Departments of 1Microbiology & Immunology and 2Haematology, Sultan Qaboos University Hospital, Muscat, Oman *Corresponding Author’s e-mail: jalila@squ.edu.om حتديد املستوى املرجعي لألجسام املناعية املضادة )نوع G( للرشاشيات أسبرييغيلوس فوميغاتوس بني السكان البالغني العمانيني منرية الرحمن، حممود الكندي، اإبراهيم كوتي، اإبراهيم الكلباين، جليلة ال�سكيلي abstract: Objectives: The presence of abnormally high levels of Aspergillus fumigatus-specific immunoglobulin (Ig) G antibodies can serve as a diagnostic criterion for severe conditions like allergic bronchopulmonary and chronic pulmonary aspergillosis. This study aimed to determine a reference range of A. fumigatus-specific IgG levels within a healthy adult Omani population. Methods: This study took place during November 2015 at the Sultan Qaboos University Hospital, Muscat, Oman. The sera of 125 healthy Omani blood donors were tested for A. fumigatus- specific IgG levels using an automated fluorescence enzyme immunoassay. Results: Initially, the data were not normally distributed; however, log transformation and the exclusion of four outliers resulted in an acceptable Gaussian distribution. The reference range was 2.0–68.7 mgA/L at the 2.5 th and 97.5th percentiles, respectively, with 90% confidence intervals of 2.0–3.0 mgA/L and 48.0–76.0 mgA/L, respectively. Conclusion: The A. fumigatus- specific IgG reference range within a healthy adult Omani population was comparable to those reported in other populations. Keywords: Aspergillus fumigatus; Allergic Bronchopulmonary Aspergillosis; Pulmonary Aspergillosis; Immuno- globulin G; Reference Range; Oman. يعترب احلالتني كلتي ويف فطري. اإلتهاب اأو ح�سا�سية اإحداث خالل من لالإن�سان خمتلفة اأمرا�ض الر�سا�سيات ت�سبب الهدف: امللخ�ص: الق�سبي الر�سا�سيات داء لت�سخي�ض الرئي�سة الركائز اأحد فوميغاتو�ض للر�سا�سيات )G )نوع امل�سادة املناعية االأج�سام م�ستوى حتديد الرئوي التح�س�سي اأو داء الر�سا�سيات الرئوي املزمن. وال يوجد حاليا معدل مرجعي �سواء اأكان حمليا اأو اإقليميا ميكن ا�ستخدامه لدعم هذه فوميغاتو�ض. للر�سا�سيات )G )نوع امل�سادة املناعية االأج�سام مل�ستوى املرجعي امل�ستوى حتديد اإىل الدرا�سة هذه هدفت الت�سخي�سات. الطريقة: اأجريت هذه الدرا�سة خالل �سهر نوفمرب2015 يف م�ست�سفى جامعة ال�سلطان قابو�ض، م�سقط، �سلطنة عمان اىلمت فح�ض جمموعة من العينات ماأخوذة من االأ�سحاء العمانيني املتربعني بالدم والبالغ عددهم 125 وذلك با�ستخدام نظام اإميونوكاب من فاديا اأوب�ساال، )G )نوع امل�سادة املناعية االأج�سام مل�ستوى االأولية القيا�سات تتبع مل النتائج: العلمية(. في�رص احلرارية با�سم االآن )املعروفة ال�سويد للر�سا�سيات فوميغاتو�ض منحنى املعدل الطبيعي مما تتطلب تطبيق التحويل اللوغاريثمي على البيانات والذى اأدى بدوره اإىل احل�سول على توزيع طبيعي ومقبول للبيانات. ومن ثم مت ح�ساب امل�ستوى املرجعي لالأج�سام املناعية امل�سادة )نوع G( للر�سا�سيات فوميغاتو�ض 97.5th وات�سح انها ترتاوح بني 2.0 مليغرام/لرت و 68.7 مليغرام/لرت بفوا�سل ثقة %90 ترتاوح 2.5th و با�ستخدام طريقة الن�سب املئوية )G )نوع امل�سادة املناعية لالأج�سام املرجعي امل�ستوى اخلال�صة: التوايل. على مليغرام/لرت 48.0–76.0 و مليغرام/لرت 2.0–3.0 بني للر�سا�سيات فوميغاتو�ض للبالغني االأ�سحاء العمانيني م�سابه ملا مت ر�سده يف جمتمعات اخرى. الكلمات املفتاحية: الر�سا�سيات فوميغاتو�ض؛ داء الر�سا�سيات الق�سبي الرئوي التح�س�سي؛ داء الر�سا�سيات الرئوي املزمن؛ امل�ستوى املرجعي؛ عمان. Determination of an Aspergillus fumigatus-Specific Immunoglobulin G Reference Range in an Adult Omani Population Munira Al-Rahman,1 Mahmood Al Kindi,1 Ibrahim Kutty,1 Ibrahim Al-Kalbani,2 *Jalila Alshekaili1 clinical & basic research Sultan Qaboos University Med J, February 2018, Vol. 18, Iss. 1, pp. e43–46, Epub. 4 Apr 18 Submitted 3 Oct 17 Revision Req. 5 Nov 17; Revision Recd. 26 Nov 17 Accepted 14 Dec 17 doi: 10.18295/squmj.2018.18.01.007 Advances in Knowledge - To the best of the authors’ knowledge, this study is the first in Oman and the Arabian Gulf region to evaluate the reference range of Aspergillus fumigatus-specific immunoglobulin G (IgG) levels in a local population. Application to Patient Care - The findings of the current study may guide clinicians in establishing diagnoses of allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis at the Sultan Qaboos University Hospital, Muscat, Oman. The Aspergillus genus is an extremely abundant type of airborne fungi constantly inhaled by humans which, for the most part, does not cause significant illness; however, individuals with pre-existing lung pathologies or weak- ened immune systems can develop Aspergillus-related diseases known as aspergillosis.1 Most cases of asper- gillosis are caused by A. fumigatus (80%), although other species can also cause infection, such as A. flavus, A. terreus and A. niger.1–3 Aspergillosis caused by Determination of an Aspergillus fumigatus-Specific Immunoglobulin G Reference Range in an Adult Omani Population e44 | SQU Medical Journal, February 2018, Volume 18, Issue 1 A. fumigatus presents with