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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 547-556
 

Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli


1 Department of Microbiology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong, Meghalaya, India
2 Department of Pediatrics, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong, Meghalaya, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Wihiwot Valarie Lyngdoh
Department of Microbiology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences, Shillong - 793 018, Meghalaya
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_277

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 ~ Abstract 

Introduction: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. Materials and Methods: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. Results: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed Conclusion: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.


Keywords: Diarrheagenic Escherichia coli, diarrheagenic, Escherichia coli, high-resolution melting, multiplex, polymerase chain reaction


How to cite this article:
Devi TS, Durairaj E, Lyngdoh WV, Duwarah SG, Khyriem AB, Lyngdoh CJ. Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli. Indian J Med Microbiol 2018;36:547-56

How to cite this URL:
Devi TS, Durairaj E, Lyngdoh WV, Duwarah SG, Khyriem AB, Lyngdoh CJ. Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli. Indian J Med Microbiol [serial online] 2018 [cited 2019 Jul 18];36:547-56. Available from: http://www.ijmm.org/text.asp?2018/36/4/547/254390



 ~ Introduction Top


Diarrhoeal disease is still a leading killer of children, although its toll has dropped by a third over the past decade worldwide due to the improvement in treatment particularly with oral rehydration therapy.[1],[2] According to the data estimated in 2015 by WHO and Maternal and Child Epidemiology Estimation Group, 1 out of 10 childhood deaths were due to diarrhoea, following pneumonia which accounted for 1 out of 4 deaths in children under 5 years of age, globally.[2],[3] This disease remains one of the leading causes of preventable deaths in developing countries, especially amongst children under 5 years of age.[4] Majority of these deaths (90%) occurred in Sub-Saharan Africa and South Asia. It has been estimated that the mean number of episodes of diarrhoea per year in children under 5 years of age from a developing region is 3.2, with the highest incidence (4.8 episodes), occurring during the first year of life, decreasing progressively to 1.4 episodes per year at 4 years of age. Furthermore, the highest age-mortality rate (8.5 children per 1000/year) occurred in children under 1 year of life.[5]

Amongst the various causes of diarrhoea, acute infectious diarrhoea is a major cause of morbidity and mortality worldwide, and it remains a major public health challenge principally in developing countries, especially in areas with poor hygiene and sanitation and with limited access to safe water.[4]

A wide assortment of organisms cause acute diarrhoea comprising viruses, bacteria and parasites.[2] Amongst the bacterial pathogens, diarrheagenic  Escherichia More Details coli (DEC) are the most frequently implicated agents in cases of epidemic and endemic diarrhoea worldwide.[6] It is the most important bacterial etiologic agent of childhood diarrhoea, with a higher incidence during the first 2 years of life.[7]

Despite the fact that E. coli are commensal bacteria which are known to be one of those predominant species of facultative anaerobes in human gut and usually harmless to the host, a group of pathogenic DEC have emerged and are responsible for diarrhoeal diseases in humans, especially amongst children under 5 years of age. They can cause debilitating and sometimes fatal diseases in this age group and are amongst the emerging enteropathogenic bacterial agents leading to major public health problem in developing countries like India.[8],[9]

The broad spectrum of pathogenic features and different clinical symptoms caused by DEC mirrors the presence of different subsets of virulence-associated genes in certain E. coli strains which are absent in commensal E. coli isolates.[10] These E. coli strains have acquired specific virulence factors through horizontal gene transfer which plays a major role in the evolution of different bacterial pathotypes and confer them the ability to adapt to new environments and make them capable of causing a broad range of infections amongst children and even in healthy adult individuals.[10],[11] Interestingly, most of these virulent factors which are frequently encoded on mobile genetic elements such as plasmids, bacteriophages and transposons, attribute to distinguish pathogenic E. coli from commensals.[10]

Based on the distinct epidemiological and clinical features, specific virulence determinants and association with certain serotypes, DEC has been categorised into six major categories: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) or verocytotoxin producing E. coli, enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli.[12]

