doi: 10.37545/haematoljbd201813

Begum F¹, Haque R², Rahman ATMA³, Yasmin F⁴, Jamal CY⁵, Islam A⁶

Authors

1. * Ferdousi Begum, Assistant Professor, Department of Paediatric Oncology, National Institute of Cancer Research and Hospital, Mohakhali, Dhaka-1212, Bangladesh.

2. Rashidul Haque, Infectious Disease Division, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka-1212, Bangladesh.

3. A.T.M. Atikur Rahman, Associate Professor, Department of Paediatric Haematology and Oncology, Bangbandhu Sheikh Mujib Medical University, Dhaka, Bangladesh.

4. Farida Yasmin, Assistant Registrar, Department of Paediatric Oncology, National Institute of Cancer Research and Hospital, Mohakhali, Dhaka-1212, Bangladesh.

5. Chowdhury Yakub Jamal, Professor, Department of Paediatric Haematology and Oncology, Bangbandhu Sheikh Mujib Medical University, Dhaka, Bangladesh.

6. Afiqul Islam, Professor, Department of Paediatric Haematology and Oncology, Bangbandhu Sheikh Mujib Medical University, Dhaka, Bangladesh.

*Correspondence

Abstract

Background: Haematological malignancies comprise 82% of all malignancies in children at the department of Paediatric Haematology and Oncology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh; Gastro-intestinal infections were the leading causes of infection in paediatric oncology patients. Objectives: This study was conducted to see the type of enteropathogens in stool samples during diarrhoeal episode in children with haematological malignancy. Methods: This observational study was conducted from April 2012 through March 2013 at BSMMU, Bangladesh. A total of 58 diarrhoeal episodes experienced by 51 children of various types of haematological malignancies were included in the study. Faecal samples from studied children were sent to Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), laboratory to do aerobic culture for common bacteria; Enzyme Immunoassay (EIA) for Clostridium difficile and multiplex real-time polymerase chain reaction (PCR) for parasitic infection. Results: Among the total 58 diarrhoeal episodes, potential pathogenic bacteria were isolated from only 5.17% sample. Faecal samples from 22.41% episodes were found positive for GDH antigen for Clostridium difficile; none of the focal samples were positive for toxin A and/ or B. Several different parasites were identified from 70.37% samples and most frequently identified protozoa was Giardia lamblia in 68.52%. Conclusion: The study found 22.41% colonization rate with Clostridium difficile but none was toxigenic. Parasitic infections were seen more frequently in children with haematological malignancy. The study also found significant association of severe neutropenia with GDH positive diarrhoeal episodes.

Key words: Enteropathogen, Diarrhoea, Children, Haematological malignancy.

Introduction

Diarrhoea is a frequent complication in children with cancer.¹ There are a number of potential causes of diarrhoea in children receiving treatment for malignant disease. In addition to bacterial, fungal, viral and parasitic infection, chemotherapy can cause small bowel mucosal damage resulting in diarrhoea.² So diarrhoea is not accepted as normal symptom of cancer chemotherapy and that stool specimens are sent for full bacteriological and viral investigations including examination for presence of Clostridium difficile and its toxin.²^,³ Whereas health-care associated infections (HAI) in paediatric cancer patients are considered to be important adverse outcome in terms of morbidity and mortality, postponing chemotherapy cycles, prolonged duration of hospital stay and additional costs are among other effects adversely affected by infections.⁴

Haematological neoplasm constitute more than 40% of malignancy in children and represent a wide range of disorders that include acute and chronic leukaemias, lymphomas and histiocytic disorders.⁵ In Paediatric Haematology and Oncology department of Bangbandhu Sheikh Mujib Medical University (BSMMU); among a total of 455 newly diagnosed childhood malignancy (January 2012 to December 2012) - haematological malignancies comprise 82% (ALL- 58%, NHL-11%, AML-10%) and solid tumours 18%.⁶

The problem of febrile neutropenia is frequently faced in the paediatric haematology and oncology department of BSMMU and infection related complication represent an important cause of morbidity and mortality in our children especially in those receiving chemotherapy. Diarrhoea is a common complication in neutropenic cancer children.⁷ Previous study from our centre also found that gastro-intestinal infections were the leading causes of infection in paediatric oncology patients of BSMMU.⁸ Environmental risk factors as well as factors related to malignancy make the children in BSMMU oncology department a vulnerable group to acquire infection.

