A Study of Blastocystis hominis in Sebha, Libya

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1 A Study of Blastocystis hominis in Sebha, Libya Rugaia Mohammed Al-Gazoiu,* Yosef Kubti,** Abdul Hafeez Khan,*** and Naji Moussa Laji,**** Abstract A total of 3645 stool samples (during the period from the 1 st of January to end of December 2005) were submitted at the Central Lab in Sebha for routine (direct smear microscopy) examination of intestinal parasites. Blastocystis hominis was found in 970 (26.61%) of all stool specimens. The prevalence rate of B. hominis was significantly more (P<0.05) in males (28.98%) compared to females (23.89%). B. hominis infection was predominat (10.56%) among 21 to 40 years old age group. The infection of B. hominis was significantly more (P<0.05) in summer than in winter among outpatients attending the Central Lab in Sebha (during the period of January 2003 to December 2005). The frequent occurrence of B. hominis in the population is probably related to the weather condition with the suggestion that hot/dry weather (summer season) of Sebha region of Libya favors the development and transmission of this organism. The total number of 950 fresh faecal specimens from outpatients attending Central Lab in Sebha, were examined for B. hominis using direct microscopy, normal saline concentration, and short-term in-vitro (42.31%) than the direct smear microscopy (26.21%), and faecal concentration (34.10%). In-vitro cultivation does seem worth in the detection of B. hominis in diagnostic laboratories. Clinical symptoms were obtained from 108 patients with stools positive only for B. hominis. The most common symptoms were diarrhea (84.94%), cramping abdominal pain (66.66%), flatulence (17.20%) and vomiting (16.12%). High concentration of B. hominis cells were found in the symptomatic patients than in asymptomatic ones (9.20 cells per high magnification field versus 4.06 respectively) with statistical significant difference (P<0.001). Treatment of 50 symptomatic patients(exclusively infected with B. hominis) with 1500 mg of metronidazole daily for 7 days showed that no B. hominis were found in stools and clinical symptoms disappeared after the therapeutic course. Introduction: Blastocystis hominis is an intestinal protozoan parasite that is frequently identified in human stool specimens and is widely distributed in both developing and developed countries. Despite a surge of information over recent years, data regarding the role of Blastocytis in causing disease in humans is still inconclusive. It is unknown whether B. hominis is a truly pathogenic organism or a commensal. Several reports have concluded that B. hominis is pathogenic. 1,2 However, other data suggest that the organism can be part of normal gastrointestinal flora. 3,4 Diagnosis of B. hominis in public health centers, and laboratories is mostly made by the demonstration of typical vacuolar form (approximately 10 to 15 µm in diameter) with a large central vacuole and 1 to 4 nuclei in peripheral cytoplasm. The smaller forms of B. hominis like multivacuolar, avacuolar, and cyst are not used in the diagnosis, which are also present in the faecal samples usually missed during laboratory examinations in direct microscopy. Moreover, symptomless infections are also common in communities and without doubt are frequently undetected or underestimated. Misdiagnosed patients or shedding of B. hominis from asymptomatic cases may be a vast potential source of infection for humans. Relatively, a little work has been done on the stool examination of intestinal parasitic infections in Libya. 5-9 However, incidence of B. hominis has been reported in patients attending the Central Lab in Sebha (8), but its clinical significance has not been investigated. *) Department of Zoology, Faculty of Science, Obari, Sebha University, Obari, Libya. **) Department of Parasitology, Faculty of Medicine, Al-Arab University of Medical Sciences, Benghazi, Libya. ***) Department of Parasitology, Faculty of Medicine, Sebha University, Sebha, Libya. ****) Department of Zoology, Faculty of Science, Sebha University, Sebha, Libya. 45

