European Journal of Scientific Research ISSN 1450-216X Vol.25 No.4 (2009), pp.606-613 EuroJournals Publishing, Inc. 2009 http://www.eurojournals.com/ejsr.

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Pathogenicity of Blastocystis Hominis in Relation to Entropathogens in Gastroenteritis Cases in Baghdad

Elham Al-kaissi Dept. of Pharmaceutics, Faculty of Pharmacy Petra University, P.O. Box 961343,Amman, Jordan E-mail: kaielham@hotmail.com Tel: 00962-6-05715546; Fax: 00962-6-5715551 Al_Magdi K J Dept. of Basic Medical Science, college of Nursing University of Baghdad, Iraq Abstract Blastocystis hominis was considered to be a member of normal intestinal flora in the past; recently it has been accepted as a controversial pathogen. In this study 200 stool specimens of patients suffering from gastroenteritis were studies by applying direct microscopic stool examination and culture. 67% of these specimens showed single entropathogen, 20% mixed and 12% were negative. These results were significant when compared with control specimens. No mixed bacterial and fungal or mixed bacterial alone were identified. As B. hominis is receiving increased attention as potential causative agent for diarrhoea, the role of this protozoon in relation to other pathogens in diarrhoeal disease in Baghdad area was investigated. Eighty two individuals whose stool examination revealed B. hominis were evaluated for clinical symptoms.12% of the cases were mixed infections with other organisms, where as 29% were infected with B. hominis only, on both occasion B. hominis showed significant relation to the presence of blood, leucocytes, and stool consistency of the specimens investigated. Metronidazole was administered for 10 days to the patients infected with B. hominis. At the end of treatment, the investigation was repeated and stools examination gave negative results for B. hominis. Keywords: Blastocystis hominis, Gastroenteritis, Metronidazole, pathogenicity.

1. Introduction
Acute gastroenteritis is one of the most common diseases in tropical and sub-tropical countries. It has been estimated that, yearly there are approximately 500 million episodes of diarrhea in children less than five years of age in Asia, Latin America and combined of these 1-4% might expected to be fatal (Cash, 1979) age, sex, nutrition, sanitation and seasonal variation are considered to be the most risk factors. Intestinal parasites associate with high incidence of diarrhea and malnutrition; mostly protozoa are considering the major causative agent (Tomkins, 1979). Infection with B. hominis has a worldwide distribution and occurs in both children and adults (Stenzel and Boreham, 1996). A Blastocystis hominis is an anaerobic unicellular protozoan parasite frequently found in the human gastrointestinal tract (Laya, et al, 2007, El-Shazly et al, 2005, Moghaddam et al, 2005). Initially discovered in 1911,

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the organism was considered for many years to be harmless yeast. Studies in the 1970s provided evidence that B. hominis is a protozoan (Zierdt,1991). B. hominis is one of the most common organisms to be detected in stool specimens of returned travelers from developing countries (Jelinek et al., 1997). B. hominis is now getting acceptance as an agent of human intestinal disease (Kaya et al, 2007)) causing intestinal disease with potentially disabling symptoms (diarrhea, nausea, flatulence, abdominal pain, lack of appetite) (Moghaddam et al 2005). Sporadic cases are reported in Europe and in the United States in immunocompetent patients (Windsor et al, 2001) and in immunosuppressed population (Rao, et al 2003, Cimerman, et al 1999), the incidence of B. hominis in different regions is reported to be between 2-65% (Doan 1998). In Iraq, diarrhoea is considered to be an important cause for infant's morbidity and mortality, bacterial diarrhea contributes to about one third of the total cases (Al-Kaissi et al, 2006). In this study, we tried to clarify the pathogenicity of B. hominis and their coexistence with other entropathogens in regarding the aetiology of diarrhoea in Iraq and to find if any synergistic or antagonistic effects which may be shown if such pathogens are sharing the aetiology of gastroenteritis disease.

2. Materials and Methods

A total of 200 adult patients of different age and sex attending the medical city outpatient clinic, suffering from diarrhoea were investigated by direct stool examination and culture. Direct stool examinations were done by using wet amount and concentrated technique (Forbes el al 2002). Microscopic examination of Giemsa- stained smears and wet preparation with India ink (to bring out the capsule surrounding the microorganism) of stool specimens confirmed the presence of a large number of B. hominis, in the absence of other parasites. Culture were done on differential and selective media as MacConkey's agar (Fulka Ag), Haektoen enteric agar (Biomerieux), Mannitol salt agar (Oxoid) and Sabouraud's agar (Biomerieux). A second culture was done on Gram Negative Broth GNB (Biomerieux) after overnight incubation at 37C, it was subcultured on to MacConkey's agar, Haektoen enteric agar. All above inoculated media were incubated overnight at 37C except for Sabouraud's media was left at room temperature (22-25)C for 3-5 days. The non-lactose colonies were identified by biochemical and serological tests according to Cowan and Steel's (1977).The golden or white coloured colonies appear on Mannitol agar were tested for DNA-ase and coagulase production using Staph-latex kit (plasmatic laboratory Products limited, Dorset, U.K.). Identification of Candida albicans from suspected colonies on Sabouraud's agar were done according to modified method of Ahearn 1973. Identification of B. hominis was done by microscopic examination of Giemsa-stained smears and wet preparation with India ink of stool specimens. Stool specimens are culture on Cleveland and collier diphasic medium (Difco) according to Zierdt and Swan 1981. Colonoscopy for all patients was negative. Fifty stool specimens were collected from healthy individuals and processed like the tested samples and used as control. The patient was treated with 1 gram of metronidazole (Flagyl) daily for 10 days. Then a stool specimen was taken and subjected to the above mentioned techniques.

