Occult Amebiasis among Pediatric with Enterocolitis in National Referral Hospital in Indonesia

Abstract

Background

Enterocolitis and gastroenteritis remain major health problems, particularly in children living in developing countries. Intestinal protozoa, such as Entamoeba histolytica, Blastocystis, and Cyclospora, are frequently associated with these conditions. Amebic colitis can cause serious complications, including fulminant necrotizing colitis, toxic megacolon, extraintestinal amebiasis, and stunting in children. The diagnosis of amoebiasis is challenging, relying on microscopic examination, which cannot distinguish E. histolytica from the nonpathogenic E. dispar and E. moshkovskii. Therefore, this study aimed to identify intestinal parasites, particularly Entamoeba, their prevalence, and the clinical characteristics of patients admitted for enterocolitis and gastroenteritis at a tertiary-referral hospital.

Material and Methods

A cross-sectional, retrospective study was conducted at a national, tertiary-referral government hospital, in Jakarta. Of the 111 retrieved medical records from hospitalized patients with enterocolitis and gastroenteritis, for which parasitology feces were examined, 54 fecal samples (48.6%) were still available in the parasitology laboratory storage. All fecal samples underwent the following tests: 1) direct stool examination, after staining with 1% Lugol’s solution, and using the water–ether concentration method; 2) modified acid-fast staining for coccidian parasites; 3) Jones’ culture medium to detect Blastocystis; 4) copro-antigen assay to detect Cryptosporidium and Giardia; and 5) a polymerase chain reaction (PCR) assay to identify Entamoeba. Clinical and demographic data were obtained from the medical records.

Results

Largely, patients (44.1%) were from the cohort of young children ≤5 years old, followed by adults aged 19–60 years old (24.3%). Both cohorts exhibited polyparasitism. Intestinal parasites were detected in 17 out of the 54 samples (31.4%). These included 6 (11.1%), 2 (3.7%),5 (9.2%), 3 (5.5%), 2 (3.7%), and 1 (1.8%) samples that were positive for Blastocystis, E dispar, E. histolytica, E. moshkovskii, Cryptosporidium, and Dientamoeba fragilis, respectively. PCR analysis revealed that 10 samples were positive for Entamoeba infection, eight of which originated from pediatric patients.

Conclusion

At a national tertiary-referral hospital in Indonesia, Entamoeba infection was the most prevalent parasite among pediatric patients with enterocolitis. E. histolytica and E. moshkovskii were the two main species identified by PCR. Therefore, PCR assays and fecal occult–blood tests are recommended in cases of enterocolitis and gastroenteritis.

Graphical Abstract

INTRODUCTION

Gastrointestinal disorders, including enterocolitis comprise diarrhea, as one of the many symptoms thereof. The etiologies of enterocolitis include autoimmune, genetic, and vascular disorders; in addition to bacterial and parasitic infections. Parasitic colitis is typically caused by the genuses Cryptosporidium, Cyclospora, Isospora, Schistosoma, and Trichuris; in addition to the species Balantidium coli, Giardia duodenalis, and Entamoeba histolytica [1,2,3]. Intestinal amebiasis, which is difficult to diagnose, has a prevalence of 3.0–10.0%, occurring particularly in tropical and sub-tropical areas with low socioeconomic conditions and poor sanitation [4].

In Indonesia, previous studies have revealed that the incidence of intestinal amebiasis ranges from 8.3 to 8.0% [5,6]. Intestinal amebiasis can result in amoebic colitis, intestinal bleeding, extra-intestinal dissemination, and fulminant necrotizing enterocolitis [7]. The symptoms of intestinal amoebiasis are non-specific; therefore, mis- and underdiagnosis frequently occur, resulting in the administration of inadequate or empiric therapy. Furthermore, complications of a mis- or underdiagnosed intestinal amoebiasis are associated with a mortality rate of approximately 40% [8].

Challenges are encountered with the laboratory diagnosis of amoebic colitis, which still relies on microscopy, lacking in sensitivity. Moreover, difficulties exist in differentiating between the morphology of Entamoeba histolytica from the non-pathogenic E. dispar and E. moshkovskii. Therefore, with a sensitivity and specificity of >70% and 90%, respectively, a polymerase chain reaction (PCR) assay of fecal samples is preferred, allowing for detection of different species of Entamoeba [9,10,11,12].

