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Scientific letter
Stability of viral RNA in clinical specimens for viral diagnosis
Estabilidad del ARN viral en muestras clínicas para el diagnóstico viral
Xana García Fernández
Corresponding author
xanagarcia2009@hotmail.com

Corresponding author.
, Marta E. Álvarez-Argüelles, Susana Rojo, María de-Oña
Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Asturias, Spain
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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">In molecular biology&#44; the conservation of genetic material from clinical samples is key when performing experiments with RNA&#46; Therefore&#44; it is important to take the maximum precautions to avoid its degradation&#44; mainly due to RNases that can cause fragmentation of genomic material&#46; These are robust proteins with enzymatic activity that participate in various physiological processes and their main function is to hydrolyse and degrade RNA&#44; and they can also be introduced exogenously into experiments&#46;<a class="elsevierStyleCrossRefs" href="#bib0040"><span class="elsevierStyleSup">1&#44;2</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">When clinical samples are kept in the laboratory for long periods of time&#44; their RNA is especially vulnerable to degradation&#44;<a class="elsevierStyleCrossRefs" href="#bib0050"><span class="elsevierStyleSup">3&#44;4</span></a> which could influence viral diagnosis by decreasing the sensitivity of amplification techniques&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Various studies have shown that ribonuclease inhibitors exhibit a wide spectrum of inhibitory activities against RNases&#44; effectively protecting RNA during laboratory procedures&#44; allowing great flexibility in experimental design&#46; Their use can be a solution to minimize these degradation problems&#46;<a class="elsevierStyleCrossRefs" href="#bib0060"><span class="elsevierStyleSup">5&#8211;7</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">The objective of this study was to evaluate the integrity of viral RNA from clinical samples after storage at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; after a standard working time period &#40;10 days&#41;&#44; which is considered sufficient to carry out all the routine processes involved in the viral diagnosis of a sample&#44; and to analyze the efficacy and performance of an RNase inhibitor to prevent its degradation&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">Of the samples processed in the Virology Unit of the Hospital Central de Asturias &#91;Central Hospital of Asturias&#93; for two months&#44; 67 exudates &#40;42 nasopharyngeal and 25 pharyngeal&#41; collected in a viral transport medium were studied&#46; Extraction and purification of the genomic material was carried out with the MagNA Pure LC Total Nucleic Acid Isolation Kit Reagent&#44; in the MagNA Pure LC 2&#46;0 automated system &#40;Roche Diagnostics&#44; Switzerland&#41;&#46; 100<span class="elsevierStyleHsp" style=""></span>&#956;l was obtained for a subsequent amplification of fragments of the viral genome and laboratory syndromic diagnostic protocols were applied&#46; In all of them&#44; the quantity and quality of the sample was evaluated&#44; and with the amplification of the human &#946;-globin gene&#44; the normalized viral load &#40;by number of cells&#41; was reported&#46; From the total nucleic extract&#44; two aliquots of 10<span class="elsevierStyleHsp" style=""></span>&#956;l were separated and in one 1<span class="elsevierStyleHsp" style=""></span>&#956;l RNase&#174; Inhibitor &#40;Applied Biosystems&#44; USA&#41; was added&#46; Both were stored at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">The 67 samples included underwent the usual laboratory diagnostic process for enterovirus &#40;<span class="elsevierStyleItalic">n</span>&#58; 53&#41;&#44; parainfluenza virus &#40;<span class="elsevierStyleItalic">n</span>&#58; 11&#41;&#44; and influenza A virus &#40;<span class="elsevierStyleItalic">n</span>&#58; 3&#41;&#46; After 10 days&#44; the two stored aliquots underwent the same PCR and the amplification cycles &#40;Ct&#41; were compared with and without inhibitor&#46; They were also divided into three groups according to their Ct &#40;&#60;20&#44; 20&#8211;25 and 25&#8211;35 cycles&#41; and type of virus&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">All statistical calculations were performed with the computer program&#44; GraphPad InStat v2&#46;04a &#40;GraphPad Software&#44; USA&#41; according to the specific requirements&#46;</p><p id="par0040" class="elsevierStylePara elsevierViewall">In <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#44; we observe that the presence of viral RNA is evident in all the samples studied&#44; and no statistically significant differences were found in any group after 10 days regardless of whether or not an inhibitor was added&#46; Furthermore&#44; the mean Ct does not vary significantly in any virus&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0045" class="elsevierStylePara elsevierViewall">The samples repeated the amplification range in 59 &#40;88&#46;1&#37;&#41; of the occasions after 10 days&#44; and in 58 &#40;86&#46;6&#37;&#41; of the cases&#44; when RNasin&#174; was added to the extracted sample&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">This study shows that&#44; although the use of RNasin&#174; is usually recommended in RNA virus management protocols&#44; it does not seem essential&#44; at least in the viruses studied&#46; In some cases&#44; the performance in the samples with and without an inhibitor decreased slightly&#44; and it is important to note that no sample was undetectable after 10 days&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall">Thus&#44; we maintain that for viral studies that are conducted in a period of 10 days stored at 4<span class="elsevierStyleHsp" style=""></span>&#176;C it does not decrease the diagnostic capacity and it would not be necessary to add preservative reagents&#44; which would decrease the handling of the samples and also would not imply an increase in the cost of the diagnostic tests&#46; It would be useful to conduct studies over longer periods of time to establish from what time possible degradation of RNA could be minimized by using an enzyme inhibitor&#46;</p></span>"
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Ct&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#60;20&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">16 &#40;16&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">2 &#40;2&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0 &#40;0&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Ns&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>18&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">20&#8211;25&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">4 &#40;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7 &#40;6&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttop\n
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                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
                  """
              ]
              "imagenFichero" => array:1 [
                0 => "xTab2302214.png"
              ]
            ]
            1 => array:2 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col">Standard&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col">24&#46;78<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;44&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col">25&#46;07<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;37&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col">25&#46;28<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;44&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&#40;21&#8211;28&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&#40;22&#8211;27&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&#40;22&#8211;27&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">IA&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">28&#46;67<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>4&#46;16&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">28&#46;34<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;78&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">28&#46;67<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;13&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#40;24&#8211;32&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#40;24&#8211;31&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#40;23&#8211;33&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
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es en pt

¿Es usted profesional sanitario apto para prescribir o dispensar medicamentos?

Are you a health professional able to prescribe or dispense drugs?

Você é um profissional de saúde habilitado a prescrever ou dispensar medicamentos