The zoonotic pathogens, Campylobacter jejuni and Campylobacter coli, are considered the leading causes of acute bacterial gastroenteritis worldwide. During 2016 in the EU, the number of reported confirmed cases of human campylobacteriosis was 246,307, with a notification rate of 66.3 per 100,000 population being the most commonly reported zoonoses, and representing almost 70% of all the reported cases, followed by salmonellosis, yersiniosis, and STEC infections (EFSA and ECDC, 2017). In the United States, a total of 303,520 culture-confirmed campylobacteriosis cases were reported during 2004–2012, with an average annual incidence rate (IR) of 11.4 cases/100,000 persons, ranging by state from 3.1 to 47.6 cases/100,000 persons (Geissler et al., 2017).

Campylobacter spp. also causes other symptoms in humans including acute exacerbation of inflammatory bowel disease and acute appendicitis. Furthermore, extra-intestinal infections such as septic thrombophlebitis, bacteremia, endocarditis, neonatal sepsis, pneumonia, bloodstream infections, brain abscesses, and meningitis may also result from Campylobacter infections (Igwaran and Okoh, 2019). Moreover, post-infectious complications associated with campylobacteriosis include reactive arthritis, severe demyelinating neuropathy, Guillain–Barré syndrome, and Miller–Fisher syndrome as well as post-infectious irritable bowel syndrome (Scallan et al., 2015; Igwaran and Okoh, 2019).

Although the epidemiology of campylobacteriosis and its long-term sequelae have been well established in developed countries, the impact of campylobacteriosis in medium- and low-income countries remains underestimated mainly due to insensitive and inconsistently applied isolation methods (Platts-Mills et al., 2014; Porte et al., 2016). In developing countries, Campylobacter epidemiology might differ compared to developed regions in that it is usually endemic, asymptomatic, and without marked seasonality (Platts-Mills and Kosek, 2014).

Campylobacter gastroenteritis is generally self-limiting, and treatments with antibiotics are not generally recommended, except in prolonged cases, systemic infections, or infections in the elderly, very young, immuno-compromised, or pregnant individuals. Campylobacter has become increasingly resistant to certain drugs, however, especially to fluoroquinolones, which are widely used for the treatment of farm animals (Fernandez and Perez-Perez, 2016). Therefore, the World Health Organization (WHO) recently promoted studies addressing the incidence and antibiotic resistance of Campylobacter spp., especially in low- and middle-income countries (LMIC) (WHO, 2013). To accomplish this goal, the development and evaluation of isolation methods, which are suitable in environments with limited resources, are crucial (WHO, 2013). Since this is hampered by economic and technical requirements, culture methods that do not require the production of artificial microaerophilic atmospheres during incubation would serve as valuable tools to determine the incidence of Campylobacter in locations that are unable to afford traditional Campylobacter culturing techniques (Hinton, 2013; Hinton, 2016).

The newly developed selective solid chromogenic medium, CAMPYAIR, supports the growth of C. jejuni and C. coli. Colonies of the pathogen are usually shiny and purple colored with or without metallic sheen after 48–72 h of incubation under aerobic conditions (Levican and Hinton, 2022). The present study aims to evaluate the ability of this novel medium to recover Campylobacter from routine clinical fecal samples compared to a commercially available medium.

A total of 191 consecutive samples were analyzed in parallel by CASA and CAMPYAIR media, and the ability of the media to recover Campylobacter from the samples was compared. Campylobacter spp. were detected in 8 (4.2%) samples on CASA medium and in 7 (3.7%) samples on CAMPYAIR medium ( Table 1 ). All isolates were confirmed as C. jejuni. Campylobacter strains grew on CAMPYAIR as large, purple, and partly confluent colonies, while on CASA, colonies were reddish, with variable sizes, but predominantly small pinpoint colonies. An almost perfect agreement was observed between both media, with a 0.93 Kappa Cohen coefficient. Compared to the reference standard, CAMPYAIR had 87.5% (95% CI 47.4–99.7%) sensitivity, 100% (95% CI 98100%) specificity, 100% PPV, and 99.45% NPV ( Table 2 ).

Growth of non-Campylobacter bacterial isolates was observed in nine samples (4.7%, 95% CI 2.5–8.7) on CAMPYAIR medium and in three samples (1.6%, 95% CI 0.5–4.5) on CASA medium. None of these isolates exhibited typical Campylobacter morphology. These isolates recovered from both media were identified by MALDI-TOF MS as mostly Enterobacterales, which are rod-shaped, Gram-negative, non-spore-forming, facultative anaerobes. Additionally, Lactobacillus spp., rod-shaped, Gram-positive, aerotolerant anaerobes or microaerophilic bacteria were also recovered on CAMPYAIR medium ( Table 1 ).