either allergies—including allergic Aspergillus sinusitis and allergic bronchopul- monary aspergillosis (ABPA)—or infections, such as local aspergillomas affecting the lungs and sinuses, necrotising chronic pulmonary aspergillosis (CPA) and systemic invasive aspergillosis.4–6 Usually, ABPA or CPA is diagnosed following assessment of the patient’s medical history for risk factors, a physical examination, radiological imaging and laboratory investigations. To this end, elevated levels of A. fumigatus-specific immunoglobulin (Ig) G antibodies are considered the main clinical criterion to support the diagnosis.4 However, there is currently no published reference range of anti-A. fumigatus-specific IgG levels for healthy adult populations in Arab countries. There- fore, the aim of this study was to establish a reference range for A. fumigatus-specific IgG antibodies in an Omani population. Hopefully, this reference range will guide clinicians in Oman when establishing diagnoses of ABPA and CPA. Methods This study took place in November 2015 at the Sultan Qaboos University Hospital (SQUH) in Muscat, Oman. Samples of sera were collected from 125 healthy adult donors attending the SQUH Blood Bank during the study period. Only healthy adult donors without any previous history of lung disease or asthma and no prior significant exposure to birds were included. Four out- liers were excluded from the initial sample; however, the final sample of 121 subjects complied with the minimum sample size recommended by the Clinical and Laboratory Standards Institute (CLSI) for establish- ing a reference range.7–9 A total of 5–10 mL of blood was obtained from each subject. The sera were immediately separated and stored at -80 °C until analysis. Specific IgG antibodies against A. fumigatus were determined using an auto- mated fluorescence enzyme immunoassay (Immuno- CAP® system, Thermo Fisher Scientific Inc., Phadia AB, Uppsala, Sweden), as per the manufacturer’s instruc- tions. The assay was performed by an experienced senior biomedical scientist in the Immunology Laboratory of SQUH. Results were expressed as milligrams of Aspergillus-specific IgG antibodies per litre (mgA/L). All reagents were received from the same lot number and each run included internal quality control samples. The interassay coefficient of variation between runs for the quality control samples was <10%, as per the manufacturer’s instructions. Data were analysed using the MedCalc® statistical software package, Version 16.4.3 (MedCalc Software, Ostend, Belgium). A non-parametric percentile method following the CLSI guidelines was used on log-trans- formed data to establish the reference range at a 90% confidence interval (CI).8,9 The level of statistical signifi- cance was set at P <0.050. Ethical approval for this study was obtained from the Research & Ethics Committee of the College of Medicine & Health Sciences, Sultan Qaboos University (MREC #1220-A). All subjects gave informed written consent prior to their participation in the study. Results The median age of the subjects was 24 years (range: 18–52 years old). Most of the donors were male, with a female-to-male ratio of 1:9.3. Initially, a frequency histogram and quantile-quantile plot to assess the distri- bution of the collected data indicated that the data were not normally distributed [Figure 1]. This finding was further supported by the results of a D’Agostino- Pearson test (P <0.001). Accordingly, the data were log-transformed for subsequent analysis. However, the data were still not normally distributed [Figure 2]. A Tukey’s test identified four outliers, the exclusion of which resulted in the normal Gaussian distribution of the log-transformed data of the remaining 121 samples [Figure 3]. The reference range of A. fumigatus-specific Figure 1: A: Relative frequency histogram of Aspergillus fumigatus-specific immunoglobulin G (IgG) antibodies in the sera of healthy Omani blood donors (N = 125). B: Quantile-quantile plot of results obtained for A. fumigatus-specific IgG antibodies in the sera of healthy Omani blood donors (N = 125). IgG = immunoglobulin G. Munira Al-Rahman, Mahmood Al Kindi, Ibrahim Kutty, Ibrahim Al-Kalbani and Jalila Alshekaili Clinical and Basic Research | e45 IgG for these 121 samples was 2.0–76.0 mgA/L, with a geo- metric mean of 10.9 mgA/L and median of 11.0 mgA/L. The 2.5th percentile was 2.0 mgA/L with a 90% CI of 2.0–3.0 mgA/L and the 97.5 th percentile was 68.7 mgA/L with a 90% CI of 48.0–76.0 mgA/L. Discussion Although uncommon, both ABPA and CPA are serious diseases that can affect immunocompetent patients with underlying lung pathologies. The former is a severe allergic and inflammatory condition that predomi- nantly affects patients with asthma or cystic fibrosis and presents with fever, cough, shortness of breath, wheezing and