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          "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Immunofluorescence analysis of the ciliary ultrastructure&#46; The first column shows the presence of cilia in the cell using acetylated tubulin &#40;in green&#41;&#44; the second shows the outcome of incubation with primary ciliary protein antibodies &#40;in red&#41;&#44; and the third shows the merge of tubulin with each ciliary protein and the DAPI-stained nucleus &#40;blue&#41;&#46; &#40;A&#41; Absence of protein DNAH5 &#40;external component of dynein arms&#41; in the ciliary axoneme&#46; &#40;B&#8211;D&#41; Presence and colocalization of tubulin and ciliary axoneme proteins &#40;in yellow&#41;&#58; &#40;B&#41; DNALI1 &#40;external component of dynein arms&#41;&#59; &#40;C&#41; RSPH4A &#40;radial spoke head component&#41;&#44; and &#40;D&#41; GAS8 &#40;nexin-dynein regulatory complex component&#41;&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">Primary ciliary dyskinesia &#40;PCD&#41; is characterized by an alteration in the ciliary structure causing problems in the clearance of respiratory secretions&#46;<a class="elsevierStyleCrossRefs" href="#bib0080"><span class="elsevierStyleSup">1&#44;2</span></a> It is an autosomal recessive hereditary disease&#44; and up to 40 causative genes have been described in &#62;70&#37; of patients&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">3</span></a> It is difficult to confirm a diagnosis of PCD using currently available techniques&#44; and the European guidelines recommend a combination of tests&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">4</span></a> The detection of low levels of nasal nitric oxide &#40;nNO&#41; is a useful screening tool&#44;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">4&#44;5</span></a> but this method is only validated in patients older than 5 years of age&#44; and may be normal in some cases&#46;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">4&#44;5</span></a> Ciliary ultrastructure analysis with electron microscopy gives false positives&#44; related to secondary changes caused by respiratory infections&#44; as well as false negatives&#44; and may be normal in 21&#37; of cases&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">4</span></a> The analysis of ciliary beat pattern using high-speed video-microscopy is very useful for diagnosis&#46;<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">4&#44;6</span></a> However&#44; it also gives false positives due to respiratory infections&#44; there is a lack of standardization in the preparation of the samples&#44; it has to be interpreted by experienced personnel&#44; and it has an element of subjectivity&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">4</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">In addition to molecular diagnostics&#44;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">3</span></a> immunofluorescence has been identified as a technique that can help define the specific PCD defect and improve diagnosis&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">7</span></a> The aim of this article is to report the cases of 2 sisters that show the usefulness of combining these techniques to reach an accurate diagnosis of the protein and molecular defect causing PCD&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">The study was approved by the Ethics Committee&#44; and authorization for inclusion in the study was requested from the parents and the patients&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Case 1</span>&#46; A 16-year-old girl&#44; born at term&#46; She was admitted at 3 days of life for bronchiolitis&#44; and subsequently developed recurrent otitis media&#44; chronic rhinitis&#44; recurrent bronchitis&#44; and middle lobe bronchiectasis&#46; At 4 years of age&#44; an electron microscopy study showed a loss of 40&#37; of the outer dynein arm &#40;ODA&#41; and 70&#37; of the inner dynein arm &#40;IDA&#41;&#44; confirming a diagnosis of PCD&#46; <span class="elsevierStyleItalic">Haemophilus influenzae</span> and <span class="elsevierStyleItalic">Pseudomonas aeruginosa</span> were habitually isolated from sputum cultures&#44; so the patient was treated with nebulized colistin&#44; respiratory physiotherapy&#44; and the administration of 7&#37; nebulized hypertonic saline&#46; Spirometry &#40;GLI-2012 reference values&#41;<a class="elsevierStyleCrossRefs" href="#bib0115"><span class="elsevierStyleSup">8&#44;9</span></a> showed FVC 3&#46;36<span class="elsevierStyleHsp" style=""></span>l &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;0&#46;08&#41;&#44; FEV<span class="elsevierStyleInf">1</span> 2&#46;23<span class="elsevierStyleHsp" style=""></span>l &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;2&#46;21&#41;&#44; FEV<span class="elsevierStyleInf">1</span>&#47;FVC 66&#37; &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;2&#46;83&#41;&#44; FEF<span class="elsevierStyleInf">25&#37;&#8211;75&#37;</span> 1&#46;42<span class="elsevierStyleHsp" style=""></span>l&#47;s &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;3&#46;19&#41;&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Case 2</span>&#46; A 13-year-old girl&#44; born at term&#46; She was admitted at 11 days of life for bronchiolitis&#44; and subsequently developed pneumonia in the right upper lobe and recurrent bronchitis&#46; At 12 months of life&#44; electron microscopy showed a 30&#37; loss of ODA and a 70&#37; loss of IDA&#44; so she too was diagnosed with PCD&#46; Chest computed tomography scan revealed atelectasis in the middle lobe and lingula&#44; peribronchial thickening and heterogeneous aeration&#46; Sputum cultures were positive for <span class="elsevierStyleItalic">H&#46; influenzae</span>&#46; She was treated with respiratory physiotherapy and 7&#37; nebulized hypertonic saline&#46; Spirometry showed FVC 1&#46;97<span class="elsevierStyleHsp" style=""></span>l &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;0&#46;91&#41;&#44; FEV<span class="elsevierStyleInf">1</span> 1&#46;45<span class="elsevierStyleHsp" style=""></span>l &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;2&#46;18&#41;&#44; FEV<span class="elsevierStyleInf">1</span>&#47;FVC 73&#37; &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;2&#46;18&#41;&#44; FEF<span class="elsevierStyleInf">25&#37;&#8211;75&#37;</span> 0&#46;98<span class="elsevierStyleHsp" style=""></span>l&#47;s &#40;<span class="elsevierStyleItalic">z</span>-score &#8722;3&#46;12&#41;&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">Last year&#44; our patients were re-evaluated using newly available diagnostic techniques&#46; The nNO value was very low in both girls&#58; 53&#46;3<span class="elsevierStyleHsp" style=""></span>ppb &#40;15&#46;2<span class="elsevierStyleHsp" style=""></span>VNO<span class="elsevierStyleHsp" style=""></span>nl&#47;min&#41; and 61<span class="elsevierStyleHsp" style=""></span>ppb &#40;17&#46;2<span class="elsevierStyleHsp" style=""></span>VNO<span class="elsevierStyleHsp" style=""></span>nl&#47;min&#41;&#46; Samples of ciliated respiratory epithelium were collected from the lower nasal meatus with a 2<span class="elsevierStyleHsp" style=""></span>mm brush for video-microscopy and immunofluorescence studies&#44; and samples of peripheral blood were obtained for genetic studies&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">We analyzed ciliary beat frequency and pattern with high-speed video &#40;MotionPro<span class="elsevierStyleSup">&#174;</span> X4&#44; IDT&#44; CA&#44; USA&#41; coupled to an optical microscope&#58; absence of ciliary motility could be seen in both sisters&#46;</p><p id="par0040" class="elsevierStylePara elsevierViewall">For the genetic study&#44; genomic DNA was extracted from the peripheral blood of both patients and their parents&#46; The samples of 1 of the sisters and the parents were analyzed with TruSight One Sequencing Panel &#40;Illumina&#44; San Diego&#44; CA&#44; USA&#41; and sequenced with the MiSeq platform &#40;Illumina&#41;&#46; This panel included 20 genes associated with PCD&#46; The results were analyzed using the VariantStudio v2&#46;2&#46;1&#44; Alamut Visual v2&#46;11&#44; and VarSome programs and different predictors of pathogenicity&#46; We consulted allele frequency in the Genome Aggregation Database and scientific evidence of pathogenicity in the Human Gene Mutation Database&#46; The candidate variants were confirmed for both this patient and her sister using Sanger sequencing&#46; The sisters show a compound heterozygous mutation in the <span class="elsevierStyleItalic">DNAH5</span> gene for variants not previously described&#44; but probably pathogenic&#58; c&#46;4625&#95;4628delGAGA&#58;p&#46;&#40;Arg1542ThrfsTer6&#41; and c&#46;12706-2A&#62;T&#46; Parents are heterozygous for 1 of the mutations&#46; This gene encodes one of the ODA heavy chains and is essential to ciliary function&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">10</span></a></p><p id="par0045" class="elsevierStylePara elsevierViewall">Immunofluorescence studies were conducted in respiratory epithelial cells to confirm expression or absence of the mutated DNAH5 protein&#46; Primary cilia anti-acetylated tubulin antibodies &#40;Sigma Aldrich&#44; St&#46; Louis&#44; MO&#44; USA&#41; and 4 ciliary structure proteins were used&#58; DNAH5 &#40;ODA&#41;&#59; DNALI1 &#40;IDA&#41;&#59; RSPH4A &#40;radial connections&#41;&#44; and GAS8 &#40;nexin-dynein-regulatory complex&#41;&#46; The