With the above background the study was conducted in the Paediatric Haematology and Oncology department of BSMMU over one-year period to find out type of the pathogens in stool samples during diarrhoeal episode in children with haematological malignancy.

Materials and Methods

This observational study was conducted at Paediatric Haematology & Oncology Department of BSMMU, Dhaka, from April 2012 to March 2013. Children, 1 to 15 years of age of both sexes with diagnosis of various types of haematological malignancies were consecutively included if they developed diarrhoea at any point during hospitalization. Diarrhoea is defined as an alteration in normal bowel pattern with the passage of at least three loose or watery stools within a 24-hour period. Children with other malignancy were not included in the study.

Data collection procedure: A total of 51 children with haematological malignancy were included in the study as per inclusion criteria. Informed consent was taken from legal guardians of individual cases. Data collection sheet was used to collect information.

Microbiological Evaluation: After primary selection of cases, stool sample were collected into closed and labelled stool containers having specific identification number and sent to the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratory for aerobic bacterial culture and Multiplex real time PCR to find out common parasitic infection. Fresh stool samples were also sent in a separate sterile container for Clostridium difficile toxin and cell wall-associated enzyme glutamate dehydrogenase (GDH) antigen identification by Enzyme Immunoassay (EIA) test (C.DIFFICILE TOX A/B II^TM and C.DIFF CHEK^TM-60 test respectively). Stool microscopy for red and white blood cells, parasitic cysts and ova examination was done within 48 hours of enrolment to exclude other infectious causes of diarrhoea. But test for virus and fungi could not be done.

Data Management: After collection of data, editing and compilation was done manually. Statistical analysis was done both manually also by using window-based software devised with Statistical Packages for Social Sciences (SPSS).

Ethical Consideration: Prior to the commencement of this study, the protocol was approved by the Institutional Review Board of BSMMU, Dhaka. The informed consent was taken from each patient’s legal guardians. Every precaution was taken so that study causes no harm or delay for treatment of cases. Anonymity of patients was maintained by coding of results. Stool samples were preserved in ICDDRB, lab for two years for further analysis if needed.

Results

During a 12-month period from April 2012 through March 2013, a total of 58 diarrhoeal episodes experienced by 51 paediatric cancer patients at BSMMU, Dhaka were studied. Age range was 13 months to 15 years with mean age 5.9+3.58. Majority of diarrhoeal episodes 32 (55.17%) were from age group 1-5 years, followed by 5-9 years age group 16 (27.60%). Among studied population, male were 32 (62.75%) and female were 19 (37.25%) with M:F =1.68:1. Acute lymphoblastic leukaemia was the most common (62.75%) haematological malignancy in the studied children. Non-Hodgkin lymphoma was 29.41% and AML was 7.84 %. (Table I)

Table I: Patients characteristics (n: 51 & Total Diarrhoeal episodes n: 58)

Characteristics		n (%)	Mean	±SD	P-value
Age			5.9	±3.58
Sex of study patients; Ratio (M:F) is 1.68:1
	Male	32 (62.75)
	Female	19 (37.25)
Diagnosis study patients
	AML	4 (7.84)
	ALL	32 (62.75)
	NHL	15 (29.41)

n=Number, SD= standard deviation, AML= Acute Myeloid leukaemia, ALL=Acute lymphoblastic leukaemia, NHL= Non-Hodgkin lymphoma

