Prevalence of Entamoeba histolytica-Like Cysts ...

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ABSTRACT: Amoebiasis, caused by Entamoeba histolytica, has a worldwide distribution and is of public health significance in many developing countries.
Prevalence of Entamoeba histolytica-Like Cysts Compared to E. histolytica Antigens Detected by ELISA in the Stools of 600 Patients from Three Socioeconomic Communities in the Metropolitan City of Lahore, Pakistan Author(s): Muhammad Azhar Alam, Azhar Maqbool, Muhammad Mudasser Nazir, Muhammad Lateef, Muhammad Sarwar Khan, Atif Nisar Ahmed, M. Ziaullah, and David S. Lindsay Source: Journal of Parasitology, 101(2):236-239. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/14-560.1 URL: http://www.bioone.org/doi/full/10.1645/14-560.1

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J. Parasitol., 101(2), 2015, pp. 236–239 Ó American Society of Parasitologists 2015

Prevalence of Entamoeba histolytica-Like Cysts Compared to E. histolytica Antigens Detected by ELISA in the Stools of 600 Patients from Three Socioeconomic Communities in the Metropolitan City of Lahore, Pakistan Muhammad Azhar Alam, Azhar Maqbool, Muhammad Mudasser Nazir*, Muhammad Lateef, Muhammad Sarwar Khan, Atif Nisar Ahmed*, M. Ziaullah†, and David S. Lindsay‡, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan; *Department of Pathobiology, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Boson Road, Multan 60800, Pakistan; †Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan; ‡Department of Biomedical Sciences and Pathobiology, Virginia–Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24060. Correspondence should be sent to: [email protected]

lysis by direct attachment and release of toxins, and phagocytic action of activated trophozoites (Devinder et al., 1996; Marie and Petri, 2014). Clinical signs and symptoms of infection vary from an asymptomatic carrier state to severe dysentery depending upon the virulence of the infecting strain of E. histolytica, the host’s nutritional and immune status, and the presence of other intestinal pathogens (Stanley, 2003). In developing countries, the diagnosis of amoebic infection is generally based on morphological identification of fecal cysts using light microscopy and a variety of fecal staining methods. Unfortunately, light microscopy cannot be used to differentiate between pathogenic E. histolytica and the morphologically identical non-pathogenic Entamoeba dispar and Ent´ amoeba moshkovskii (Ximenez and Nosaki, 2014), and microscopy has less sensitivity and specificity than do the other diagnostic methods (Haque et al., 2003). Several fecal antigen ELISAs and PCR-based assays have been developed and used worldwide (Hamzah et al., 2006; Fotedar et al., 2007) for the detection of true E. histolytica infections and the identification of other Entamoeba species. About 40 million people worldwide are infected with E. histolytica, and invasive E. histolytica infection accounts for death in between 40,000 and 100,000 patients each year (Al-Harthi, 2004; Haque et al., 2006). Amoebiasis is considered to be the third most-important cause of death due to parasitic disease in humans after malaria and schistosomiasis (Walsh, 1986). The disease is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty and associated poor sanitation infrastructure. Because little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries, we undertook the present study in the city of Lahore, Pakistan. Our hypothesis was that the prevalence of E. histolytica-like cysts and E. histolytica stool antigen would be lower in a group of patients from an upper socioeconomic group than in individuals from middle or lower socioeconomic groups. Fecal samples (n ¼ 600) from individuals belonging to 3 socioeconomic communities with different economic resources were obtained from volunteers at 3 hospitals (Mayo Hospital, Children’s Hospital, and Jinnah Hospital) in Lahore, Pakistan. The Directorate of Advance Studies and Research Board, UVAS, Lahore approved the study, and written or oral consent was obtained from each patient. Participants were placed in economic classes based on a medium per capita income of $806.38 (US dollars) in 2013 in Pakistan. Socioeconomic group 1 (upper income) consisted of 286 individuals that were above the median income level of Pakistan and lived in modern housing with electricity, indoor plumbing, and toilet facilities. Socioeconomic group 2 (middle income) consisted of 172 individuals that were at the median income level of Pakistan and most lived in modern housing with electricity, indoor plumbing, and toilet facilities. Socioeconomic group 3 (lower income) consisted of 141 individuals that were below the median income level of Pakistan and most lived in housing without electricity, indoor plumbing, and toilet facilities. Patients were not undergoing specific anti-E. histolytica treatment when stool samples were collected. About 3 g of fecal sample was fixed in

ABSTRACT :

Amoebiasis, caused by Entamoeba histolytica, has a worldwide distribution and is of public health significance in many developing countries. It has a fecal–oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica-like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica-like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica. Samples were from individuals living under conditions deemed to be upper socioeconomic class (n ¼ 287), middle socioeconomic class (n ¼ 172), and lower socioeconomic class (n ¼ 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P , 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirtynine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple test and by antigen ELISA, respectively. We accept our hypothesis based on these findings. We also demonstrated that fecal samples collected from the youngest age group (1 mo–5 yr) were more likely to be positive for E. histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P , 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

