Respiratory Pathogens Screening and Clinical Characteristics Analysis for Pediatric Acute Rhinosinusitis Patients

MAI Ai,LUO Ya-sha,ZHONG Guo-yu,WEI Feng-gui,CHEN De-hui,WEI Xiao-ping,ZHOU Qiang,LIN Yong-ping

1.Department of ENT,the First Affiliated Hospital of Guangzhou Medical University，Guangzhou,Guangdong Province,510120 China; 2.Department of Laboratory,Guangdong Provincial Maternity and Child Care Center,Guangzhou,Guangdong Province,511442 China; 3.Department of Pediatrics,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong Province,510120 China;4.Department of Clinical Laboratory,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong Province,510120 China;5.The Second Affiliated HospitalClinical Laboratory of Guangzhou University of Chinese Medicine, Guangzhou,Guangdong Province,510120 China

Respiratory Pathogens Screening and Clinical Characteristics Analysis for Pediatric Acute Rhinosinusitis Patients

MAI Ai1,LUO Ya-sha2,ZHONG Guo-yu3,WEI Feng-gui3,CHEN De-hui3,WEI Xiao-ping4,ZHOU Qiang5,LIN Yong-ping4

1.Department of ENT,the First Affiliated Hospital of Guangzhou Medical University，Guangzhou,Guangdong Province,510120 China; 2.Department of Laboratory,Guangdong Provincial Maternity and Child Care Center,Guangzhou,Guangdong Province,511442 China; 3.Department of Pediatrics,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong Province,510120 China;4.Department of Clinical Laboratory,the First Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong Province,510120 China;5.The Second Affiliated HospitalClinical Laboratory of Guangzhou University of Chinese Medicine, Guangzhou,Guangdong Province,510120 China

Objective Acute upper respiratory tract infection (AURI)is a common disease in most pediatric outpatients,and viral etiologies have been shown to play an important role.However,the URI infection following to result most pediatric sinus infections,which is known to damage the nasal/sinus epithelial cells and cilis.Therefore,the pathogens screening and clinical characteristics analysis were conducted to determine the prevalence of viruses in acute rhinosinusitis (ARS)and controls groups.Methods Nasal swab(NS) samples were collected from 564 pediatric outpatients,which were 259 ARS patients(group I),219 AURIs patients(group II)and 86 controls(group III)respectively.Multiplex PCR was used to identify respiratory viruses in each group and the pathogens were compared with each other.Results The datas were shown that infected with respiratory viruses would increase 3.104-fold risk become RS (RR=3.104,95%CI=2.134～4.517).Only RV and ADV had a higher rate in group I than group II(P＜0.05),the rest pathogens shown no significant difference either(P＞0.05).RV infection rate in group Ia(ARS only)was absolutely higher than other groups(P＜0.01), ADV was more prevalent in the AURI population with ARS(group Ib)(P＜0.05).Effusion of immune cells have been found in 49.6% group I samples by cell smearing analysis,which nearly equal to the pathogen detection rate of group I(47.4%).Conclusion This investigation proved that RV and ADV contribute more on ARS development,especially RV,which made children have more chance developing to ARS with the neutrophils and lymphocyte effusion in nasal mucosa.Further studies should be done to generalize the research by incresing sample capacity and collecting ARS patients from different hospital during continuous seasons.

Acute sinusitis in Children；Rpathogens;Screenin

Acute rhinosinusitis(ARS)is a common problem in children with acute upper respiratory tract infections(AURIs), while AURIs is the largest number of disease in pediatric outpatients.Recent evidence shows that most URIs are caused by viruses,but the viral screening and clinical characteristics analysis for pediatric of acute RS are not well understood.Since rhinitis and sinusitis in pediatric patients are often a continuous disease according to the anatomical characteristics in children[1],it is not possible to distinct rhinitis from sinusitis on clinical espression individually.Recognition of history and symptoms are important to establish a diagnosis of RS due to which is still often under-diagnosed in practical pediatric.Research has shown that the the most common RS symptoms were rhinorrhea,cough,post-nasal drip and nasal congestion,and most symptoms and signs in acute and chronic RS were similar[2].In addition,RS is a multifactorial disease,which is the several predisposing factors changes with increasing age[3-4].Therefore,more continued research in these fields should be done to determine the most safe and effective methods to prevent and treat RS in children.

