) using FEC (Figure 1). High detection rate of helminthes (51%) (Figure 2) by FEC and of protozoa (10.2%) (Figure 3) by DS was found.

Figure 1.

The performance of DSM, FEC and KK techniques to identify intestinal parasites.

Figure 2.

The performance of DSM, FEC and KK techniques to identify helminthes parasites.

Figure 3.

The performance of DSM, FEC and KK techniques to identify protozoa parasites.

The detection rate when all three techniques (‘gold’ standard method) used together was 59.8% (Figure 1) for intestinal parasitosis and 43.4% for S. mansoni and geohelminths (43.4%) (Table 1). FEC had the highest sensitivity and NPV in the detection of intestine parasitosis (Table 2) and S. mansoni and geohelminths (Table 3).

Table 1.

The distribution of S. mansoni and geohelminths identified in each diagnostic test.

Types of methods	n	A. lumbricoids Pos (n (%))	Hw species Pos (n (%))	T. tricuria Pos (n (%))	S. mansoni Pos (n (%))	Total Pos (n (%))
DSM	778	45 (26)	99 (57.2)	2 (1.2)	27 (15.6)	173 (22.2)
FEC	778	99 (35.6)	160 (57.6)	4 (1.4)	15 (5.4)	278 (35.8)
KK	778	69 (28.3)	156 (63.9)	4 (1.6)	15 (6.2)	244 (31.4)

DSM, direct saline stool microscopy; Pos, positive.

Table 2.

The performance of diagnostic techniques to diagnose intestinal parasitosis against the gold standard method.

Types of techniques	Gold standard Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	P value	Kappa value
DSM	61 (60–63)	100 (98.5–100)	100 (98.7–100)	63.4 (59–67	<0.005	0.56
FEC	92.3 (91–93)	100 (98.6–100)	100 (98.7–100)	89.7 (89–91)	<0.005	0.91
KK	59 (57–60)	100 (98.5–100)	100 (98.3–100)	62 (61–66)	<0.006	0.53

DSM, direct saline stool microscopy; NPV, negative predictive value; PPV, positive predictive value.

The agreement of DS, FEC and KK techniques on diagnosis of intestinal parasitosis was moderate (k = 0.56), almost perfect (k = 0.91) and moderate (k = 0.53), respectively. Their agreement on the diagnosis S. mansoni and geohelminths was moderate (k = 0.52%), almost perfect (k = 0.98) and fair (k = 0.72), respectively (Table 3).

Table 3.

The performance of diagnostic techniques to diagnosis geohelminths and S. mansoni against the gold standard method.

Types of techniques	Gold standard Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	Kappa value
DSM	48.5 (47–49)	100 (99–100)	100 (97–100)	72 (71–72)	0.52
FEC	98.5 (97–99)	100 (99–100)	100 (99–100)	98.9 (98–99)	0.99
KK	69.5 (68–70)	100 (99–100)	100 (98–100)	81 (80–81)	0.72

DSM, direct saline stool microscopy.

Discussion

In this study, a wide variety of parasites were detected by the FEC method. Our results confirmed that FEC is considerably more reliable than either DS or KK. This result agrees with previous studies.^14,15 In our study, DS proved more effective than KK in contrast to earlier reports.¹⁶ However, this difference may be due to poor KK preparation and a greater proportion of protozoal parasites which this method picks up less readily.

Taking the combined results of three techniques as a standard test for intestinal parasitic infection, the sensitivity and NPV of FEC was higher than DS or KK, possibly for similar reasons, in contrast to findings in Gondar.¹⁶ The detection rate by DS for S. Mansoni and geohelminths was poor compared to either FEC or KK in our study, as suggested in previous studies performed in northwest Ethiopia,¹⁴ Gondar¹⁶ and Nigeria.¹⁷

The detection rate of hook worm species using FEC and KK was high in our present study unlike in Zanzibar¹⁸ and Northwest Gondar.¹⁴ This may be due high endemicity in Bahir Dar.

Furthermore, DS was poor in detecting A. lumbricoides, T. trichiura and hookworm species as compared to KK and FEC, as previously recognised.^14,19

Despite this, DS remains the preferred stool parasitological detection technique in almost all health institutions in Ethiopia notwithstanding its limitations. Undeniably the serious under-reporting of intestinal parasitosis will therefore continue to the detriment of our population.

FEC has, however, a high sensitivity, and it is recommended that FEC is, at least, used for samples initially found negative for parasites and as a confirmatory test, if its use cannot be universally introduced.

Conclusion

The present study revealed that FEC diagnosis methods showed better performance than the KK and DSM techniques. Thus, FEC technique can be used as a routine diagnostic method to examine intestinal parasites.