results showed a complete absence of DNAH5 protein in the ciliary axoneme &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#41; and colocalization of DNALI1&#44; RSPH4A and GAS8 with the ciliary acetylated tubulin &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B&#8211;D&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0050" class="elsevierStylePara elsevierViewall">The results of the immunofluorescence test are consistent with the genetic study and confirm that the previously described mutations produce a complete lack of expression of the protein DNAH5 in the ciliary axoneme and cause an ODA defect&#46;<a class="elsevierStyleCrossRefs" href="#bib0130"><span class="elsevierStyleSup">11&#44;12</span></a> In our analysis of video-microscopy&#44; ciliary immobility was observed&#44; which is consistent with the findings in these cases&#46;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">11</span></a> The observation on electron microscopy of an alteration in IDA as well as ODA could be explained by IDA changes due to respiratory infections&#44;<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">13</span></a> the fact that in healthy subjects IDA might not occur in more than 50&#37; of the doublets&#44;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">14</span></a> or the presence of processing artifacts&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">15</span></a></p><p id="par0055" class="elsevierStylePara elsevierViewall">The immunofluorescence test has limitations<a class="elsevierStyleCrossRefs" href="#bib0095"><span class="elsevierStyleSup">4&#44;7</span></a>&#58; it does not provide antibodies to all defective proteins&#44; and the technique may fail due to the absence of cilia or interference of mucus or blood in the sample&#46; However&#44; in combination with the molecular study&#44; it offers a useful approach to the diagnosis of this disease and to identifying the specific causative defect&#46; This conclusion will have to be confirmed in more extensive studies&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Funding</span><p id="par0060" class="elsevierStylePara elsevierViewall">This work has been partially funded by a Strategic Action in Health grant from the <span class="elsevierStyleGrantSponsor" id="gs1">Instituto de Salud Carlos III</span>&#40;<span class="elsevierStyleGrantNumber" refid="gs1">PI16&#47;01233</span>&#41;&#44; a grant from the <span class="elsevierStyleGrantSponsor" id="gs2">Spanish Society of Pediatric Pulmonology</span>&#44; and a grant from the <span class="elsevierStyleGrantSponsor" id="gs3">Catalan Foundation of Pulmonology &#40;FUCAP&#41;</span>&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Conflict of interests</span><p id="par0065" class="elsevierStylePara elsevierViewall">AMG has received funding for participation in Abbvie advisory boards&#44; and has received assistance for travel and registration at medical congresses from Abbvie&#44; Actelion&#44; and Novartis&#44; all activities unrelated with this work&#46; SR has received assistance for travel and registration at medical congresses from Abbvie&#44; Teva&#44; and Novartis&#44; all activities unrelated with this work&#46; The other authors state that they have no conflict of interests&#46;</p></span></span>"
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        "nota" => "<p class="elsevierStyleNotepara" id="npar0005">Please cite this article as&#58; Baz-Red&#243;n N&#44; Rovira-Amigo S&#44; Camats-Tarruella N&#44; Fern&#225;ndez-Cancio M&#44; Garrido-Pontnou M&#44; Antol&#237;n M&#44; et al&#46; Papel de la inmunofluorescencia y el diagn&#243;stico molecular en la caracterizaci&#243;n de la discinesia ciliar primaria&#46; Arch Bronconeumol&#46; 2019&#59;55&#58;439&#8211;441&#46;</p>"
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          "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Immunofluorescence analysis of the ciliary ultrastructure&#46; The first column shows the presence of cilia in the cell using acetylated tubulin &#40;in green&#41;&#44; the second shows the outcome of incubation with primary ciliary protein antibodies &#40;in red&#41;&#44; and the third shows the merge of tubulin with each ciliary protein and the DAPI-stained nucleus &#40;blue&#41;&#46; &#40;A&#41; Absence of protein DNAH5 &#40;external component of dynein arms&#41; in the ciliary axoneme&#46; &#40;B&#8211;D&#41; Presence and colocalization of tubulin and ciliary axoneme proteins &#40;in yellow&#41;&#58; &#40;B&#41; DNALI1 &#40;external component of dynein arms&#41;&#59; &#40;C&#41; RSPH4A &#40;radial spoke head component&#41;&#44; and &#40;D&#41; GAS8 &#40;nexin-dynein regulatory complex component&#41;&#46;</p>"
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