鼻及鼻竇炎免疫學及微生物學之研究

背景與目的：鼻及鼻竇炎是人類最常見的健康問題之一，雖然許多研究深入探討鼻及鼻竇炎的病理生理學，然而該疾病的致病機轉在許多方面仍然不清楚。正常的免疫功能在鼻及鼻竇黏膜的防禦機轉上扮演重要的角色，為了研究鼻及鼻竇黏膜的免疫狀態，我們採用非侵入性的方法收集正常人、鼻過敏病患與鼻及鼻竇炎病患的鼻腔黏液並計算IgA及IgG的含量。除了免疫學之外，鼻及鼻竇炎的微生物學也相當重要，因為大部分鼻及鼻竇炎的發生都是因為ostiomeatal complex阻塞引發細菌性感染。為了知道受損的鼻及鼻竇黏膜發生細菌感染時是否會有不同的型態，我們收集患有急性鼻及鼻竇炎的鼻咽癌病患的鼻竇培養結果，以闡明鼻咽癌病患鼻及鼻竇炎的微生物學並解釋受損的鼻及鼻竇黏膜對引發鼻及鼻竇炎的影響。關於鼻竇的培養方法，一般而言上頜竇穿刺為公認獲得鼻竇細菌培養的準則，雖然證據顯示在成人使用內視鏡中鼻道培養的結果與上頜竇穿刺所獲得的培養結果相關性很高，不過目前兒童使用內視鏡中鼻道培養的可信度依然不明，因此我們的研究也比較鼻及鼻竇炎病童上頜竇穿刺與內視鏡中鼻道培養的結果，以釐清在兒童使用內視鏡中鼻道採樣培養鼻竇致病菌的可信度。最後，因為有關現今兒童慢性鼻及鼻竇炎致病菌抗藥性形式的研究仍然有限，而且鼻及鼻竇炎致病菌抗藥性的形式經常會隨著時間與不同地區而有所改變，因此我們以上頷竇穿刺蒐集罹患慢性鼻及鼻竇炎兒童的鼻竇分泌物以培養病菌株，並分析其抗藥性。這些即時及在地的資料可以提供本地臨床醫師在治療兒童頑固性慢性鼻及鼻竇炎時的重要參考。方法：根據研究的目的，我們將實驗分成四個部分：1) 我們收集20位鼻及鼻竇炎病患、25位鼻過敏病患、與24位正常人，用indicator-PBS溶液灌入鼻腔以收集鼻腔黏液，並以enzyme-linked immunosorbent assay (ELISA)的方式測量鼻腔黏液中total IgA (tIgA)、secretory IgA (sIgA) 及IgG subclasses的含量。 2) 我們蒐集鼻咽癌放射線治療後患有急性上頷竇炎的病人，以上頷竇穿刺蒐集這些病患的鼻竇分泌物並沖洗其患病的鼻竇，收集到的鼻竇分泌物則送往實驗室做細菌培養。3) 我們執行前瞻性研究，收集患有社區感染鼻及鼻竇炎的兒童，這些病童會同時接受內視鏡中鼻道培養與上頜竇穿刺培養，為了釐清兒童使用內視鏡中鼻道培養的可信度，我們計算內視鏡中鼻道培養與上頜竇穿刺培養結果的關聯性與診斷測試(diagnostic test)。若是上頜竇穿刺培養出多種菌種，則內視鏡中鼻道培養只要有一種相同的菌種即算認定是相同的培養結果。4) 以回顧病歷的方式蒐集2001年到2006年間患有社區感染鼻及鼻竇炎的兒童，以上頷竇穿刺蒐集這些病童的鼻竇分泌物並沖洗其患病的鼻竇，收集到的鼻竇分泌物則送往實驗室做細菌培養，並分析致病菌株的抗藥性。結果：1) 病患與對照組鼻腔黏液中，我們以ELISA的方式測量到tIgA、sIgA及IgG subclasses的含量。這三組中，tIgA、sIgA、IgG1、IgG2 及IgG4 的含量並沒有差異，而相較於對照組與鼻過敏病患，IgG3的含量在鼻及鼻竇炎病患這一組則有顯著的升高。 2) 本研究蒐集了20位鼻咽癌放射線治療後患有急性上頷竇炎的病人，其中14位患有單側的急性上頷竇炎，6位患有雙側的急性上頷竇炎。在這26個鼻竇採樣樣本中，22個樣本培養出一株或一株以上的細菌。最常見的嗜氧菌為α-hemolytic streptococcus及Staphylococcus aureus，其他常見的嗜氧菌包括Pseudomonas aeruginosa 及Streptococcus constellatu。 3) 本研究共收集到41套樣本做嗜氧菌培養，在這41套樣本中有32套(78.0%)內視鏡中鼻道培養的結果與上頜竇穿刺培養一致。當以上頜竇穿刺培養的結果為準則計算內視鏡中鼻道培養的診斷測試，則得到敏感度(sensitivity)為75.0%，專一度 (specificity) 為88.9%，predictive value of a positive result為96.0%，predictive value of a negative result 為50.0%，而正確率(accuracy)為78.0%。 4) 本研究分析169名兒童的資料，一共獲得301份檢體，其中分離出403支菌株。最常見的菌種依序是α-hemolytic streptococcus，Hemophilus influenzae，Streptococcus pneumonia，coagulase-negative Staphylococcus以及Staphylococcus aureus，厭氧菌則佔所有菌株的7.9%。在前三年，發現對ampicillin不具敏感性的Hemophilus influenzae佔55.3%，對co-trimoxazole不具敏感性的佔57.9%，而在後三年，對ampicillin不具敏感性的Hemophilus influenzae佔75%，對co-trimoxazole不具敏感性的佔60% 。此外，在前三年對penicillin不具敏感性的Streptococcus pneumonia佔16.7%，對erythromycin不具敏感性的佔100%，對clindamycin不具敏感性的佔66.7%，而在後三年，對penicillin不具敏感性的Streptococcus pneumonia佔26.3%，對erythromycin不具敏感性的佔94.7%，對clindamycin不具敏感性的佔71.1%。結論：我們的研究顯示，相較於對照組與鼻過敏的病患，IgG3的含量在鼻及鼻竇炎病患這一組則有顯著的升高。這表示鼻及鼻竇黏膜對外來微生物或是致病抗原的局部防禦機轉是靠誘發適應性體液免疫反應(adaptive humoral immune response)來增加免疫球蛋白的含量，其中以IgG3是主要被提升的抗體。另外我們藉由研究鼻咽癌病患鼻及鼻竇炎的微生物學而闡明鼻及鼻竇黏膜纖毛功能的重要性，我們的結果顯示與鼻咽癌病患鼻及鼻竇炎最有關的細菌是黏膜上的共生菌，如 α- hemolytic streptococcus及Staphylococcus aureus。這表示在鼻咽癌病患因放射線治療受損的鼻及鼻竇黏膜狀況下，僅有低毒性的共生菌便可成為鼻及鼻竇炎的致病菌。我們的研究也探討了幾項兒童鼻及鼻竇炎的重要課題，首先，我們比較鼻及鼻竇炎病童上頜竇穿刺與內視鏡中鼻道培養的報告，結果顯示，在兒童鼻及鼻竇炎的培養上，內視鏡中鼻道採樣與上頜竇穿刺的培養結果一致性沒有在成人運用時理想，特別是在年紀小的病童。有了這樣的概念，便可以知道上頜竇穿刺在兒童鼻及鼻竇炎致病菌培養上的重要性，採用這一方法，我們得到的結果顯示小兒慢性鼻及鼻竇炎培養出的主要菌株為α-hemolytic streptococcus，Hemophilus influenzae，Streptococcus pneumonia，coagulase-negative Staphylococcus以及Staphylococcus aureus，除了與成人慢性鼻竇炎的菌株不同外，也顯示了與先前類似研究不同的抗生素抗藥性形態。因此，對罹患慢性鼻及鼻竇炎的小兒族群施與抗生素治療時需考慮此一疾病的微生物特色及其抗藥性形式，而這些菌種以及抗藥性形態在不同的時間與地區則可能會有明顯的差異。

Background and objectives: Rhinosinusitis is among the most common health problems in human. Although intensive researches have been conducted to elucidate the pathophysiology of rhinosinusitis, the pathogenesis of this disease is still elusive in many parts. A normal functioning of immune system, especially the humoral immunity, is important in the defense mechanism of sinonasal mucosa. To investigate the immunological conditions of sinonasal mucosa, we adopted a noninvasive method to collect nasal secretions and determined the levels of IgA and IgG subclasses antibodies in healthy subjects and patients