mjweb.indd  F-   ,  water, food and hands become contaminated with faecal material, and then come in contact with mouth, is the most common mode of gastrointestinal infections.1 Studies indicate that parasitic infections aff ect the health of the population through malabsorption, diarrhoea, stunted growth in children and impaired work capac- ity.2–5 Morbidity caused by enteric parasites has always been a public health problem, especially in the tropics. Th e incidence and severity of intestinal parasitic infections may vary with place and time.6–9 Estimates of prevalence Usefulness of Kato-Katz and trichrome staining as diagnostic methods for parasitic infections in clinical laboratories *Mohamed A Idris, Ayda M Al-Jabri Department of Microbiology and Immunology, College of Medicine, Sultan Qaboos University, P.O. Box: , Al-Khod , Muscat, Sultanate of Oman. *To whom correspondence should be addressed. midris@squ.edu.om. ABSTRAC T. Objectives: To assess the effi cacy of the Kato-Katz technique and to re-evaluate other routine procedures conducted in the Microbiology Clinical Laboratory at Sultan Qaboos University Hospital (SQUH) and to throw light on the prevalence of intestinal para- sitic infections among a small group of food handlers in Muscat. Method: Faecal samples collected from food handlers were examined using fi ve parasitological techniques. Results: Out of  faecal samples,  were positive for one or more of  intestinal parasites. The Kato- Katz and trichrome stain methods were found superior to the other techniques in detecting helminthic and protozoan infections, respec- tively. The auramine stain was useful only in detecting Cryptosporidium parvum oocysts. Conclusion: A combination of trichrome stain and Kato-Katz techniques for stool examination is suffi cient and recommended for busy laboratories; auramine stain should be applied only to samples with suspected cryptosporidal infections. Key Words: Kato-Katz, wet preparation, concentration, trichrome and auramine stains, intestinal parasites, food handlers, Oman. of intestinal parasitic diseases are at best extremely rough, especially in regions were these diseases are endemic, due to poor reporting.10 Th e prevalence of intestinal parasites has been studied in many regions of Oman, especially in Dhofar.11,12 In a study conducted in  among adults and children in Dhofar,  had parasites.13 Studies of schoolchildren in Muscat14 and in Nizwa15 and of patients from Musandam16 have shown percentage rates of , , and  respectively. Recent studies from Sur17 and AI-Hamra Wilayat18 also reported high prevalence. A survey among food handlers,     :   , : , : , – ©   :: . : .: . . .: .  mostly Indians, revealed that . were infected with intestinal parasites.19 Although numerous methods for diagnosing parasitic infections by stool examination are available, no single technique is satisfactory as none is equally applicable for trophozoites and cysts of Protozoa, and eggs or larvae of helminths. For this reason, an optimum combination of two or more techniques of stool examination is desir- able.6,10 Accordingly, the objective of this study was to compare the sensitivity, reliability and practicability of the Kato- Katz technique in detecting intestinal parasites, with the stool tests routinely performed at the clinical microbiology Laboratory in SQUH, namely, the direct wet smear, con- centration technique, trichrome stain and auramine-phe- nol staining, in order to recommend the most appropriate procedures. A subsidiary objective of this study was to throw light on the prevalence of enteric parasitic infec- tions among a small group of food handlers in the capital city, Muscat, because of the potential of transmitting these infections to the general population. M E T H O D S During January to March , stool samples of  food handlers, mostly Indians, were collected in labelled plastic containers with covers at the Communicable Diseases Centre in Muscat. Th e samples were investigated in the Clinical Microbiology Laboratory, SQUH. Each sample was subjected to the following fi ve diff erent techniques. . Direct saline and iodine mount (wet preparation) An applicator stick was used to mix about  mg of faeces with one or two drops of normal saline or iodine placed on a clean slide. A uniform thin suspension was made and covered with a  mm square cover slip. Th e entire fi lm was screened systematically for the presence of helminth ova and larvae or protozoan cysts and trophozoites. . Formalin-ether