Although DEC strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli.[7] Until the 1970s, serotyping was the only means of distinguishing DEC strains from those of normal flora, bacause no biochemical, microbiological or animal tests were available for their differentiation.[13] However, it is not sufficient to identify an E. coli strain as diarrheagenic based on the serotyping, as in most cases it does not correlate with the presence of virulence factors.[7]

Understanding the different pathogenic mechanisms and specific virulent factors present in DEC has led to the development of numerous molecular diagnostic techniques such as polymerase chain reaction (PCR) assays for rapid identification and categorisation of these E. coli strains.[9] Thus, the advent of PCR technology has greatly simplified the detection of DEC associated with diarrhoea.[7] Multiplex real-time PCR assay is now established to be a sensitive, specific and inexpensive technique for rapid detection of DEC pathotypes, which leads to rapid availability of results and helping clinicians make timely appropriate therapeutic decisions because some DEC pathotypes warrant antibiotic therapy in addition to oral rehydration therapy.[6],[7]

Considering the importance of DEC as an emerging diarrhoeal agent especially amongst the children and increase in antibiotic resistance reported in all the pathotypes, there is a need for a rapid, sensitive and inexpensive detection technique. Prompt diagnosis and treatment with the appropriate antibiotics when indicated has led to decrease in complications. Therefore, it is important to determine not only the prevalence of DEC as a cause of diarrhoea but also to study the antibiotic resistance patterns amongst the DEC strains.

In India, especially in the North-Eastern part, there is a paucity of information on the epidemiology of DEC pathogens and their association with diarrhoeal disease. This may be attributed to the lack of molecular diagnostic facilities as diagnosing DEC in the laboratories is not only confined to conventional methods but also requires molecular techniques for detecting the specific virulent genes. Hence, this study was conducted with an objective to study the prevalence of DEC from children using multiplex PCR. In the present study, we have standardised and evaluated the application of real-time multiplex PCR for detecting the virulent genes associated with DEC pathotypes.


 ~ Materials and Methods Top


Study design

This was a hospital-based prospective study conducted in the department of microbiology of a tertiary care centre over a period of 1 year (January to December, 2015).

Inclusion criteria for study participants

Case

Any paediatric patients (<18 years of age) with acute diarrhoea which was defined as an increase in fluidity, volume and number of stools passed relative to usual bowel habits of each individual within 24 h and lasting not longer than 14 days were enrolled in the study. Fever was defined as a temperature (Tm) of ≥37.5°C. If the parents or legal guardians accepted participation in the study, patients with acute diarrhoea attending the outpatient and inpatient department of paediatrics were enrolled in this study. Demographic information for each patient including age, sex and clinical symptoms were collected.

Control

Children (<18 years of age) with no history of diarrhoea for at least 1 month were included as controls.

Exclusion criteria

Children whose diarrhoea could be attributed to classic pathogens such as  Salmonella More Details spp/Shigella spp or gross infestation with parasites were excluded from the study. In addition, either cases or controls treated with antibiotics 1 week before the collection of stool samples were excluded.

Ethical approval

Ethical approval was obtained from the institution ethics committee.

Sample collection

Stool samples were obtained from 170 children with diarrhoea (cases) and 47 from children without diarrhoea (controls) and further processed and analysed for the detection of DEC pathotypes as follows.

Morphological and biochemical identification of Escherichia coli

Fresh stool sample from the participants was inoculated and streaked onto the surface of MacConkey agar (Himedia, India) for isolated colonies. Characteristic discrete lactose fermenting colonies produced after 24 h of incubation aerobically at 37°C were streaked onto fresh sterilised Nutrient agar (Himedia, India) and identified by conventional biochemical tests such as indole, methyl red, Voges–Proskauer, citrate and urease tests. The isolates that were positive to indole and methyl red tests but negative to Voges–Proskauer, citrate and urease tests were identified as E. coli.

Maintenance of isolates

Biochemically confirmed E. coli isolated from the stool samples were maintained in Trypticase Soy broth supplemented with 20% glycerol (Himedia, India) and nutrient agar slants (Himedia, India) for the investigation of the genes encoding pathogenicity of at molecular level. Confirmed E. coli strains harbouring the specific virulent genes for DEC were used as positive controls for this molecular study.

