|Year : 2020 | Volume
| Issue : 1 | Page : 32-36
Prevalence of campylobacter enteritis in children under 5 years hospitalised for diarrhoea in two cities of Northeast India
Biswajyoti Borkakoty1, Aniruddha Jakharia1, Mandakini Das Sarmah1, Rahul Hazarika1, Pranjal Jyoti Baruah1, Chandra Jyoti Bora2, T Temsu3, Munmun Gohain2, Utpala Devi1, Dipankar Biswas1
1 Indian Council of Medical Research-Regional Medical Research Centre, NE Region, Dibrugarh, Assam, India
2 Department of Paediatrics, Assam Medical College and Hospital, Dibrugarh, Assam, India
3 Department of Pathology, District Hospital, Dimapur, Nagaland, India
|Date of Submission||31-Dec-2019|
|Date of Decision||27-Mar-2020|
|Date of Acceptance||27-Apr-2020|
|Date of Web Publication||25-Jul-2020|
Dr. Biswajyoti Borkakoty
Indian Council of Medical Research-Regional Medical Research Centre, NE Region, Dibrugarh - 786 010, Assam
Source of Support: None, Conflict of Interest: None
Background: Campylobacter enteritis is the major cause of bacterial gastroenteritis worldwide. In recent years, there has been a rise in global incidence of campylobacteriosis. There are no available data on prevalence of Campylobacter diarrhoea from Northeast India. Materials and Methods: The study investigated archival stool samples collected between 2014 and 2016 from two hospitals of Northeast India. A total of 407 archival stool samples from cases of diarrhoea under 5 years of age were screened for Campylobacter spp. using commercial probe-based real-time polymerase chain reaction assay. Results: Campylobacter spp. was detected in overall 10.1% (41/407; 95% confidence interval: 7.4%–13.3%) in children under 5 years hospitalised for diarrhoea. The prevalence was significantly higher from Dibrugarh, Assam, compared to Dimapur, i.e., 13.4% (27/201) versus 6.8% (14/206), respectively (P = 0.02). Campylobacter detection was highest in the month of June and July compared to December and January (20%–18.8% vs. 8.9%–6.2%, respectively). Further, Campylobacter infection was higher in the age group below 24 months (11.7%) compared to above 24 months (7.0%). Campylobacter jejuni was detected in 80.5% of the positive cases. Conclusion: The present study reveals that Campylobacter infection is endemic in the studied regions of Northeast India and microbiological laboratories of the region should actively pursue the isolation or detection of Campylobacter spp. in cases of diarrhoea in routine stool cultures.
Keywords: Campylobacter jejuni, Campylobacter, gastroenteritis, Northeast India, U5
|How to cite this article:|
Borkakoty B, Jakharia A, Sarmah MD, Hazarika R, Baruah PJ, Bora CJ, Temsu T, Gohain M, Devi U, Biswas D. Prevalence of campylobacter enteritis in children under 5 years hospitalised for diarrhoea in two cities of Northeast India. Indian J Med Microbiol 2020;38:32-6
|How to cite this URL:|
Borkakoty B, Jakharia A, Sarmah MD, Hazarika R, Baruah PJ, Bora CJ, Temsu T, Gohain M, Devi U, Biswas D. Prevalence of campylobacter enteritis in children under 5 years hospitalised for diarrhoea in two cities of Northeast India. Indian J Med Microbiol [serial online] 2020 [cited 2020 Aug 11];38:32-6. Available from: http://www.ijmm.org/text.asp?2020/38/1/32/290681
| ~ Introduction|| |
Campylobacter infection is the leading cause of acute bacterial gastroenteritis worldwide. It is estimated that Campylobacter species are responsible for over 172 million episodes of diarrhoea annually worldwide and account for over 75,000 deaths due to diarrhoea as per the Global Burden of Disease Study 2016.Campylobacter infection with an incubation period of 2–4 days is characterised by diarrhoea (frequently bloody), abdominal pain, fever and occasionally nausea and vomiting.Campylobacter generally is transmitted to humans mainly through the consumption of contaminated or undercooked poultry, unpasteurised dairy products, contaminated water or from contact with faeces of a dog or cat, whereas person-to-person spread of Campylobacter is infrequent. The disease frequently occurs as sporadic cases, but it can also occur in outbreaks, which are usually associated with unpasteurised milk or contaminated water. Campylobacteriosis is considered to be self-limiting disease, however, timely use of antimicrobials can reduce the duration of illness. In developing countries, it is estimated that Campylobacter isolation rates for food-borne illness are between 5% and 20%. Although campylobacteriosis can occur in all age groups, it affects predominately young children compared to other age groups. Further, complications such as bacteraemia can arise due to an inadequate therapy, and sequelae in the form of Guillain–Barré syndrome (the virulent form may lead to ascending paralysis which can rapidly lead to respiratory paralysis and death) are estimated to occur in 1 out of every 1000 reported Campylobacter gastroenteritis.
