Augmentation of DTH reaction of mycobacterial antigenic cocktail using synthetic mycobacterial 19-kDa lipoprotein as a TLR-stimulant.

The current study proposed that previously characterized individual antigenic proteins could represent potential replacement for conventional purified protein derivative (PPD) in tuberculosis skin testing when used in cocktails triggered by suitable TLR-stimulants that would provide the missing pro-inflammatory stimulus. Three different cocktails of previously selected antigens, including C1 (ESAT-6/CPF-10/MPB-83); C2 (ESAT-6/MPB-64/MPB-83); and C3 (CPF-10/MPB-64/MPB-83), were evaluated in vitro using lymphocytic proliferation and IFN-γ production assays, as well as mRNA and protein expression levels of TNF-α, IL-12p40, and IL-2 as pro-inflammatory molecules. C1 showed the highest significant induction of pro-inflammatory molecules as compared to other cocktails, yet still significantly lower than that induced by conventional PPD. Interestingly, inclusion of the synthetic Mycobacterium tuberculosis 19-kDa lipoprotein (Pam3Cys-SSNKSTTGSGETTTA) as a TLR-stimulant resulted in obvious augmentation of C1-induced pro-inflammatory molecules to levels comparable to that of PPD. In addition, skin testing using sensitized guinea pig model revealed comparable significant reaction to that of conventional PPD. ESAT-6/CPF-10/MPB-83 cocktail is suggested as a potential alternative skin-testing reagent when used in combination with the M. tuberculosis 19-kDa lipoprotein as a TLR-stimulant.

Characterisation of vitamin D-related molecules and calcium-sensing receptor in human Fallopian tube during the menstrual cycle and in ectopic pregnancy.

This is a prospective observational study that measures the expression of vitamin D (VD) metabolising and signalling molecules and Ca2+ sensing receptor (CaSR) in human Fallopian tube (FT) during the menstrual cycle and ectopic pregnancy (EP). Fresh FTs were obtained during total abdominal hysterectomy at the follicular (n = 16) and midluteal (n = 16) phases. Specimens from remote and implantation sites as well as trophoblastic tissues were also freshly collected from each FT with EP (n = 10). All women had normal serum VD and ionised Ca2+. The expression of VD synthesising (CYP27B1) and catalysing (CYP24A1) enzymes, binding protein (VDBP), receptor (VDR), retinoid X receptor (RXR) and CaSR was measured by immunohistochemistry and quantitative RT-PCR. All molecules, except VDBP, were significantly increased (P < 0.05) in midluteal compared with follicular samples. Remote EP sites showed significantly (P < 0.05) lower expression of CYP27B1, CYP24A1, VDR and RXR and a higher expression of VDBP and CaSR (P < 0.05) compared with midluteal samples. Significant differences were observed by immunohistochemistry between implantation and remote sites from EP for all molecules, which were also localised in the trophoblastic tissues. In conclusion, VD and calcium are under cycle-dependent regulations within human FT and they appear to play a role in tubal biology through paracrine/autocrine mode of signalling. Furthermore, EP was associated with alterations in the expression of all the studied molecules by the tubal epithelium. Further studies are needed to explore the roles of VD in tubal biology and pathogenesis of EP.

The prevalence of dengue virus serotypes in asymptomatic blood donors reveals the emergence of serotype 4 in Saudi Arabia.

BACKGROUND: Transmission of dengue virus (DENV) through blood transfusion has been documented and hence screening for DENV during blood donation has been recently recommended by the American Association of Blood Banks and Centres of Disease Control and Prevention. DENV is endemic in the Western province of the Kingdom of Saudi Arabia (KSA) and serotypes 1, 2 and 3, but not 4, have been detected. However, little is known regarding the rates of DENV during blood donation in the kingdom. The aim of this study was therefore to measure the prevalence of dengue virus and its serotypes in eligible Saudi blood donors in the endemic Western region of KSA. METHODS: This was a cross-sectional study and serum samples were collected from 910 eligible Saudi male blood donors. DENV IgM and IgG antibodies were measured serologically by ELISA while viral serotypes were detected by a single step IVD CE certified multiplex RT-PCR kit. RESULTS: The overall prevalence was 39 and 5.5% for IgG+ and IgM+, respectively. There were 12 (1.3%) with exclusively IgM+, 317 (34.8%) exclusively IgG+ and 38 (4.2%) with dual IgM+/IgG+ donors. The overall prevalence was 3.2% (n = 29) and 2.3% (n = 21) for primary and secondary infections. PCR was positive in 5.5% (n = 50) and, DENV-2 (n = 24; 48%) was the most frequent serotype and was significantly higher than DENV-1 (20%; P = 0.02) and DENV-3 (2%; P = 0.1 × 10-5) but not DENV-4 (30%; P = 0.2). There was no significant difference between both DENV-4 and DENV-1 (P = 0.4). The combination of the PCR and serology findings showed that 22 (2.4%) and 28 (3.1%) donors had primary and secondary viremic infections, respectively. CONCLUSIONS: The detected rates of DENV by PCR suggest a potential high risk of viral transmission by blood transfusion. To the best of our knowledge, this study is the first to report the detection of DENV-4 serotype in Saudi Arabia. More studies are required to measure the precise prevalence of DENV serotypes and their potential transmission rate during blood donation in the kingdom.