Molecular analysis for screening diarrheagenic Escherichia coli virulent genes

DNA extraction

DNA was extracted from an overnight pure culture of E. coli using QIAamp DNA Mini Kit (Qiagen, Germany). The DNA concentration and its purity were checked by measuring absorbance in spectrophotometer. DNA eluted in 200 μl of buffer solution was stored at −20°C for further analysis.

Primers

The specific primer nucleotide sequences of different DEC pathotypes designed by Sigma Aldrich, Bengaluru, based on the previously published sequences [14],[15] were used in this study. Details of nucleotide sequences of the specific primer pairs used and the predicted product sizes are listed in [Table 1].
Table 1: Primer sequences used in multiplex real-time polymerase chain reaction (type it high-resolution melting polymerase chain reaction) assay

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Multiplex real-time polymerase chain reaction with high-resolution melting technology

Multiplex real-time PCR was performed for detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-Resolution Melt analyser. The Rotor-Gene Q instrument provides 5plex high-resolution melting (HRM) platform having 5 channels – green, yellow, orange, red and crimson. HRM-curve analysis was done using Type-it HRM PCR master mix (Qiagen) containing EvaGreen fluorescent intercalating dye.

1 μl of each reconstituted primer (100 μM) was diluted in 9 μl of RNase free water to make 10 times dilution (10 μM). The concentration of the primers was checked by spectrophotometry.

First, each 1 μl of the extracted DNA from the positive control strains was subjected to the specific PCR conditions followed by HRM analysis to identify melting Tm of each amplicon. Then, multiplex real-time PCR assay 1 (Quadruplex) was standardised for the detection of elt and stlA for ETEC, ial for EIEC and eaeA for EPEC. Multiplex real-time PCR assay 2 (Triplex) was standardised for the detection of CVD432 for EAEC, hlyA for EHEC and bfpA for EPEC.

Multiplex real-time polymerase chain reaction assay 1 (Quadruplex)

It was done in a 25 μL reaction volume containing 12.5 μL of Type-it HRM PCR master mix (HotStarTaq ® Plus DNA Polymerase, Type-it HRM PCR Buffer [with EvaGreen ® dye], Q-Solution ®, dNTP mix [dATP, dCTP, dGTP and dTTP]), 1 μL of primer mix, 1 μl of DNA template and 10.5 μL of RNase free water (Qiagen, India) [Table 2]. Each assay was performed following the optimal cycling condition as shown in [Table 3]. Primer mix used in this assay consisted of each forward and reverse primers of elt and stlA for ETEC isolates, ial for EIEC, eaeA for EPEC. The DNA samples carrying the relevant virulence gene (s) served as positive controls in each reaction and sterile distilled water served as negative control.
Table 2: Polymerase chain reaction reaction mixture

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Table 3: Cycling conditions for polymerase chain reaction Assay 1 (quadruplex)

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Multiplex real-time polymerase chain reaction assay 2 (Triplex)

Multiplex PCR assay 2 was performed individually using each primer pair CVD432 for EAEC, hlyA for EHEC and bfpA for EPEC and other constituents as described above. The optimal cycling condition of this assay is shown in [Table 4].
Table 4: Cycling conditions for polymerase chain reaction Assay 2 (triplex)

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High-resolution melting-curve analysis

Melting curves were analysed using Rotor-Gene Q Series Software v2.3.1 (Build 49) interpreted according to the specific melting Tm of each target amplicon.

Statistical analysis

The data were collected and recorded using MS-Excel for Windows v2013®. The basic descriptive statistics, frequency charts graphs, trend analysis and related graphs were computed using the same. Summary statistics and analysis of significance were done using MedCalc ® v12.5.0 for Windows (MedCalc Software, Ostend, Belgium). The comparison of single and two proportions were done using Chi-square test and Fisher's exact test as applicable. The threshold for significance was considered at P < 0.05. The data analysis and generation of graphs for the PCR was performed using Rotor-Gene Q Series Software v2.3.1 (Build 49, Qiagen, Germany).


 ~ Results Top


The present study was conducted to study the prevalence of DEC, their associated virulent genes and antibiotic resistance pattern amongst the paediatric age group (<18 years) with and without diarrhoea.