Campylobacter genus with 26 species and 9 subspecies is a Gram-negative spiral, rod-shaped or curved microaerophilic bacillus with a single polar flagellum, bipolar flagella or no flagellum, depending on the species. They belong to a distinct group of specialised bacteria designated under rRNA superfamily Class VI of proteobacteria, family Campylobacteraceae and genus Campylobacter.Campylobacter is a fastidious organism, uses menaquinones as their respiratory quinones, does not ferment or oxidise carbohydrates and requires microaerophilic environment (5%O2, 10% CO2 and 85% N2) for growth, and isolates causing human gastroenteritis are primarily of the thermotolerant variety which can also grow at 42°C–43°C., Charcoal-based selective media, modified charcoal cefoperazone deoxycholate agar and Skirrow's medium are effective for isolating Campylobacter spp. from clinical specimens. The requirement of a microaerophilic condition, i.e., 5%–10% O2, and requirement of selective media limit many microbiology diagnostic laboratories in resource-limited settings from regularly screening for Campylobacter spp. in diarrhoeal stools. However, cost-effective culture and isolation comparable to use with selective media have been found with the use of blood agar media using candle jar for creating microaerophilic conditions. Over the past few decades, culture-independent-based diagnostics, i.e., nucleic acid test, especially qualitative polymerase chain reaction (qPCR) performed directly from diarrhoeal stools, have provided a rapid and a highly sensitive method of diagnosis in laboratories with molecular diagnostic facilities.
Although Campylobacter enteritis is a major public health problem in both developed and developing countries, there is still paucity of data related to the prevalence and burden of Campylobacter diarrhoea across vast regions of India. Moreover, there are no available data on Campylobacter diarrhoea from north-eastern states of India. This study was undertaken to get baseline data on the prevalence of Campylobacter infections in under-five children (U5) hospitalised for acute gastroenteritis in two major hospitals of Northeast India.
| ~ Materials and Methods|| |
This study was approved by the Institutional Ethics Committee of the ICMR-Regional Medical Research Centre for NE region, Dibrugarh, in 2017. The study was performed on leftover diarrhoeal stool samples collected from January 2014 to December 2016 for National Hospital-based Rotavirus Surveillance Network conducted in two hospitals of Northeast India (Assam Medical College and Hospital, Dibrugarh, Assam, and District Hospital, Dimapur, Nagaland) as a part of an all India Rotavirus Surveillance Network. Informed and written consent for the use of leftover stool samples for future research was taken at the time of sample collections. Briefly, 0–59 months (U5) old with acute gastroenteritis (≥3 unformed stools in any 24 h) requiring inpatient treatment for clinical management and parents consenting for written and informed consent were recruited. A total of 1407 processed diarrhoeal stools stored in −80°C deep freezer were systematically randomised to screen a representative 407 samples.
For screening of Campylobacter spp., a commercial CE-IVD approved probe-based quantitative reverse transcription PCR (qRT-PCR) kit panel for bacterial gastroenteritis covering six bacterial pathogens (Campylobacter jejuni/coli/lari, Clostridium difficile, enterohaemorrhagic Escherichia coli, Salmonella spp., Shigella spp./enteroinvasive E. coli and Yersinia enterocolitica) was used (FTD Bacterial gastroenteritis kit, Fast-Track Diagnostics, Luxembourg). In brief, total nucleic acid was extracted from processed stool samples using a commercial spin column-based nucleic acid extraction kit (RTP Pathogen Kit, Stratec Molecular, Berlin, Germany) based on manufacturer's instruction. Extracted nucleic acids were subjected to multiplex qRT-PCR reactions in two wells per sample including internal controls, and detection wavelength was set at 520, 550, 610 and 670 nm as per manufacturer's instructions in an ABI 7500 Real-Time PCR machine. As the test detects the targeted Campylobacter spp.(C. jejuni/coli/lari) simultaneously at single wavelength (670 nm), species discrimination could not be performed. However, to identify C. jejuni amongst the screened samples, a conventional PCR was performed targeting the amidohydrolase (hippuricase) gene. In brief, conventional PCR was performed in a total reaction volume of 25 μl using 2x PCR Master Mix (Promega, Madison, USA), 5 μl of template and 0.2 μM each of reverse and forward primers (hipO-R 5'-GACTTCGTGCAGATATGGATGCTT and hipO-F (5'-GCTATAACTATCCGAAGAAGCCATCA) based on the method described by Persson and Olsen. Primers amplified a 344-bp fragment of the hipO gene characteristic of C. jejuni. Amplified products were visualised using 2.5% agarose gel in a gel documentation imager (myECL imager, Thermo Fisher Scientific).