急性上呼吸道感染是兒科門(mén)診中就診數(shù)量最多的疾病，而急性鼻竇炎是小兒急性上呼吸道感染中的常見(jiàn)問(wèn)題。最新研究表明大多數(shù)的上呼吸道感染是由病毒引起的，但對(duì)小兒急性鼻竇炎的病毒篩查和臨床特點(diǎn)分析尚不明確。根據(jù)兒童的解剖特點(diǎn)，鼻炎和鼻竇炎在兒科患者中往往是一種連續(xù)性疾病，因此，兩者的臨床表現(xiàn)難以區(qū)分。由于鼻竇炎在目前的兒科就診患兒中容易漏診，所以對(duì)其疾病史和癥狀的認(rèn)知對(duì)診斷該疾病很重要的。研究表明最常見(jiàn)的鼻竇炎癥狀是流鼻涕，咳嗽，鼻塞，鼻后滴流，急性鼻竇炎和慢性鼻竇炎的大多數(shù)癥狀和體征相似。此外，鼻竇炎是由多因素引起的疾病，隨著年齡的增加多種誘因也隨之改變。因此，應(yīng)該開(kāi)展更多關(guān)于該領(lǐng)域的研究來(lái)決定預(yù)防和治療小兒鼻竇炎的最安全最有效的方法。

Viral etiologies have shown virus play an important role in the AURI pediatric which following to result most sinus infections,was known to damage the nasal/sinus epithelial cellsandcilis[5].After samples were collected from control groups without ARS,the respiratory pathogens rate was compared with that in ARS subjects.These data proved that some specific virus would made children have more chance developing to ARS with plenty of neutrophils effusion in nasal mucosa.

病毒病因?qū)W表明病毒在小兒急性上呼吸道感染中起很大作用，會(huì)導(dǎo)致進(jìn)一步的鼻竇感染，而鼻竇感染會(huì)損傷鼻竇黏膜上皮細(xì)胞。從沒(méi)有患急性鼻竇炎的對(duì)照組中采集樣本之后，該研究將患有急性鼻竇炎的受試組的呼吸系統(tǒng)病原體感染率與其相比較。研究數(shù)據(jù)表明一些特定的病毒會(huì)增加兒童感染急性鼻竇炎的風(fēng)險(xiǎn)，并且鼻黏膜會(huì)有大量的中性粒細(xì)胞滲出。

MATERIALS AND METHODS

Patient population.Five hundred sixty-four pediatric out-patients were recruited between July 2013 and June2014 at 1st Affiliated Hospital of Guangzhou Medical University,China,of whom with a ARS diagnosis were divided into group I(259),including a subgroup Ia (95)only with ARS and a subgroup Ib(164)both with ARS and AURIs.The other 219 simple AURIs children who didn’t have any nasal symptoms were divided into group II,and group III(86)were control volunteers who have neither ARS nor AURIs.All rhinosinusitis patients met the established diagnostic criteria[6]for ARS,which the most common symptoms were rhinorrhea, cough,post-nasal drip and nasal congestion,and the persistent symptoms for more than 10 days but less than 4 weeks. The diagnosis of AURIs is usually made from the history and presenting symptoms,which can include cough,wheeze and fever,among others,also the persistent symptoms for more than 10 days but less than 4 weeks.Asthma and allergic disease were excluded from the study.All the legally authorized representatives of children provided informed consent.

1 資料與方法

1.1 研究對(duì)象

選取該院564例兒科門(mén)診病人，收取時(shí)間是2013年7月—2014年7月，并根據(jù)診斷結(jié)果將患者分為3組。I組有259例病人，包括Ia組（僅患有急性鼻竇炎）95例患者和Ib組 （同時(shí)患有急性鼻竇炎和急性上呼吸道感染）164例患者；其他219例沒(méi)有鼻癥狀的兒童被分為II組；III組作為健康對(duì)照組，共86名。所有的鼻竇炎患者均符合診斷標(biāo)準(zhǔn)。鼻竇炎的常見(jiàn)癥狀是流鼻涕，咳嗽，鼻塞，鼻后滴流，癥狀持續(xù)時(shí)間是10 d～4周；而通常根據(jù)既往和現(xiàn)有的癥狀對(duì)急性上呼吸道感染進(jìn)行診斷，包括咳嗽、氣喘、發(fā)熱。該研究不包括哮喘與過(guò)敏性疾病患者；所有的知情同意書(shū)均向兒童的法定監(jiān)護(hù)人出示。

Specimens collection.Nasal swab(NS)samples were collected from five hundred sixty-four participants from July 2013 to June 2014.To obtain nasal specimen,a Dacron swab with a plastic shaft(Copan,Italy)was placed 2 to 2.5 cm into the right nostril and rotated three times against the surface of the nasal cavity.Specimens were placed into transport tubes containing 2 mL of sterile phosphate-buffered saline medium. The samples were frozen immediately after collection and stored at-80°C until use.In addition,455 of these children had also been done with nasal secretions cell analysis by smearing observation.

1.2 標(biāo)本采集

從2013年7月—2014年6月，在564例研究對(duì)象中收集鼻腔黏膜拭子樣本。將一塑料軸滌綸拭子（Copan，意大利）置入右鼻孔2～2.5 cm，然后沿鼻腔表面旋轉(zhuǎn)3次；將標(biāo)本置于包含2 mL無(wú)菌磷酸鹽緩沖鹽水培養(yǎng)基的透明管中，標(biāo)本采集后立即冷凍并貯藏在-80°C直到使用。此外，對(duì)其中455例兒童的鼻分泌物涂片，并進(jìn)行細(xì)胞染色分析。