The length of hospital stay of children before diarrhoeal episode was 0 to 43 days with mean duration 7+9 days. Total Thirty-one (n=31; 53.45%) diarrhoeal episodes of studied children had history of hospital stay of >72 hours and 27 samples (46.55%) of other children had hospital stay of <72hours. (Table II)

Table II: Hospital stay in days in each episode of diarrhoea (Total episodes:58)

Characteristics		n (%)	Mean	±SD	P-value
	<72 hours	27 (46.55)
	>72 hours	31 (53.45)
	Total	58 (100.00)	7	±9

n=Number, SD= standard deviation

Regarding blood parameter, majority of the children (81.03%) were neutropenic (n=47 episode out of 58) with Absolute Neutrophils Count (ANC) count < 0.5 X10⁹/L of blood at the time of enrolment for diarrhoeal episode. (Table III)

Table III: Complete blood count (CBC) in each episode of diarrhoea (n:58)

Characteristics	n (%)	Mean	±SD	P-value

Haemoglobin in gm/dl	GDH Positive	8.21	±1.96	0.790^ns
Haemoglobin in gm/dl	GDH Negative	8.81	±1.97	0.790^ns
Total count of WBC X 10⁹/L	GDH Positive	0.61	±0.47	0.023^s
Total count of WBC X 10⁹/L	GDH Negative	1.52	±2.04	0.023^s
Actual Neutrophils Count X 10⁹/L	GDH Positive	0.11	±0.14	0.005^s
Actual Neutrophils Count X 10⁹/L	GDH Negative	0.76	±1.44	0.005^s
Platelets Count X 10⁹/L	GDH Positive	42	±31	0.058^ns
Platelets Count X 10⁹/L	GDH Negative	82	±87	0.058^ns

WBC= White blood cells, GDH= glutamate dehydrogenase antigen, P-value extracted from independent sample student t-test.

In the study, 22.41% of focal samples were found (n=13/58) positive for GDH antigen by C.DIFF CHEK^TM-60 TEST (Table I). But C.DIFFICILE TOX A/B II^TM test used for detection of both toxins A and /B by enzyme immunoassay (EIA) showed negativity for all faecal samples. (Table IV)

Table IV: Results of EIA to detect GDH-antigen and toxins of Clostridium difficile from focal samples of each episode. (Total episode: 58)

Type of malignancy	Clostridium difficile			Total (%)
Type of malignancy	GDH Positive; n (%)	GDH Negative; n (%)	TOX A/B Positive; n (%)	Total (%)
AML	02 (3.45)	02 (3.45)	00	04 (6.90)
ALL	08 (13.79)	29 (50.00)	00	37 (63.79)
NHL	03 (5.17)	14 (24.14)	00	17 (29.31)
Total (%)	13 (22.41)	45 (77.59)	00	58 (100)

n=Number, AML= Acute Myeloid leukaemia, ALL=Acute lymphoblastic leukaemia, NHL= Non-Hodgkin lymphoma, GDH= glutamate dehydrogenase antigen, TOX A/B= toxins A and/B.

Potential pathogenic bacteria were isolated from 3 focal sample, Campylobacter jejuni in 2 sample (3.45) and Salmonella in 1 sample (1.72) by aerobic stool culture. Several different parasites were identified from 70.37% focal samples by multi-plex real time PCR. No parasite was found in 29.63% focal samples. The most frequently identified protozoa were Giardia lamblia (68.52%), then Entamoeba histolytica (12.96%) and Cryptosporidium (5.56%). Giardia was identified as a single agent in 29 (53.70%) samples out of 54 samples. All three parasites were simultaneously identified from one sample. Presence of leucopenia and severe neutropenia were significantly associated with Clostridium difficile GDH positive samples. (Table V)

Table V: Microbial evaluation of stool samples of studied children in addition to Clostridium difficile. (n:58)