with allergic rhinitis or chronic rhinosinusitis. Beside immunology, microbiology of the rhinosinusitis is also essential because obstruction of ostiomeatl complex and subsequent bacterial infections represent the general scenario in the development of most rhinosinusitis. To answer if different patterns of bacterial infections might occur in damaged sinonasal mucosa than in that of normal individuals, we collected culture data obtained from nasopharyngeal carcinoma (NPC) patients with acute rhinosinusitis in order to elucidate the microbiology of rhinosinusitis in irradiated NPC patients and to address the effect of sinonasal mucosa damage on the development of rhinosinusitis. Among the sampling methods used to investigate the pathogens responsible for rhinosinusitis, maxillary sinus puncture is considered to be the gold standard. Although endoscopic middle meatal culture seems to correlate well with the results obtained by maxillary sinus puncture, the reliability of documentation of bacteria by endoscopic culture in children with rhinosinusitis is still unknown. Finally, because studies on current bacterial resistance patterns in pediatric patients are limited, we employed maxillary sinus puncture to obtain sinus secretions for bacterial culture and analysis of antibiotic resistance in children with chronic rhinosinusitis. This may provide clinicians important information in managing refractory rhinosinusitis in children.Methods: According to the objectives of the current study, the protocol was designed into four parts: 1) Twenty patients with chronic rhinosinusitis and 25 patients with perennial nasal allergy were included. Another 24 healthy medical students were recruited as the control group. Nasal secretions were collected by lavage with phosphate-buffered saline containing 0.001% phenol red indicator (indicator-PBS). Total IgA, secretory IgA and IgG subclasses in nasal secretions were assayed by an enzyme-linked immunosorbent assay (ELISA) and their concentration was calculated. 2) We recruited irradiated NPC patients with acute maxillary sinusitis. Patients were considered for inclusion if they presented with typical symptoms of sinusitis. Maxillary sinus puncture was performed on these patients both for sampling of sinus contents and irrigation of the diseased sinuses. Aspirate contents of the sinuses were collected for aerobic and anaerobic cultivation. 3) A prospective study of pediatric patients with community-acquired rhinosinusitis was conducted. Bacterial culture was performed on the specimens obtained from maxillary sinus by puncture and middle meatus by endoscopic sampling. The correlation and diagnostic usefulness of endoscopic middle meatal culture versus maxillary sinus puncture were determined. 4) A retrospective analysis of children with community-acquired rhinosinusitis was conducted from 2001 through 2006. Maxillary sinus puncture was performed to collect secretions for bacterial culture. Examination of antimicrobial resistance was also performed for specific antibiotics according to different bacteria by using disc diffusion test.Results: 1) Total IgA, sIgA and IgG subclasses in the nasal secretions were assayed by ELISA and were detectable in these nasal secretions. There was no difference in the levels of tIgA, sIgA, IgG1, G2 and G4 among the three groups, while a significant elevation of IgG3 was noted in the group of chronic rhinosinusitis when comparing to control group and patients with allergic rhinitis. 2) There were 20 NPC patients with acute rhinosinusitis received maxillary sinus puncture in the study. Fourteen of the patients presented with unilateral maxillary sinusitis and 6 patients had bilateral maxillary sinusitis. Of the 26 samples, 22 recovered one or more organisms. The most frequently identified aerobes were α-hemolytic streptococcus and Staphylococcus aureus. Other common aerobic bacteria recovered from these specimens included Pseudomonas aeruginosa and Streptococcus constellatus. 