Pathogen screening.After vortex shocked and centrifuged,aqueous phase were recover for nucleic extracted by QIAamp viral RNA kit(Qiagen),according to the manufacturer’s instructions.Super Script III Reverse Transcriptase (Invitrogen)was used to synthesize cDNA.Each cDNA was added to a multiplex PCR reaction system,at a final volume of 25 μL,including 12.5 μL Premix Taq (TaKaRa),4 μL primer mix,5.5 μL dd H2O and 3 μL cDNA.Samples were subjected to initial denaturation at 94°C for 5 min;35 cycles of 94°C for 30 s,56°C for 30 s,and 72°C for 30 s;and a final extension at 72°C for 3 min.The products were visualized by electrophoresis on 3%agarose gel.The multiplex PCR system included 4 groups for viral screening,parainfluenza viruses 1,2,3,and 4,influenza viruses A and B,res-piratory syncytial viruses (RSV),rhinoviruses (RV)and enteroviruses(EV),coronaviruses 229E,NL63 and OC43,adenovirus(ADV),human metapneumovirus(HMPV),and human bocaviruses[7-9]were included.

1.3 病因篩查

對(duì)樣本進(jìn)行旋渦震蕩、離心，提取上清液，按照試劑盒說(shuō)明書(shū)進(jìn)行操作。使用逆轉(zhuǎn)錄酶III(Invitrogen)合成cDNA，按試劑盒要求配置25 μL反應(yīng)體系。PCR的反應(yīng)條件為94°C 5 min，35個(gè)循環(huán) （94°C 30 s，56°C 30 s、72°C 30 s），72°C 3 min進(jìn)行最終擴(kuò)增。PCR產(chǎn)物通過(guò)2%瓊脂糖進(jìn)行凝膠電泳。多重PCR反應(yīng)體系對(duì)以下病毒進(jìn)行篩查，包括副流感病毒1、2、3、4，流感病毒A和 B，呼吸道合胞病毒，鼻病毒，腸病毒，冠狀病毒229E、NL63和OC43，腺病毒，肺炎病毒和人博卡病毒。

Analysis of cells in nasal secretions.455 nasal secretion samples from these children were smeared to slide and dyed by Wright's stain after them dried,observed and imaged through microscrope.A sample would be considered to positive as if immune cells were observed under the microscope. Serum C reaction protein level and peripheral blood white cells analysis.

352CRP and WBC clinical laboratory test results of the children were collected though the hospital information system.

1.4 鼻分泌物細(xì)胞分析

對(duì)455個(gè)鼻分泌物樣本進(jìn)行滑動(dòng)涂片及瑞氏染色，干燥后通過(guò)顯微鏡觀察，如果顯微鏡下可見(jiàn)免疫細(xì)胞，則該樣本就被視為陽(yáng)性樣本。

Statistical analysis.Pearson’s chi-squared tests and Fisher’exact tests were used to compare the positive rate of viruses and cells in nasal secretions for different groups,risk ratios(RR)and 95%confidence intervals(CI)were also calculated.ANOVA was used to compare the serum CRP level and blood white cells counts in each group.Statistical analysis was performed using SPSS version 19.0 and a P value＜0.05 was regarded as significant.

1.5 血清C反應(yīng)蛋白水平和外周血白細(xì)胞分析

通過(guò)醫(yī)院檢驗(yàn)信息系統(tǒng)收集352例兒童的CRP和WBC臨床實(shí)驗(yàn)室結(jié)果。

1.6 統(tǒng)計(jì)方法

使用SPSS 19.0統(tǒng)計(jì)學(xué)軟件對(duì)本研究數(shù)據(jù)進(jìn)行分析。對(duì)不同組間的鼻分泌物病毒和細(xì)胞的陽(yáng)性率分別應(yīng)用χ2檢驗(yàn)和Fisher精確檢驗(yàn)法比較，同時(shí)計(jì)算風(fēng)險(xiǎn)率（RR）和95%置信區(qū)間（CI）。對(duì)每組的血清CRP水平和白細(xì)胞數(shù)目應(yīng)用ANOVA法進(jìn)行比較。以P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。

RESULTS

The study recruited five hundred sixty-four pediatric out-patients from July 2013 to June 2014,including 259 children with ARS,219 with AURIs and 86 control subjects.The mean age of all subjects was(3.61±2.55)years. Age and gender were not shown differences among the three groups.

Pathogen etiologies.179 (31.7%)positive for one or more pathogens of all samples were detected,and the positive rate for group I (44.4%),group II (27.4%)and group III (4.7%)were significant different(P＜0.001).It was shown that infected with respiratory viruses would increase 3.104-fold risk become rhinosinusitis(RR=3.104,95%CI=2.134～4.517). The most prevalent virus was rhinovirus,24 samples (9.3%) were positive in group I and 9(4.1%)in group II(P＜0.05, RR=2.27);secondarily,16 samples(6.2%)were adenovirus positive in group I and 4(1.6%)in group II(P＜0.05,RR= 3.374),the rest pathogens shown no significant difference in group I and II(Table 1).Four viruses have been detected in group III were RSV,MP,IFA and ADV respectively.While, PIV3,PIV4,NL63,229E and MPV haven’t been detected in this study.