Identified pathogen	GDH +ve n (%)	GDH –ve n (%)	Total n (%)
Bacteria by aerobic stool culture
Salmonella	01 (1.72)	00 (00)	01 (1.72)
Shigella	00 (00)	00 (00)	00 (00)
C. Jejuni	02 (3.45)	00 (00)	02 (3.45)
V. cholerae	00 (00)	00 (00)	00 (00)
E. coli	00 (00)	00 (00)	00 (00)
*Total*	03 (5.17)	00 (00)	03 (5.17)
Total Parasites by Multiplex real time PCR (n = 54)*
E. histolytica	01 (1.85)	6 (11.11)	7 (12.96)
G. lamblia	6 (11.11)	31 (57.41)	37 (68.52)
Cryptosporidium	00 (00)	03 (5.56)	03 (5.56)
Multiple Parasites found by Multiplex real time PCR (n = 54)*
E. histolytica + G. lamblia	01 (1.85)	5 (9.26)	6 (11.11)
Cryptosporidium + G. lamblia	00 (00)	1 (1.85)	1 (1.85)
E. histolytica + G. lamblia + Cryptosporidium	00 (00)	1 (1.85)	1 (1.85)
**Total Parasite found by Multiplex RT- PCR**	6 (11.11)	32 (59.26)	38 (70.37)

*In 4 cases multiplex real time PCR not done. n=Number, GDH= glutamate dehydrogenase antigen, RT- PCR= Real time polymerase chain reaction.

Discussion

Haematological malignancies are the most common cancers affecting the paediatric cancer patients. Diarrhoeal episodes occur frequently among these patients as a complication of disease itself or consequences of treatment related complications. In fact, diarrhoea is a frequent complication in children with cancer while neutropenic.

The findings of GDH positivity in children with diarrhoea but without findings of toxin in GDH positive sample denotes that children have colonization or are carriers of C. difficile and diarrhoea may be due to other cause. Though there is some explanation that failure to detect toxins may be due to host antibody binding of toxins or low in vivo toxin levels from lack of toxin production, a low bacterial burden of C. difficile at the time of testing, or a relative predominance of spores versus vegetative cells.⁹

The carrier rate of C. difficile in hospitalized children and adults approximate 20% by Cohen et al.¹⁰ Armin et al. showed 25% rate of colonization with Clostridium difficile in children with cancer.¹¹ Some other studies however, indicate that prevalence of asymptomatic colonization may be more on the order of 20-50% in facilities where C. difficile infection is endemic.¹² The C. difficile colonization rate of 22.41% in our study at BSMMU, Bangladesh, is closely similar to above mentioned studies. In 77.59% of diarrhoeal episodes, we found no relation with Clostridium difficile or its toxins, which is consistent with the findings of Wolfhagen et al., who also found no relation with Clostridium difficile or its toxin in 75% of the period with diarrhoea in immunocompromised children.¹³

In our study we have found that most (94.83%) focal samples did not show any common bacterial growth. Potential pathogenic bacteria were isolated from only 5.17% sample by aerobic stool culture. The findings of bacterial growth of 5.17% from stool culture among paediatric cancer is much lower than other studies like EL Mahalawaya et al (2004) study conducted at National Cancer Institute, Cairo University where bacterial pathogens were the commonest causes (39.4%) encountered – with C. difficile detected in 14.4% cases.¹⁴ In other study stool culture for bacteria was positive in 40.5% cases of diarrhoea in neutropenic children with cancer.⁷ The lower incidence of bacterial growth in our study may be due to the fact that our studied children were getting antibiotics empirically for febrile neutropenia in some cases and in some cases diarrhoea were might be due to causes other than bacteria.