Thus, accurate diagnosis is important for patients presenting with either dysentery or asymptomatic amoebic infections [13,14]. Consequently, in this study, we aimed to determine the proportion of different species of intestinal parasites, particularly focusing on the Entamoeba infection, in patients who had been hospitalized with gastroenteritis or enterocolitis. Moreover, we investigated which clinical characteristics associated with the fecal parasitology profile.

MATERIALS AND METHODS

1. Study population and design

This cross-sectional, retrospective study was conducted at Rumah Sakit Cipto Mangunkusumo (RSCM), a national tertiary-referral government hospital, in Jakarta. Fecal samples from 190 patients clinically diagnosed with gastroenteritis and enterocolitis were collected and referred to the parasitology laboratory, Faculty of Medicine Universitas, Indonesia, during the period of 2020 to 2022, for parasitic identification.

Eligibility criteria consisted of available frozen fecal sample in the laboratory storage and, accessible medical records from the hospital. As many as 111 medical records could be retrieved and 54 fecal samples (48.6%) were still available in the laboratory storage. These samples were included in the final data analysis. The research flow is illustrated in Figure 1.

Figure 1. Schematic diagram of the study flow is depicted.

PCR, polymerase chain reaction.

2. Ethical Statement

Ethical approval to conduct the study was obtained from the Health Research Ethics Committee, FKUI-RSCM (approval number: KET.445/UN2.F1/ETIK/PPM.00.02/2022). Moreover, this study was conducted in accordance with the principles of the Declaration of Helsinki.

3. Parasitology stool examination

The fecal samples were examined macroscopically for color, consistency, and the presence of mucus and blood. Intestinal parasites were screened microscopically by performing the following tests: 1) direct stool examination with 1% Lugol solution, and fecal water–ether concentration method; 2) modified acid-fast staining for coccidian parasites; 3) Jones’ culture medium to detect Blastocystis; 4) copro-antigen assay for the detection of Cryptosporidium and Giardia; and 5) a PCR assay to identify Entamoeba.

The water–ether concentration method was performed as follows: 1) approximately 1 g of the fecal sample was dissolved in 3 mL of aquadest; and 2) 1 mL of ether was added, mixed, and subsequently centrifuged at 2,500 rpm for 2 min. The resulting four layers included ether, debris, distilled water, and sediment. The top layer was discarded; and the bottom layer was preserved, for microscopic examination and modified acid-fast staining.

4. Modified acid-fast staining

Modified acid-fast staining was used to screen for coccidian parasites, including Cryptosporidium, Cyclospora, and Isopora. The fecal concentrate was smeared onto an object-glass and fixed by passing it over the burner two to three times, followed by immersion in methanol for 3 min. Thereafter, the samples were stained with carbol fuchsin for 15 min and counter-stained with 0.4% malachite green for 30 s.

5. Jones’ culture medium

Samples were cultured in Jones’ medium to detect Blastocystis. Before the inoculation of the samples, 10% of horse serum was added to the medium.

6. Cryptosporidium and Giardia detection by the copro-antigen assay

Screening for Cryptosporidium and Giardia involved the use of immunodiagnostic methods to detect copro-antigens in samples. The Cryptosporidium/Giardia Copro-antigen kit (Cortez Diagnostics, Inc., Woodland Hills, CA, USA) was used, as per the manufacturer’s instructions. The absorbance value was read at wavelengths of 450 nm and 620–650 nm, using an enzyme-linked immunosorbent assay reader.

7. Stool deoxyribonucleic acid (DNA) extraction and PCR

DNA was extracted using the Favor Prep stool DNA isolation Mini Kit (Favorgen Biotech Corp., Ping Tung, Taiwan), as per the manufacturer's instructions. The extracted DNA was stored at -20 ⁰C, prior to performing PCR.

Conventional PCR was performed, using a single round of PCR, as per a study by Hamzah et al. in 2006 [15]. Primers were designed based on a small subunit of ribosomal ribonucleic acid gene sequences from E. histolytica, E. dispar, and E. moshkovskii (GenBank accession nos. X64142, Z49256, and AF149906, respectively).

The primer sequences used were as follows: 1) forward common Entamoeba primer, EntaF (5'-ATGCACGAGAGCGAAAGCAT-3’); 2) reverse primer, EhR (5'-GATCTAGAAACAATGCTTCTCT-3'); 3) reverse primer, EdR (5'-CACCACTTACTATCCCTACC-3'); and 4) reverse primer, EmR (5'-TGACCGGAGCCAGAGACAT-3’).