2 結(jié)果

該研究在2013年7月—2014年6月收集564例兒科門(mén)診患者，包括259例患有ARS的兒童，219例患有AURIs的兒童和86名健康對(duì)照患兒。所有研究對(duì)象的平均年齡是（3.61±2.55）歲，3組間年齡和性別差異無(wú)統(tǒng)計(jì)學(xué)意義。

2.1 病原學(xué)病因

該研究樣本中，其中179例（31.7%）測(cè)得有1種以上病

原感染，其中I組（44.4%），II組（27.4%）和III組（4.7%），3組間的陽(yáng)性率差異具有高度統(tǒng)計(jì)學(xué)意義（P＜0.001），提示感染了呼吸道病毒的患兒有3.104倍的風(fēng)險(xiǎn)會(huì)進(jìn)一步發(fā)

展為鼻竇炎(RR=3.104,95%CI=2.134～4.517)。其中最常見(jiàn)的是鼻病毒，I組有24例是陽(yáng)性（9.3%），II組有9例是陽(yáng)性(4.1%)（P＜0.05,RR=2.27）；其次是腺病毒，I組有16例陽(yáng)性(6.2%)，II組有4例陽(yáng)性(1.6%)（P＜0.05,RR=3.374）；其余的病原體在兩組間的差異無(wú)統(tǒng)計(jì)學(xué)意義，見(jiàn)表1。在III組中發(fā)現(xiàn)4種病毒，分別是RSV，MP，IFA和ADV，此外，該研究中并未檢測(cè)出PIV3,PIV4,NL63,229E和 M PV。

Table 1 Comparison of pathogen detection rate between group I with II[n(%)]

表1 I組和II組病原菌檢測(cè)率的比較[n(%)]

According to the above results,RV and ADV positive rate shown difference in group I and II,so we compared their detection rate in group Ia,Ib and group II further,the results both shown difference rate of RV and ADV among group Ia, Ib and II(P＜0.05).RV infection rate in Ia was absolutely higher than the other groups(P＜0.01),however,ADV was more prevalent in the AURI population with RS(group Ib) (P＜0.05)(Table 2).

根據(jù)上述結(jié)果，I組和II組的RV和ADV陽(yáng)性率差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）；再進(jìn)一步的比較Ia組、Ib組和II組的檢測(cè)率，結(jié)果表明3組間的RV和ADV的檢測(cè)率差異有統(tǒng)計(jì)學(xué)意義（P＜0.05），其中Ia組的RV感染率明顯高于其他組 （P=0.01）。然而，ADV在同時(shí)患有AURI和RS的患兒中更為常見(jiàn)（Ib組），P＜0.05，見(jiàn)表2。

Table 2 Comparison the positive rate of RV and ADV among group Ia,Ib and II[n(%)]

表2 Ia組,Ib組和II組的RV與ADV陽(yáng)性率比較I[n(%)]

Cell smears analysis.455 samples were analyzed the cells of nasal secretions,epithelial cells have been found in every sample.Immune cells(neutropils,lymphocyte or monocyte)also been observed in 140 smear samples,113(49.6%) in group I,22 (13%)in group II and 5(8.6%)in group III, which has significant difference among three groups(P= 0.000).Addictional,immune cells were also positive in 78 samples(74.3%)of group I in which PCR were positive(P= 0.000).Figure 2 was shown several pictures of the smears.

2.2 細(xì)胞涂片分析

對(duì)455例鼻腔分泌物樣本進(jìn)行涂片分析，每一個(gè)樣本在顯微鏡下均可見(jiàn)鼻黏膜上皮細(xì)胞，其中的140例涂片樣本中可同時(shí)觀察到免疫細(xì)胞（中性粒細(xì)胞，淋巴細(xì)胞和單核細(xì)胞）（圖 2），包括I組的 113例 (49.6%),II組的 22 (13%)和III組的5(8.6%)，并且3組間差異有統(tǒng)計(jì)學(xué)意義（P=0.000）。此外，I組有78例樣本(74.3%)的免疫細(xì)胞在PCR檢測(cè)中同樣也是陽(yáng)性（P=0.000）。

Figure 2 Images for the scrape content smear(×1000)

圖2 鼻腔分泌物涂片的顯微鏡下圖像(×1 000)

Serum CRP and WBC analysis.352 children have been tested serum CRP and white blood cells count examinations, the values of these tests were compared among each group by ANOVA,CRP level and monocyte of rhinosinusitis patients were higher than other children,white blood cells analysis shown no significant difference in each group(table 3).