The organism commonly reported to cause nosocomial diarrhoea in less developed areas of the world include salmonella species, Shigella species, Vibrio cholera, enteropathogenic E.coli, and Campylobacter species.¹⁵^-¹⁷ The present study showed isolated organism similar with Albert et al (1999) study.¹⁷Several different parasites were identified from 70.37% of focal samples of our study samples by Multiplex real time PCR which is more sensitive than microscopy for identification of parasite specific-DNA.¹⁸^,¹⁹ This result is higher than the study of Umit A et al (2003) where parasite identified in 42.0% of children with malignancy.²⁰

In the present study, most frequently identified protozoan was Giardia lamblia (n:37; 68.52%), followed by Entamoeba histolytica (n:7; 12.96%) and Cryptosporidium (n:3; 5.56%). The incidence of cryptosporidium (5.56%) match with the study of Umit et al (2003) where Cryptosporidium were found in 2 (5.2%) of the 38 patients of haematological malignancy.²⁰Multiple co-existing organisms were also found in some episodes (3/58) of the present studied children, similar findings were also observed by EL-Mahallawy et al (2004).¹⁴ So parasitic infestations should be considered in the diagnostic work-up of diarrhoea in our settings as immunosuppressed patients are at increased risk of serious complications associated with some parasitic disease. In children with malignancy intestinal parasitic infections can follow a severe course, which might in some cases result in death.²¹

Diarrhoea is a frequent complication in children with cancer while neutropenic, as observed by Sherief et al (2012). They found 55.5% diarrhoeal episode in neutropenic children; another study found 18.6% episodes of diarrhoea while following the neutropenic adult cancer patients.⁷^,²² We have found higher incidence of neutropenia in 81.03% cases of children with diarrhoeal episode. The present study found significant association of severe neutropenia and C. difficile colonization among paediatric cancer children. All the GDH positive sample of studied children was severely neutropenic.

Conclusion

The present study found colonization rate of C. dificile 22.41% but none was toxigenic. The findings of GDH positivity in children with diarrhoea but without findings of toxin in GDH positive sample denotes that children are carriers of C. difficile and diarrhoea may be due to other cause. Parasitic infections are seen more frequently in children with malignancy. The possibility of a parasitic infection should always be considered when making a clinical assessment of children with immune-suppression associated with haematological malignancy. Small sample size and single centre study were the limitations of the study. Also tests for viral and fungal causes could not be done in this study.

Authors’ disclosures of potential conflicts of interest

The author(s) indicated no potential conﬂicts of interest and this research work did not involve any financial support from any funding agencies.

Acknowledgement

Infectious Disease Division of ICDDR,B, Dhaka, provided laboratory supports for conducting this study.

References

1. Madani TA. Clinical Infections And Bloodstream Isolates Associated with Fever in Patients Undergoing Chemotherapy for Acute Myeloid Leukaemia. Infection. 2000; 28(6):367-74.

2. Brunetto AL, Pearson AD, Craft AW, Pedler SJ. Clostridium Difficile in an Oncology Unit. Archives of Disease in Childhood. 1988; 63(8):979-81.

3. Schuller I, Saha V, Lin L, Kingston J, Eden T, Tabaqchali S. Investigation and Management of Clostridium Difficile Colonisation in A Paediatric Oncology Unit. Archives of Disease in Childhood. 1995;72(3):219-22.

4. Coffin SE, Zaoutis TE. Infection Control, Hospital Epidemiology and Patient Safety. Infectious Disease Clinics of North America. 2005; 19(3):647-65.

5. Heerema-Mckenney AMY, Cleary M L, Arber, D A. Pathology and Molecular Diagnosis of Leukemias and Lymphomas. In: Pizzo, P A, Poplack, D G edditors. Principles and Practice of Paediatric Oncology. 6^th ed. Philadelphia: Wolters Liuwer-Lippincott-Williams & Wilkins; 2011. p-138.

6. Islam A, Jamal CY, Nahar K, Siddique R, Begum F, Begum M, Yasmin F, Ara Z, Rahman AA, Khaleque A, Hafiz G. Paediatric Oncology Data Based Network (POND) Registry Initiated In Bangladesh. Paediatric Blood & Cancer. 2013; 60:144-5.

7. Sherief LM, Beshir MR, Kamal NM, Gohar MK, Gohar GK. Diarrhoea in Neutropenic Children with Cancer: An Egyptian Centre Experience with Emphasis on Neutropenic Enterocolitis. Indian Journal of Medical and Paediatric Oncology. 2012; 33(2):95.