Complete genome sequencing and phylogenetic analysis of dengue type 1 virus isolated from Jeddah, Saudi Arabia.

BACKGROUND: Dengue viruses (DENVs) are mosquito-borne viruses which can cause disease ranging from mild fever to severe dengue infection. These viruses are endemic in several tropical and subtropical regions. Multiple outbreaks of DENV serotypes 1, 2 and 3 (DENV-1, DENV-2 and DENV-3) have been reported from the western region in Saudi Arabia since 1994. Strains from at least two genotypes of DENV-1 (Asia and America/Africa genotypes) have been circulating in western Saudi Arabia until 2006. However, all previous studies reported from Saudi Arabia were based on partial sequencing data of the envelope (E) gene without any reports of full genome sequences for any DENV serotypes circulating in Saudi Arabia. FINDINGS: Here, we report the isolation and the first complete genome sequence of a DENV-1 strain (DENV-1-Jeddah-1-2011) isolated from a patient from Jeddah, Saudi Arabia in 2011. Whole genome sequence alignment and phylogenetic analysis showed high similarity between DENV-1-Jeddah-1-2011 strain and D1/H/IMTSSA/98/606 isolate (Asian genotype) reported from Djibouti in 1998. Further analysis of the full envelope gene revealed a close relationship between DENV-1-Jeddah-1-2011 strain and isolates reported between 2004-2006 from Jeddah as well as recent isolates from Somalia, suggesting the widespread of the Asian genotype in this region. CONCLUSIONS: These data suggest that strains belonging to the Asian genotype might have been introduced into Saudi Arabia long before 2004 most probably by African pilgrims and continued to circulate in western Saudi Arabia at least until 2011. Most importantly, these results indicate that pilgrims from dengue endemic regions can play an important role in the spread of new DENVs in Saudi Arabia and the rest of the world. Therefore, availability of complete genome sequences would serve as a reference for future epidemiological studies of DENV-1 viruses.

Prevalence of 7 sexually transmitted organisms by multiplex real-time PCR in Fallopian tube specimens collected from Saudi women with and without ectopic pregnancy.

BACKGROUND: Ectopic pregnancy (EP) is associated with maternal morbidity and occasionally mortality during the first trimester. A history of sexually transmitted infection (STI) and pelvic inflammatory disease have been implicated as major risk factors for EP. Our aim was to measure the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae, Mycoplasma genitalium (MG), Ureaplasma parvum/urealyticum, Gardnerella vaginalis, Trichomonas vaginalis and herpes simplex virus (HSV)-1&2 in Fallopian tubes collected from EP and the results were compared with those obtained from total abdominal hysterectomy (TAH) and tubal ligation. METHODS: This was a prospective case-control study and tubal samples were collected from 135 Saudi women recruited from 3 centres in the Western region as follow: 84 EPs, 20 TAH and 31 tubal ligations. Multiplex TaqMan PCR was performed using an IVD CE kit for the simultaneous detection of candidate pathogens following DNA extraction. RESULTS: Infections were detected in 31.8 % of the 135 participants either as single (11.1 %) or co-infections (20.7 %) and the frequencies were significantly higher in EP (42.85 %) compared with control (13.72 %). The rates of CT (27.4 %; P = 0.001); MG (20.2 %; P = 0.009) and HSV-1/2 (21.4 %; P = 0.01) were significantly higher in EP. No significant difference between the study groups was observed for the other pathogens (P > 0.05). Binary logistic regression also showed that infection with ≥ 2 pathogens (OR 4.9; 95 % CI: 2.2 - 11.6; P = 0.006), CT (OR 3.07; 95 % CI: 1.3 - 12.3; P = 0.002), MG (OR 2.3; 95 % CI: 1.1 - 8.6; P = 0.03) and HSV-1/2 (OR 1.7; 95 % CI: 0.75 - 5.7; P = 0.004) were associated with a significantly higher risk of developing EP. CONCLUSIONS: STIs are frequent in the upper genital tract of Saudi women during the reproductive age and, CT, MG and HSV-1/2 were more prevalent in EP. The observed high rates of co-infection advocate the necessity of establishing national guidelines and/or screening program utilising multiplex PCR approach for the detection of common STIs among high risk groups in the kingdom. Further studies are needed to measure the adverse reproductive outcomes associated with STIs in Saudi Arabia.