血清CRP和WBC分析：通過(guò)ANOVA法對(duì)352例兒童的血清CRP和白細(xì)胞計(jì)數(shù)進(jìn)行檢測(cè)比較，可見(jiàn)鼻竇炎患兒的CRP水平和單核細(xì)胞均高于其他組患兒，而組間的白細(xì)胞計(jì)數(shù)差異無(wú)統(tǒng)計(jì)學(xué)意義，見(jiàn)表3。

TABLE 3 Serum CRP and white blood cells analysis（±s）

TABLE 3 Serum CRP and white blood cells analysis（±s）

Group I (n=162) Group II (n=135) Group III (n=55)FP CRP(mg/L) WBC(×109/L) Neutrophils(%) Lymphocyte(%) Monocyte(%) 12.16±16.93 10.10±4.19 57.07±17.44 32.59±16.10 8.80±2.88 6.69±8.10 9.79±4.33 57.23±19.34 32.99±18.44 8.24±4.19 5.48±9.41 10.61±4.12 52.39±19.25 36.66±18.03 7.64±2.68 8.711 0.678 0.347 1.045 3.213 0.000 0.508 0.261 0.353 0.041

表3 血清CRP和WBC分析（±s）

表3 血清CRP和WBC分析（±s）

I組 (n=162)II組 (n=135)III組 (n=55)FP CRP(mg/L) WBC(×109/L)中性粒細(xì)胞(%)淋巴細(xì)胞(%)單核細(xì)胞(%) 12.16±16.93 10.10±4.19 57.07±17.44 32.59±16.10 8.80±2.88 6.69±8.10 9.79±4.33 57.23±19.34 32.99±18.44 8.24±4.19 5.48±9.41 10.61±4.12 52.39±19.25 36.66±18.03 7.64±2.68 8.711 0.678 0.347 1.045 3.213 0.000 0.508 0.261 0.353 0.041

Discussion

Rhinosinusitis(RS)is a common disease among children and complicates 5%～10%of pediatric upper respiratory infections[10].Most patients were first diagnosed as RS at ambulatory pediatric clinics,with similar symptoms such as nasal discharge,fever,and cough.So far,the diagnosis of RS is mainly established on the history and symptoms of patients. Nevertheless, the symptoms or severity are subjective and vary from each individual.Although imaging examination such as X-ray,CT or MRI can assist in diagnosis,the performance of distinguishing ARS from mild or recovered URTI is not sensitive enough[11],and the imaging examination can only confirm the absence of RS when the result is negative[12].While,under-diagnosis or delayed treatment of ARS may lead to CRS or complications,viral infection has been described in many existing studies as vital cause of cold and may be the trigger of RS,combinedsymptoms with the infection information on nasal cavity would contribute to diagnosis of RS.It is known that the procedure of obtaining a nasopharyngealaspirate (NPA) specimen is uncomfortable and frightening to children,it’s also unpleasant for medical staff who have to carry out the process in struggling,crying and coughing children.Many studies have demonstrated that NS might prove suitable for obtaining respiratory viral specimens.The collection of a NS is easy and convenient,it requires no additional devices,and it is less costly and causes less distress for the patient than the NPA[13].Some research considered mucosal scraping as more clinically significant and specific method than nasal lavage fluid,since the turbinate epithelial cells may better reflect sinus mucosa.In addition,nasal lavage fluid might have an increased chance of trapping viruses floating in ambient air,and of containing a more mixed viral infection than the scraping samples[14].Then in clinical practice,the optimal sampling methods must be balanced with patient’s comfort,costs,effectiveness and risk to others to achieve the best cooperation of children[15],so we used nasal swab for collecting sample in outpatient children with ARS.Respiratory tract infections are common in children and diagnosis is usually made from the history and presenting symptoms, which can include cough,wheeze and fever,among others. And viral etiologies are the most common causes for AURI, including RS[16].

3 討論

鼻竇炎是兒童一種常見(jiàn)疾病，占小兒上呼吸道感染的5%～10%。許多病人在兒科門(mén)診中初步被診斷為鼻竇炎，具有流鼻涕，發(fā)熱，咳嗽等癥狀。迄今為止，鼻竇炎的診斷主要是建立在病人的病史和癥狀的基礎(chǔ)上；然而，癥狀及其輕重程度是主觀的并且因人而異。盡管X-ray,CT或者M(jìn)RI的影像檢查有助于診斷，但對(duì)于急性鼻竇炎與輕度或恢復(fù)中的急性上呼吸道感染的比較仍然不夠敏感；同時(shí)，急性鼻竇炎診斷不足或延誤治療可能會(huì)導(dǎo)致發(fā)展為慢性鼻竇炎及其并發(fā)癥。目前，許多研究把病毒感染描述為感冒的重要起因或者鼻竇炎的誘因，因此，關(guān)于鼻腔感染的聯(lián)合癥狀將有助于鼻竇炎的診斷。眾所周知，獲取鼻咽分泌物樣本的過(guò)程會(huì)使兒童感到不舒適和恐懼，同樣，面對(duì)掙扎，哭泣和咳嗽的兒童，收集標(biāo)本的醫(yī)務(wù)人員也會(huì)手足無(wú)措。已經(jīng)有許多研究表明鼻腔黏膜拭子可能是獲取上呼吸道病毒標(biāo)本的合適手段，該采集方法簡(jiǎn)單方便，無(wú)需額外的裝置，成本低廉，比起獲取鼻咽分泌物樣本更能減少患兒痛苦。由于鼻甲粘膜上皮細(xì)胞可以更好地反映鼻竇粘膜，因此一些研究認(rèn)為與鼻灌洗液相比，黏膜拭子采樣是更具體、更有效的臨床方法。在臨床實(shí)踐中，最佳的抽樣方法必須與病人的舒適度、成本、有效性和風(fēng)險(xiǎn)達(dá)到一個(gè)平衡，因此，對(duì)患有急性鼻竇炎的門(mén)診病人采用這種簡(jiǎn)便無(wú)痛的鼻黏膜拭子的樣本采集方法。

