Campylobacter spp. Prevalence in Santiago, Chile: A Study Based on Molecular Detection in Clinical Stool Samples from 2014 to 2019

Campylobacter spp. is an emerging cause of infectious diarrhea worldwide. In South American countries such as Chile, its prevalence is underestimated due to inadequate detection methods. Gastrointestinal multiplex PCR panels (GMP) permit rapid and sensitive detection of bacterial pathogens and provide important epidemiological information. This study aimed to analyze Campylobacter epidemiology using the results of molecular methods and to compare molecular detection results to those of culture methods. We performed a retrospective, descriptive analysis of Campylobacter spp. detected in clinical stool samples between 2014–2019 by GMP and culture. Within 16,582 specimens examined by GMP, Campylobacter was the most prevalent enteropathogenic bacteria (8.5%), followed by Salmonella spp. (3.9%), Shigella spp./enteroinvasive Escherichia coli (EIEC) (1.9%), and Yersinia enterocolitica (0.8%). The highest Campylobacter prevalence occurred in 2014/2015. Campylobacteriosis affected more males (57.2%) and adults from 19–65 years (47.9%) and showed a bimodal seasonality with summer and winter peaks. In 11,251 routine stool cultures, Campylobacter spp. was detected in 4.6%, mostly C. jejuni (89.6%). Among 4533 samples tested by GMP and culture in parallel, GMP showed a superior sensitivity (99.1% versus 50%, respectively). The study suggests that Campylobacter spp. is the most frequent bacterial enteropathogen in Chile.


Introduction
Campylobacter spp. are emerging zoonotic pathogens inhabiting the gastrointestinal tract of different warm-blooded animals [1,2]. Human campylobacteriosis is endemic worldwide and usually manifests as acute diarrhea. Campylobacter jejuni is the most prevalent human pathogen. Campylobacteriosis has been the most frequent gastrointestinal infection reported in Europe during the last decade, with an incidence of 59.7 cases per 100,000 persons in 2019 [3,4]. Similar results have been reported from the USA, with an incidence of 19.5/100,000 during the same period [3].
Stool culture is the diagnostic standard for Campylobacter spp.; however, the high costs and complexity of this method have prevented its routine implementation in many resource-limited countries, although the pathogen seems to be of emerging epidemiological relevance in these regions [5,6]. The epidemiology and spectrum of campylobacteriosis in South America are uncertain [7]. Studies of different populations using various techniques found a prevalence ranging from 2.2% in Colombian children to 30.1% in children from Argentina [7]. Epidemiological studies in Chile demonstrated prevalence rates from 0.4%, Pathogens 2023, 12, 504 2 of 8 using microscopy, to 18%, using culture [8]. A clinical study showed an increase in the detection of campylobacteriosis from 0.4% to 6.1% after the implementation of culture methods within routine stool workflow [5].
Gastrointestinal multiplex panels (GMP) based on polymerase chain reaction (PCR) have recently emerged as a sensitive, user-friendly, and rapid alternative to conventional culture methods [9]. This study aimed to analyze Campylobacter prevalence rates after the introduction of GMP and to compare molecular test performance to traditional culture methods.

Materials and Methods
We conducted a retrospective, descriptive study at the Clinical Laboratory of Clínica Alemana, a private nonprofit hospital in Santiago, Chile, between January 2014 and December 2019. The health center is located in an upper-income neighborhood and mainly serves this segment of the Metropolitan Region. Information was extracted from the clinical laboratory databases Sisalud (SONDA, Santiago, Chile) and KernMIC (bioMérieux, Marcy-l'Étoile, France). The diagnostic tests were applied according to the sender's request. The data included demographics and the results of routine detection of Campylobacter spp. and other enteric bacterial pathogens (Salmonella, Shigella, and Yersinia enterocolitica) by two GMP, FilmArray gastrointestinal panel ® (bioMérieux, version 2.3) and A.I.I. Screen (Sacace Biotechnologies, Como, Italy). The former detects 13 bacterial, five viral, and four parasitic pathogens and was used from 2015-2019; the latter detects three bacterial (Campylobacter, Salmonella, and Shigella) and four viral enteropathogens, and was offered from 2014-2019. For their genetic similarity, Shigella spp. and enteroinvasive Escherichia coli (EIEC) were not distinguishable with either GMP. Assays were performed and interpreted according to the manufacturers' instructions. If a pathogen was detected in the same patient more than once within a 30-day interval, only the first result was considered. The GMP detection rates of Campylobacter spp. and the three main bacterial enteropathogens (Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica) were compared. Samples consisted of fresh stool, transported at ambient temperature, and processed within 2 h after collection.
In addition, the results of Campylobacter stool cultures performed during the study period were analyzed. Culturing was performed on CASA agar (bioMérieux, Marcy-l'Étoile, France) and plates were incubated at 42 • C under microaerobic conditions (Anaerocult ® C, Merck, Darmstadt, Germany) for 48 h. Quality control for each batch of culture medium was performed using C. jejuni ATCC 33291 (growth at 48 h) and E. coli ATCC 25922 (growth inhibition). Campylobacter isolates were confirmed and identified by MALDI-TOF mass spectrometry (Vitek MS, bioMérieux), as described previously [8]. Samples in which both GMP and culture were performed were used to analyze the diagnostic performance (sensitivity and specificity) of each method against two reference standards: (A) culture (true positive = culture positive) and (B) a composite standard (true positive = culture and/or GMP positive).
Analyse-it software (5.66.0 for MS Excel 10) was used for the statistical analysis and GraphPad version 9 (October 2020) for graph design.