Activins and Follistatin in Chronic Hepatitis C and Its Treatment with Pegylated-Interferon-α Based Therapy.

Pegylated-interferon-α based therapy for the treatment of chronic hepatitis C (CHC) is considered suboptimal as not all patients respond to the treatment and it is associated with several side effects that could lead to dose reduction and/or termination of therapy. The currently used markers to monitor the response to treatment are based on viral kinetics and their performance in the prediction of treatment outcome is moderate and does not combine accuracy and their values have several limitations. Hence, the development of new sensitive and specific predictor markers could provide a useful tool for the clinicians and healthcare providers, especially in the new era of interferon-free therapy, for the classification of patients according to their response to the standard therapy and only subscribing the novel directly acting antiviral drugs to those who are anticipated not to respond to the conventional therapy and/or have absolute contraindications for its use. The importance of activins and follistatin in the regulation of immune system, liver biology, and pathology has recently emerged. This review appraises the up-to-date knowledge regarding the role of activins and follistatin in liver biology and immune system and their role in the pathophysiology of CHC.

Pegylated Interferon-α Modulates Liver Concentrations of Activin-A and Its Related Proteins in Normal Wistar Rat.

AIMS: To measure the expression of activin ßA-subunit, activin IIA and IIB receptors, Smad4, Smad7, and follistatin in the liver and the liver and serum concentrations of mature activin-A and follistatin in normal rat following treatment with pegylated interferon-α (Peg-INF-α) and ribavirin (RBV). MATERIALS AND METHODS: 40 male Wistar rats were divided equally into 4 groups: "control," "Peg-only" receiving 4 injections of Peg-INF-α (6 µg/rat/week), "RBV-only" receiving ribavirin (4 mg/rat/day) orally, and "Peg & RBV" group receiving both drugs. The expression of candidate molecules in liver was measured by immunohistochemistry and quantitative PCR. The concentrations of mature proteins in serum and liver homogenate samples were measured using ELISA. RESULTS: Peg-INF-α ± RBV altered the expression of all candidate molecules in the liver at the gene and protein levels (P < 0.05) and decreased activin-A and increased follistatin in serum and liver homogenates compared with the other groups (P < 0.05). There were also significant correlations between serum and liver activin-A and follistatin. CONCLUSION: Peg-INF-α modulates the hepatic production of activin-A and follistatin, which can be detected in serum. Further studies are needed to explore the role of Peg-INF-α on the production of activins and follistatin by the liver and immune cells.

Seroprevalence of Asymptomatic Dengue Virus Infection and Its Antibodies Among Healthy/Eligible Saudi Blood Donors: Findings From Holy Makkah City.

BACKGROUND: Threat to blood transfusion-transmitted dengue virus (DENV) and its antibodies has recently emerged worldwide. Dengue fever is an endemic disease in Saudi Arabia, particularly in its Western region. The aim of this study was to estimate the seroprevalence of asymptomatic DENV infection and its antibodies among eligible Saudi blood donors. METHODS: Serum samples from 910 healthy/eligible adult male Saudi blood donors, who reside in Holy Makkah City of Saudi Arabia, were collected between March 2015 and August 2016 and screened for the detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM and IgG antibodies using commercial enzyme-linked immunosorbent assay kits (Panbio, Brisbane, QLD, Australia). RESULTS: Among the tested donors, 48 (5.3%) were seropositive for DENV-NS1 antigen, whereas 50 (5.5%) and 354 (38.9%) were seropositive for anti-DENV IgM and IgG antibodies, respectively. Seropositivity for DENV-NS1 antigen and/or anti-DENV IgM antibody among the tested donors reflects their ongoing asymptomatic viremic infectious stage with DENV during their donation time, whereas high prevalence of anti-DENV IgG seropositivity reflects the high endemicity of dengue disease in this region of Saudi Arabia. CONCLUSIONS: These results show high prevalence of asymptomatic DENV infection and its antibodies among Saudi blood donors, raising the importance of establishing blood screening for dengue disease at different blood donation services and units in Saudi Arabia to improve the guarantee of blood transfusions and to control DENV dissemination.