Then with the advantages on sensitivity,specificity and rapidly,multiplex PCR was used to detecting the common respiratory pathogens.Accordingly,our study screened the existence of 15 common respiratory viruses on nasal cavity of 564 children.Finally,the total positive rate for these 16 pathogens shown significant difference on each group,which means the children who suffered respiratory viruses infection would have more chance (3.104-fold)turn to ARS.Only RV and ADV had a higher rate in group I than group II(P＜0.05). However,distinguishing ARS and AURI absolutely is hard, ARS always be an early stage or a complication of AURI,so it is still difficult to evaluate viral infection would be a key reason for RS or just as a common cause for URI with

rhinosinuisitis or sinusitis complication.In our investigation, there is a difference on viral positive rate between the population of ARS patients and the AURI children without nasal symptoms,we can see that RV infection rate in group Ia(ARS only)was absolutely higher than other groups,ADV was more prevalent in the AURI population with ARS than which without ARS,it’s prove that RV and ADV contribute more on ARS development,especially RV.

由于多重PCR檢測(cè)方法的敏感性、特殊性和快速性方面的優(yōu)勢(shì)，臨床上用于檢測(cè)常見(jiàn)的呼吸道病原體，因此，該研究采用該檢測(cè)方法對(duì)564例兒童的鼻腔進(jìn)行常見(jiàn)呼吸道病毒的篩查。最終，16種病原菌的總陽(yáng)性率在組間差異有統(tǒng)計(jì)學(xué)意義，這意味著感染了這部分呼吸道病毒的兒童有3.104倍的機(jī)會(huì)發(fā)展為急性鼻竇炎。I組與II組比較，有更高的RV和ADV感染率（P＜0.05）。然而，急性鼻竇炎與急性上呼吸道感染的鑒別很困難，前者總是后者的早期階段或是其并發(fā)癥，因此，病毒感染是鼻竇炎的一個(gè)關(guān)鍵原因。在該次調(diào)查中，急性鼻竇炎與沒(méi)有鼻部癥狀的急性上呼吸道感染兒童的病毒陽(yáng)性率之間存在差別，我們可以發(fā)現(xiàn)僅患有急性鼻竇炎組的RV感染率明顯高于其他組患兒；伴有急性鼻竇炎癥狀的急性上呼吸道感染患兒與沒(méi)有鼻竇炎癥狀的患兒比較，ADV的檢出更常見(jiàn)。研究表明，RV和ADV促進(jìn)了ARS的發(fā)展，尤其是RV。

Effusion of immune cells have been found in 49.6% group I samples by cell smearing analysis,which nearly equal to the pathogen detection rate of group I(47.4%).The data of this research showed that 78 (74.3%)in 105 PCR positive samples were immune cells observed,it means viral infection was related to the immune cells effusion on the nasal mucosa.It is suggest that a cell-mediated immune response will be activated after viral infection,mainly dominated by neutrophils,Van[17]found the same results. Epithelialcellscan inducetheproduction ofseveral cytokines after viral infected,deposition in the nose the virusistransported to the posteriornasopharynx and attaches to some specific receptor,then reduces the release of proinflammatory cytokines such as IL-1 beta,platelet activating factor and IL-8,initiates the host immune response by enhancing the recruitment of more immune effector cells into the inflammation site[18],that’s the reason why nasalmucosa and secretionswere teeming with neutrophils and(or)lymphocyte,monocyte.On the other hand,nasal epithelial cells and cilia damaged[19],cytokine increased and several inflammatory pathways induced can provide an environment for bacteria to adhesion,and make the ARS step into secondary bacterial infection or chronic diseaseworsestill.In case of rhinoviruses infection,ICAM-1 was a specific receptor as a trigger of a serial cascade reactions has been proved[20].Matrix metalloproteinase-2, matrix metalloproteinase-9,and vascular endothelial growth factor expression can also be upregulated by rhinoviruses, which may contribute to the pathogenesis of nasal polyps formation in patients with chronic rhinosinusitis[20]. Adenovirus is one of the most common viral reason to cause children upper respiratory tract infections,the patients presented common symptoms including fever(97.9%),cough and rhinitis(74%)[21],but the pathogenesis mechanism was still unclear.Some studies revealed[22]that the increasing counts of neutrophil and monocyte had been seen in peripheral blood within a couple of days after inoculation,

but we only found the increase of monocyte,in addition to barriers defence and a cell-mediated immune response,soluble chemical factors such as C-reactive protein concentrations were shown increased slightly in our study.