2 Concentration methods, as used for other protozoa, faecal parasites, generally appear unsuitable for B. hominis. 1 Whether short-term in-vitro cultivation would increase the sensitivity of detection of B. hominis remains a matter of controversy. 10 In the present study, attempts were made to record the prevalence of B. hominis among patients attending the Central Laboratory in Sebha, to evaluate the sensitivity of direct smear, concentration and culture method for the detection of B. hominis and to describe the clinical presentations of patients parasitized only with B. hominis infection. Attempts were also made for the treatment of symptomatic blastocytosis patients. Materials and Methods: Patient Population During the period from the beginning of January up to the end of December 2005, a total of 3645 faecal samples were collected from randomized Libyan patients, attending the Central Laboratory in Sebha for routine examinations of stool for parasites. Age, sex and clinical presentations of patients were recorded. The patients were from Outpatients of different clinics and the 2 nd March Hospital, Sebha. The prevalence of B. hominis was collected from the patients who attended the Central Lab in Sebha from January 2003 to December 2004 was also included in the present study to ascertain the seasonal variation of B. hominis. A comparison of direct smear microscopy, concentration technique and short-term in-vitro culture was carried out for the detection of B. hominis among 950 randomized patients. Examination of stool specimens: During the routine stool examination of parasites among patients attending the Central Lab in Sebha, both direct wet unstained and stained preparations were employed to record the incidence of B. hominis. Culture of B. hominis: -D) Locke-Egg- Serum (LES) Medium: A total of 950 faecal specimens collected from patients, were cultured at the same time after the routine (direct smear) examination of inspissated egg medium as described by Zierdt and Swan. 11 The solid B-D medium was covered with a ution with 20 % human serum. In this study, the medium was medium. Faecal concentration for B. hominis: Soon after direct smear microscopy, the same samples (collected from 950 individuals) were concentrated by sedimentation technique using normal saline. Approximately 29 of faeces were emulsified in 10 ml of normal saline, strained through two layers of guaze and centrifuged at 2000 rpm for three minutes. The supernatant was discarded, and sediment was examined under the microscope for B. hominis. Clinical features of patients infected with B. hominis: The clinical presentations of 108 patients exclusively parasitized by B. hominis were obtained by sending a survey questionnaire to the practicing physicians at the Al-Jadeed clinic, Sebha. Pharmacotherapy in patients infected with B. hominis: Fifty patients (harboring only B. hominis) presented with gastrointestinal symptoms (diarrhea with or without abdominal pain and flatulence or vomiting) were treated for seven days with prescribed doses of metronidazole (500 mg three times per day for 7 days) by the physicians of Health Center of Al-Jadeed in Sebha. Stool samples from treated patients were re-investigated for B. hominis after they had completed metronidazole therapeutic course. Statistical analysis: The positive rates of B. hominis were expressed as percentage and statistical analysis was carried out by using independent sample-ttest. A probability value of less than 0.01, 0.05 and was considered statistically significant whenever appropriate. Results: Prevalence of B. hominis: During the period from 1 st of January to the end of December 2005, a total of 3645 stool specimens were collected and examined by direct smear microscopy in the Central Lab, Sebha. B. hominis was detected in 970 (26.61%). Of the 3645 patients examined, 1925 (52.81%) were males and 1720 (47.18%) 46

3 were females. 558(28.98%) males and 411(23.89%) females were found harboring B. hominis. The highest positivity (10.28%) rate was found in the age group of 21 to 40 years. Seasonal variation of B. hominis: Seasonal variation of B. hominis in the stool samples of patients attending the Central Lab, Sebha are presented in Table 1. Prevalence of B. hominis was found to be significantly more (P < 0.05) in summer than in winter. Detection methods for B. hominis: All 950 faecal specimens were examined with the three different detection methods for the demonstration of B. hominis. Sensitivity results are shown in Table 2. Both direct smear and concentration showed significantly low sensitivity (P < 0.01) compared to in-vitro cultivation for the detection of B. hominis. Direct smear microscopy was positive for B. hominis in 26.21% (249 of 950). Short-term invitro