Aberrant expression of interleukin-6 and its receptor in Fallopian tubes bearing an ectopic pregnancy with and without tubal cytomegalovirus infection.

Cytomegalovirus (CMV) has recently been suggested as a potential risk factor for the development of ectopic pregnancy (EP) following upper genital tract infection in women. However, little is known about its associated underlying pathogenic mechanisms. This was a prospective case-control study that measured the prevalence of CMV infection in Fallopian tubes (FT) bearing an EP and its effects on the tubal expression of interleukin (IL)-6 and its signaling molecules, which are known to play significant roles in the immune response against CMV infection as well as embryo implantation. Fresh FTs from 96 EPs during salpingectomy and another 61 women at the midluteal phase during total abdominal hysterectomy (TAH) were collected to measure the rate of CMV by an IVD CE PCR kit. The participants were then classified to measure the expression of IL-6, its receptor (IL6R) and intracellular mediators (gp-130, STAT3) by immunohistochemistry and quantitative RT-PCR. The results showed significantly higher (P = 0.01) rates of CMV in FTs obtained from EP (22.9 %) compared with controls (8.2 %). IL-6 (P = 0.003), IL6Rα (P = 0.02), gp 130 (P = 0.008) and STAT3 (P = 0.03) were significantly higher in TAH-positive (n = 5) compared with TAH-negative FTs by immunohistochemistry. Furthermore, the expression in the non-infected EP samples was significantly higher for IL-6 (P = 0.004), IL6R (P = 0.007), gp130 (P = 0.006) and STAT3 (P = 0.007) compared with negative TAH. Similar results were observed by quantitative PCR. CMV-positive EP samples showed the highest significant increase of the studied molecules by all techniques. In conclusion, Fallopian tubal infection with CMV is higher in EP and could predispose to embryo implantation by up-regulating the expression of IL-6 and its related molecules as part of tubal innate immune response. Further in vitro and in vivo studies are compulsory to illustrate the roles of IL-6 and CMV in the pathogenesis of EP.

The prevalence of Chlamydia trachomatis and Mycoplasma genitalium tubal infections and their effects on the expression of IL-6 and leukaemia inhibitory factor in Fallopian tubes with and without an ectopic pregnancy.

This was a prospective case-control study that measured the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG) by an IVD CE multiplex PCR kit in fresh Fallopian tubes (FT) obtained from 96 ectopic pregnancies (EP) and 61 controls in the midluteal phase of the cycle. We later measured the expression profile of IL-6, leukaemia inhibitory factor (LIF) and their signalling molecules, in respect to the type and number of infections, by immunohistochemistry, ELISA and quantitative RT-PCR. The frequencies of CT, and MG mono- and co-infections were significantly higher in EP. IL-6, LIF, their receptors and intracellular mediators were significantly up-regulated at the gene and protein levels in positive compared with negative FTs within each group (P < 0.05). EP tubal samples with co-infections showed the highest significant expression of the candidate cytokines by all techniques (P < 0.05). CT and MG are frequent in EP and up-regulate the tubal expression of IL-6, LIF and their signalling molecules. Both cytokines could be involved in the tubal immune response against bacterial infections, as well as the pathogenesis of EP. Further studies are needed to explore the roles of IL-6 family in infection-induced tubal inflammation and EP.

Vitamin D and chronic hepatitis C: effects on success rate and prevention of side effects associated with pegylated interferon-α and ribavirin.

