Abstract

Background

Every year more than 2 million pilgrims from different countries in the world including Iran participate in the annual Hajj in Saudi Arabia. Respiratory diseases have been the most common cause of illnesses among Iranian pilgrims.

Methods

Direct fluorescent staining and viral culture were performed on nasal wash specimens of Iranian Hajj pilgrims with symptoms of acute respiratory tract infections at Shiraz (a city in southern Iran) airport on return from the Hajj during December 2006 to January 2007. They were screened for influenza A and B, parainfluenza 1 to 3, adenovirus, and respiratory syncytial virus (RSV) by viral culture and immunofluorecent staining. Rhinovirus and enterovirus were diagnosed based on reverse transcription polymerase chain reaction methods.

Results

The patients aged between 19 and 82 years (mean: 52.4 years) consisting of 135 females and 120 males. Cough in 213(83.5%) and sore throat in 209 (82%) were the most common symptoms. Eighty‐three patients (32.5%) had viral pathogens: influenza in 25 (9.8%), parainfluenza in 19 (7.4%), rhinovirus in 15 (5.9%), adenovirus in14 (5.4%), enterovirus in 5 (2%), and RSV in 4 (1.6%) and coinfection with two viruses in 1 patient (0.4%). Influenza virus was identified more in unvaccinated than in vaccinated pilgrims (16.5% vs. 9.2%) but statistically insignificant (p= 0.19).

Conclusions

According to the results, each of the above‐mentioned viruses played a role in the development of respiratory diseases among Iranian pilgrims, with influenza virus as the commonest one. Because influenza vaccine could not prevent respiratory infections in Hajj pilgrims statistically, the possibility of the appearance of new drift variants not included in vaccine and also inappropriate vaccine handling and storage might be considered. So it is also advisable to check if the circulating influenza strains were different from the vaccine strains.

Every year more than 2 million pilgrims from different countries in the world including 100,000 pilgrims from Iran (of whom about 4,200 from Shiraz) participate in the annual Hajj in Saudi Arabia. Overcrowding and continuous close contacts greatly increase the spread of respiratory infections, and respiratory diseases have been the most common cause of illnesses among Iranian pilgrims.1

It has been estimated that more than one in three pilgrims will experience respiratory symptoms during the stay.2 Although most of Iranian pilgrims received influenza vaccine 2 weeks before departure, respiratory infections affected considerable percentage of these pilgrims. Since in our country, there has been just a limited etiologic study3 of viral respiratory pathogens among Hajj pilgrims, this study can serve as an attempt to determine the incidence of the common respiratory viruses among Iranian pilgrims.

Patients and Methods

This study was a cross‐sectional one in which nasal wash sampling was performed for all pilgrims with symptoms of acute respiratory tract infections at Shiraz (a city in southern Iran) airport on return from the Hajj during December 2006 to January 2007 (Mecca to Shiraz is about 3 hours by air). The inclusion criterion was the presence of two of the conditions: fever, cough, sore throat, congestion, and sinus pain within the past 3 weeks during the Hajj.4 A questionnaire was distributed among the trained physicians of the eight caravans (dispatching groups of pilgrims, each consisting of 150–250) of Hajj pilgrims before departure from Iran. The items contained in the questionnaire included demographic and clinical information including the documentation of influenza vaccination. The physicians of caravans were instructed to send the name of patients with acute respiratory infections (ARI) by short message service. Ethical approval was obtained from the research ethics committee of Shiraz University of Medical Sciences. A trained research team was placed at Shiraz airport several hours before the arrival of each group of Iranian Hajj pilgrims. Having received the informed consents, nasopharyngeal wash specimens were obtained using 10 mL normal saline; placed in viral transport media containing 50% DMEM, 1% bovine serum albumin, and antimicrobial agents including penicillin (250 IU/mL), streptomycin (100 μg/mL), and amphotericin B (1 μg/mL) in phosphate‐buffered saline; and transported rapidly in 4°C to Prof. Alborzi Clinical Microbiology Research Center, Shiraz, Iran (in less than 2 h). Then, all nasal wash samples were inoculated onto MDCK, HeLa, Vero, and Hep‐2 cell lines using conventional tissue culture methodology.5 They were observed for 10 days for cytopathic effects. The duplicates of cultures with positive results were stained using virus‐specific monoclonal antibodies (IMAGEN, Dakocytomation, Cambridge, UK).