Data were entered into a statistical statistical software (IBM SPSS statistics version 26, IBM, Armonk, New York, USA), and associations between clinical variables including number of diarrhoeal episodes, fever, abdominal pain, vomiting, age and gender of the participants were examined with Campylobacter enteritis. All variables were examined by univariate analysis and odds ratios, and their 95 per cent confidence intervals were documented to indicate magnitude and direction of associations. Statistical significance level was set at P ≤ 0.05.
| ~ Results|| |
A total of 407 under 5 years (U5) hospitalised children (age range of 3–59 months) with acute gastroenteritis from two hospitals of NE India, i.e., Assam Medical College, Dibrugarh (201 cases), and District Hospital, Dimapur, Nagaland (206 cases), were screened for Campylobacter diarrhoea. The median age of participants was 20 months (standard deviation ± 11.7) with an interquartile range of 12–29 months. Males constituted 61.7% of the hospitalised diarrhoea cases.
The overall point prevalence of Campylobacter spp. (C. jejuni/coli or lari) detected in the U5 hospitalised cases of diarrhoea was 10.1% (41/407; 95% confidence interval [CI]: 7.4%–13.3%), with U5 children from Assam recording a significantly higher prevalence compared to Dimapur, i.e., 13.4% (27/201) versus 6.8% (14/206), respectively (P = 0.02). Although the overall admitted cases of diarrhoea were less for the female child, the prevalence of Campylobacter was slightly higher amongst the female child compared to males, i.e., 12.8% (20/156) versus 8.4% (21/251) respectively (P = 0.14). There was no statistically significant trend in distribution of cases of Campylobacter across seasonality covering 2 years. However, it was highest in the month of June and July compared to December and January (20%–18.8% vs. 8.9%–6.2%, respectively).
The prevalence of Campylobacter diarrhoea was higher in children up to 24 months (11.7%; 31/265) compared to 7.0% (10/142) above 24 months in the U5 hospitalised cases, but it was insignificant statistically (P = 0.13). Further, no statistical significance was found with severity of diarrhoea with Campylobacter infections, though the prevalence of Campylobacter infection was slightly higher (11.3% vs. 8.7%) amongst participants who had reported more than 6 episodes of loose motions compared to 6 or less in any 24 h (P = 0.38). Further, it was seen that U5 children with restlessness had a lower prevalence of Campylobacter compared to those children which did not have (2.9% vs. 11.5%, respectively, P = 0.03) [Table 1]. Further, amongst the 41 positive cases detected by real-time probe-based PCR method [Figure 1]a, C. jejuni was detected in 80.5% (33/41) based on conventional PCR specific for detection of C. jejuni [Figure 1]b. The other bacterial pathogens detected amongst the 407 diarrhoea cases screened were C. difficile detected in 1/407 (0.25%), enterohaemorrhagic E. coli was detected in 2/407 (0.5%), Salmonella spp. was detected in 6/407 (1.5%), Shigella spp./enteroinvasive E. coli was detected in 175/407 (43%) and Y. enterocolitica was detected in 1/407 (0.25%).