Chronic hepatitis C (CHC) is one of the most common causes of liver diseases worldwide, affecting 3% of the world population and 3 to 4 million people acquire new infection annually. Despite the recent introduction of novel antiviral drugs for the treatment of CHC, these drugs are expensive and the access to them is not an option for many patients. Hence, the traditional therapy by pegylated interferon-α (Peg-IFN-α) and ribavirin may still have a role in the clinical management of CHC especially in developing countries. However, this standard therapy is associated with several severe extra-hepatic side effects and the most common adverse events are hematological abnormalities and thyroid disorders and they could result in dose reduction and/or termination of therapy. Vitamin D has been shown to be a key regulatory element of the immune system, and its serum concentrations correlate with the severity of liver damage and the development of liver fibrosis/cirrhosis. Furthermore, supplementation with vitamin D with Peg-IFN-α based therapy for the treatment of CHC could be beneficial in increase the response rate to Peg-INF-α based therapy. Vitamin D has also been shown to regulate the thyroid functions and the process of erythropoiesis. This review appraises the data to date researching the role of vitamin D during the treatment of CHC and the potential role of vitamin D in preventing/treating Peg-IFN-α induced thyroiditis and anemia during the course of treatment.

The effects of pegylated interferon-α and ribavirin on liver and serum concentrations of activin-A and follistatin in normal Wistar rat: a preliminary report.

BACKGROUND: Activin-A and follistatin regulate the liver and the immune system. AIMS: To measure the effects of treatment with pegylated-interferon-α (Peg-IFN-α) and ribavirin on the concentrations of mature activin-A and follistatin in serum and liver tissue homogenates in rats. METHODS: A total of 28 male Wistar rats were divided equally into four groups as follow: 'Control group' (n = 7), 'PEG only group' consisted of those that only received a weekly injection of Peg-IFN-α (6 µg/rat) for 4 weeks, 'RBV only group' received ribavirin only (4 mg/rat/day) orally for 35 days and the last group received both Peg-IFN-α and ribavirin 'PEG & RBV group'. The concentrations of candidate proteins in serum and liver samples were measured using ELISA. RESULTS: Pegylated-interferon-α decreased activin-A and increased follistatin significantly in serum and liver of 'PEG only' and 'PEG & RBV' groups compared with the 'Control' and 'RBV only' groups (P < 0.05). There was no significant difference between the 'RBV only' and 'Control' groups (P > 0.05) in the concentrations of candidate proteins. A significant positive correlations between serum and liver activin-A (r = 0.727; P = 0.02 × 10(-3)) and follistatin (r = 0.540; P = 0.01) was also detected. CONCLUSION: Pegylated-interferon-α modulates the production of activin-A and follistatin by the liver, which is reflected and can be detected at the serum level. Further studies are needed to explore the role of Peg-IFN-α based therapy on the production of activins and follistatin by the liver and immune cells.

Effects of chronic hepatitis C genotype 1 and 4 on serum activins and follistatin in treatment naïve patients and their correlations with interleukin-6, tumour necrosis factor-α, viral load and liver damage.

The importance of activins and follistatin in liver diseases has recently emerged. The aim of the present study was to measure the influence of chronic infection with viral hepatitis C (CHC) genotype 1 and 4 on serum levels of activin-A, activin-B and follistatin, and to determine their correlations with viral load, liver damage, interleukin-6 (IL-6) and tumour necrosis factor (TNF)-α. Sera samples collected from 20 male and 20 female treatment naïve CHC genotype 1 and 4 Saudi patients (ten males and ten females for each genotype), and 40 gender- and age-matched healthy participants were analysed for activin-A, activin-B and follistatin using enzyme-linked immunosorbent assay and their levels were correlated with IL-6, TNF-α, viral load and AST platelet ratio index (APRI). Serum activin-A, activin-B, IL-6 and TNF-α were significantly increased, while serum follistatin was significantly decreased, in both genders of CHC patients compared with control subjects, In both viral genotypes, activin-A was strongly and positively correlated with the viral load, APRI, IL-6 and TNF-α, and negatively with albumin (P < 0.01). Activin-B showed the same correlations of activin-A only in CHC genotype 1 patients, but it was weaker than activin-A. No correlation was detected with follistatin. Serum activins, particularly activin-A, and follistatin are significantly altered by CHC genotype 1 and 4. This dysregulation of activins/follistatin axis may be associated with viral replication, host immune response and liver injury. Further studies are needed to illustrate the definite role(s) and clinical value of activins and follistatin in CHC.

Detection of human cytomegalovirus, human herpesvirus type 6 and human herpesvirus type 7 in urine specimens by multiplex PCR.