Also on arrival in the laboratory, respiratory epithelial cells in the specimens were washed by centrifugation, pelleted, and then spotted onto 10 microscope slides (Thermo Shandon, cytospine 4, Shandon, UK). After drying and fixation, slides were stained using antibodies (IMAGEN, Dakocytomation, Cambridge, UK) specific for seven viruses [influenza A and B, parainfluenza 1–3, adenovirus, and respiratory syncytial virus (RSV)].

Ribonucleic acid was extracted from respiratory specimens using RNX purification kit (Cinnagen, Tehran, Iran). Multiplex nested polymerase chain reaction (PCR) assays were performed according to Billaud and colleagues6 with primers for human rhinovirus and enterovirus.

Statistical methods

Analysis of data was performed using the SPSS version 11.5 statistical package. Differences in proportions were analyzed using the chi‐square test. A p value of ≤0.05 was considered as significant.

Results

A total of 255 Iranian pilgrims with clinical criteria for ARI during the Hajj were enrolled in the study. The patients ages were between 19 and 82 years (mean: 52.4 years). There were 135 females (52.9%) and 120 males (47.1%). More than three fourths (76.5%) of the patients had developed ARI within the past 2 weeks in Mecca (the Holy Pilgrimage city for Muslims, located in the Kingdom of Saudi Arabia) and the remaining in Medina (the second holiest city in Islam located in the Kingdom of Saudi Arabia) (23.5%). Cough in 213(83.5%) and sore throat in 209 (82%) were the most common symptoms (Table 1). Inactivated influenza vaccine was received in 218 (85.5%) of the patients before leaving for the Hajj pilgrimage.

Table 1

Symptoms of acute respiratory infections among Iranian Hajj pilgrims 2006 (n= 255)

Frequency (%) Symptom 
155 (60.9) Fever 
213 (83.5) Cough 
209 (82.0) Sore throat 
58 (22.7) Sinus pain 
191 (74.9) Congestion 
Frequency (%) Symptom 
155 (60.9) Fever 
213 (83.5) Cough 
209 (82.0) Sore throat 
58 (22.7) Sinus pain 
191 (74.9) Congestion 

Viral agents were identified in 83 (32.5%) of nasal wash specimens of the patients including influenza in 25 (9.8%), parainfluenza in 19 (7.4%), rhinovirus in 15 (5.9%), adenovirus in 14 (5.4%), enterovirus in 5 (2%), and RSV in 4 (1.6%) and coinfection with two viruses in 1 patient (0.4%) (Table 2). Influenza virus was identified more in unvaccinated (6 of 37, 16.5%) than in vaccinated (20 of 218, 9.2%) pilgrims but statistically insignificant (p= 0. 19).

Table 2

Viral etiology of acute respiratory infections in nasal wash specimens of Iranian Hajj pilgrims 2006 (N= 83)

 Cases diagnosed via 1 method  
Virus Culture Immunofluorescent staining PCR Cases diagnosed via multiple methods 
Influenza virus A 12 ND 1* 
Influenza virus B 11 ND 1* 
Parainfluenza virus 1 12 ND — 
Parainfluenza virus 2 ND — 
Parainfluenza virus 3 ND — 
Adenovirus ND 3* 
Respiratory syncytial virus ND — 
Rhinovirus ND ND 15 — 
Enterovirus ND ND — 
Influenza virus B and parainfluenza virus 1 ND — 
Total 54 20 5* 
 Cases diagnosed via 1 method  
Virus Culture Immunofluorescent staining PCR Cases diagnosed via multiple methods 
Influenza virus A 12 ND 1* 
Influenza virus B 11 ND 1* 
Parainfluenza virus 1 12 ND — 
Parainfluenza virus 2 ND — 
Parainfluenza virus 3 ND — 
Adenovirus ND 3* 
Respiratory syncytial virus ND — 
Rhinovirus ND ND 15 — 
Enterovirus ND ND — 
Influenza virus B and parainfluenza virus 1 ND — 
Total 54 20 5* 

N = number of patients; ND = not determined; PCR = polymerase chain reaction.

*

Immunofluorescent staining and culture.

Discussion

In our study, respiratory viruses were detected in about one third (32.5%) of nasal wash samples of Iranian Hajj pilgrims, and influenza virus was identified in 25 (9.8%) of the patients and was the most common virus detected by culture and immunofluorecent staining.