Gastrointestinal Multiplex Panels (GMP)
During the study period, a total of 16,582 GMP exams were performed and analyzed; the majority (11,278; 68%) by FilmArray. Campylobacter spp. was detected in 1412/16,582 samples (8.5%), while Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica were identified in 653/16,582 (3.9%), 323/16,582 (1.9%), and 95/11,274 (0.8%) specimens, respectively. Campylobacter detection rates were highest in 2015 (12.6%) and subsequently declined, reaching their lowest rate in 2019 (4.8%) ( Table 1). In contrast, the prevalence of Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica remained stable throughout the study period. Campylobacteriosis had a bimodal distribution with peaks during the summer months of January/February and winter months of July/August; such a winter peak was not observed with other bacteria (Figure 1).  Campylobacteriosis had a bimodal distribution with peaks during the sum months of January/February and winter months of July/August; such a winter peak not observed with other bacteria (Figure 1). The study population had an equal sex distribution (49.3% female; 50.7% male). pylobacter spp. was significantly more frequent in male patients (57.2%). All bacterial p ogens analyzed were most frequently detected in the adult age group (19-65 years). C pylobacteriosis cases had a median age of 20 years (range 0-99), which was higher Salmonella spp., but lower than Shigella spp./EIEC and Yersinia enterocolitica spp.; Cam obacter spp. was less frequent in children aged 0-5 years than Salmonella spp. (Table The study population had an equal sex distribution (49.3% female; 50.7% male). Campylobacter spp. was significantly more frequent in male patients (57.2%). All bacterial pathogens analyzed were most frequently detected in the adult age group (19-65 years). Campylobacteriosis cases had a median age of 20 years (range 0-99), which was higher than Salmonella spp., but lower than Shigella spp./EIEC and Yersinia enterocolitica spp.; Campylobacter spp. was less frequent in children aged 0-5 years than Salmonella spp. (Table 2).