3) There were 41 specimen sets sent for aerobic cultivation. Correlation obtained from the middle meatus with those from the maxillary sinus puncture was demonstrated in 32 of 41 specimens (78.0%). When looking at the diagnostic usefulness of endoscopic middle meatus sampling versus maxillary sinus puncture, endoscopic sampling provided a sensitivity of 75.0%, a specificity of 88.9%, a predictive value of a positive result of 96.0%, a predictive value of a negative result of 50.0%, and an accuracy of 78.0%. 4) Data on 169 children were analyzed. There were 301 total cultures sent, yielding 403 isolates. The most common isolates were α-hemolytic streptococcus, Hemophilus influenzae, Streptococcus pneumonia, coagulase-negative Staphylococcus, and Staphylococcus aureus. Anaerobes were recovered from 7.9% of all isolates. We found rates of nonsusceptibility of Hemophilus influenzae were 55.3% for ampicillin and 57.9% for co-trimoxazole in the first 3 years of the study, and 75% for ampicillin and 60% for co-trimoxazole in the later 3 years. The rates of nonsusceptibility of Streptococcus pneumoniae were 16.7% for penicillin, 100% for erythromycin, and 66.7% for clindamycin in the first 3 years, and 26.3% for penicillin, 94.7% for erythromycin, and 71.1% for clindamycin in the later 3 years.Conclusion: We demonstrated a significant elevation of IgG3 in the nasal secretions of patients with chronic rhinosinusitis when comparing to the control group and patients with allergic rhinitis. This suggested that the local defense mechanism of sinonasal mucosa reacts to microorganisms and pathogenic antigens by inducing the adaptive humoral immune response to increase the amount of immunoglobulins, with IgG3 being the major up-regulated antibody. We also demonstrated the importance of mucociliary clearance by investigating microbiology of acute rhinosinusitis in irradiated NPC patients. Our data showed that sinonasal commensal flora, such as α-hemolytic streptococcus and Staphylococcus aureus, are the major bacteria involved in acute rhinosinusitis of irradiated NPC patients. The results implied that commensal flora with lower virulence can readily cause inflammation and become pathogenic because of an impaired mucociliary function in irradiated NPC patients. Important issues about pediatric rhinosinusitis were also addressed in our studies. First, we tried to compare the results obtained by endoscopic middle meatal culture and maxillary sinus punctures in children with rhinosinusitis. Our data demonstrated that, when performed in pediatric patients (especially in children with younger age), the correlation between endoscopic middle meatal culture and maxillary sinus puncture was not as favorable as in the case of adult patients. With this notion in mild, we can appreciate the importance of maxillary sinus puncture in documenting microbiology of pediatric rhinosinusitis. By this methodology, we demonstrated that α-hemolytic streptococcus, Hemophilus influenzae, Streptococcus pneumonia, coagulase-negative Staphylococcus, and Staphylococcus aureus are the major organisms found in pediatric rhinosinusitis. We also demonstrate a different pattern of antibiotic resistance when comparing to previous studies performed in both children and adults. Therefore, administration of antimicrobial therapy in the pediatric population with chronic rhinosinusitis should consider the current patterns of antibiotic resistance, which might be variable according to different geographic areas.