Amoebic dysentery is caused by Entamoeba histolytica and the parasite is distributed throughout the world, especially among humans and other primates (Suzuki et al., 2008). The parasite has a fecal–oral life cycle comprised of infectious, tetranucleate cysts (passed out in the feces) and uninucleate trophozoites (replicating feeding stages infecting the large intestine) (Okafor and Azubike, 1992). Humans become infected through the ingestion of cysts on contaminated food or in water. The risk factors are similar to other diseases transmitted by the fecal–oral route. The pathogenesis of E. histolytica is not well understood; however, it is believed to involve competition with intestinal microflora, target host cell DOI: 10.1645/14-560.1 236

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TABLE I. Prevalence of Entamoeba histolytica-like cysts and E. histolytica antigen in stool samples among 3 economic groups.* Triple fecal test Economic group

ELISA

No. examined

Positive

% infection

P-value

Positive

% infection

P-value

287 172 141 600

44 39 52 135

15.3 22.6 36.8 22.5

0.001

32 29 40 101

11.1 16.8 28.3 16.8

0.0012

Upper Middle Lower Total

* Significant difference (P , 0.05) of prevalence was observed between 3 socioeconomic class groups. methanol and examined in the triple fecal test (Van Gool et al., 2003) to detect fecal cysts with morphology consistent with E. histolytica (referred to herein as E. histolytica-like). A portion of fecal sample was not fixed but kept refrigerated at 4 C until examined for fecal antigens of E. histolytica using a commercially available antigen detection kit (E. histolytica II). ELISAs were performed according to the directions of the manufacturer (E. histolytica II, TechLab, Blacksburg, Virginia). The E. histolytica II kit is based on monoclonal antibody–peroxidase conjugate specific for E. histolytica adhesion (Fadeyi et al., 2009; Dawah et al., 2010). Because cyst morphology cannot be used to definitively diagnosis E. histolytica, we refer to samples positive for cysts by the triple test as being E. histolyticalike. Only samples positive by ELISA were considered to be true E. histolytica positives (Table I). Triple fecal test positive samples were stained with permanent stain (chlorazole black stain). Samples examined using the fecal antigen ELISA were read using an ELISA plate reader operating at 450 nm wavelength. Information about patient age and clinical symptoms were also recorded on a separate Performa sheet at the time of sampling. The date of sample collection was recorded and used to evaluate the influence on season of the year with parasite prevalence. Data regarding clinical status, (i.e., patients with diarrhea and dysentery having a fever of 37.8–38.9 C) were also recorded prior to the collection of samples from each patient. All patients had gastrointestinal problems, and stool samples were collected from 463 patients that had diarrhea and 137 patients that had no diarrhea. Pearson’s chi-square test was used to compare positive status with respect to economic group, age, and season by using SPSS for Windows, version 17.0 (SPSS, Chicago, Illinois), and P , 0.05 was considered as significant. One hundred and thirty-five (22.5%) of the 600 samples examined were positive for E. histolytica-like cysts in the triple test fecal examination while 101 (16.8%) of the 600 samples were positive for antigens of E. histolytica in the antigen ELISA (Table I). Prevalence was negatively associated with economic group. Individuals in the lower socioeconomic group had the highest prevalence of E. histolytica-like parasite cysts (52/ 141, 36.8%) and E. histolytica antigens (40/141, 28.3%), the middle socioeconomic group had the next highest prevalence of E. histolytica-like cysts (39/172, 22.6%) and E. histolytica antigens (29/172, 16.8%), while the upper socioeconomic group had the lowest prevalence of E. histolyticalike cysts (44/287, 15.3%) and E. histolytica antigens (32/287, 11.1%).

These prevalences were significantly different (P , 0.05) between the socioeconomic groups. We found that 15 (11.1%) of 135 positive triple test positive samples were negative by fecal antigen ELISA. Five (11.3%) samples from upper class, 3 (7.6%) from middle class, and 7 (13.4%) from lower class were triple fecal test-positive samples that were negative for fecal antigen ELISA. It is possible that this prevalence represents the prevalence of E. dispar or E. moshkovskii in our population. The prevalence of E. histolytica-like cysts and for E. histolytica antigens by ELISA was highest in the youngest groups and decreased with patient age (Table II). Prevalence of amoebic infection based on age group was significantly different using either test. The prevalence of E. histolytica-like cysts and E. histolytica fecal antigen positive samples was associated with season of the year. Most positive stool samples were observed during the summer months and the fewest in the winter months (Table III). The prevalence of E. histolytica-like cyst-positive and E. histolytica-like cystnegative samples or E. histolytica antigen-positive and E. histolytica antigen-negative stool samples was not statistically different in patients with diarrhea or no diarrhea (Table IV). Entamoeba histolytica infection is often widespread in the residents of developing countries. It affects populations living under poor socioeconomic conditions associated with inadequate hygienic and sanitary conditions and often malnutrition because of lack of public health resources. Our data demonstrate that a high prevalence of E. histolyticalike cysts was observed in patients, based on a triple fecal test, and a comparatively moderate prevalence of true E. histolytica infection was detected in patients based on stool antigen ELISA. Because the triple fecal test detects cysts of amoeba and other parasites, and it cannot distinguish between the potentially pathogenic E. histolytica and the nonpathogenic, morphologically identical E. dispar, positive samples are reported as E. histolytica-like cysts in the present study. The stool antigen ELISA is specific for E. histolytica and does not cross-react with E. dispar and is reported as true E. histolytica infection in the present report. The overall high prevalence (22.5%) of E. histolytica-like cysts among different socioeconomic groups of humans is comparable to the previous studies based on microscopy made by others, Tasawar et al. (2010), who reported a high prevalence of E. histolytica (21.7%) in humans at Multan, Pakistan. The high prevalence gives us a clue that E. histolytica may be considered a major risk factor of intestinal diseases in Pakistan. We found the highest