3. Results
Microscopic examination of 200 diarrhoeic stool specimens revealed that, blood were found in7.5%, pus (leukocytes) in 11% and mucus in 6% in stool specimens of liquid consistency as compared to semi-liquid (Table 1).

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Table 1:

Elham Al-kaissi and Al_Magdi K J

Macroscopic appearance and microscopic finding of faecal specimens of 200 patients with diarrhea in the medical city teaching hospital
Stool consistency Semi - liquid % No % 13 98 49.0 7.5 5 2.5 11 13 6.5 6 4 2.0 2 1 0.5 Total No 124 20 35 16 5 % 62.0 10.0 17.5 8.0 2.5

Macroscopic, Microscopic Examination Negative* Blood Pus Mucus Blood+ Pus+ +Mucus
* Samples without blood, pus, and mucus

Liquid No 26 15 22 12 4

On the other hand the 50 control stool specimens showed no association with blood, pus, mucus. B. hominis was found in only 6% of control stool specimens while other pathogenic parasites were found in 24% of control stool specimens. Out of 200 diarrhoeic specimens processed in this study 41% showed B. hominis, 8% showed pathogenic bacteria, 25 showed pathogenic fungi, while 37% showed pathogenic parasites and 12% were negative for any pathogens (Table 2, Fig.1).
Table 2: Entropathogens isolated from stool specimens of 200 patients with diarrhoea in the medical city teaching hospital
Number 82 16 4 74 24 200 % 41 8 2 37 12 100

Causative organism B. hominis Bacteria Fungi Other parasites Negative Total

Figure 1: B. hominis isolated from 200 patients with diarrhoea in relation to other pathogens
200 180 160 140 Percentage 120 100 80 60 40 20 0 B. hominis Bacteria Fungi Other parasite negative Total

Bacterial and fungal species isolated are listed in Table 3.

Pathogenicity of Blastocystis Hominis in Relation to Entropathogens in Gastroenteritis Cases in Baghdad

Table 3:

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Pathogenic bacteria and fungi isolated from stool specimens of 200 patients with diarrhoea in the medical city teaching hospital
Number 1- bacteria 4 1 1 1 3 3 1 2 16 2- Fungi 4 20 % 2.0 0.5 0.5 0.5 1.5 1.5 0.5 1.0 8.0 2.0 10.0

Organisms S. typhi Salmonella. paratyphi A Salmonella. arezonae Salmonella. enteritidis Pseudomonas aeruginosa Staphyloccoci. aureus Shigella flexneri Aeromonas Total Candida albicans Total

Out of 8% bacterial infections, one case was associated with blood, 5 cases with pus and 8 cases with blood and pus together. The most common bacterial species associated with blood, pus and liquid in consistency was salmonella. Fungal infections were associated with pus in only 2 out of 4 cases. B. hominis was associated with pus and rarely blood, the stool consistency was mostly semiliquid (Table 4).
Table 4: Isolation of B. hominis and other microorganisms in relation to the presence of blood, pus and stool consistency from stool of 200 patients with diarrhoea.
Number 4 7 3 3 1 2 82 102 % 2 3.5 1.5 1.5 0.5 1.0 41.0 51.0 Blood 0 0 0 1 0 0 0 1 Pus 2 3 0 1 0 1 4 11 Blood + Pus 0 4 2 1 1 0 1 9 Stool consistency Liquid Semi-Liquid 3 1 5 2 2 1 3 0 1 0 0 2 18 64 32 70

Microorganism C. albicans Salmonella Pseudomonas aeruginosa Staphyloccocus aureus Shigella flexneri Aeromonas B. hominis Total

Out of the 125 mixed infections diagnosed, 11% of them showed B. hominis in association with other parasites, while only 1.5% of them showed B. hominis in association with bacteria and no association with fungi (Table 5).
Table 5: The relation of B. hominis to other entropathogens isolated from 200 patients
Number 1- Single 58 2- Mixed 21 3 0 82 % 29 10.5 1.5 0 41

Type of infection B. hominis B. hominis + other parasites* B. hominis + bacteria B. hominis + fungi Total

* B. hominis with Entamoeba histolytica in 16 cases (8%), B. hominis with Giardia lamblia in 4 case (2%) and B. hominis with S. stercoralis in 1 case only (0.5%)

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Elham Al-kaissi and Al_Magdi K J Out of 200 specimens, 12% showed negative results as demonstrated in Table 6.