During the study period of 1 year (January 2015 to December 2015), 170 children with diarrhoea (cases) and 47 children without diarrhoea (controls) were included in this study. A total of 217 non-duplicated biochemically confirmed E. coli isolates were obtained from the stool samples of these children.

The demographic profile of 217 study participants is presented as follows:

The mean age of participants with diarrhoea was 6.59 (±5.22) and that of the controls was 7.87 (±4.82). The two groups were comparable in terms of age (P = 0.1320). The gender distribution was also comparable (P = 0.6810); 60% of the participants with diarrhoea were male and amongst the controls 55.31% were male.

Multiplex real-time polymerase chain reaction assay with high-resolution melting analysis

A total of 217 non-duplicated biochemically confirmed E. coli isolates obtained from the stool samples of children were subjected to two multiplex real-time PCR assays in Rotor-Gene Q instrument (Qiagen) having High-Resolution Melt analyser. Out of the 217 E. coli isolates, 42 DEC were detected based on the specific melting Tm of the amplicons observed in HRM analysis curve. In this study, the predicted Tm values of all target genes were: elt: 85.8 ± 0.18°C, stla: 88.0 ± 0.15°C, ial: 84.5 ± 0.15, eaeA: 82.2 ± 0.06°C, bfpA- 93.40 ± 0.15°C, CVD 432–91.2 ± 0.08°C, hlyA 89.8 ± 0.02°C. Melting peaks were automatically calculated by Rotor-Gene Q Series Software 2.3.1 (Build 49).

Multiplex real-time polymerase chain reaction with high-resolution melting analysis graphs

Amplification and HRM analysis graphs of one set of samples are shown in the following figures. A triplex PCR amplification assay is shown in [Figure 1], where Sample 14 and Sample 19 had shown amplification. In [Figure 2], two distinct separate melting peaks for Sample 14 and Sample 19 are shown at 93.4°C and 91.2°C which correspond to the predicted melting Tm for bfpA and CVD432 DNA templates, respectively. A Quadruplex PCR amplification assay is shown in [Figure 3], where sample 14 had shown amplification. In [Figure 4], melting peak for the Sample 14 are shown at 82.2°C correspond to the predicted melting Tm for eaeA DNA template.
Figure 1: Amplification plot for triplex polymerase chain reaction assay. X-axis: No. of cycles; Y-axis: Normalised fluorescence; PPC: Positive polymerase chain reaction control; NC: Negative control; S14: Sample 14; S15: Sample 15; S17: Sample 17: S18: Sample 18; S19: Sample 19

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Figure 2: High-resolution melting analysis plot for triplex polymerase chain reaction assay. X-axis: Tm; Y-axis: negative derivative of fluorescence with respect to temperature, (-dF/dT vs. T); S14: Sample 14; S15: Sample 15; S17: Sample 17; S18: Sample 18; S19: Sample 19

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Figure 3: Amplification plot for quadruplex polymerase chain reaction assay. X-axis: No. of cycles; Y-axis: Normalised fluorescence; PPC: Positive polymerase chain reaction control; NC: Negative control; S14: Sample 14; S15: Sample 15; S17: Sample 17; S18: Sample 18; S19: Sample 19

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Figure 4: High-resolution melting analysis plot for quadruplex polymerase chain reaction assay 2. X-axis: Temperature (T); Y-axis: negative derivative of fluorescence with respect to temperature, (-dF/dT vs. T); S14: Sample 14; S15: Sample 15; S17: Sample 17; S18: Sample 18; S19: Sample 19

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Prevalence of diarrheagenic Escherichia coli

The detection rate of virulent genes of DEC was significantly more from the cases i.e., 39 (22.94%) as compared to controls 3 (6.39%) (P = 0.0195). EPEC, EAEC, ETEC and EIEC were the only DEC pathotypes detected in this study [Table 5].
Table 5: Diarrheagenic Escherichia coli with specific virulence genes detected by multiplex real-time polymerase chain reaction

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Out of the 39 DEC isolated from 170 diarrhoeal cases, EPEC had a high proportion accounting for 10% (n = 17), followed by EAEC-8.82% (n = 15), ETEC-2.94% (n = 5) and EIEC-1.18% (n = 2). While amongst the 3 DEC pathotypes detected from the 47 controls, EPEC accounted for 4.26% (n = 2) followed by EAEC-2.13% (n = 1).