Footnotes

Acknowledgments

We would like to thank all the children, parents and school teachers who cooperated with us in this study. We are grateful to the Institute of Health Carlos III, Madrid, Spain, for financial support and especially to Dr. Jose Maria for his valuable comments.

Declaration of conflicting interests

All the authors have seen the manuscript and approve it for submission. The authors have no competing interest in the publication of the manuscript to declare.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

World Health Organization. Control of Tropical Diseases, Geneva: WHO, 1998.

Faust

Antoni

Odom

. A critical study of clinical laboratory technicians for the diagnosis of protozoan cycts and helminth eggs in feces. Am J Trop Med 1938; 18: 169–183.

Shimelis

Berhanu

Girmay

Zelalem

Techalew

. Intestinal parasitic infections in relation to HIV/AIDS status, diarrhea and CD4 T-cell count. BMC Infectious Diseases 2009; 9: 155–155.

Knopp

Rinaldi

Khamis

Stothard

Rollinson

Maurelli

. A single FLOTAC is more sensitive than triplicate Kato-Katz for the diagnosis of low-intensity soil-transmitted helminth infections. T Roy Soc Trop Med Hyg 2009; 103: 347–354.

Tarafder

Carabin

Joseph

Balolong

Olveda

McGarvey

. Estimating the sensitivity and specificity of Kato-Katz stool examination technique for detection of hookworms, Ascaris lumbricoides and Trichuris trichiura infections in humans in the absence of a ‘gold’ standard. Int J Parasitol 2010; 40: 399–404.

Levecke

Wilde

Vandenhoute

Vercruysse

. Field validity and feasibility of four techniques for the detection of Trichuris in Simians: a model for monitoring drug efficacy in public health. PLoS Neg Trop Dis 2009; 3: e366–e366.

Ritchie

. An ether sedimentation technique for routine stool examination. Bull US Army Med Dep 1948; 8: 326–326.

Mcmillan

Mcneillage

GJC

. Comparison of the sensitivity of microscopy and culture in the laboratory diagnosis of intestinal protozoal infection. Clin Pathol 1984; 37: 809–811.

Santos

Luciano

Cerqueira

EJL

Soares

. Comparison of the thick smear and Kato-Katz techniques for diagnosis of intestinal helminth infections. Revista da Sociedade Brasileira de Med Trop 2005; 38: 196–198.

10.

Federal Democratic Republic of Ethiopia. Population Census Commission: Summary and statistical Report of the 2007 Population and Housing Census: Population size by age and sex, Addis Ababa: Federal Democratic Republic of Ethiopia, 2008.

11.

Dendukuri

Rahme

Bélisle

Joseph

. Bayesian sample size determination for prevalence and diagnostic test studies in the absence of a gold standard test. Biometrics 2004; 60: 388–397.

12.

Tungtroncher

Chiworaporn

Praewanich

Radomyos

Biotano

. The potential usefulness of modified Kato thick smear technique in the detection of intestinal sarcocystosis during field survey. Asian J Trop Med Public Health 2007; 38: 232–238.

13.

World Health Organization Expert Committee. Prevention and control of schistosomiasis and soil-transmitted helminthiasis, World Health Organization Technical Report Series 912, Geneva: WHO, 2002.

14.

Moges

Belyhun

Tiruneh

Kebede

Mulu

Kassu

. Comparison of formol-acetone concentration method with that of the direct iodine preparation and formol-ether concentration methods for examination of stool parasites. Ethiopian J Health Dev 2010; 24: 148–151.

15.

Taye

. Comparison of Kato-Katz and Formol-Ether concentration methods for the diagnosis of intestinal helminthic infections among school children of Wonji Shoa town, Eastern Ethiopia: A school based cross-sectional study. American Journal of Health 2014; 2: 271–274.

16.

Endris

Tekeste

Lemma

Kassu

. Comparison of the Kato-Katz, Wet Mount, and Formol-Ether concentration diagnostic techniques for intestinal helminth infections in Ethiopia. ISRN Parasitology 2013; 2013: 5–5.

17.

Sheyin

Bigwan

Galadima

. Comparison of Formol-ether, direct smear and nigrosine methylene blue for the diagnosis of human intestinal parasites. J Microbiol Res Rev 2013; 1: 30–34.

18.

Goodman

Haji

Bickle

. A comparison of methods for detecting the eggs of ascaris, trichuris, and hookworm in infant stool, and the epidemiology of infection in zanzibari infants. Am J Trop Med Hyg 2007; 76: 725–731.

19.

Paraemeshwarappa

Chandrakanth

Unil

. The prevalence of intestinal parasitic infections and the evaluation of different concentration techniques of the stool examination. J Clin Diagn Res 2012; 6: 1188–1191.