In a previous study in Iran, gargled pharyngeal secretions of 105 Iranian Hajj pilgrims were tested just by immunologic assay for respiratory viruses. In contrast to our study, adenovirus (38 pilgrims; 36.2%) was the most frequently detected virus followed by influenza virus (24 pilgrims; 21.5%) and RSV (2 pilgrims; 1.9%).3 In another recent study involving pilgrims who attended the 2006 Hajj, nasal swabs from 260 pilgrims from the UK and Saudi Arabia were tested by real‐time reverse transcription PCR tests for varieties of respiratory viruses. Of these pilgrims, 38 (25%) from the UK and 14 (13%) from Saudi had respiratory infections. Rhinovirus in 19 UK pilgrims (13%), and influenza virus in 11 Saudi pilgrims 10%, were the most frequent detectable viruses. Influenza rates were similar in both groups (10%) like in our study.7 In two other studies in Saudi Arabia, influenza virus was the most common virus detected by viral culture and immunofluorescence.8–9

Most Iranian pilgrims (85.5%) were vaccinated against influenza 2 weeks before departure. In a cohort of 461 Hajj pilgrims to Mecca from Marseille, only 6% received vaccination against influenza in 2005 on the basis of vaccination certificates produced.10 Influenza virus was identified more in unvaccinated (6 of 37, 16.5%) than in vaccinated (20 of 218, 9.2%) pilgrims but statistically insignificant (p= 0.19). The reasons for the development of influenza among vaccinated pilgrims might be as follows: the appearance of new drift variants not included in vaccine and also inappropriate vaccine handling and storage. In a report on the efficacy of influenza vaccination in Iranian pilgrims on the basis of clinical manifestations rather than lab findings, it was revealed that vaccine was effective in the year 2003 (51%) but not effective in 2004.11 In a previous study about influenza among 115 UK pilgrims to the Hajj, the attack rate of influenza was 30% among the vaccinated and 41% among the nonvaccinated pilgrims (p= 0.28).12 Considering the above findings and since the pilgrimage is not changeable, it seems more supportive personal hygienic measures are needed to reduce the incidence of respiratory infections and promote health conditions during Hajj pilgrimage.

The evolution in the demographics and figures of international travel is raising the importance of movements from areas of low disease prevalence to high incidence destinations and vice versa.13 Several factors contribute to the widespread transmission of ARIs including direct contact with affected people, change in climate, and crowded places, all of which are potentially present in the Hajj environments.14 More than three fourths (76.5%) of the patients had developed ARI within the past 2 weeks in Mecca, which confirmed the high transmission of ARI in Hajj pilgrims in Mecca.

Cough (83.5%) and sore throat (82%) were the most common symptoms in Hajj pilgrims but fever was present in only 60% of patients, like the other study addressing Pakistani pilgrims.

In this report, the influenza‐like illnesses attack rates among 2,070 Pakistani pilgrims were 36 and 62% in vaccinated persons and controls, respectively. This study was based on only clinical manifestations. Compared to influenza‐like illness, fewer people developed fever while almost all the studied subjects reported upper respiratory tract infection symptoms.15

Culture was not found to be a suitable method for the detection of respiratory viruses in our study. This might be due to the interval between the appearance of symptoms and performing viral culture. The results would have been different if the samples were collected at the Hajj spots during the acute phase of the illness. We did not detect viral pathogens in 67.5% of nasal wash specimens in Iranian pilgrims with ARI and other causes like bacteria, environmental allergens, and mycotic spores might be considered. Furthermore, to identify more viruses and promote etiologic diagnosis, PCR could be helpful.

Conclusions

According to the results of this study, viral respiratory tract infections seem to constitute an important portion of respiratory symptoms in Iranian pilgrims, and influenza virus plays a critical role in the development of such symptoms, as demonstrated in some other studies conducted on the pilgrims from other nations. Because influenza vaccine could not prevent respiratory infections in Hajj pilgrims statistically, the possibility of the appearance of new drift variants and also inappropriate vaccine handling and storage might be considered. It is also advisable to check if the circulating influenza strains were different from the vaccine strains. Since rhinovirus and RSV are spread by direct hand contact, personal hygiene, especially hand washing and regular use of alcohol‐based hand scrubs, should be emphasized.16

Our special thanks to Dr. Mohammad Salah, the chief of security at Shiraz airport, for his close cooperation. We also express our deep gratitude to the board of Hajj physicians affiliated with Iranian Red Crescent, for their highly appreciated practical collaboration with the study. Finally, we would also thank Hassan Khajehei for valuable care and skill in copy editing the article.

Declaration of interests

The authors state that they have no conflicts of interest.

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