Of the total 19 EPEC (cases-17, control-2) identified according to the specific genotypes, atypical EPEC harbouring only eaeA was present in 12 (2.68%) and typical EPEC harbouring both eae and bfpA genes was present in 7 (1.60%) as shown in [Table 5]. Amongst the 12 atypical EPEC strains harbouring only eaeA gene, 10 were from diarrhoea group and 2 were from control group. However, all the 7 typical EPEC strains harbouring both eae and bfpA genes were from diarrhoea group.

Age distribution of diarrheagenic Escherichia coli pathotypes amongst the cases and controls

The age distribution of the children harbouring virulent genes for DEC is shown in [Table 6]. The age distribution of DEC amongst the cases and controls was observed varyingly in the four age groups as shown in [Table 6]. DEC strains were isolated maximum from the 1–5 years of age group i.e., 20 (47.62%) followed by 17 (40.48%) in <1 years of age group and 1 (2.38%) from the >10 years of age group.
Table 6: Recovery rate of different diarrheagenic Escherichia coli pathotypes in various age strata of cases and control groups

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Gender variations of diarrheagenic Escherichia coli and non-diarrheagenic Escherichia coli isolates

The gender variation of DEC versus non-DEC was found to be non-significant (P = 0.9242). The proportion of males in DEC was 57.14% which was similar to non-DEC (59.42%).

Clinical presentation of diarrheagenic Escherichia coli and non-diarrheagenic Escherichia coli cases

Acute diarrhoea without blood or mucus was the most common presentation amongst the study participants with DEC 35 (83.33%) in comparison to the non-DEC 115 (65.71%) cases, as shown in [Table 7]. The associated manifestations such as fever with vomiting, abdominal pain and presence of severe dehydration are shown in [Table 8].
Table 7: Types of diarrhoea amongst diarrheagenic Escherichia coli versus non-diarrheagenic Escherichia coli study participants

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Table 8: Associated clinical manifestations amongst diarrheagenic Escherichia coli versus non-diarrheagenic Escherichia coli study subjects

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Seasonal variation

The monthly distribution of DEC isolates and non-DEC isolates was analysed for assessing the seasonal variation and presented in [Table 9] and [Figure 5].
Table 9: Monthly distribution of non-diarrheagenic Escherichia coli isolates versus diarrheagenic Escherichia coli isolates

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Figure 5: Monthly distribution of non-diarrheagenic Escherichia coli isolates versus diarrheagenic Escherichia coli isolates

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 ~ Discussion Top


Diarrhoeal diseases remain one of the leading causes of preventable death in developing countries, especially amongst children under 5 years of age.[4] Amongst the various causes of diarrhoea, acute infectious diarrhoea is a major cause of morbidity and mortality worldwide and it remains a major public health challenge principally in developing countries like India. Amongst the bacterial pathogens, DEC are the most frequently implicated agents in cases of epidemic and endemic diarrhoea worldwide.[6]

The epidemiological features of these DEC pathotypes as causative agents of diarrhoea vary from region to region. This variation is also seen between and within countries in the same geographical area.[11],[12] In India, especially in the North-Eastern region, there are only few studies regarding the prevalence of DEC pathotypes which may be attributed to the lack of molecular diagnostic facilities. In the laboratories, detection and identification of DEC is not only confined to conventional methods but also requires molecular techniques for detecting the specific virulent genes.

Numerous molecular methods have been developed to identify and classify DEC pathotypes based on the presence of the specific virulent genes.[7] Conventional PCR methods are too expensive and labour-intensive for routine detection of these organisms since these require amplification of DNA templates in a thermocycler followed by post-amplification product separation by gel electrophoresis which is time-consuming and laborious.[16] Multiplex real-time PCR which depends on the detection of the specific virulent genes has provided a practical and rapid method for the detection of DEC. This technique is simple, rapid, sensitive and inexpensive system as compared to other molecular technologies-TaqMan PCR assay and sequencing.[6],[7],[17]

Therefore, this study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using multiplex real-time PCR with HRM technology. The study was conducted in the Department of Microbiology, NEIGRIHMS, a tertiary care hospital in North-East India.