The expected PCR products were 166 bp, 752 bp, and 580 bp for E. histolytica, E. dispar, and E. moshkovskii, respectively. A total volume of 25 µL of PCR reaction mix comprised 12.5 uL of GoTaq^® Master Mix (Promega, Madison, WI, USA), 10 pMol of primers, 2 µL of DNA template, with a concentration <250 ng/uL, and nucleus-free water (to bring the total volume to 25 µL).

PCR reaction condition was initiated at 94°C for 2 min for the initial denaturation. Thereafter, 30 cycles of denaturation were performed at 94°C for 1 min, with annealing at 58°C for 1 min, and an extension at 72°C for 1 min. The final extension occurred at 72°C for 7 min.

Electrophoresis was performed using a 100-bp DNA ladder (BenchTop DNA Ladder, Promega Corporation, Madison, WI, USA) and nucleic acid staining (GelRed, Biotium, Inc., Fremont, CA, USA). Subsequently, the gel was covered with aluminum foil, and placed on a rotator shaker (Rotamax 120, Heidolph, Schwabach, Germany) for 30 min. Thereafter, the gel was visualized on an ultraviolet transilluminator (Vilber Loumart, Collégien, France).

8. Data analysis

Clinical and demographic data were obtained from the medical records. The data were examined, coded, tabulated, and recorded on a Microsoft Excel spreadsheet for further analysis, using SPSS software (version 25.0; IBM Corp., Armonk, NY, USA).

RESULTS

1. Demographic and clinical profile of study population

The demographic profile of the study population from 111 medical records showed 69.4% of patients were male, aged 1-month to 74-years-old. Largely, patients (44.1%) were from the cohort of young children ≤5-years-old, followed by adults aged 19–60-years-old (24.3%). The nutritional status of most patients was poor. Moreover, 15.3% of patients were infected with human immunodeficiency virus infection/acquired immune deficiency syndrome (HIV/AIDS), and approximately 50% of these patients were children aged 4-months to 15-years-old (n=8).

Clinical symptoms retrieved from the medical records included diarrhea, abdominal distension, nausea, and vomiting. Nausea and vomiting were present in 29.7% and 24.3% of patients, respectively (Table 1). All patients diagnosed with gastroenteritis or enterocolitis had a median duration and frequency of diarrhea of 4 days and 4 times per day, respectively. The maximum frequency of defecation was 15 times per day, and the longest duration of diarrhea was 180 days. Macroscopic characteristics of the fecal samples largely revealed a soft stool consistency (83.8%). Fecal mucus and blood were observed in 44.1% and 8.1% of fecal samples, respectively.

Table 1. Demographic and clinical characteristics of patients.

Variables		Prevalence (%) (n=111)
Age group
	Young children (0 - 5-years-old)	49 (44.1)
	School-aged children (6 - 11-years-old)	14 (12.6)
	Adolescents (12 - 18-years-old)	15 (13.5)
	Adults (19 - 60-years-old)	27 (24.3)
	Older adults (>60-years-old)	6 (5.5)
Sex
	Male	77 (69.4)
	Female	34 (30.6)
Nutritional status
	Poor	70 (63.1)
	Normal	35 (31.5)
	Obese	6 (5.4)
HIV/AIDS status
	Positive	17 (15.3)
	Negative	94 (84.7)
Frequency of defecation (day)a		4
Duration of diarrhoea (day)a		4
Abdominal distension
	Present	8 (7.2)
	Absent	103 (92.8)
Nausea
	Present	33 (29.7)
	Absent	78 (70.3)
Vomiting
	Present	27 (24.3)
	Absent	84 (75.7)
Fecal consistency
	Soft	93 (83.8)
	Watery	18 (16.2)
Fecal mucus
	Present	49 (44.1)
	Absent	62 (55.9)
Fecal blood
	Present	9 (8.1)
	Absent	102 (91.9)

^aMedian (min-max).

HIV/AIDS, human immunodeficiency virus infection/acquired immune deficiency syndrome.

2. Microscopic results

Fecal microscopic examination revealed that 16.7% (n=9) were positive for intestinal protozoal infection; in which Blastocystis, Cryptosporidium, and concomitant Dientamoeba fragillis and Trichuris trichiura were detected in six, two, and one fecal samples, respectively.