8. Rahman ATM, Mannan MA, Islam A, Jamal CY, Karim MA, Begum H. Outcome of Two Empirical Antibiotic Regime in the Management of Febrile Neutropenia in Children with Cancer, DS (Child) HJ. 2006; 22(2):49-53.

9. Delmée M, Van Broeck J, Simon A, Janssens M, Avesani V. Laboratory Diagnosis of Clostridium Difficile-Associated Diarrhoea: A Plea for Culture. Journal of Medical Microbiology. 2005; 54(2):187-91.

10. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, Pepin J, Wilcox MH. Clinical Practice Guidelines for Clostridium Difficile Infection in Adults: 2010 Update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infection Control & Hospital Epidemiology. 2010; 31(5):431-55.

11. Armin S, Shamsian S, Drakhashanfar H. Colonization with Clostridium Difficile in Children with Cancer. Indian Journal of Pediatrics. 2013; 23(4):473-6

12. Riggs MM, Sethi AK, Zabarsky TF, Eckstein EC, Jump RL, Donskey CJ. Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents. Clinical infectious diseases. 2007; 45(8):992-8.

13. Wolfhagen MJ, Meijer K, Fluit AC, Torensma R, Bruinsma RA, Fleer A, Verhoef J. Clinical Significance of Clostridium Difficile and Its Toxins in Faeces of Immunocompromised Children. Gut. 1994; 35(11):1608-12.

14. El‐Mahallawy HA, El‐Din NH, Salah F, El‐Arousy M, El‐Naga SA. Epidemiologic Profile of Symptomatic Gastroenteritis in Paediatric Oncology Patients Receiving Chemotherapy. Paediatric Blood & Cancer. 2004; 42(4):338-42.

15. Paton S, Nicolle L, Mwongera M, Kabiru P, Mirza N, Plummer F, Wamola I. Salmonella and Shigella Gastroenteritis at a Public Teaching Hospital in Nairobi, Kenya. Infection Control & Hospital Epidemiology. 1991; 12(12):710-7.

16. Hernández JE, Mejía CR, Cazali IL, Arathoon EG. Nosocomial Infection due to Vibrio Cholerae in Two Referral Hospitals in Guatemala. Infection Control & Hospital Epidemiology. 1996; 17(6):371-2.

17. Albert MJ, Faruque AS, Faruque SM, Sack RB, Mahalanabis D. Case-Control Study of Enteropathogens associated with Childhood Diarrhoea in Dhaka, Bangladesh. Journal of Clinical Microbiology. 1999; 37(11):3458-64.

18. Haque R, Ali IK, Akther S, Petri WA. Comparison of PCR, Isoenzyme Analysis and Antigen Detection for Diagnosis of Entamoeba Histolytica Infection. Journal of Clinical Microbiology. 1998; 36(2):449-52.

19. Roy S, Kabir M, Mondal D, Ali IK, Petri WA, Haque R. Real-Time-PCR Assay for Diagnosis of Entamoeba Histolytica Infection. Journal of Clinical Microbiology. 2005; 43(5):2168-72.

20. Aksoy U, Erbay A, Akisu C, Apa H, Ozkoc S, Ozturk S. Intestinal Parasites in Children with Neoplasms. The Turkish Journal of Paediatrics. 2003; 45(2):129-132.

21. Clark DP. New Insights into Human Cryptosporidiosis. Clinical Microbiology Reviews. 1999; 12(4):554-63.

22. Aksoy DY, Tanriover MD, Uzun O, Zarakolu P, Ercis S, Ergüven S, Oto A, Kerimoglu U, Hayran M, Abbasoglu O. Diarrhoea in Neutropenic Patients: A Prospective Cohort Study with Emphasis on Neutropenic Enterocolitis. Annals of Oncology. 2006; 18(1):183-9.