|Table 1: Distribution and association of Campylobacter diarrhoea with demographic and clinical variables (n=407)|
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|Figure 1: Laboratory diagnosis of Campylobacter enteritis. (a) The real-time polymerase chain reaction amplification curve based on TaqMan probe detection of Campylobacter spp. (b) Conventional polymerase chain reaction gel picture for identification Campylobacter jejuni showing a specific 344-bp fragment amplicon of hippuricase gene|
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| ~ Discussion|| |
Campylobacter species originally under Vibrio genus was assigned a new genus in 1963 following the renaming of Vibrio fetus to Campylobacter fetus. Since its initial description, the genus has grown to include important human and animal pathogens amongst which Campylobacter jejuni is one of the most important causes of gastroenteritis worldwide. Campylobacteriosis is a zoonotic disease most often transmitted from meat, poultry or dairy products contaminated with animal faeces and is one out of the four key causes of diarrhoeal disease worldwide. It is estimated that about 50%–70% of the strains associated with diarrhoea are attributed to poultry reservoirs. Although the main route of transmission is believed to be food-borne usually through undercooked meat, poultry products, raw milk or contaminated milk products or contaminated water, the transmission from household pets may also contribute. Pets such as cats and dogs can carry Campylobacter, and young children, especially under the age of 5 years, should not be allowed to kiss their pets or lick their hands after touching the pets. Based on a meta-analysis of 34 studies, it has been seen that the prevalence of campylobacter is high in petting household animals most commonly in dogs and cats (mean: 24.7%) compared to 6.7% in petting zoo animals (cattle, sheep, horses, swine, etc.). Apart from responsible for gastroenteritis, Campylobacter infection is associated with extraintestinal manifestations including bacteraemia, lung infections, reactive arthritis, meningitis, brain abscess, Barrett's oesophagus, colorectal cancer and inflammatory bowel disease in small cohort studies. Further, antecedent Campylobacter infection is considered the most common risk factor for development of Guillain–Barré syndrome (GBS) or demyelinating neuropathy. It is estimated that between 25% and 40% of GBS patients worldwide suffer from an antecedent Campylobacter diarrhoea 1–3 weeks before the illness. Globally, it is the most common cause of culture-proven bacterial gastroenteritis estimated to have caused 96 million food-borne cases of gastroenteritis in 2010. The predominant Campylobacter spp. responsible for 90% human infection is Campylobacter jejuni, with less common species, such as C. coli, Campylobacter upsaliensis, C. fetus and C. lari, being also implicated. Another important emerging Campylobacter spp. is Campylobacter concisus which has been implicated in human intestinal infections. In a study from Cape Town using the Cape Town protocol (membrane filtration technique), Campylobacter spp. were detected in 21.1% of the over 19,000 stools samples screened. Further, amongst the Campylobacter spp. detected, 23.5% were C. concisus. The sensitivity of detection by culture methods is low compared to PCR. A study by Platts-Mills et al. based on the detection of Campylobacter genus-specific 16S rRNA and CadF gene (specific for C. jejuni/C. coli) by TaqMan assay real-time PCR found that PCR had a high sensitivity and can distinguish Campylobacter at species level easily than culture-based methods.
The present study based on TaqMan assay real-time PCR, Campylobacter diarrhoea due to C. jejuni or C. lari or C. coli in under-five children hospitalised for gastroenteritis in two different hospitals in Northeast India was found to be between 6.8% and 13.4%. Furthermore, in the present study, it was found that C. jejuni (80.5%) was the predominant species detected in Campylobacter diarrhoea in the region. In a recent study from Bhubaneswar city, Campylobacter diarrhoea in acute diarrhoea cases in children aged 2–5 years was found to be much higher at 23% based on PCR detection methods. Another study from Kolkata based on culture isolation technique detected an isolation rate of 7% amongst hospitalised cases of diarrhoea with a slightly higher prevalence of 10% amongst U5 diarrhoea children, which had a similar prevalence with our present study. A study from North India (Chandigarh) detected a low prevalence (2.6%) Campylobacter diarrhoea in adult diarrhoea cases. A study from Vellore, South India, recorded a prevalence of 4.5% Campylobacter infection based on PCR methods amongst cases of diarrhoea in under 5 years. In the present study, it was seen that infection was higher amongst <2 years or less which is also recorded from other studies elsewhere including a study from Denmark which recorded a higher prevalence in toddlers than in other age groups. As per the WHO, in developing countries, Campylobacter infections are frequent in children under the age of 2 years, sometimes resulting in death. It is inferred that in developing countries where Campylobacter is endemic, symptomatic infection may be limited or common in childhood, suggesting development of protective immunity due to early or repeated exposure in childhood, and asymptomatic Campylobacter infections may be common in adults.,
There was limitation of the present study on performing antibiogram as the study was based on stored archival stool samples and detection was based on molecular probe-based technique. However, reports from elsewhere documents high level of resistance to fluroquinolones in recent years and macrolide antibiotics are still recommended for treating Campylobacter diarrhoea in severe, persistent or relapsing diarrhoea including in immunocompromised persons and pregnant women.,
| ~ Conclusion|| |
The present study from Northeast India documents the endemicity of Campylobacter enteritis in U5 cases of gastroenteritis in the region. This is the first documented report of Campylobacter diarrhoea from Northeast region of India. There is a need for microbiology laboratories of the region to upgrade capacity to detect Campylobacter spp in cases of diarrhoea in routine stool cultures. Campylobacteriosis is still one of the major infectious diseases, and along with its predisposition for causing serious extraintestinal sequels such as reactive arthritis and GBS, it will remain an important global disease for years to come.
The authors will like to acknowledge the National Rotavirus Surveillance Network for initial support in recruitment of the participants for the project.
Financial support and sponsorship
This study was supported by the Intramural fund of ICMR-RMRC, Dibrugarh, and Regional VRDL, Dibrugarh, Department of Health Research, New Delhi.
Conflicts of interest
There are no conflicts of interest.
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