Table 6:

Negative* stool of 200 patients with diarrhoea in relation to the presence of blood, pus and stool consistency
Cases Number 24 24 Blood 3 3 Pus 7 7 Blood + pus 14 14 Stool consistency Liquid Semi - liquid 14 10 14 10

Negative Total

% 12 12

* Sample without any detectable entropathogens.

The relationship of entropathogens and negative cases to the presence of blood, leukocytes and the stool consistency was statically significant (X2 9.23, P< 0,05), (X2 31.3, P< 0,05), respectively (Table7).
Table 7: Entropathogens isolated from stool samples of 200 patients with diarrhea in relation to the presence of blood, pus and stool consistency
cases Number % 16 8 4 2 197 98.5 24 12 241 120.5 Presence of Pus Blood + pus 5 6 2 0 17 9 7 14 31 31 Stool consistency Liquid Semi liquid 11 5 3 1 43 154 14 10 71 170

Entropathogens* Bacteria Fungi Parasites Negative Total

* Including mixed infection.

Blood 1 0 1 3 5

2 X =9.23, Relation of entropathogens to presence of blood, pus and blood+ pus. 1 2 X =31.3, Relation of entropathogens to stool consistency 2 P< 0.05 There was no sex differences in the incidence of diarrhea, the highest incidence of diarrhea occurred among age group18-20 and 20-30 years (Table 8).
Table 8: The percentage of B. hominis isolated from male and female patients with diarrhea in relation to other entropathogens.
Male % 18-20y 21-30y 31-40y 41-50y 51-60y 1 6.5 3.5 1.5 2.5 0.5 0.5 0.5 7.5 15.0 Female % 31-40y 41-50y 1 2 0.5 0.5 5.0 6.0 1.5 5.0 0.5 3.5 5.5 10.0 9.5 16.0 0.5 2.5 4.0 1.5 4.5 1 2.0 Total 29.0 1.5 1.0 0.5 1.0 34.5 67.5

Entropathogens B. hominis C albicans S. typhi Shig. flexneri Staph. aureus Other parasites Total

18-20y 4 0.5

21-30y 6 0.5

51-60y 1

1.5

A second stool specimen was obtained from the positive patients with B. hominis after metronidazole therapy. The consecutive parasitological investigation revealed no intestinal protozoa in 38 (92.7%) of 41 B. hominis positive stool specimens.

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4. Discussion
Acute gastroenteritis can cause by various pathogens, one of them is Blastocystis hominis (Svenungsson et al, 2000). However the role of B. hominis and its possible pathogenicity in human intestine has been debated in the medical literatures. During the last decade, there have been many reports supporting the pathogenic potential of this organism (Windsor et al, 2001, Nimri and Batchoun 1994). Several methodologies are available to detect intestinal parasite, with the most common being microscopic examination of direct wet-mount or concentrated stool using iodine, giemsa, trichrome, or safranin-methylene blue stains (Windsor et al, 2002). Khalifa in 1999 suggested that safraninmethylene blue stains produce the best results. More recently, however, Suresh et al, (2004) suggested that microscopy following in vitro culture is a reliable method for detecting B. hominis in stool samples. A similar finding was reported in this work were detection of B. hominis by culture was higher than that by microscopic examination. In a study in Jordan, stool specimens were collected from 180 patients who presented with acute or persistent diarrhoea and other symptoms. B. hominis was identified from 54(30%) patients (Nimri and Meqdam 2004), in this investigation B. hominis were detected in 82 out of 200 patients(41%), this may be due to seasonal and medical care variation. The high percentage of positive parasitic infection (30%) in the control group may suggest the existence of causal carriers which are the foci of the infection within population. One of the major questions is whether B. hominis induces leukocytosis, but there are very few reports currently supporting this idea (Levy et al 1996), In this study B. hominis were associated with leukocytes in 8.6% (table 7), rarely associated with R,B.Cs and stool consistency was mostly semiliquid. This may suggest that B. hominis may play a role in development and persistence of diarrhea, especially when large numbers of parasite are seen in the stool. High percentage of mixed infections (12%) was recognized in this work, mixed infections suggest the possibility that some type of synergism can be exist. The damage done by parasite to the lining of the intestinal wall, give a chance for pathogenic bacteria to invade the intestine. Metronidazole has been reported as a successful treatment for blastocystosis (Nigro et al. 2003), patients were treated with 1 gram of metronidazole daily for 10 days. On the day after the end of therapy, B. hominis had disappeared from their faeces

5. Conclusions
B. hominis should be search for in all stool specimens and considered a potential pathogen of human disease in symptomatic patients with diarrhea accompanied with leukocytes in the absence of other known viral, bacterial causes and should be treated accordingly.

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