African Health Sciences Makerere University Medical School ISSN: 1680-6905 EISSN: 1729-0503 Vol. 7, Num. 1, 2007, pp. 33-36

African Health Sciences, Vol. 7, No. 1, March, 2007, pp. 33-36

Substitution of Malachite Green with Nigrosin - Eosin Yellow Stain in the Kato-Katz method: microscopical appearance of the helminth eggs

Emmanuel I.Odongo-Aginya¹; Narcis Kabatereine²; Siefert Ludwig³; HenryWabinga⁴; Alan Fenwick⁵; Antonio Montresor⁶.

1) Uganda Virus Research Institute P.O.Box 42 Entebbe Uganda (East Africa).
2) Vector Control Department Ministry of Health P.O.Box 1666 Kampala Uganda.
3) Faculty of Veterinary Medicine Makerere University,Wild Life Conservation Department P.O.Box 7062 Kampala
4) Faculty of Medicine Department of Pathology Makerere Medical School P.O Box 7062 Kampala Uganda.
5) St.Mary^,s Campus, Norfolk Place, Paddington, London W2 1PG, UK
6) WHO Vietnam PO Box 52 10000 Hanoi Vietnam.

Corresponding author Antonio Montresor World Health Organization 63 Tran Hung Dao Street Mail P.O. Box 52 Ha Noi - Vietnam Tel +(84 4) 943 3734 /5 /6 ext 29 Fax +(84 4) 943 3740 e-mail montresora@vtn.wpro.who.int

Code Number: hs07007

Abstract

Background:The Kato-Katz thick smear technique is the standard technique recommended by the World Health Organisation for the quantitative diagnosis of Schistosoma mansoni and other intestinal helminth infections.The major problem of the technique is that a few hours after the preparation of slides hookworm eggs over clear and disappear due glycerin.
Objective:To illustrate clear visibility of different helminth eggs microscopically in Odongo-Aginya method, substitution of malachite green with 7.5% nigrosin in 10% formalin and 5% eosin in 10% formalin.
Method: Measured, strained stool specimen was stained with mixture of nigrosin/ eosin and covered with cellophane cover slips.The prepared slide was examined immediately microscopically.
Result: Slides prepared with Odongo-Aginya method can be examined immediately or later without compromising the visibility of parasite eggs and larvae. Hookworm eggs remain visible for a long time.
Conclusion:The present publication shows microscopic appearance of the helminth eggs using the Odongo-Aginya modification.

Keywords: Kato - Katz method, pictorial illustration, Odongo-aginya modification, helminth eggs, Uganda.

Introduction

The Kato-Katz thick smear technique is the standard technique recommended by World Health Organisation (WHO) for the quantitative diagnosis of Schistosoma mansoni and other intestinal helminth infections¹.

The glycerin, in the malachite green in Kato Katz’s technique, functions as a clearing agent while the malachite green besides being a dye, is bactericidal ^2,3 . The Kato-Katz methods require between 1 to 2 hours before the glycerin clears the background of the stool smear on the slide for accurate visualization of most helminth eggs ¹. The major problem of the technique is that few hours after the preparation of the slide hookworm eggs are difficult to recognize due to overclarification by glycerin ¹.

The aim of this paper is to show the appearance of the helminth eggs when malachite green is replaced with a stain comprised of nigrosin and eosin yellow in formalin⁴. Several field studies confirm the simplicity, quality, and cost effectiveness of the proposed modification^5-9, a visual reference of the results of the method can be useful to facilitate the recognition of parasite eggs by microscopists willing to adopt this methodology.

Material and Method

The malachite green in the Kato-Katz technique is substituted by the following compound stain: 5% eosin yellow in 10% formalin, mixed 1:1 with 7.5% nigrosin in 10% formalin4. Cellophane cover slips cut in 25x40 mm pieces are soaked in 50% glycerin. Stool specimens are strained through a stainless steel sieve as used in Kato- Katz’s method. Through a template, providing 41.7 mg of feces, the strained stool is delivered on a microscope slide and a drop of the compound stain (about 10-50μl) is added to the stool on the slide and stirred using the corner of another slide. A cellophane cover slip presoaked in 50% glycerin is picked with a pair of forceps and excess glycerin on it is blotted out on an absorbent paper. The cellophane cover slip is then placed on the stained stool on the slide. The slide is inverted upside down and pressed down gently on tissue paper or any absorbent paper to spread out the stool smeared on the slide and to remove excess of the stain from the slide. A well-prepared slide has a pinkish background and is thin enough to read the face of a wristwatch through it.

