Prevalence of Campylobacter spp. in human clinical samples

Aruna Devi

    Research output: ThesisDoctoral Thesis

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    Diarrhoea remains a significant public health problem and is a leading cause of mortality and morbidity globally, particularly, among children in developing countries. Campylobacter species are the most commonly reported bacterial cause of gastroenteritis in developed and developing countries with Campylobacter jejuni and Campylobacter coli frequent causes of foodborne gastroenteritis. The current study has focused on addressing knowledge gaps of Campylobacter spp. infections in the human populations from two jurisdictions where this disease is not currently notifiable, namely Fiji and New South Wales (NSW), Australia. This study reports the prevalence of laboratory confirmed cases of Campylobacter spp. in diarrhoeal disease in Fiji as 176 cases per 100,000 population, an estimate which is consistent with data from similar developing countries. The estimate was based on the results of testing extracts from human diarrhoea samples (n = 408) using conventional PCR, targeting 16S rRNA genes, with 59.1% of samples being reactive. Samples were also subjected to Campylobacter spp. culture, with only one sample returning a positive result. When patient age was considered, samples from children below the age of 5 years and those aged from 15 to 44 years were at the highest risk of reacting with the PCR assay. Overall, these data suggest that Campylobacter spp. play an essential role in diarrhoeal disease in Fiji. The data indicated that Fiji should consider implementing routine testing of Campylobacter spp. and adding this disease to the list of notifiable diseases. These actions would facilitate the collation of comprehensive data on Campylobacter spp. which could be used to inform the development and implementation of specific control strategies to monitor emerging antibiotic resistance in these bacteria. This study also investigated the reactivity of human diarrhoeal samples (n = 1,422) collected from patients in NSW (Australia) using conventional PCR with 10.8% (n = 154) of samples determined to be reactive. A subset of the NSW sample extracts was interrogated with a duplex (qPCR) assay to identify those sample extracts that were reactive for Campylobacter spp. and/or the C. jejuni. Targeted genes were 16S rRNA and map. Testing of the extracts from the clinical diarrhoea samples (n = 400) with this duplex qPCR assay determined that 52.3% (n = 211) of samples were reactive for the Campylobacter spp.