To validate a novel simplified Campylobacter culture technique, the CAMPYAIR medium protocol was compared head-to-head to a standard culture medium for the ability to recover Campylobacter from clinical stool samples. Results indicated that CAMPYAIR had a 0.93 Kappa Cohen coefficient. The new medium also showed high agreement with the traditional, reference method, exhibiting a sensitivity of 87.5% and an NPV of 99.45%, which is in accordance with a previous study (Le Bars et al., 2011). Although there was one was false-negative sample recovered on CAMPYAIR medium, a better growth of Campylobacter was observed on this medium with larger colonies being produced. The only sample that was positive by CASA™ agar and negative by CAMPYAIR was the first one included in this study. Therefore, the possibility that this disagreement may be due to the lack of experience of the technician who performed this analysis at the beginning of the study cannot be ruled out.

C. jejuni was the only Campylobacter species isolated from the stool samples, but C. coli showed the same morphological characteristics when grown on CAMPYAIR in previous tests (Levican and Hinton, 2022). Campylobacter culture has shown a poor sensitivity compared to molecular and immunological detection, and this poor sensitivity has been explained by the loss of Campylobacter viability in clinical specimens stored in transport medium, the number of bacteria present in the sample, and the fact that the typical laboratory culture methods are optimized for C. jejuni and C. coli and are not set up to detect additional pathogenic Campylobacter species like C. lari and C. upsaliensis (Buss et al., 2019). However, the World Health Organization (WHO, 2013) has promoted studies addressing incidence and antibiotic resistance of Campylobacter spp., especially in LMIC, by the development and evaluation of isolation methods by culturing, which are suitable in environments with limited resources. Moreover, it has been stated that the uptake of molecular and immunological detection threatens the utility of the surveillance systems, and their incorporation should be done strategically to maintain and improve surveillance for antimicrobial resistance, outbreaks, and the role of various Campylobacter species in human illness (Geissler et al., 2017). In this line, future studies aimed to assess the performance of the medium CAMPYAIR in comparison to non-cultural methods and the growth of other species different to C. jejuni and C. coli are warranted.

Although both media compared in the present study were able to inhibit the growth of most non-Campylobacter bacteria from intestinal flora, CAMPYAIR medium did allow the growth isolates of Enterobacterales and Lactobacillus; these last isolates were probably related to probiotics prescription to the patients. However, those bacteria showed their typical morphology on blood agar, i.e., large, smooth, shiny, circular, and raised white colonies for Enterobacterales, and small to medium gray colonies that were alpha or non-hemolytic for Lactobacillus. In contrast, Campylobacter produced colonies with a very different morphology (large, purple, and partly confluent colonies, with or without metallic sheen). In a previous study, Le Bars et al. (2011) have also reported non-specific growth of non-fermenters Gram-negative bacteria and yeasts in 24 of 260 samples (9.2%) on CASA™ medium, while in the present study, less growth of non-Campylobacter bacterial isolates was observed, i.e., 4.7% and 1.6% on CAMPYAIR and CASA media, respectively. The selectiveness of CAMPYAIR requires further studies with higher sample numbers and different settings. However, due to the chromogenic properties of CAMPYAIR, Campylobacter colonies were easy to differentiate, which is crucial for routine microbiological diagnosis.

In this study, the typical Campylobacter colonies on CAMPYAIR obtained after 48–72 h of incubation under aerobic conditions were confirmed by MALDI-TOF mass spectrometry (Vitek MS, Biomérieux). However, in laboratories with limited resources, the isolates can be identified by submitting them to Gram staining, oxidase and hippuricase determination, and motility observation. The isolates of Campylobacter spp. are Gram-negative bacilli, curved, S or seagull shaped, positive for oxidase and motility. Moreover, those isolates that are positive for hippuricase can be certainly identified as C. jejuni, which account for 90% or more of the cases (Levican et al., 2019).

The novel CAMPYAIR medium had a very high agreement with the standard technique based on a commercial Campylobacter medium on the ability to isolate Campylobacter from fecal samples. CAMPYAIR showed a high degree of performance for the selective isolation of Campylobacter spp. from stool samples without the need of incubating containers under microaerophilic atmospheres. Because of these lower technical requirements, CAMPYAIR could be useful to implement Campylobacter culture in countries with limited resources.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

Conceptualization: AL, AH, LP, IB, and TW. Methodology: AL, CV, FG, and BM. Formal analysis: AL, AH, CV, and LP. Writing—original draft preparation: AL. Writing—review and editing: AL, AH, LP, IB, and TW. Funding acquisition: AL. All authors contributed to the article and approved the submitted version.