Discussion
In our analysis based on molecular detection methods, Campylobacter had a prevalence of 8.5% (CI95% 4.6-8.1) in clinical stool samples and represented the most common bacterial enteropathogen. Salmonella was second with a detection rate of 3.9%. To our knowledge, the present data set is the largest on human campylobacteriosis detected by molecular methods in South America. Results of deeper molecular analysis by whole genome sequencing of some of our strains were reported separately [10]. C. jejuni predominated, in accordance with previous local and global data [5,11].
In Chile, the true burden of campylobacteriosis is unknown, most probably reflecting the insufficient availability of diagnostic techniques [8,12]. Consequently, there is a lack of surveillance data and most available information has been generated within research projects with limited sample numbers. Between 2000 and 2019, Chilean studies using different culture strategies and/or rapid antigen detection tests reported a prevalence of campylobacteriosis ranging from 2.1% to 18% [5]. In 2013, an analysis in southern Chile using an in-house PCR in 140 patients with diarrhea found C. jejuni in 10.7% of cases [13]. Our study detected a prevalence of 8.5% using GMP, which is consistent with other GMP-based reports from Santiago, with rates between 7.3% and 12.7% [14,15].
Epidemiological information from other countries in South America is mainly based on different techniques for the culture and identification of Campylobacter, resulting in a wide range of prevalence rates (reviewed in [7]). For example, rates from Argentina varied from 9.1% in 2003, in a study performed in a low-income population, to 30.1% in 2010 in children younger than 15 years. In 2001, Bolivia reported a 4.4% frequency of detection for C. jejuni and a 7.3% frequency of detection for C. coli in patients with diarrhea. In 2002, a study from Peru found a prevalence of 13% for C. jejuni in infants between 0-2 years; in 2003, another study found C. coli (5%) to be more frequent than C. jejuni (2.9%) in patients with gastroenteritis. In 2007 and 2010, Paraguay and Uruguay, respectively, reported high rates of detection in children of 18.4% and 14.3%. In 2003, using a filtration culture technique, a study from Venezuela found a prevalence of 6.5% for C. jejuni in stool samples [7]. Colombia reported a low rate of campylobacteriosis in preschool children (0-5 years) with 2.3% in 2006 and 3.5% in 2013-2014 [7,11]. In 2010, Brazilian children with diarrhea had a prevalence of 9.6% based on a PCR method of detection [7]. These results are difficult to interpret due to the variability in methods, socio-economical settings, and age distributions; however, Campylobacter is likely an important gastrointestinal pathogen in most countries of the region.
The prevalence of Campylobacter showed significant differences over time, exhibiting a peak in 2015 followed by a decrease from 2016 to 2019. The reasons for this decline are unknown, since no other epidemiological information, e.g., outbreaks, is available for this period in Chile [16]. A bias due to the initial tendency to use GMP in more severe cases is unlikely since the prevalence of other organisms remained stable.
Interestingly, the seasonal distribution of Campylobacter spp. was bimodal, with peaks in summer (January-February) and winter (July-August). An increased prevalence during summer months has been reported worldwide [2,11,17]. Some studies have tried to identify the drivers of this seasonality, including improved survival and replication of microorganisms under warmer temperatures, transmission by flies, variations in animal colonization, seasonal changes in eating behavior and human recreational activities, and travel to endemic regions [11,17,18]. However, the mechanisms for the association between campylobacteriosis and warmer climate are still unclear [18,19]. Distinct but smaller annual winter peaks have been observed in some European countries (Austria, Belgium, Finland, Germany, Luxembourg, the Netherlands, Switzerland, and Sweden) and China (Beijing) [2,11]. Food habits or cooking trends could be a possible explanation. In Chile, until now, seasonal trends have not been reported. The observed summer and winter peaks, however, suggest that food preparation practices rather than environmental factors, such as temperature, might be more relevant.
We found a predominance of infection in males, which is similar to previous reports from Chile [20] and industrialized countries [11,21,22]. Louis et al. found that males represented 53.7% of campylobacteriosis cases in England and Wales between 1990 and 1999 in all age groups and all regions, suggesting that this might be related to a higher sex-specific susceptibility to infection [23]. Interestingly, a study from 2000-2003, in the same region, found a higher incidence of campylobacteriosis in males from birth to 17 years Pathogens 2023, 12, 504 6 of 8 old, especially in the 13 to 15 years group. The authors postulated a relationship between hormonal changes during puberty in boys with increased growth of Campylobacter in the intestine. However, the male gender also predominated in patients 50 years and older, disempowering the hypothesis [24]. In a review of the global epidemiology of Campylobacter from 2014 to 2021, the infection was also more commonly found in males in most countries, with few exceptions [11]. A recent meta-analysis including national data from seven countries found that the incidence rates for campylobacteriosis were higher in males throughout all age groups, especially during puberty and in senior age. The authors speculated on a combination of multiple factors. In the older age group, men could be more prone to eat outside the home, getting exposed to undercooked meat, which, combined with a more common use of proton pump inhibitors at that age, could increase their risk of infection. They also referred to animal studies demonstrating that estrogen has a protective role against some enteropathogens and influences immune response through the gut microbiota [25].
Our data revealed a higher prevalence of Campylobacter in the adult group between 19 and 65 years old with a median age in cases of 20 years. This age distribution is in accordance with observations from several industrialized countries [4,11,26]. Similar trends have been found in urban communities from developing countries [11]. In developing countries, symptomatic infections usually affect children under 4 years of age, with the highest incidence in infants younger than 1 year [2]. In these settings, the frequency of clinical diseases caused by Campylobacter infections is assumed to decrease with age, reflecting the acquisition of immunity as a consequence of repeated exposure [27].
In addition, we used our databases to compare the performance of GMP and Campylobacter culture, using results from over 4500 samples, which were examined in parallel by both methods. As in the analysis of all samples, FilmArray GMP showed a higher Campylobacter prevalence than culture in this sample subset (13.7% versus 6.7%) [28]. Older studies evaluating molecular methods to detect Campylobacter in stool samples have used culture as a reference method [3]. Using this approach in our population, GMP showed a very high sensitivity (99.9%), but reduced specificity (92.7%). However, the interpretation of PCR-positive/culture-negative results is controversial [29][30][31]. On one side, molecular techniques detect Campylobacter DNA (not viable bacteria), which could lead to false-positive results. On the other hand, culture is limited by the fastidious nature of Campylobacter, possible antibiotic pretreatment, and the inability to detect species such as C. upsaliensis, which are inhibited by antibiotic supplements in culture media, leading to false-negative results [31]. More recently, PCR has been applied as a reference standard in some studies, resulting in a low sensitivity of culture methods [13,28,32]. Due to the absence of an accepted reference method, we applied a composite reference standard to evaluate the sensitivity of both methods. Using this approach, GMP displayed very high sensitivity (99.5%; CI95% 98.6-99.8) versus culture (50.7%; CI95% 46.8-54.6). A large North American prospective study comparing culture to molecular methods showed similar findings and concluded that PCR tests should have a major role in diagnostic testing for Campylobacter [31]. Despite the higher diagnostic sensitivity, GMP might not replace traditional culture methods, since they allow species identification and antimicrobial susceptibility testing. In addition, multianalyte PCRs often produce polymicrobial results of uncertain clinical relevance [9]. A preliminary analysis in our laboratory, however, showed that Campylobacter-positive samples had a lower co-infection rate than most other intestinal pathogens [33].
The study is limited by its retrospective nature and lack of clinical data. As a monocentric study in a high-income setting in Santiago, the results might not represent the Chilean population. Further multicentric studies are needed to confirm these findings.

Conclusions
This study provides important information on the epidemiological profile of campylobacteriosis in our population such as age groups, sex, and seasonality. The use of commercial gastrointestinal multiplex panels led to a significantly higher detection rate