    The highest number of Campylobacter spp. reactive samples were in the three age groups ≥55 years (55 to 64 years, 65 to 74 years and ≥75 years). The lowest number of Campylobacter spp. reactive samples were from children aged between 0 and 4 years, 5 to 14 years and young adults in the category 25 to 34 years. This study also demonstrated that in the age range 0-4 years and 5-14 years, higher prevalence rates were detected in samples from males compared to females 6.4% and 1.0%, 8.2% and 0%, respectively. In contrast, in the age group 35 to 44 years, females had a higher prevalence than males. Based on the data from the duplex qPCR assay, the estimated prevalence of laboratory confirmed cases of Campylobacter spp. in human diarrhoeal samples collected from Western Sydney and Wagga Wagga, was 52.3%. These findings were compared with the national data from the OzFoodnet report (2010), and it was found that this estimate suggests that NSW may have the highest number of laboratory confirmed cases of Campylobacter spp. infection of any Australian State. This study had some limitations. Firstly, only diarrhoeal samples from the participating laboratories (Wagga Wagga and Sydney) were available for analysis. Consequently, this study did not investigate asymptomatic infections or those cases where individuals did not seek medical care. This limitation may have resulted in the current research underestimating the true prevalence of Campylobacter spp. in the study population. Secondly, the prevalence of Campylobacter spp. was estimated based on reactive samples during the period of study and did not cover all the seasons. The evidence of seasonal trends of campylobacterosis has been reported with an increase in prevalence in warmer months. This may represent possible links between seasonal trends in the exposure of humans to animal faecal material and any seasonal trends in human campylobacter disease. Hence, human behaviour during different seasons needs to be considered to estimate the true prevalence of Campylobacter spp. Further, due to differences in diagnostic methodologies applied in various studies, such as culture, conventional PCR, qPCR, multiplex qPCR and immunoenzymatic methods, this could also influence the prevalence estimate. These methodologies have different specificities and sensitivities, and there is no standardised testing method recommended for studies estimating Campylobacter spp. prevalence. A larger sample size, collected over a more extended period of time, is required to improve the estimate of the true prevalence of campylobacteriosis in NSW (Australia) human population. Ideally, this would include samples of people with asymptomatic infections and those who do not seek medical care to improve the accurate prevalence estimate. The logistics of identifying suitable participants and collecting such samples may render this impractical. As such, any Campylobacter prevalence estimates are likely always to be restricted to those patients presenting to medical facilities, with subsequent extrapolations made to the broader population. Individuals of all age ranges in this study group were identified as being at risk of Campylobacter spp. disease whether or not it contributed to the disease during the time of sample collection. Females, particularly those living in Sydney, were shown to have the highest risk of acquiring Campylobacter spp. disaese. To further investigate the role of specific members of the Campylobacter genus in human cases of diarrhoea, samples from patients in NSW from Sydney (n = 400) and Wagga Wagga (n = 400) were interrogated with a multiplex qPCR assay with the capacity to specifically detect C. jejuni and C. coli. Targeted genes were 16S rRNA, map and lpxA for Campylobacter spp., C. jejuni and C. coli respectively. Samples were deemed to be qPCR reactive or negative based on the estimated number of C. jejuni and/or C. coli genomes present in the analysed samples by using this assay. Consequently, the overall prevalence estimate was lower compared to the results of the duplex qPCR assay. When the assay was used to interrogate extracts from the 800 diarrhoeal samples, the prevalence estimate for Campylobacter spp. was 10.9% (n = 293). At the species level, 3.9% (n = 31) and 4.1% (n = 33) of samples were deemed qPCR reactive for C. jejuni and C. coli respectively. These analyses suggest the patient location was a risk factor for testing qPCR reactive to C. coli the prevalence in Sydney (6.5%) being significantly higher compared to Wagga Wagga (1.8%) (P = 0.001). Based on the results of this study, it was evident that interpretation of the multiplex qPCR assay in terms of pathogen genome copy number is potentially problematic in the absence of additional patient information regarding disease progression. The emergence of antimicrobial resistance is rapidly becoming a critical issue in the management of human bacterial infections across the world. To the best of the researcher’s knowledge, no recent studies have investigated antimicrobial resistance in clinical isolates of C. jejuni in patients residing in NSW. Consequently, a study was conducted to address this knowledge gap by testing the susceptibility of C. jejuni isolates (n = 119) to four antimicrobials. The investigation revealed that the degree of resistance to these antimicrobials was variable with 16.1% of isolates resistance to ampicillin. Further, resistance to ciprofloxacin and tetracycline antimicrobials were detected in 13.6% and 5.1% of isolates respectively. Importantly, all isolates tested were sensitive to erythromycin, except for three isolates which exhibited intermediate resistance. A total of 11 isolates exhibited resistance and/or intermediate resistance to more than one antimicrobial and were classed as having multidrug resistance. Relevant isolates were further examined by PCR for the presence of well described resistance genes and/or mutations associated with antimicrobial resistance. Resistance to ciprofloxacin in C. jejuni was associated with the previously reported gyrA gene point mutation C257T, which results in the Thr-86-Ile substitution in DNA gyrase subunit A. While the blaOXA-61 and tet(O) genes were detected in all C. jejuni isolates resistant to ampicillin and tetracycline respectively. Analyses of the partial sequences of the genes linked to resistance identified several synonymous and nonsynonymous mutations which may prove useful in any future studies to determine the origin(s) of these resistant strains. The findings of this study have addressed essential knowledge gaps with respect to Campylobacter spp. in human diarrhoea samples in the populations of Fiji and NSW (Australia). Further studies are required to fully understand the epidemiology of Campylobacter spp. in these populations. Moreover, these data also demonstrate the importance of adding Campylobacter spp. to the list of notifiable infections in Fiji and NSW to facilitate the collection of accurate and more detailed information on the factors influencing human diseases. This more in-depth knowledge of Campylobacter spp. in these two populations could then be used to inform the development of more effective control programmes in both jurisdictions to reduce the overall disease burden.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • Charles Sturt University
    • Vanniasinkam, Thiru, Principal Supervisor
    • Wilkinson, Jenny, Co-Supervisor
    • Mahony, Timothy, Co-Supervisor, External person
    Place of PublicationAustralia
    Publication statusPublished - 2019


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