None of the fecal samples including those containing blood and mucus, were positive for E. histolytica. Four out of the 17 patients with HIV/AIDS, of which three were children and one was an adult, harbored intestinal parasites. Moreover, the fecal samples of two patients with HIV/AIDS were positive for Cryptosporidium on microscopy. This was concordant with the results of the Cryptosporidium/Giardia copro-antigen test. The PCR assay of the fecal samples of the other two patients with HIV/AIDs were positive for Blastocystis and E. dispar.

3. Molecular diagnosis

The PCR assay of fecal samples was positive for Entamoeba in 10 cases (18.5%), principally with E. histolytica (n=5) and followed by E. moshkovskii (n=3) and E. dispar (n=2) (Fig. 2). Two fecal samples were both positive microscopically and by PCR analysis for intestinal parasites. The first sample was positive for Cryptosporidium sp. and E. histolytica; while the second sample was positive for T. trichiura and D. fragilis, in addition to E. moshkovskii, respectively. Cryptic Entamoeba infections were clearly evident in the rest of the fecal samples. The total percentage of patients with intestinal parasites was 31.5% (17/54), of which 13 out of 17 subjects (76.4%) were children aged between 1-month and 15-years-old. The fecal parasitology profile and detection methods are presented in Table 2.

Figure 2. Agarose gel electrophoresis revealing samples positive for Entamoeba histolytica (166 bp), E. moshkovskii (580 bp), and E. dispar (752 bp).

Table 2. Spectrum of intestinal parasites in patients with enterocolitis diagnosed by microscopic, PCR, and copro-antigen detection methods.

Method			Prevalence (%) (n=54)
Microscopy
	Negative		45 (83.3)
	Positive		9 (16.7)
		Blastocystis	6
		Cryptosporidium	2a
		Dientamoeba fragilis + Trichuris trichiura	1b
PCR for Entamoeba
	Negative		44 (81.5)
	Positive		10 (18.5)
		E. histolytica	5
		E. moshkovskii	3
		E. dispar	2
Copro-antigen: Cryptosporidium/Giardia
	Negative		52 (96.3)
	Positive		2 (3.7)

^aCryptosporidium + E. histolytica (n=1).

^bD. fragilis + T. trichiura + E. moshkovskii (n=1).

PCR, polymerase chain reaction.

4. Clinical characteristics of patients with Entamoeba infections

The clinical characteristics of the patients with Entamoeba infections are depicted in Table 3. Largely, all patients with Entamoeba infection were children <11-years-old. Patients infected with E. histolytica had a longer duration of diarrhea than that of those infected with E. dispar or E. moshkovskii, with median values of 10 and 3–4 days, respectively.

Table 3. Clinical characteristics and stool macroscopy of patients with enterocolitis infected with either Entamoeba or Cryptosporidium or both.

Characteristics		Entamoeba histolytica (n=5)	Entamoeba moshkovskii (n=4)	Entamoeba dispar (n=1)	Cryptosporidium (n=2)a
Age group
	Young children (0–5-years-old)	3	1	0	0
	School-aged children (6–11-years-old)	1	2	1	1
	Adolescents (12–18-years-old)	0	0	1	1
	Older adults (>60-years-old)	1	0	0	0
Sex
	Male	1	3	0	0
	Female	4	0	2	2
Nutritional status
	Poor	4	1	2	1
	Obese	1	2	0	1
Comorbidities
	HIV/AIDS	1	0	1	2
	Medulloblastoma	1	0	0	0
	SLE	1	0	0	0
	Polyposis, non-Hirschsprung’s disease	0	2	0	0
	Thalassemia	0	0	0	0
	None	2	1	0	0
Frequency of defecation, dayb		4 (2–6)	4 (2–6)	4.5 (4–5)	4.5 (3–6)
Duration of diarrhea, dayb		10 (1–28)	3 (2–3)	5 (3–7)	104 (28–180)
Fecal consistency
	Soft	5	2	0	2
	Watery	0	1	2	0
Fecal mucus
	Present	3	2	1	2
	Absent	2	1	1	0
Fecal blood
	Present	1	1	0	0
	Absent	4	2	2	2

^aCo-infection: Cryptosporidium + E. histolytica (n= 1).

^bMedian (min–max).

HIV/AIDS, human immunodeficiency virus infection/acquired immune deficiency syndrome; SLE, systemic lupus erythematosus.