haemoptysis; moreover, patients become sensitised to A. fumigatus, causing significant morbidity and mortality.10 Immunological mechanisms in ABPA that result in pathology are mainly type I immediate and type III immune complex hypersensitivity reactions mediated by IgE and IgG antibodies, respectively.4 The primary treatment for ABPA is oral cortico- steroids to control the inflammatory process, while anti-fungals are used only as an adjuvant therapy in order to reduce the fungal load.9 In contrast, CPA is a chronic lung infection that can progress to pulmonary fibrosis and loss of lung function; the disease presents with constitutional and respiratory symptoms including weight loss, fatigue, shortness of breath and haemoptysis.10 In general, CPA affects immunocompetent patients with pre- existing lung conditions as opposed to invasive asper- gillosis, which usually occurs in immunocompromised patients.11 Underlying lung comorbidities which may predispose a patient to CPA include chronic obstruct- ive pulmonary disease, pre-existing pulmonary cavitary tuberculosis, fibrotic sarcoidosis and ABPA. In some cases, CPA can manifest as a simple aspergilloma, chronic cavitary pulmonary aspergillosis, chronic fibro- sing pulmonary aspergillosis, subacute invasive asper- gillosis and aspergillosis nodules.11,12 For most patients, CPA is treated with antifungal agents and surgery. However, interferon-γ has been recently proposed as an adjuvant therapy.4 In the current study, the reference range of A. fumigatus-specific IgG levels among a sample of healthy adult Omani blood donors was found to be 2.0–68.7 mgA/L using the percentile method. This refer- ence range was comparable to other published ranges Figure 2: A: Relative frequency histogram of log-transformed Aspergillus fumigatus-specific immunoglobulin G (IgG) antibodies in the sera of healthy Omani blood donors (N = 125). B: Quantile-quantile plot of log-transformed data for A. fumigatus-specific IgG antibodies in the sera of healthy Omani blood donors (N = 125). IgG = immunoglobulin G. Figure 3: A: Relative frequency histogram of log-transformed Aspergillus fumigatus-specific immunoglobulin G (IgG) antibodies in the sera of healthy Omani blood donors (N = 121). B: Quantile-quantile plot of log-transformed data for A. fumigatus-specific IgG antibodies in the sera of healthy Omani blood donors (N = 121). IgG = immunoglobulin G. Determination of an Aspergillus fumigatus-Specific Immunoglobulin G Reference Range in an Adult Omani Population e46 | SQU Medical Journal, February 2018, Volume 18, Issue 1 and can potentially be used in conjunction with other parameters to diagnose patients suspected of having ABPA or CPA. Utilising the same immunoassay as that of the current study, Watkins et al. found the reference range of A. fumigatus-specific IgG levels to be 2.79–66.45 mgA/L in a group of 122 healthy adults in South Africa.13 The same method and assay techno- logy was used to detect an A. fumigatus-specific IgG range of 2.01–70.10 mgA/L in a group of 42 healthy individuals in Belgium.14 An enzyme-linked immuno- sorbent assay yielded similar results when assessing A. fumigatus IgG levels among 100 healthy Finnish children between 7–13 years old to the 95th percentile (67.8–68.3 mg/L); in addition, the researchers observed that A. fumigatus-specific IgG concentrations increased with age to reach a plateau at 6–7 years old, although the concentrations were not affected by gender.15 Agarwal et al. compared 67 patients with ABPA to 32 asthmatic controls in India; using a receiver operated characteristic curve, the researchers determined the ideal cut-off value for A. fumigatus-specific IgG to be 26.9 mgA/L. 16 However, it is important to note that the controls in this study were a small cohort of diseased rather than healthy individuals.16 To the best of the authors’ knowledge, this study is the first in the Arabian Gulf region to derive a reference range for A. fumigatus-specific IgG levels in a cohort of healthy donors. In addition, the sample size was adequate in comparison to other studies and complied with CLSI recommendations.8,9,17 Neverthe- less, this study was limited in that patients with ABPA or CPA were not included. Such patients should there- fore be included in future research so as to validate the reference range determined. In addition, due to a lack of female patients within the cohort, no differ- ences in A. fumigatus-specific IgG levels were noted between genders. Conclusion In summary, A. fumigatus-specific IgG levels are an important diagnostic parameter for patients with ABPA or CPA. In the current study, the reference range of A. fumigatus-specific IgG levels in healthy Omani donors was found to be 2.0–68.7 mgA/L at the 2.5 th and 97.5th percentiles, respectively. 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