INCUBATION OF PARASITIC NEMATODE USING HARADA-MORI FILTER PAPER STRIP CULTURE METHOD OF THE FECAL SAMPLE AMONG CHILDREN

IN TERESA ST. STA. MESA MANILA

Garcia, Carla Mae D.C, Gumban, John Daniel P., Ocasla, Donitalyn D, and Penetrante, Gerson Kim O. Department of Biology, College of Science Polytechnic University of the Philippines Group 9
ABSTRACT: Strongyloidiasis is recognized to life-threatening to the patients. Diagnosis rests on the microscopic identification of larvae (rhabditiform and occasionally filaform) in the stool . Culture techniques are the most sensitive, and one of the culture technique is the Harada-Mori filter paper technique. A total of 30 stool samples are collected among children from Teresa St. Sta. Mesa Manila. The primary goal of this study is to detect parasitic nematode (Strongyloides stercoralis) and also to detect other hookworms (Ancylostoma duodenale, and Necator americanus) and Ttrichostrongylus spp. Definitely, it intends to apply Harada-Mori Filter Paper Strip Culture for the recovery and observation of parasitic nematodes in fecal samples. Hookworm or nematodes was seen in none of the stool samples after incubation. The children in the study benefit the most because knowledge about nematode will enable them to be treated and prevent possible infection. And the parents knowledge regarding nematodes would be increased and prevention could be done to avoid more serious damage to their childrens health.

INTRODUCTION Strongyloidiasis has been recognized as one of the life-threatening parasitic infections in the immune compromised patients. The intestinal nematode, Strongyloides stercoralis, is a soil-transmitted nematode endemic in many countries throughout tropical and temperate regions, and is responsible for a wide range of symptoms (Grove, 1989). As the life cycle of Strongyloides stercoralis in warm climates like Philippines, the parasite lives the freeliving stage generation in humid soils. When the microenvironment changed to

unfavorable circumstances (dry and/or cold temperature), rhabditiform larvae transform to filaform larvae, and an infective larva penetrates into the human skin and takes into superficial veins. In immune competent hosts, the infection of S. stercoralis is largely confined to the intestinal tract and is asymptomatic or induces nonspecific complaints such as moderate abdominal pain, nausea and diarrhea (Tanaka et al., 1996). In immune compromised hosts, the autoinfection results in dissemination of the filaform larvae into various tissues and organs, which is unfortunately very fatal

(Venizelos et al., 1980; Gotuzzo et al., 1999). In Philippines, the intestinal strongyloidiasis in human has not been uncommon in terms of larval positive rate in the stool examination (Belizario, 2005). In accordance to the decrease of prevalence of soil-transmitted helminths over last three decades (MHW and KAH, 1997), human infection by S. stercoralis have been recorded as clinical cases, about 20 cases in number. The human cases recorded were of intestinal or hyper infections (Kim et al., 1992; Lee et al., 1994; Chung et al., 1995). Diagnosis rests on the microscopic identification of larvae (rhabditiform and occasionally filaform) in the stool. Examination of samples may be necessary, and not always sufficient, because direct stool examination is relatively insensitive. Culture techniques are the most sensitive, but are not routinely available in the West. In the UK, culture is available at either of the schools of tropical medicine in Liverpool or London. One of the culture technique is the Harada-Mori filter paper technique (Harada and Mori, 1955). This filter paper test tube culture technique was initially introduced by Harada and Mori in 1955 (Harada and Mori, 1955) and was later modified by others (Hsieh, 1962; Sasa, 1958). The principle of this technique was employs a filter paper to which fecal material is added and a test tube into which the filter paper in inserted. Moisture is provided by

adding water to the tube. The water continuously soaks the filter paper by capillary action. Incubation under suitable conditions favors hatching of ova and/or development of larvae. Fecal specimens to be cultured should not be refrigerated, since some parasites (especially Necator americanus) are susceptible to cold and may fail to develop after refrigeration. The primary goal of this study is to detect parasitic nematode (Strongyloides stercoralis) and also to detect other hookworms (Ancylostoma duodenale, and Necator americanus) and Ttrichostrongylus spp. Definitely, it intends to apply Harada-Mori Filter Paper Strip Culture for the recovery and observation of parasitic nematodes in fecal samples among children in Teresa St. Sta. Mesa Manila. The result of the present study would provide descriptions and data regarding to parasitic nematodes. Furthermore, this study will produce significant and relevant information for future studies. METHODOLOGY Sample Site The study will be conducted at Teresa St., Sta. Mesa Manila City, which is located at 140 35 59.85 N latitude and 1210 0 43.86 E latitude. The residential areas at Teresa St. with the ocular inspection, are characterized with good level of environmental sanitation, because the barangay officials make sure that the environment are clean and green. Also,