TABLE II. Prevalence of Entamoeba histolytica-like cysts and E. histolytica fecal antigen by age groups.* Triple fecal test

ELISA

Age group

No. examined

Positive (n)

% infection

P-value

Positive (n)

% infection

P-value

1 mo–5 yr 6–20 yr 21–35 yr 36 and above Total

90 92 251 167 600

39 28 44 24 135

43 30 17 14 22.5

0.009

27 23 32 19 101

30 25 13 11 16.8

0.011

* Age-wise prevalence was significant (P , 0.05) among all groups.

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TABLE III. Prevalence of Entamoeba histolytica-like and true E. histolytica based on season. Triple fecal test

ELISA

Season

Number examined

Positive

% infection

P-value

Positive

% infection

P-value

Winter Spring Summer* Autumn Total

200 100 200 100 600

34 18 62 21 135

17 18 31 21 22.5

0.005

25 13 48 15 101

12.5 13.0 24.0 15.0 16.8

0.010

* Prevalence in summer was significantly higher than in other months, P , 0.05. prevalence of both E. histolytica-like cysts and E. histolytica antigenpositive stool samples in the lower socioeconomic group in the present study (Table I). This higher prevalence in lower socioeconomic group patients is likely due to their having limited access to modern sanitation and water sources and a lack of awareness of preventive measures that can be taken to decrease exposure to cysts (Chacin-Bonilla et al., 1992; Karaman et al., 2006). Our patients demonstrated that the infection prevalence with E. histolytica-like cysts and E. histolytica stool antigens decreased as patient age increased (Table II). This age-related difference in prevalence has been observed by others in Pakistan (Waqar et al., 2003) and in Malaysia (Noor Azian et al., 2007). This age difference may be due to a less-developed immune system in children, lack of prior exposure, and increased exposure due to lack of personnel hygiene in children (Abu Mourad, 2004; Oguntibeju, 2006). Young children are more-frequently exposed to infectious stages because of activities such as playing in contaminated soil in and around wastewater sites. Amoebic infection among the younger age group may be due to inherited primary deficiencies of the immune system that may result in infections with common pathogens as well as with opportunistic organisms (Davies, 2006). Higher prevalence observed in the summer may be due to favorable hot and humid climatic conditions for cyst survival and parasite transmission, as described by others (Ahmed et al., 1996; Dawah et al., 2010). Our results indicated that the gross consistency of the patients’ stool was not associated with the occurrence of Entamoeba-like cysts or positive E. histolytica antigen tests (Table IV). Others have reported a higher prevalence of E. histolytica in patients with diarrhea (Chung et al., 1959; Fadeyi et al., 2009), but in 1 study stool samples from HIV patients had a higher prevalence than did diarrheic samples in patients with HIV (Sachin et al., 2011). These results are probably due to cyst shedding frequency and the sensitivity and specificity of the diagnostic test used. The current study demonstrated that E. histolytica infection is more prevalent in lower socioeconomic groups when compared to middle and upper socioeconomic groups. The high prevalence of this parasite is probably due to lack of modern facilities, low levels of hygiene, and poor sanitary conditions that increase the probability of transmission. Improvement in the quality and availability of sanitary facilities and TABLE IV. Prevalence of Entamoeba histolytica-like cysts and E. histolytica fecal antigen-positive stool samples from diarrheic and non-diarrheic patients.* Triple fecal test

Diarrhea Yes No Total

ELISA

Number examined

Number positive

% infected

Number positive

% infection

463 137 600

112 23 135

24.2 16.8 22.5

82 19 101

17.7 13.9 16.8

* No significant differences (P . 0.05) were present using either diagnostic test.

public health education measures should be undertaken to lower the prevalence of E. histolytica in lower socioeconomic groups in the metropolitan city of Lahore, Pakistan. These measures should also decrease the prevalence in other socioeconomic groups in the city. The present study was economically supported by a grant from the Higher Education Commission, Islamabad, Pakistan. The authors would like to acknowledge Dr. Aftab Ahmed Anjum, Dr. Haroon Akbar, Dr. Imran Rasheed, Dr. Muhammad Oneeb, Dr. Ahsan Mustafa, Asif Ali, Noman Nazir, Umer Bacha, and Muhammad Kashif Nazir for their technical help regarding this study.

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