cultivation was positive for B. hominis in 42.31% (402 of 950). All stool specimens found positive in direct smear microscopy were also found positive in stool culture. Of the 701 direct smear negative specimens, 153 (21.82%) yielded positive results for B. hominis in culture method. In-vitro cultivation appeared more sensitive than direct smear microscopy and concentration method. Faecal concentration was positive for B. hominis in 34.10% (324 of 950). None of the stool samples were found negative in concentration method was found positive in direct smear microscopy. Faecal concentration method had a higher positive yield for B. hominis than direct smear microscopy (34.10% versus 26.2%). Out of 626 stool samples (negative in concentration method for B. hominis), only 78 (12.46%) were found positive in stool culture for the organism. Almost all of the B. hominis detected in the direct smear were of the vacuolar form. After incubation of cultures for 48 hours, only vacuolar forms were seen in culture smears. After 72 hours of incubation of cultures, most of the B. hominis were of vacuolar forms but some were granular (Fig.1). Amoeboid forms of B. hominis were also seen in few culture positive samples. Mostly granular forms were observed in 96 hours old culture (Fig. 2). The vacuolar forms in culture smears were generally larger and in greater numbers than those seen in direct smears. Only vacuolar forms were detected after faecal concentration of fresh stool specimens, and were smaller and less in number, compared to culture positive faecal smears. Clinical symptoms of patients infected with B. hominis: Clinical presentation of 108 patients, parasitized exclusively by B. hominis is presented in Table 3. The most frequent manifestation of blastocystosis was diarrhea (84.94%). Clinical manifestations of symptomatic patients are shown in Table 4. These patients had no other parasitic infection. Patients had two or more gastrointestinal disorders. Twenty-nine patients (31.18%) had only one clinical feature (25 had diarrhea and 4 had abdominal pain). Forty-nine patients (52.68%) had two gastrointestinal disorders (33 had diarrhea with cramping abdominal pain, 6 had diarrhea with vomiting, 2 had vomiting with cramping abdominal pain and 8 had cramping abdominal pain with flatulence). Fifteen patients (16.12%) had three clinical symptoms of blastocystosis (7 had diarrhea with cramping abdominal pain and vomiting, 8 had diarrhea with cramping abdominal pain and flatulence). Intensity of B. hominis infection among symptomatic and asymptomatic individuals is shown in Table 5. The mean number of B. hominis was significantly higher, (P < 0.001) in symptomatic patients, than asymptomatic individuals (9.20 ± 2.41 organisms per high magnification (40 X) field versus 4.06 ± 1.86 respectively). In two infected persons, B. hominis cells were found to be 10 and 12 per high magnification (40 X) field, but were entirely asymptomatic. Therapeutic response in patients infected with B. hominis: Only fifty patients presented with gastrointestinal symptoms and exclusively parasitized by B. hominis were treated with metronidazole. B. hominis were not fond in stool from patients who have completed a therapeutic course of metronidazole for 7 days. All patients showed clinical improvement and were fully cured using 1500 mg of metronidazole daily for 7 days. Discussion: Prevalence of B.hominis: 47

4 The results of this study show that B. hominis was detected in 26.61% of stool specimens submitted to the Central Lab in Sebha. Almost similar prevalence's have been described in other parts of the world In the present study, prevalence of B. hominis was found comparatively more, compared to that reported by Al-Fellani et al 8 among patients attending the Central Lab in Sebha. This may be due to more awareness of B. hominis infection in the Central lab, and/or infection of this organism might have also increased in the population. Comparatively higher prevalence of B. hominis (46.9 %) was found in patients from health centers of Soledad of Venezuela. 21 Moreover, the incidence of B. hominis was found higher, compared to any other intestinal parasites reported in Sebha, Libya. 