In this study, we had standardised and evaluated two multiplex real-time PCR assays with High-Resolution DNA Melt Analysis. The sequences of the virulence genes that were amplified in the two multiplex real-time PCR assays were from highly conserved regions and thus the assays had low risk of false negatives related to genotypic variation within the different pathotypes. However, initially, it was time-consuming to standardise the two multiplex PCR assays as the targeted amplicons had wide range of product sizes. Selecting the appropriate virulence genes of the DEC pathotypes having specific amplicon sizes for detection by the Quadruplex and Triplex PCR assays was of utmost importance to enable amplification following the same cycle protocols standardised.

In this study, a total of 42 DEC strains were detected from the total 217 E. coli isolates recovered from the cases and control groups by real-time multiplex PCR. A total of 39 (22.94%) DEC were isolated from 170 children with diarrhoea. DEC was detected in 3 (6.39%) out of 47 stool samples from children without diarrhoea. The overall recovery rate of DEC strains was significantly higher in children with diarrhoea, compared to the control group (P = 0.0195) which was supported by a study conducted by Shetty et al., in India.[6] The significant association of DEC with diarrhoea observed in this study had also been similarly reported by other studies carried out in other locations such as Ghana,[18] Brazil [19] and Nigeria.[20] This study recorded a similar rate of recovery of DEC (22.94%) from the diarrhoeal children in comparison to a study from South India (21.4%).[21] However, this study had a lower prevalence rate in comparison to a study in Brazil where DEC was detected in 36.8%.[19] The low prevalence of DEC amongst the diarrhoeal cases also suggests that other causative agents such as Rotavirus, Salmonella spp and Shigella spp that were not included in this study might be other causes of diarrhoea. The recovery of DEC from 6.69% of the control group in this study suggested that these pathogenic organisms were rarely encountered in healthy children and the few from whom the DEC were isolated might be recovering from diarrhoea or were in the presymptomatic stage of the infection.

The distribution of DEC amongst the various children age groups in this study was highest amongst the age group 1–5 years (47.62% n = 20) followed by <1 year of age (40.48%, n = 17). The prevalence of DEC genes was significantly high in younger age strata. Our findings were in agreement with other various studies conducted in India and other countries.[2],[6],[22]

It was observed that the overall prevalence of diarrhoea and detection of DEC pathotypes from both diarrhoeal and non-diarrhoeal group were significantly higher during March and July (P = 0.0071–0.0186). The peak prevalence was observed in July, a period considered as the hottest month in the study area, which is favourable for the proliferation of infectious agents in the tropics.[23] This observation was similar to previous studies on seasonal variation of DEC infection reported by El Metwally et al. in Egypt [24] and Albert et al. in Kuwait.[25] Correspondingly, in similar studies conducted in India, the incidence of diarrhoeal diseases was observed to be maximum during the summer months followed by rainy or winter months.[22]

Amongst the DEC isolated from children with diarrhoea (n = 170), EPEC (n = 17, 10%), EAEC (n = 15, 8.82%), ETEC (n = 5, 2.94%) and EIEC (n = 2, 1.18%) were detected and in children without diarrhoea (n = 47) only EAEC (n = 1, 2.13%) and EPEC (n = 2, 4.26%) were isolated.