there are ready schedule in collecting the garbage. Each house has latrines or toilets. But the sewage system is poor because a rain of about 30 minutes can cause flood. And also, street children playing and go around the area barefoot and eating without washing hands. Sampling Size A total of 30 children from Teresa St. is the total sample size of this study. It is a random sampling that whosoever children that the researchers meet or interviewed and kindly ask them for their fecal samples with an exchange of tokens for them (candies, mini chocolates, cookies and junk foods) and with the consent of their parents. Sample Collection Thirty (30) stool samples will be collected of children. A fecal container for collection and storage will be used. These containers will be labeled with unique identifiers including the name of the patient, sex, age, data and the consistency of the stool. The patient will place the stool specimen directly into the container or on a piece of paper. Specimens will be placed inside ice chest for transport to the laboratory. Sample Processing Upon the arrival of stool samples in the laboratory, they will be classified according to their physical characteristics. The stool samples that contain blood will be examined first, followed by watery specimens.

Harada-Mori Paper Strip Culture Method was employed for the culture of parasitic nematode. Cut a narrow strip of filter paper and taper it slightly at one end. Smear 0.5 to 1 g of feces in the center of the strip. Then, add 3 to 4 mL of distilled water to a 15 mL conical centrifuge ttube. Insert the filter paper strip into the tube so that the tapered end is near the bottom of the tube. Make sure that the water level should be slightly below the fecal spot. It s necessary to cap the tube. However, a cork or stopper or cottn plug may be used. Afterwards, allowe the tube to stand upright in a rack at 25 to 18o C. Add distilled water to maintain the original level. Kepp the tube for 10 days and check daily by withdrawing a small amount of fluid from the botoom of the tube. Preapare a smear on a glass slide, cober with a cover slip and examine. This technique allows both parasitic and free-living forms of nematodes to develop. If specimens have benn contaminated with soil or water containing these forms, it is necessary to distuinguish parasitic from free-living forms. This distinction is possible since parasitic forms are more resitant o slight acidity than are free-living forms. Add 0.3 mL of concentrated hydrochloric acid per 10 ml of water containing larvae. RESULTS AND DISCUSSION A total of 30 stool samples of children were collected from Teresa St., Sta. Mesa Manila. Stool sample were investigated for the presence of nematode worm using Baermann Funnel Method. Of

a total of 30 stool samples, hookworm or nematodes was seen in none of the stool samples of children from Teresa St., Sta. Mesa Manila. Present study showed no detection of nematodes in the stool samples of the children at the end of the incubation. Interviewing the children, it is reported that at their school, they attend, a deworming program that is given free at the public schools by the Manila City government and in the Philippines, a proposed model for school-based worm control involving mass treatment and health education has been well documented (Belizario, V. et al., 200). And this study suggests that the hookworm infection are controlled by periodic mass treatment in the form of chewable and attractive preparations of albendazole or mebendazole tablets given single dose, two to three times a year (Belizario, V. et al., 2007). Also, the most important factor for sustaining hookworm control is the support of the childrens parents and families. And of course, the Teresa St. as a community in general, with the help of information, families should be tapped as important resources to provide for the cost of the drugs, to improve hygiene and sanitation, and to reinforce health education activities. The result from this study provides us a glimpse of nematode status in Teresa St. Sta. Mesa Manila. The children in the study benefit the most because knowledge about nematode will

enable them to be treated and prevent possible infection. Also, Strongyloidiasis should be one of the problems given priority in all countries, to enhance quality of life, health and productivity of children. And through this study, the parents knowledge regarding nematodes would be increased and prevention could be done to avoid more serious damage to their childrens health. LITERATURE CITED Grove DI. (1989). Strongyloidiasis: A Major Roundworm Infection of Man. London, UK: Taylor and Francis. pp. 1-11, pp. 155-173

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