concentration technique (FEC) Using an applicator stick, about  g of faeces was placed in a clean  ml conical centrifuge tube containing  ml formalin saline. Th e sample was dissolved and mixed thor- oughly with a vortex mixer. Th e resulting suspension was fi ltered through a sieve into a beaker and the fi ltrate was poured back into the same tube. Th e debris trapped on the sieve was discarded. Aft er adding  ml of diethyl ether to the formalized solution, the contents were centrifuged at , rpm for  minutes. Th e supernatant was poured away and the tube was replaced in its rack. Iodine stain preparation was made. Th e entire area under the cover slip was systematically examined using × and × objective lenses. . Trichrome staining of faeces (Gomori’s trichrome stain) About  g of faeces was placed into sodium acetate forma- lin (SAF), mixed well and left overnight. Th e contents were centrifuged at , rpm for  minutes. Th e supernatant SAF was poured off , the deposit re-suspended and replaced with normal saline, and then centrifuged for  minutes. A smear was prepared using an orange stick and a small drop of Myer’s albumin to bind a drop of concentrated faeces to the slide. Th e smear was dried for – minutes. Th e prepared smear was then washed in  ethanol for  minutes and then in  ethanol for  minutes. Th ereaft er the smear was stained with Gomori’s trichrome for  minutes, diff erentiated in  ethanol for – sec- onds, and rinsed in  ethanol for  second. It was dehy- drated in two changes of ethanol, each lasting  minutes. Th e slide was cleaned twice in xylene, each time for  min- utes, and mounted in DPX mounting medium with a large × mm cover slip. Th e slide was left overnight before examining with the × oil immersion objective lens. . Kato-Katz technique, cellophane faecal thick smear A small amount of faecal material was placed on newspa- per or scrap paper and a piece of nylon screen was pressed on top so that some of the faeces sieved through the screen and accumulated on top. A fl at-sided spatula was scraped across the upper surface of the screen to collect the sieved faeces. A template was placed on the slide and the sieved faeces was added with the spatula so that the hole in the template was completely fi lled. Th e spatula was passed over the fi lled template to remove excess faeces from the edge of the hole. Th e template was removed carefully so that a cylinder of faeces was left on the slide. Th e faecal material was covered with a pre-soaked cellophane strip. Th e slide was inverted and the faecal sample was pressed fi rmly against the hydrophilic cellophane strip to spread evenly. Th e slide was placed on the bench with cellophane upwards to enable the evaporation of water while glycerol cleared the faeces. For all helminths, except hookworm eggs, the slide was kept for one or more hours at room temperature to clear the faecal material, prior to micro- scopic examination. . Auramine-phenol staining Th e air-dried faecal smear was fi xed for  minutes in abso- lute methanol and the slide was fl ooded with auramine for  minutes, washed in tap water and decolorized in  acid alcohol for a minimum of  minutes. Th e slide was again         -      washed in tap water, stained with potassium permanganate solution for  minutes, washed and dried. Th e slide was then examined under ultra/violet microscope to detect Cryptosporidium oocysts that fl uoresce brightly against a dark red background. R E S U L T S Out of the  stool samples,  were positive for one or more species of  intestinal parasites ( helminths and  Protozoa). Th e prevalence and percentages of parasites among food handlers obtained by using the fi ve proce- dures are shown in Table . Ascaris lumbricoides showed the highest overall prevalence (), followed by Blastocystis hominis (), Trichuris trichiura (), hookworm (), Giardia lamblia and Entamoeba coli ( each), Endolimax nana (), Cryptosporidium parvum, Dientamoeba fragilis and Iodamoeba butschlii () each and Entamoeba his- tolytica (). Single, double and multiple (– species of parasites) infections showed positive rates of , , and , respectively. Out of the  cases of ascariasis recovered by Kato-Katz during this study, direct smear and FEC recovered  and  cases, respectively. Out of  cases of trichiurasis recovered by Kato-Katz,  and  cases were recovered by direct smear and FEC, respectively. Out of  hookworm cases recovered by each of Kato-Katz and FEC, one case was recovered by direct smear. Th e two