Photographs of the helminth eggs are presented in figure 1, 2 and 3. The photographs were taken using an Olympus Photomicroscope B 20 from slides prepared in the Department of Pathology Makerere Medical School according to Kato-Katz and Odongo-Aginya’s methods. The stool specimens were collected from Kigungu residents as a part of Odongo-Aginya, Doctor of Philosophy (Ph.D) study. Kigungu is a fishing village on Lake Victoria shoreline, Entebbe Uganda. Photographs were taken immediately after preparations of slides, after one hour and one week later.

Ethic: Faculty of Medicine, Higher Degree and Ethic Committee; Uganda National Council of Science and Technology, approved the study. Consent was requested before the residents were recruited in the study. All ailments were treated. For S.mansoni, praziquantel was used. Privacy was observed. The doctor in the treatment room released results to individuals and risk was minimal.

Discussion

The aim of this paper is to illustrate pictorially the improvements made on Kato-Katz^’s technique by replacing malachite green in 50% glycerin with a compound stain comprising of 7.5% nigrosin in 10% formalin and 5% eosin yellow in 10% formalin mixed in proportion of 1:1 as proposed by Odongo-Aginya.⁴ Odongo-Aginya method has the following advantages: Firstly it is quick because the slide prepared by this method can read immediately. Secondly, it is simple and easy to learn.Thirdly, hookworm eggs do not over-clear, as it is the case in Kato/Katz method. The Kato/Katz method requires between 1 to 2 hours before good visualization can be made. After this period, hookworm eggs over clear and identification become difficult. Besides, eggs of other parasites tend to lose their characteristic morphology in slides kept for a long period ¹. Mahdi and a team of experts from the World Health Organisation evaluated the Odongo-Aginya modification of Kato/Katz method in the field elsewhere to compare the simplicity, quality, and cost effectiveness of the two methods ^{6, 7}.

Although the malachite green in Kato/Katz is weakly bactericidal, it does not provide protection against viral and other microorganism infections provided by formalin in 5% eosin/ 7.5% nigrosin method. This is because formalin is a strong fixative at the concentration used. This property could be significant when working with stool specimens from Human Immunodeficiency Viruses (HIV) infected patients.⁴

We conclude that the slides prepared using the eosin/ nigrosin stain can be microscopically observed immediately after preparation. Hookworm eggs remain visible up to six months later⁴.Therefore, this method is ideal in the study of intestinal helminth especially where the examination of the slides cannot be performed immediately or shipped to other examination centre.

Acknowledgement

We are grateful to the patients who provided the stool specimens without which this exercise would not have been accomplished. The photographic work was done using the microscope in the Pathothology Department Makerere Medical School. We are grateful for their technical support.

References

1. Basic Laboratory methods in medical Parasitology. World Health Organisation. 1991 Geneva 25- 29
2. Kato, K., Miura, M. Comparative examination of faecal thick smear technique with cellophane paper covers .Japanese Journal of Parasitolology. 1954, 3: 35-37.
3. Katz, K., Chaves,A., & Pellegrino,J.A simple device for quantitative tool thick smears technique in schistomiasis mansoni. Revista do institut De medicine Tropical de Sao Paulo. 1972; 4: 397-400
4. Odongo-Aginya, EI.,Taylor, MG., Sturrock, RF.,Ackers, JP & Doehring, E.A field evaluation of Aginya stain in the Kato Technique for quantification of Helminth eggs in the faeces. Tropical Medicine and Parasitology 1995; 46: 275-277.
5. Martin, LK., Beaver, PC. Evaluation of Kato^’s thick technique for quantitative diagnosis of helminth infections. American Journal of tropical Medicine and Hygiene. 1968; 17: 382-391.
6. Mahdi, RM., Montresor, A., Foum, A., Ameri, H., Di Matteo, L., Albonico, M., & Savioli L. Independent evaluation of the Nigrosin-Eosin of the Kato-Katz technique. Tropical Medicine and International Health, 1999; 4 1: 46 -49.
7. Mahdi, RM., & Luigi DM. Preliminaryevaluations of Aginya stain (substitution of malachite green/glycerin in Kato technique by 7.5% nigrosin / 5% eosin stain method in Wonji Area (Ethiopia).World Health Organisation Technical Report 1996; A16-181-15.
8. Sleigh, AC, Mott, KE, Hoff R, Barreto ML, Mota EA, Hmaguire J, Sherlock I, & Weller TH. Three years prospective study of evaluation of mansoni’s schistosomiasis in North-East Brazil. Lancet 1985; II 63-66.
9. Sleigh AC, Rodyney H, Mott K, Barreto M, Maisk de Paiva T, De Souza, PJ & Sherlock I. Comparison of filtration staining (Bell) and thick smear Kato for the determination and quantitation of Schistosoma mansoni egg in faeces. Transaction of Royal Society of Tropical Medicine Hygiene. 1982; 76:403-407.

Copyright © 2007 - Makerere Medical School, Uganda

The following images related to this document are available:

Photo images