Among the patients, the clinical symptoms and macroscopic appearance of the fecal samples stool were comparable. Two patients who had the longest duration of diarrhea at 180 and 28 days, respectively, were children with HIV/AIDS. The first patient had been infected with Cryptosporidium, and the second patient had been co-infected with Cryptosporidium and E. histolytica.

Most patients had not been administered treatment with metronidazole, cotrimoxazole, or ciprofloxacin, which could have potentially affected the parasitology test results. However, one patient had been treated with metronidazole, prior to the fecal parasitology test; subsequently testing positive for a co-infection of Cryptosporidium and E. histolytica.

DISCUSSION

In this study, we investigated the prevalence and species of intestinal parasites, particularly focusing on the genus Entamoeba, in patients who had been hospitalized with gastroenteritis or enterocolitis. Moreover, we investigated the clinical characteristics correlated with the fecal parasitology profile. Our findings highlighted that the cohort of young children ≤5-years-old had the largest prevalence of Entamoeba infections. Thus, Entamoeba and other intestinal parasites should be considered in patients with enterocolitis, particularly in cases of diarrheal complaints. Diarrhea was the etiology for mortalities in 10.3% and 14.0% of toddlers and neonates, respectively [16]. Several studies have reported cases of amebiasis in infants. Moreover, in South Asia, amebiasis is one of the top seven pathogens causing dysentery in children <5-years old [17,18].

The presence of persistent diarrhea could interfere with food absorption, which may have resulted in undernutrition in four of our patients in this study. Furthermore, E. histolytica infection has previously been shown to cause weight loss [8,19,20,21].

The identification of the particular Entamoeba species is important for clinical purposes. E. histolytica has pathogenic properties and requires treatment. Conversely, E. dispar and E. moshkovskii are non-pathogenic [22]. Nonetheless, they are morphologically similar; thus, microscopic differentiation is challenging. In this study, Entamoeba was detected after performing a PCR assay, revealing that microscopic examination has a low level of sensitivity for the identification of E. histolytica and is highly dependent on the examiner [22]. However, the microscopic examination of multiple samples can increase the sensitivity of detecting Entamoeba infection [21,23]. The diagnostic ability of the PCR assay possesses a sensitivity of 100%, specificity of 98.4%, positive predictive value of 66.7%, and negative predictive value of 100%, and diagnostic accuracy of 98.4%. Therefore, the PCR assay can be used as a confirmatory test post-microscopy [22].

Approximately 90% of E. histolytica infections are asymptomatic; nonetheless, parasitic colonization can endanger the population, due to the risk of infection. Accordingly, treatment is urgently needed. One-third of the patients with amebic colitis present with fever, and fecal blood is rarely observed on macroscopic examination. Nevertheless, positive fecal occult–blood tests (FOBT) are observed [9,24,25]. Similarly, in our study, out of the five patients with fecal samples positive for E. histolytica, only one patient had macroscopic fecal blood. However, as per the medical records, FOBTs were not performed in this study; therefore, occult blood could not be identified.

In this study, findings revealed that E. moshkovskii was the second most dominant species. E. moshkovskii is postulated to have a role in provoking human diseases, causing diarrheal and other intestinal disorders [26,27]. In the present study, the frequency and duration of diarrhea were more severe in patients with E. histolytica infection than that in patients with E. moshkovskii infection. However, one of the two patients with fecal blood harbored E. moshkovskii.

Diarrhea could have been additionally attributed to concomitant bacterial or viral infections, the data of which were not available in the medical records in this study. In the cohort of patients with E. histolytica infection in this study, some patients had comorbid HIV/AIDS or malignancy. Findings of a study that has been conducted in a population of patients with HIV has revealed that HIV is an independent risk factor for infection with intestinal parasites, such as G. duodenalis and E. histolytica [28]. Finally, a limitation of this research was the retrospective, cross-sectional design thereof, with the limited availability of data from accessed medical records and the number of stored fecal samples. A prospective study involving a larger number of institutions and fecal samples is required, to assess the actual burden of amebiasis among cases of enterocolitis. This will result in the early detection and treatment and will prevent further complications.

In summary, at the national, tertiary-referral hospital in Indonesia, the most prevalent intestinal parasite was an occult Entamoeba infection that largely occurred among pediatric patients with enterocolitis. Thus, a high burden of amebiasis among children was revealed. E. histolytica and E. moshkovskii were the two main species identified by PCR. Accordingly, PCR assays and FOBTs are recommended in cases of enterocolitis.