cases of Cryptosporidium were recov- ered only by auramine stain. Th e Kato-Katz technique and auramine stain did not recover any other protozoan spe- cies, which were all partly recovered by trichrome stain, direct smear and FEC methods. Table  shows that the total number of helminthic infec- tions was . All of them were detected by Kato-Katz fol- lowed by the FEC () and direct smear (.). Out of the  protozoan infections,  () were recovered by trichrome stain,  () by FEC and  (.) by smear wet preparation. D I S C U S S I O N Many pathogenic intestinal parasites are transmitted through the faecal-oral route. Since unhygienic prepara- tion, storage and handling of food by infected individuals are a major cause for food-borne diseases, food handlers need to be screened before they are allowed to work in food establishments such as restaurants, hotels, food stores, factories or as helpers and cooks in private houses. Unfortunately, such screening is not simple, since no single technique of stool examination detects cysts and trophic forms of Protozoa and helminth eggs or larvae equally well. As our results indicate, a combination of two or more tech- niques is more likely to be eff ective. Our results indicate that the Kato-Katz technique is more sensitive for detecting helminthic parasites, followed by the FEC technique and the smear wet preparation. Th ese fi ndings agree with those of previous studies that showed the Kato-Katz technique to be an effi cient means of diagnosing intestinal schistosomiasis and intestinal helminths.20 Auramine stain was useful only in the detec- tion of Cryptosporidium oocysts. Th e trichrome stain was superior to all other procedures in the detection of cysts and trophozoites of Protozoa. Trichrome stain detected comparatively high numbers of seven diff erent species of Protozoa while FEC and direct smear could detect only small numbers of four protozoan species [Table ]. Trophozoites can be diffi cult to detect in faeces par- ticularly when they are no longer motile and the cysts may be confused with pus cells or macrophages. Th e use of Gomori’s trichrome stain helps to overcome these problems and greatly assists in distinguishing between pathogenic and non-pathogenic Entamoeba. Furthermore, some parasites such as D. fragilis may only be seen using a staining procedure.20 Th is study generally revealed low prevalence for all intestinal parasitic infections, especially for ascariasis, hook worm and trichiurasis which are considered to be the most prevalent in man, especially in developing countries.6,21-23 - Table 1. Number of parasites recovered from 100 faecal samples using five different techniques Techniques used for parasite identification Parasite Total cases WP FEC TRICH AURAM KATO A. lumbricoides 24 9 15 - - 24 T. trichuria 9 3 6 - - 9 Hookworm 7 1 7 - - 7 G. lamblia 6 1 2 4 - - E. histolytica 1 0 0 1 - - E. coli 6 4 4 6 - - I. butschlii 2 0 0 2 - - B. hominis 17 1 0 17 - - D. fragilis 2 1 1 2 - - E. nana 6 0 1 6 - - C. parvum 2 - - - 2 - WP: Direct saline and iodine mount (wet preparation), FEC: Formalin-ether concentration technique, TRICH: Trichrome staining of faeces, AURAM: Auramine-phenol staining, KATO: Kato-Katz technique  However, the small sample inspected in this study may not represent the whole community of food handlers in Oman. It is also possible that some of our subjects had been inspected and treated in their home countries before travelling to Oman, or in Oman before they came for screening, which may account for the low infection rates. Inspection and treatment of food handlers are neces- sary to reduce food borne infections and should be an integral part of public health education. We recommend that whenever possible more than one parasitological technique should be used in screening faecal samples before an individual can be considered free from intestinal infection. We also suggest that all food handlers should particularly be screened using multiple parasitological techniques and, if found infected, should be eff ectively treated before they are licensed, and re-examined annually. C O N C L U S I O N Th e current fi ndings show that the Kato-Katz and tri- chrome stain are superior to other stool examination tech- niques for detecting helminthic and protozoan infections, respectively, and the authors recommend them for the clinical laboratory. Th e auramine stain method should be used only when Cryptosporidium is suspected. 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