通過(guò)細(xì)胞涂片分析發(fā)現(xiàn)，I組49.6%的樣本中有免疫細(xì)胞的滲出，與PCR方法檢測(cè)的I組病原檢測(cè)率比例大致相同(47.4%)。研究數(shù)據(jù)表明，105個(gè)PCR陽(yáng)性樣本中78個(gè)樣本檢測(cè)出免疫細(xì)胞(74.3%)，這就意味著病毒感染和鼻粘膜免疫細(xì)胞滲出是相關(guān)的。有學(xué)者也發(fā)現(xiàn)了同樣的結(jié)果，上皮細(xì)胞被病毒感染后，也會(huì)產(chǎn)生多種細(xì)胞的滲出，鼻內(nèi)病毒沉積輸送至后鼻咽并且依附于某個(gè)特定的受體，隨后會(huì)發(fā)生IL-1β、血小板活化因子和IL-8前炎性細(xì)胞活素的釋放減少，通過(guò)使炎癥部位吸附更多的免疫效應(yīng)細(xì)胞啟動(dòng)宿主免疫反應(yīng)，這就是鼻粘膜和分泌物充滿中性粒細(xì)胞和（或）淋巴細(xì)胞和單核細(xì)胞的原因。另一方面，鼻黏膜上皮細(xì)胞與纖毛的損傷，細(xì)胞因子的增加，若干炎癥途徑為細(xì)菌依附提供了環(huán)境，導(dǎo)致急性鼻竇炎發(fā)展為繼發(fā)性細(xì)菌感染或使慢性疾病加重。

Above all,the investigation found that RV and ADV make children have more chance developing to ARS with the neutrophils and lymphocyte effusion in nasal mucosa,and then trigging the immune cascade reactions at the nasal local site,serum C-reactive protein concentrations and monocyte will increase slightly in peripheral blood which is different from the sharp increasing when bacterium infection.

On the other hand,there were some shortcomings in this study,sample capacity was small and patients were recruited from a single hospital,which may limit the generalizability of the research and pending in further study.

該調(diào)查發(fā)現(xiàn)，RV和ADV更容易使兒童發(fā)展成為在鼻粘膜內(nèi)有中性粒細(xì)胞和淋巴細(xì)胞滲出的急性鼻竇炎，從而導(dǎo)致鼻部出現(xiàn)一系列免疫反應(yīng)。此外，周圍血液的血清C反應(yīng)蛋白濃度和單核細(xì)胞會(huì)有輕微增加，這有別于急性細(xì)菌感染的病例。

另一方面，該研究仍存在不足，樣本容量小，而且僅僅只從一家醫(yī)院獲取門(mén)診患兒的樣本，這使調(diào)查研究的普及受限，因此，有待在未來(lái)的研究中進(jìn)一步完善。

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[2]Poachanukoon O,Nanthapisal S,Chaumrattanakul U.Pediatric acute and chronic rhinosinusitis:comparison of clinical characteristics and outcome of treatment[J].Asian Pac J Allergy Immunol,2012,30(2):146-151.

[3]van der Veken P J,Clement P A,Buisseret T,et al.CT-scan study of the incidence of sinus involvement and nasal anatomic variations in 196 children[J].Rhinology,1990,28 (3):177-184.

[4]Yaniv E,Oppenheim D,Fuchs C.Chronic rhinitis in children[J].Int J Pediatr Otorhinolaryngol,1992,23(1):51-57.

[5]Pedersen M,Sakakura Y,Winther B,et al.Nasal mucociliary transport,number of ciliated cells,and beating pattern in naturally acquired common colds[J].Eur J Respir Dis Suppl,1983,128(Pt 1):355-365.

[6]Fokkens W J,Lund V J,Mullol J,et al.European Position Paper on Rhinosinusitis and Nasal Polyps 2012[J].Rhinol Suppl,2012(23):291-298.

[7]Brittain-Long R,Nord S,Olofsson S,et al.Multiplex realtime PCR for detection of respiratory tract infections[J].J Clin Virol,2008,41(1):53-56.

[8]Aguilar J C,Perez-Brena M P,Garcia M L,et al.Detection and identification of human parainfluenza viruses 1,2,3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR[J].J Clin Microbiol,2000,38(3): 1191-1195.

[9]Zhang G,Hu Y,Wang H,et al.High incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in Shanghai,China[J]. PLoS One,2012,7(9):e44568.

[10]Wald E R.Sinusitis in children [J].N Engl J Med, 1992,326(5):319-323.

[11]Gwaltney J J.Acute community-acquired sinusitis[J].Clin Infect Dis,1996,23(6):1209-1225.

[12]Brook I.Acute sinusitis in children[J].Pediatr Clin North Am,2013,60(2):409-424.

[13]Meerhoff T J,Houben M L,Coenjaerts F E,et al.Detec-tion of multiple respiratory pathogens during primary respiratory infection:nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction[J].Eur J Clin Microbiol Infect Dis,2010,29(4):365-371.

[14]Cho G S,Moon B J,Lee B J,et al.High rates of detection of respiratory viruses in the nasal washes and mucosae of patients with chronic rhinosinusitis[J].J Clin Microbiol, 2013,51(3):979-984.