There was a significant association of DEC with diarrhoea group in comparison to non-diarrhoea group (P = 0.0195). The most common DEC prevalent amongst cases in this study was EPEC (10%) followed by EAEC (8.82%). These findings were similar to a study conducted earlier in Mangalore, South India by Shetty et al., in the year 2004, where the most prevalent DEC was EPEC (atypical) accounting for 12 (10.4%) cases followed by 4 cases of EAEC (3.4%).[6] In another study conducted in the year 2006, in Kashmir, EPEC was also the most common DEC detected in 7.6% of faecal samples from patients with diarrhoea.[6],[21] Similarly, in a study conducted on incidence of bacterial enteropathogens amongst hospitalized diarrhoea patients from Orissa, India, most of the pathogenic E. coli spp. isolated were EPEC (40.6%), followed by ETEC (52.7%), and EAEC (40.6%).[26]

EPEC remains one of the most important pathogens infecting children, and they are one of the main causes of persistent diarrhoea worldwide.[27] EPEC are classified into two types: Type I or typical EPEC which are positive for both eaeA gene and the bfp gene and Type II or atypical EPEC which are positive for eaeA gene only.[28] A significant association of typical EPEC strains with diarrhoea was reported previously from developing countries.[29] However, in recent years, several studies have shown that atypical EPEC are more prevalent than typical EPEC strains in developed countries as well as in resource-limited countries including India.[6],[21]

Correspondingly, in this study, it had been found that most of the EPEC detected were of atypical type. Of the total 19 EPEC (cases-17 and controls-2), atypical EPEC harbouring only eaeA was present in 12(2.68%) and typical EPEC harbouring both eaeA and bfp genes in 7(1.60%).

EAEC are a heterogeneous emerging pathogen affecting all ages, prevalent in both resource-rich and resource-limited settings and are associated with acute and persistent watery diarrhoea in children, travellers and in individuals infected with HIV/AIDS.[6]

In our study, EAEC was the second most common DEC pathotype detected contributing to 10.95% of the total DEC (cases and control). Similar findings had been reported from Brazil where EAEC (4%) was the 3rd most common DEC pathotype after EPEC (10%) and ETEC (7.5%).[6] In contrast, EAEC had been reported as the most common DEC pathotype detected from Tanzania (33%) and several other regions in India, including Northern India (12.3% in acute diarrhoea and 34.5% in persistent diarrhoea) and Manipal (22%).[6] Another study on children affected by diarrhoea in Nigeria revealed a percentage of 7.84% of this DEC.[30]

In our study, ETEC was detected in 2.94% of the cases and it was not found amongst the control group. In contrast, several countries in the world, including Bangladesh, Egypt, Iran and Mexico, had reported ETEC as the most common cause of diarrhoea amongst all E. coli intestinal pathotypes.[9] However, from India, previous reports have reported varied findings ranging from 0.92% in Kashmir in the year 2006 to 12% in Calcutta.[6] In a similar study conducted in Mangalore, India, by Shetty et al., no ETEC strains were detected.

Lack of epidemiological attention to EIEC was related to the low incidence of this pathogen as a cause of diarrhoea when compared to other pathotypes of DEC. Another reason for the low isolation rate of EIEC may be related to the fact that they were missed when only lactose fermentation is used as a preliminary screening tool for diarrheagenic E. coli since over 70% E. coli in this group did not ferment lactose.[29] In our study, EIEC was present in 1.17% (n = 2) cases of total DEC and was not detected in the control group. Similarly, low frequencies of EIEC strains had been reported in other studies performed in different parts of the world. This result was in agreement with the low prevalence rate of 3 (1.5%) EIEC isolates reported by Hegde et al., in 2012 in India.[7]

In the present study, neither cases nor controls harboured genes for EHEC. These results agree with the low prevalence of EHEC infection in developing countries.


 ~ Conclusion Top


DEC strains are amongst the important causative bacterial agents of diarrhoea amongst children in developing countries like India and are now being recognised as emerging enteropathogens in the developed world. Because conventional phenotypic and serological tests cannot differentiate and identify DEC pathotypes, molecular methods such as DNA hybridisation and PCR play a major role in detecting and identifying DEC pathotypes. However, DNA hybridisation and conventional PCR assay are proven to be extremely time-consuming and too laborious as compared to multiplex real-time PCR.

Hence, in this study, we had successfully standardised two multiplex PCR assays which were found to be simple, rapid and cost-effective for direct detection of EPEC, EAEC, ETEC and EIEC from both the diarrhoeal and non-diarrhoeal groups. This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis. This study has also highlighted the importance of emerging antibiotic resistance amongst the DEC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

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