Pilgrims who planned to participate in the 2013 Hajj were recruited on September 15, 2013, at a private specialized travel agency in Marseille, France, which organizes travel to Mecca. Potential participants were asked to participate in the study on a voluntary basis if they were ≥18 years of age and were able to provide consent.

In this prospective cohort study, participants were sampled and followed up before departing from France (on October 2, 2013) and immediately before leaving Saudi Arabia (on October 24, 2013). Upon inclusion in the study, participants were interviewed by Arabic-speaking investigators who used a standardized pre-travel questionnaire that collected information on the demographic characteristics and medical history of each participant. A post-travel questionnaire that collected clinical data and information on vaccination status and compliance with preventive measures was completed during a face-to-face interview 2 days before the pilgrims returned to France by a single investigator who joined the pilgrims after the Hajj. Health problems that occurred during the pilgrims’ stay were also recorded by a physician who traveled with them during the entire stay in Saudi Arabia, including during the rituals.

Subjective fever was defined as a feverish feeling according to the pilgrims’ report. Influenza-like illness (ILI) was defined as the presence of cough, sore throat, and subjective fever (15). The study protocol was approved by the Aix Marseille Université institutional review board (July 23, 2013; reference no. 2013-A00961–44) and by the Saudi Ministry of Health Ethical Review Committee. The study was performed in accordance with the good clinical practices recommended by the Declaration of Helsinki and its amendments. All participants gave written informed consent.

Paired nasal and throat swab specimens were collected from each participant by using rigid cotton-tipped swab applicators (Medical Wire and Equipment, Corsham, UK) 10 days (September 22, 2013) before participants departed from France (pre-Hajj specimens) and only 1 day (October 23, 2013) before they left Saudi Arabia (post-Hajj specimens). Nasal and throat swab specimens collected from participants were placed in viral transport media (Virocult and Transwab, respectively; Sigma, St. Louis, MO, USA) at the time of collection and kept at 20°C before being transported to a laboratory in Marseille for storage at −80°C within 48 h of collection.

Nasal swab samples were independently tested as described (5) for influenza virus A/H3N2 (16), influenza B virus (16), influenza C virus (17), and A(H1N1)pdm09 virus (18); human adenovirus (19); human bocavirus (20), human cytomegalovirus (21); human coronaviruses (HCoVs); human enterovirus (22); human metapneumovirus (23); human parainfluenza viruses (HPIVs); human parechovirus (24); human respiratory syncytial virus (25); and human rhinovirus (HRV) (26) by using real-time reverse transcription PCRs. HCoVs and human HPIVs were detected by using an HCoV/HPIV R-Gene Kit (Argene/bioMérieux, Marcy l’Etoile, France) (27). HCoV-positive samples were then genotyped by using the FTD Respiratory Pathogens 21 Kit (Fast Track Diagnostics, Luxembourg, Luxembourg).

Throat swab samples were independently tested as described (12) by using quantitative real-time PCRs for Streptococcus pneumoniae, Neisseria meningitidis, Bordetella pertussis, and Mycoplasma pneumoniae. Sequences of all primers and probes have been reported (28). In the present study, reactions were performed by using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

The Pearson χ² and Fisher exact tests, as appropriate, were used to analyze categorical variables. Statistical analyses were performed by using SPSS software package version 17 (SPSS Inc., Chicago, IL, USA). p values ≤0.05 were considered significant.

A total of 129 persons were invited to participate in the study. All persons agreed to participate in the study and responded to the pre-travel questionnaire. The participants were 77 women (59.7%) and 52 men (40.3%) who had a mean (SD) age of 61.7 (9.8) years (age range 34–85 years) (Table 1). Although most (94.6%) participants were born in northern Africa, most (94.5%) had lived for years in Marseille or the surrounding cities. More than half of the participants (52.7%) reported having ≥1 chronic disease, as described (14).

All post-travel questionnaires were completed. During the 3-week stay in Saudi Arabia (October 3–24, 2013), most (90.7%) pilgrims had ≥1 respiratory symptom, including cough (86.8%), sore throat (82.9%), rhinorrhea (72.1%), myalgia (50.4%), fever (49.6%), and dyspnea (21.7%), and 47.3% met the criteria for self-reported ILI (41.3% in 2012 vs. 47.3% in 2013; p = 0.325). Onset of respiratory symptoms peaked in the second week (week 41) after the arrival of the pilgrims in Mecca and decreased thereafter. However, 90 (69.8%) pilgrims still had respiratory symptoms before leaving Saudi Arabia at the time of sampling (week 43). Only 1 pilgrim (0.8%) was hospitalized during the stay in Saudi Arabia (for undocumented pneumonia). No deaths occurred.