[15] Sung R Y,Chan P K,Choi K C,et al.Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection[J].J Clin Microbiol,2008,46(9):3073-3076.

[16] Subauste M C,Jacoby D B,Richards S M,et al.Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure[J].J Clin Invest, 1995,96(1):549-557.

[17]Van Cauwenberge P B,Vermeiren J S,van Kempen M J. Viral rhinitis and asthma[J].Curr Opin Allergy Clin Immunol,2001,1(1):21-25.

[18]Brook I.Microbiology of sinusitis[J].Proc Am Thorac Soc, 2011,8(1):90-100.

[19]Pedersen M,Sakakura Y,Winther B,et al.Nasal mucociliary transport,number of ciliated cells,and beating pattern in naturally acquired common colds[J].Eur J Respir Dis Suppl,1983,128(Pt 1):355-365.

[20] Winther B,Gwaltney J J,Mygind N,et al.Sites of rhinovirus recovery after point inoculation of the upper airway[J].Jama,1986,256(13):1763-1767.

[21] Chau S K,Lee S L,Peiris M J,et al.Adenovirus respiratory infection in hospitalized children in Hong Kong: serotype-clinical syndrome association and risk factors for lower respiratory tract infection[J].Eur J Pediatr,2014,173 (3):291-301.

[22] Hansen J G,Hojbjerg T,Rosborg J.Symptoms and signs in culture-proven acute maxillary sinusitis in a general practice population[J].Apmis,2009,117(10):724-729.

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小兒急性鼻竇炎呼吸道病原體篩查與臨床特征分析

麥艾1，羅婭莎2，鐘幗鈺3，衛(wèi)鳳桂3，陳德輝3，魏小平4，周強(qiáng)5，林勇平4

1.廣州醫(yī)科大學(xué)附屬第一醫(yī)院耳鼻喉科，廣東廣州 510120；2.廣東省婦幼保健院檢驗(yàn)科，廣東廣州 511442；3.廣州醫(yī)科大學(xué)附屬第一醫(yī)院兒科，廣東廣州 510120；4.廣州醫(yī)科大學(xué)附屬第一醫(yī)院檢驗(yàn)科，廣東廣州 510120；5.廣州中醫(yī)藥大學(xué)第二附屬醫(yī)院檢驗(yàn)科，廣東廣州 510120

目的 急性上呼吸道感染（URI）是兒科門(mén)診最常見(jiàn)的疾病之一，其病毒學(xué)病因發(fā)揮著重要的作用。由于病毒對(duì)鼻腔、鼻竇上皮細(xì)胞及纖毛的損害，URI的感染多數(shù)會(huì)進(jìn)一步導(dǎo)致小兒的鼻竇炎感染。因此，通過(guò)病原體篩選和臨床特征分析，來(lái)比較急性鼻竇炎（ARS）組與對(duì)照組的病毒發(fā)生率。方法 對(duì)該組564例兒科患者進(jìn)行鼻粘膜拭子采樣，其中259例為ARS患兒（I組），219例為急性URI患兒（II組），86名為對(duì)照組（III組）。應(yīng)用多重PCR技術(shù)對(duì)每組的呼吸道病毒及病原體進(jìn)行比較。 結(jié)果 感染了呼吸道病毒的患兒進(jìn)一步發(fā)展為鼻竇炎（RS）的風(fēng)險(xiǎn)將增加3.104倍（RR=3.104,95%CI=2.134～4.517）。 I組中的鼻病毒（RV）與腺病毒（ADV）含量均高于II組（P＜0.05），其余的病原體在兩組間的差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。Ia組（單純ARS組）中RV的感染率明顯高于其他組（P＜0.01）；與其他組比較，ADV的感染率在Ib組（急性URI伴ARS）患兒中更常見(jiàn)（P＜0.05）。鼻腔分泌物的涂片分析結(jié)果顯示，49.6%的I組樣品中可見(jiàn)免疫細(xì)胞的滲出，此結(jié)果幾乎與該組的病原體檢出率（47.4%）相等。結(jié)論小兒急性URI發(fā)展為ARS的的患者中，病毒RV與ADV擔(dān)當(dāng)了比較重要的作用，特別是RV，這部分患兒的鼻腔分泌物中可見(jiàn)中性粒細(xì)胞和淋巴細(xì)胞的滲出。應(yīng)該通過(guò)不同的采樣醫(yī)院，在連續(xù)季節(jié)收集更多的ARS樣本量，以便更好地推廣該研究。

小兒急性鼻竇炎；呼吸道病原體；篩查

R765.4+1

A doi 10.11966/j.issn.2095-994X.2016.02.03.08

（

）

2016-07-20；

2016-08-15

廣東省科技計(jì)劃項(xiàng)目（2011B061300038）。

麥艾（1972.1-），女，廣東臺(tái)山人，博士研究生，副主任醫(yī)師，研究方向：鼻科學(xué)研究。

林勇平（1973.12-），男，廣東人，博士，副教授，研究方向：臨床病毒學(xué)檢驗(yàn)，Email：lin_y_p@hotmail.com。