ACCURACY OF DIAGNOSIS OF INTESTINAL HELMINTH PARASITES IN A REFERENCE DIAGNOSTIC LABORATORY IN THE ASHANTI REGION OF GHANA

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INTRODUCTION
Intestinal helminth infection is one of the most prevalent and widespread chronic infections of man [1] with over a quarter of the world's population being infected.Roundworm, hookworm and whipworm infections are estimated at 1.47, 1.05, and 1.30 billion people respectively [2,3].Many intestinal helminths trigger immune responses in man and these may drain the body's ability to fight other diseases, making affected individuals more prone to co-infection [4].There are reports that helminthiasis is contributing to the unrelenting prevalence of AIDS and tuberculosis in many developing countries particularly in Africa [5].The majority of soil-transmitted helminth infections are found in the third world countries as a result of low standard of living due to poor personal hygiene and environmental sanitation [6,7,8,9].In many West African communities, prevalence of helminth infections exceeds 70% [10].School-age children are particularly vulnerable to these parasites because of their high mobility and lower standards of hygiene [11].In Ghana, Annan et al. [12] reported up to 63% of intestinal helminth infections in school-age children.Nevertheless, the levels of helminth infections diagnosed in hospital laboratories in Ghana are very low and do not highlight the high prevalence as reported by many researchers in the sub-region [13].This study seeks to find out if there is some critical diagnostic factor responsible for the low recovery of helminth parasites from stool specimens.[14], NCCLS [15], Melvin and Brooke [16] and WHO [17].

Parasitologic examination:
The direct wet mount, Kato-Katz and Formol-ether concentration, methods were employed.Each specimen was first examined macroscopically and its consistency or nature was recorded in accordance with the description by Ash and Orihel [18], Estevez and Levine [19] and Smith and Schad [20].The test procedures were carried out in accordance with standard protocols as described by Garcia [21,22] and WHO [17,23].

Helminth Infection intensity determination by Kato Katz technique:
The faecal sample was pressed through a Kato nylon screen mesh (HelmR test kits, Belo Horizonte, Brazil) of size 200 μm and the sieved stool was transferred into the 6 mm diameter hole of the cardboard template on a microscope slide.The template was then removed and the remaining sample (approximately 42 mg) was covered with the glycerol soaked cellophane strip of size 25 × 35 mm and 50 ųm thick.The prepared slide was microscopically examined and the eggs of each of the helminth species were recorded.The number of eggs per gram of faeces was calculated and used to estimate the infection intensity of the parasites based on the classification reported by WHO (2002) for major soil-transmitted helminths and S. mansoni infections.Loose and watery stool specimens were not analyzed by the Kato-Katz method because of the technical difficulties that limit analysis of such specimens by this method [10,25].

Helminth infection intensities
The intensity of helminth infection was measured on the basis of egg counts per gram (epg) of stool [26], determined by the Kato-Katz method.Infection intensities for the soil transmitted helminths (STHs) and S. mansoni cases detected in the study were classified in accordance with the criteria prescribed by WHO [24].All cases of STHs found in the study were of low intensity.It was evidenced that a significantly high number of individuals infected with medium and high S. mansoni burden belonged to 10-15 age group (p < 0.05; Table 4).

RESULTS AND DISCUSSION
Routine diagnosis of intestinal helminth infections is normally based on detection of the helminth eggs and/or larvae in stool samples examined through a variety of parasitologic methods [27].In most laboratories in Ghana and many other developing nations, the Direct Wet Mount is the preferred stool parasitological detection technique because it is simple and fast to perform and the most affordable [21,22,28,29,30,31].The study showed that 506 (25.3%) out of the 2000 patients had provisional diagnoses that could be related to intestinal parasitic diseases (Table 1) and most of the helminth-positive specimens (75 %) were from these patients.On the other hand the laboratory tests requested for 30.4% of the population studied had no provisional diagnosis or clinical summaries related to the investigations ordered.Most of the stool specimens (95 %) from these patients tested negative for helminth parasites, which implied that the request for these stool examinations were probably inappropriate and did not significantly (p<0.05)contribute to patient care [32].
The total prevalence of intestinal helminth infections detected by the formol-ether concentration, Kato-Katz and the direct wet mount methods were 11.1%, 5.2% and 3.25%, respectively.The direct wet mount exhibited the lowest performance (29.2%); being about three times less sensitive than the formol-ether concentration (Table 2).Kato-Katz method (43.4%) is generally about twice as less sensitive than the formol-ether concentration (Fig. 1) but exhibited comparable sensitivity in the detection of hookworm, T.trichiura and S. mansoni (Table 3).
The differences (p <0.05) observed in the sensitivities of the three screening methods have been reported in other studies [27,33,34,35,36,37].This study therefore appears to unveil underestimation of helminth infection prevalence in this diagnostic laboratory and other medical laboratories in Ghana since the direct wet mount method is the commonest employed helminth parasitological detection test in the country.The study also highlighted a high occurrence of low worm burden (Table 4) among the population which cannot be easily detected by the direct wet mount technique.
The Kato-Katz exhibited very low sensitivity for the detection of intestinal helminth parasites such as S. stercoralis, Dicrocoelium species, Taenia species and H. nana.Similar observations have been reported elsewhere [33,35,38,39,40].The choice of the direct wet mount over the Kato-Katz method is desirable when there is a good indication of infection with S. stercoralis, Dicrocoelium species, Taenia species and H. nana.At any rate, the use of the formol-ether concentration as a confirmatory test will significantly reduce misdiagnosis of intestinal helminth infections and its attendant public health consequences.

CONCLUSION
The study has shown that the intensity of intestinal helminth infections reported at the Reference Copyright © 2011, Bioinfo Publications Laboratory were mostly of low grade and can only efficiently be detected by the formol-ether concentration method which exhibited superior sensitivity to Kato Katz technique and the traditional Direct Wet Mount method.The employment of formol-ether concentration technique as a confirmatory test in routine laboratory examination of stool will significantly reduce misdiagnosis of intestinal helminth infections and its attendant public health consequences.

Table 1 -
Provisional clinical diagnosis of patients studied for helminth infections at KATH = included malaria, enteric fever, urinary tract infection and chronic kidney and liver diseases. †

Table 2 -
Performance of Direct wet mount test against formol-ether concentration in detection of intestinal helminth parasites

Table 3 -
Comparison of Kato-Katz versus Formol-ether concentration tests for detection of intestinal helminth parasites.

Table 4 -
Infection Intensity of STHs and S. mansoni using the Kato-Katz