8 In accordance with other reports, 17, 29 vacuolar forms of B. hominis was most frequently found in stool of patients. Seasonal variation of B. hominis: The parasitological examination of faecal samples revealed that the incidence of B. hominis was widespread throughout the year with particular summer seasonal incidence peak among the patients attending the Central Lab in Sebha. The high incidence of B. hominis in Sebha region may be due to dry climatic, condition of this region, which favours the survival, and transmission of the organism throughout the year in the population. Infections of B. hominis were also most common during summer (warm and hot) season of the year in the other parts of the world. 23,29 Detection methods for B. hominis: Kukoschke et al 30 stated that in-vitro cultivation appeared to have no advantages over direct smear microscopy of fresh faecal samples for the diagnosis of B. hominis. It was suggested that cultures would only be scored positive if they had been set up using stool specimens that contained large numbers of B. hominis. 31 Zaman and Khan 32 suggested the usefulness of cultivation (like that of concentration technique) might depend on the exact reagents and procedures employed. In particular, the use of different culture media may have led to different points of view on the usefulness of B. hominis culture. In the present study, culture method detected significantly more (42.31%) infection of B. hominis than direct smear microscopy (26.21%) among patients (P < 0.01). Similar observations had been reported by others These workers had reported that short-term cultivation of faecal material was effective, and had advantage over direct smear microscopy for the detection of B. hominis. The increase in the numbers of B. hominis using stool culture method appears to be the result of the changing of cysts and multivacuolar forms into vacuolar forms (as mostly vacuolar forms are seen in culture smears). Moe et al (36) also observed that cyst forms of B. hominis were isolated from human faeces, developed into a large number of vacuolar forms in short-term in-vitro culture. Increase in number and size of this organism was also observed in cultures of the present study. These results were the same as those of, Moe et al 36, Leelayoova et al 10 and Zhang et al. 37 Although the examination of direct wet smears is convenient and inexpensive, it frequently leads to false-negative results. The main problem with simple direct smear is that small numbers of B. hominis which are present in the stool sample may go undetected or at least unrecognized. All stool samples screened for B. hominis in direct smear microscopy, were checked for the organism using normal saline sedimentation concentration technique. The results showed that concentration method was found relatively more sensitive (34.10%) than direct smear microscopy (26.21%). This increase in the detection efficiency of B. hominis in concentration method, compared with direct smear is probably due to the presence of small numbers of B. hominis cells in the faecal specimens of patients, which are missed during routine direct smear microscopy. The results are in accordance with Hussain Qadri et al, 38 Logar et al 39 and Taamasri et al, 40 who reported that concentration methods were beneficial, compared with simple smear. Moreover, Guimares and Sogayar 41 suggested that spontaneous sedimentation concentration in normal saline was a suitable method for separating B. hominis from faecal material. However, other authors had reported that concentration methods had no advantages over direct smear microscopy for the detection of B. hominis in the stool samples. 10,20,29,34 They assumed that low detection efficiency appeared 48

5 to be the necessary steps of shaking and centrifugation in formalin ether technique that led to rupture of the vacuolar, multivacuolar and granular forms of B. hominis. In the present study, there is also increase in the detection of B. hominis in concentration negative stool samples using stool culture method from the same patients. This may be due to the presence of smaller forms (other than vacuolar) of B. hominis in the faecal material, which successfully grow, and multiply in Boeck and Drbo Leelayoova et al, 10 Suresh and Smith 29 and Tungtrongchitr et al 34 also reported that stool specimens found negative in concentration (using formalin-ether technique), were found positive for B. hominis in stool culture. So far, there are two reporters 10, 34 who used direct smear concentration and culture method for the detection of B. hominis in the same fresh faecal specimens. These workers have reported that stool culture is more sensitive than direct