Regarding preventive measures, 51.2% of participants reported receiving pneumococcal vaccination (Pneumo 23) in the past 5 years, which was significantly higher than the rate in 2012 (35.9% in 2012 vs. 51.2% in 2013; p = 0.013). None had received the 2013 influenza vaccine before departing for the Hajj, but 44.2% reported having received the seasonal influenza vaccine in 2012 (31.8% among participants <65 years of age vs. 65.8% among participants >65 years of age; p = 0.001). During the stay in Saudi Arabia, 53.5% of pilgrims reported either frequent use (9.3%) or occasional use (44.2%) of facemasks; 93.0% used disposable handkerchiefs; 49.6% reported frequent handwashing; and 67.4% used hand sanitizer. ILI symptoms were less frequently reported by persons who reported receiving the influenza vaccine in 2012 compared with reports by unvaccinated persons (34.1% vs. 61.5%, respectively; p = 0.009) (odds ratio 0.32, 95% CI 0.14–0.76). In contrast, none of the other preventive measures was found to be effective in preventing ILI symptoms during the stay in Saudi Arabia.

Pre-Hajj and post-Hajj nasal swab specimens were obtained from 121 (93.8%) and 129 (100%) participants, respectively. A total of 26 (21.5%) of 121 pre-Hajj specimens tested were positive for ≥1 virus compared with 50 (38.8%) of 129 post-Hajj specimens tested (p = 0.003) (Table 2). Moreover, 36 (29.8%) participants had acquired ≥1 virus during the stay in Saudi Arabia (Figure 1). The prevalence of human coronavirus E229 (HCoV-E229) was significantly higher in post-Hajj specimens than in pre-Hajj specimens (12.4% vs. 0%; p<0.001). A high prevalence of HRV was observed in pre-Hajj and post-Hajj specimens (14.0% and 14.7%, respectively; p = 0.88). Of 19 participants whose post-Hajj specimens were positive for HRV, 17 (89.5%) had acquired the infection during their stay in Saudi Arabia (Figure 1).

The prevalence of influenza A and B viruses was significantly higher in post-Hajj specimens than in pre-Hajj specimens (7.8% vs. 0%; p = 0.002); further details are described elsewhere (14). Coronaviruses HKU1, NL63, and OC43; human enterovirus; human metapneumovirus; HPIV; and human respiratory syncytial virus were also acquired during the stay in Saudi Arabia by a low proportion of participants (Table 2). Of 50 participants whose post-Hajj specimens were positive for ≥1 respiratory virus, 43 (86.0%) reported ≥1 respiratory symptom during their stay in Saudi Arabia, of whom 37 (86.0%) still had respiratory symptoms at the time of sampling. Also, of 79 participants whose post-Hajj specimens were negative for respiratory viruses, 74 (93.7%) reported ≥1 respiratory symptom during their stay Saudi Arabia, of whom 53 (71.6%) still had respiratory symptoms at the time of sampling. None of the preventive measures was found to be effective in preventing respiratory viruses in post-Hajj specimens.

Pre-Hajj and post-Hajj throat swab specimens were obtained from 126 (97.7%) and 129 (100%) participants, respectively. None of the participants were positive for N. meningitidis, B. pertussis, or M. pneumoniae at any point in the study period (Table 2).

A total of 63 (50.0%) of 126 pre-Hajj specimens tested and 80 (62.0%) of 129 post-Hajj specimens tested were positive for S. pneumoniae (p = 0.053) (Table 2; Figure 2). Of 80 participants whose post-Hajj specimens were positive for S. pneumoniae, 29 (36.3%) had acquired the infection during their stay in Saudi Arabia (Figure 2). In addition, of 63 participants whose pre-Hajj specimens were positive for S. pneumoniae, 12 (19.0%) subsequently had post-Hajj specimens that were negative for S. pneumoniae (Figure 2), of whom 10 (83.3%) reported having received antimicrobial drugs during their stay in Saudi Arabia: 7 received amoxicillin, 2 received amoxicillin and ciprofloxacin, and 1 received azithromycin. Of 80 participants whose post-Hajj specimens were positive for S. pneumoniae, 73 (91.2%) reported ≥1 respiratory symptom during their stay in Saudi Arabia, of whom 56 (76.7%) still had respiratory symptoms at the time of sampling.

Among 66 participants who reported having received a pneumococcal vaccination in the 5 years before traveling to Saudi Arabia, 37 (56.1%) had post-Hajj specimens that were positive for S. pneumoniae. The prevalence of S. pneumoniae in post-Hajj specimens was significantly lower in persons who reported using hand sanitizer during their stay in Saudi Arabia than in remaining participants (55.2% vs. 76.2%; p = 0.021) (odds ratio 0.39, 95% CI 0.17–0.88) and slightly lower in persons who reported more frequent handwashing than usual during their stay in Saudi Arabia than in persons who reported usual handwashing (54.7% vs. 69.2%; p = 0.08).

Of 80 participants whose post-Hajj specimens were positive for S. pneumoniae, 27 (33.8%) were co-infected with ≥1 virus (Figure 2). Of 49 participants whose post-Hajj specimens were negative for S. pneumoniae, 23 (46.9%) were infected with ≥1 virus (33.8% vs. 46.9%; p = 0.14) (Figure 2).