OBJECTIVES: To develop a sensitive multiplex PCR to detect HCMV, HHV6 and HHV7, to test this PCR on urine specimens sent to the virus diagnostic laboratory and on stored urine samples from HIV-positive patients and their HIV-negative partners and to compare the sensitivity of the multiplex PCR with the diagnostic laboratory's routine service for the detection of HCMV. STUDY DESIGN: Primers specific for each of the three viruses were combined in a multiplex PCR that was then optimised for sensitivity. This PCR was applied prospectively to 413 unselected routine urine specimens over a 1 year period and retrospectively to 258 urine specimens from 63 HIV-positive patients and 10 HIV-negative partners. METHODS: In the prospective study, the multiplex PCR detected 40 specimens positive for HCMV alone, 10 for HHV6, 3 for HHV7 and 3 with a dual infection of HCMV and HHV6. The sensitivity for HCMV was 93.5% by multiplex PCR compared to 28.3% by culture. HHV6 DNA was detected in 6 neonates (2-21 days) and HHV7 DNA in 2 neonates (4 and 20 days). In the retrospective study of HIV patients, HCMV was the most commonly detected virus (55.6%) compared to HHV6 (7.9%) and HHV7 (4.8%). CONCLUSIONS: . The multiplex PCR was significantly more sensitive than non-DNA based procedures for the detection of HCMV. Urine may be a useful non-invasive specimen for the detection of HHV6 and HHV7 and their presence in neonates suggest perinatal transmission or the possibility of in utero infection.

Serum Activins and Follistatin during the Treatment of Chronic Hepatitis C Genotypes 1 and 4 and Their Correlations with Viral Load and Liver Enzymes: A Preliminary Report.

Aims. To measure the effect of pegylated interferon- α therapy on serum activin-A, activin-B, and follistatin and their correlation with viral load and liver fibrosis in chronic hepatitis C (CHC). Methods. This study was cross-sectional and sera were collected from 165 participants classified into 7 groups: 40 healthy negative control, 33 treatment naïve patients as positive control, 19 patients at week 4, 22 at week 12, and 19 at week 24 of treatment initiation and 21 responders and 11 nonresponders at the end of 48-week treatment protocol. Serum candidate proteins were measured using ELISA and liver fibrosis was assessed by AST platelet ratio index (APRI). Results. CHC significantly increased activins and decreased follistatin compared to negative control (P < 0.05). Activin-A and follistatin levels returned to the levels of negative control group at weeks 4, 12, and 24 following treatment initiation and were significantly different from positive control (P < 0.05). Both proteins were significantly different between responders and nonresponders. Activin-A correlated positively and significantly with the viral load and APRI. Conclusion. CHC modulates serum activin-A and follistatin and they appear to be influenced by pegylated interferon- α therapy. Further studies are needed to explore the role of activins in CHC.

Demographic distribution and transmission potential of influenza A and 2009 pandemic influenza A H1N1 in pilgrims.

INTRODUCTION: The World Health Organization's persistent reporting of global outbreaks of influenza A viruses, including the 2009 pandemic swine A H1N1 strain (H1N1pdm09), justified the targeted surveillance of pilgrims during their annual congregation that pools more than two million people from around 165 nations in a confined area of Makkah city in the Kingdom of Saudi Arabia (KSA). METHODOLOGY: A total of 1,600 pilgrims were included in the targeted surveillance of influenza A and the 2009 pandemic swine H1N1 strain in the Hajj (pilgrimage) season of 2010. Each pilgrim responded to a demographic and health questionnaire. Collected oropharyngeal swabs were analyzed by real-time PCR for influenza A viruses, and positive samples were further analyzed for the presence of H1N1pdm09. Fisher's exact test was applied in the analysis of the significance of the distribution of influenza-positive pilgrims according to demographic characters. RESULTS: A total of 120 pilgrims (7.5%) tested positive for influenza A viruses by real-time PCR. Nine out of the 120 influenza-A-positive pilgrims (7.5%) were positive for H1N1pdm09. Demographics played a significant role in those pilgrims who tested positive for influenza A. CONCLUSIONS: The detection of H1N1pdm09 in pilgrims at their port of entry to the KSA was alarming, due to the high potential of trans-boundary transmission. This situation necessitates the implementation of specific prevention and control programs to limit infection by influenza A viruses.