smear, and concentration method leads to an even lower sensitivity, compared to direct microscopy. Moreover, Leelayoova et al 10 observed that the stool samples found positive in direct smear and concentration method, did not show the growth of organism in short-term in-vitro cultivation. They observed that out of 142 direct smear positive and 64 concentrations positive stool samples, 32 and 12 samples respectively did non give positive result for B. hominis in culture method. To avoid false-negative results, routine diagnosis of B. hominis in symptomatic patients in parasitological laboratories should be done by culture method, although stool culture is time-consuming and more costly. The routine diagnosis of B. hominis by direct smear microscopy is not reliable when dealing with patients with gastrointestinal symptoms or irritable bowel syndrome. Clinical symptoms in blastocystosis patients: B. hominis is one of the most common intestinal protozoa in humans. Now B. hominis is getting acceptance as an agent of human intestinal disease. 27 The reason why B. hominis has been found in both symptomatic and asymptomatic individual is largely unknown. 42 Although several reports have suggested that B. hominis could cause gastrointestinal disorders, the specific pathogenicity of this organism has not yet been defined. 35,43 In the present study, the most prominent gastrointestinal symptoms in 93 patients (parasitized exclusively by B. hominis) were diarrhea (45.93%), cramping abdominal pain (36.04%) and nonspecific gastrointestinal symptoms, such as flatulence (9.30%) and nausea or vomiting (8.72%). The majority of these patients had two or more gastrointestinal symptoms. The results of this study are in agreement with the clinical presentation of B. 14, 27, 42, and 44 hominis infection. There is still controversy over the relation between the number of B. hominis cells present in stool specimens from patients and the gastrointestinal disorders. In the present study, the number (more than seven) and mean number (9.20 ± 2.41) of B. hominis was significantly higher (P < 0.001) in symptomatic patients than in asymptomatic individuals (less than five, and 4.06 ± 1.86). These results were the same as those of Sheehan et al, 24 Zuckerman et al, 45 Nimri 14 and El-Shazly et al, 27 who reported more than five numbers of B. hominis per high magnification (40 X) field in stool samples from symptomatic patients. Moreover, Barahona et al 46 and El-Shazly et al 27 also reported significantly more mean number of B. hominis cells in symptomatic patients than asymptomatic ones. Five or more B. hominis cells per oil immersion field (100 X) are also detected in symptomatic patients More severe symptoms have been noticed with an increase in number of B. hominis in stool samples from patients. 26, 27, 50 However, Dolye et al, 51 Martin-Sanchez et al 52 and Shilm et al 4 did not find a statistically significant association between the number of B. hominis cells in the faeces of patients and disease state. Moreover, Sun et al, 53 designated five or nine and ten or more organisms of B. hominis per high magnification (40 X) field as moderate number of B. hominis and numerous parasites respectively. They concluded that B. hominis was probably not responsible for clinical symptoms in patients. In the present study, among asymptomatic subject (except two cases) the number of B. hominis cells was found less than five per high 49

6 magnification (40 X) field. Similar findings have been reported by Nimri 14 and Requena et al, 16 who found less than five (fewer than three) per high magnification (40 X) microscopic fields among stool samples of apparently healthy subjects. Moreover, large numbers of B. hominis were observed in the faeces of some asymptomatic individuals. 43 In conclusion, gastrointestinal symptoms in blastocystosis patients supported that B. hominis infection might have a role in some pathologic conditions involving the gastrointestinal tract and resulting into diarrhea, cramping abdominal pain and other nonspecific symptoms. Further research of B. hominis, to evaluate the pathogenic potential of this organism especially in patients with gastrointestinal symptoms, should be conducted in the absence of other identified causes of symptoms (enteric viruses or bacterial pathogens), and also by inclusion of large number of patients. Therapeutic response in blastocystosis patients: In this prospective study, the physicians of Health Central Al-Jadeed in Sebha had successfully treated fifty patients infected with B. hominis with metronidazole for 7 days. In all these patients, no other parasites were demonstrated in the faecal specimens. All patients responded to doses of metronidazole (500 mg three times per day for 7 days) recommended by the physicians and symptoms disappeared after 7 to 10 days. Moreover, B. hominis were not seen in the stools of patients after 7 days. The results of this study showed that gastrointestinal symptoms in all these patients (positive only for B. hominis) were probably due to B. hominis infection as symptoms disappeared after treatment with metronidazole. These findings are in accordance with Hussain Qadri et al, 38 Rao et al 54 and Nasirudeen et al, 55 who observed that metronidazole was the first choice of chemotherapeutic agent for the treatment of B. hominis infection, and clinical manifestation responded to therapy. However, some authors reported that metronidazole did not eradicate the B. hominis completely and was effective in some individuals. 56,57 Based on the results of the present study, symptomatic patients in whom only B. hominis was found, responded to metronidazole therapy and that response indicated elimination of B. hominis among patients. Conclusion: In-vitro cultivation can be used as the gold standard method for the detection of B. hominis in stool specimens. Short-term cultivation in Boeck and Drbohlav s medium of faecal specimens would be particularly beneficial when faecal samples are negative in the routine direct smear microscopy or when subject is excreting small numbers of B. hominis and also when a microscopic examination of simple smear is uncertain. For those who are looking for human pathogenicity (or clinical outcome), and true prevalence of B. hominis among Libyan communities, it may be necessary to examine faecal samples both as simple, direct smear microscopy and by the short-term in-vitro cultivation. False-negative faecal specimens in the direct smear microscopy should not be regarded as negative and not to be considered as insignificant for B. hominis infection, especially among symptomatic patients. Table1: Seasonal variation of B. hominis among patients attending the Central Lab in Sebha. Year Summer season* Winter season** Total investigated No. No. Positive No. No. Positive No. No. Positive*** (17.80) (15.04) (16.64) (21.95) (19.09) (20.86) (27.39) (25.13) (26.61) Figures in parentheses indicates percentages. * April to October. ** November to March. *** P<0.05 versus comparison with summer and winter season. 50

7 Table 2.: Detection of B. hominis in stool specimens by direct smear microscopy, and concentration technique, compared with in-vitro cultivation. Detection No. of cases Results method Positive by culture Negative by culture Investigated Direct smear microscopy Positive Negative 249(61.94) 153(38.05) NIL 548(100) 249(26.21) 701(37.78) Concentration method Positive Negative 324(80.59) 78(19.4) NIL 548(100) 324(34.10) 626(65.89) Direct smear or Concentration Any 402(42.31) 548(57.69) 950(100) Figure in parentheses indicates percentage. Table 3. Clinical manifestation of 93 symptomatic patients parasitized exclusively by B. hominis. Clinical symptoms No. of cases Diarrhea 79(84.94) Cramping abdominal pain 62(66.66) Flatulence 16(17.20) Vomiting/Nausea 15(16.12) Figure in parentheses indicates percentage. Table 4: Different gastrointestinal symptoms among 93 blastocystosis patients. Clinical symptoms No. of cases Diarrhea 25(26.88) Diarrhea with cramping abdominal pain 33(35.48) Diarrhea with vomiting 6(6.45) Diarrhea with cramping abdominal pain and vomiting 7(7.52) Diarrhea with cramping abdominal pain and flatulence 8(8.60) Cramping abdominal pain with vomiting 2(2.15) Cramping abdominal pain with flatulence 8(8.60) Cramping abdominal pain 4(4.30) Figure in parentheses indicates percentages. Table 5 Intensity of B. hominis infection among blastocystosis patients. Category No. of cases and (%) Intensity No. organisms Mean of No.± SD Positive for B. hominis with More than 7 93(86.11) gastrointestinal symptoms (8 to 30)* 9.20±2.41 ** Positive for B. hominis without Less than 5 15(13.88) gastrointestinal symptoms (2 to 12)* 4.06 ±1.86 * Range of No. of B. hominis cells per 40 X field. ** P < versus comparison with number of B. hominis cells among asymptomatic individuals. 51

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