Santral sinir sistemi enfeksiyonu etkeni enterovirusların RT-PCR ve hücre kültür yöntemleri ile saptanması
Küçük Resim Yok
Tarih
2011
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info:eu-repo/semantics/openAccess
Özet
Aseptik menenjit/ensefalitlerin en önemli etkenleri viruslar olup, etiyolojinin belirlendiği aseptik menenjit olgularının %80’inden fazlasından polio dışı enteroviruslar sorumludur. Bu çalışmada, viral santral sinir sistemi (SSS) enfeksiyonu şüpheli hastaların beyin omurilik sıvısı (BOS) örneklerinde, viral kültür ve ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) yöntemleri ile enterovirus varlığının araştırılarak epidemiyolojiye katkıda bulunulması ve ticari bir RT-PCR kitinin rutin laboratuvarımız için uygunluğunun araştırılması amaçlanmıştır. Çalışmaya, Ocak 2007-Aralık 2008 tarihleri arasında Ege Üniversitesi Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı Laboratuvarları’na klinik ve BOS bulguları açısından viral SSS enfeksiyonu şüphesi olan hastalardan gönderilen ve rutin laboratuvar değerlendirmesinde mikrobiyolojik yöntemler ile bakteriyel, mikobakteriyel, fungal ve viral (HSV, sitomegalovirus) etkenlerin saptanmadığı 66 BOS örneği dahil edilmiştir. Örneklerin 34 (%51.5)’ü kadın, 32 (%48.5)’si erkek hastalara ait olup, hastaların 23 (%34.8)’ü çocuk (5 ay-18 yaş), 43 (%65.2)’ü yetişkin (19-86 yaş) yaş grubunda yer almaktadır. Örneklerden virus izolasyonu için Vero, Hep-2 ve RD hücre serilerini içeren “shell vial” hücre kültürü yöntemi kullanılmış, 48 saatlik inkübasyon sonunda hücreler floresanla işaretli poliklonal antikorlar (Pan-Enterovirus Blend, Light Diagnostics, ABD) ile boyanarak değerlendirilmiştir. Enterovirus RNA’sını saptamak amacıyla ticari bir RT-PCR yöntemi (Enterovirus Consensus Kit, Argene, Fransa) kullanılmıştır. Viral SSS enfeksiyonu şüpheli 66 BOS örneğinin 61 (%92.4)’i, RT-PCR ve hücre kültürü yöntemleri ile enterovirus negatif olarak bulunmuş; 3 (%4.5) BOS örneğinde hücre kültürü ile pozitiflik saptanmıştır. Bu örneklerden biri RT-PCR yöntemi ile de pozitif olarak bulunurken, kalan iki örnek negatif olarak tespit edilmiştir. Hücre kültürü ile negatif olarak saptanan iki örnekte (%3) RT-PCR ile ara değer elde edilmiştir. Hücre kültüründe pozitiflik saptanan üç örnekten ikisi tiplendirme sonucu echovirus olarak tanımlanmış; diğer örneğin miktarı yeterli olmadığından tiplendirme yapılamamıştır. Sonuç olarak, RT-PCR ile ara değer olarak saptanan sonuçlar yorum ve değerlendirmeyi engellemiş ve ayrıca hücre kültürü ile pozitif bulunan iki örneği saptayamadığı için Enterovirus Consensus Kit’in laboratuvarımızda rutin kullanım için pratik olmadığı ve hücre kültürüne üstünlük göstermediği kanısına varılmıştır. Diğer taraftan nükleik asit amplifikasyon testlerinin (NAT) yüksek duyarlılıkları nedeniyle enteroviral SSS enfeksiyonu tanısındaki yararı bilindiğinden, alternatif bir NAT yönteminin özellikle çocuk yaş grubu hastaların BOS örneklerinde hücre kültürü ile paralel değerlendirilmesinin yararlı olacağı düşünülmüştür.
Viruses are the major causes of aseptic meningitis and encephalitis. Enteroviruses account for more than 80% of the aseptic meningitis cases for which an etiologic agent is identified. The aims of the present study were to identify agents of enteroviral meningitis by viral culture and reverse transcriptase polymerase chain reaction (RT-PCR) methods, to evaluate the appropriateness of a commercial RTPCR kit for its use in routine laboratory, and to obtain epidemiological data about enteroviral meningitis. Sixty six cerebrospinal fluid (CSF) samples from patients with suspected viral central nervous system (CNS) infection by clinical and CSF biochemical findings, sent to Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. The CSF samples were all negative for tested bacteria, mycobacteria, fungi, herpes simplex virus and cytomegalovirus. Thirty-four (51.5%) of the samples were from female and 32 (48.5%) were from male patients. Twenty-three (34.8%) patients were children (5 months-18 years) and 43 (65.2%) were adults (19-86 years). Shell vial rapid cell culture method by using Vero, HEp-2 and RD cell lines was performed for virus isolation and the results were evaluated on $48^ {th}$ hours after staining the cells with fluorescein labeled polyclonal antibodies (Pan-Enterovirus Blend, Light Diagnostics, USA). Enteroviral RNA in the samples was detected by a commercial RT-PCR kit (Enterovirus Consensus Kit, Argene, France). Sixty-one (92.4%) of 66 samples from patients with suspected viral CNS infection were found to be negative for enterovirus both with RT-PCR and shell vial cell culture methods. Three samples (4.5%) were positive by shell vial culture method. In one CSF sample that was culture positive, RT-PCR was also positive. However, the remaining two culture positive samples yielded negative result by RT-PCR. Intermediate results with RT-PCR were obtained in two samples (3%) that were identified as negative by cell culture. Two of the three positive samples in cell culture were identified as echovirus, however, the remaining sample could not be identified due to small sample amount. As a result, the commercial assay was found non-practical and labor intensive, giving indeterminant results in some cases and missing two culture positive samples. Since it didn’t have an advantage over the cell culture method used, it was found inappropriate for routine diagnosis in our laboratory. On the other hand, it has been known that nucleic acid amplification tests (NAT) have markedly improved the diagnosis of enterovirus infections by increasing the sensitivity compared with cell culture methods. An alternative NAT method should be evaluated in parallel with cell culture method especially in CSF samples of children with suspected viral central nervous system infections.
Viruses are the major causes of aseptic meningitis and encephalitis. Enteroviruses account for more than 80% of the aseptic meningitis cases for which an etiologic agent is identified. The aims of the present study were to identify agents of enteroviral meningitis by viral culture and reverse transcriptase polymerase chain reaction (RT-PCR) methods, to evaluate the appropriateness of a commercial RTPCR kit for its use in routine laboratory, and to obtain epidemiological data about enteroviral meningitis. Sixty six cerebrospinal fluid (CSF) samples from patients with suspected viral central nervous system (CNS) infection by clinical and CSF biochemical findings, sent to Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. The CSF samples were all negative for tested bacteria, mycobacteria, fungi, herpes simplex virus and cytomegalovirus. Thirty-four (51.5%) of the samples were from female and 32 (48.5%) were from male patients. Twenty-three (34.8%) patients were children (5 months-18 years) and 43 (65.2%) were adults (19-86 years). Shell vial rapid cell culture method by using Vero, HEp-2 and RD cell lines was performed for virus isolation and the results were evaluated on $48^ {th}$ hours after staining the cells with fluorescein labeled polyclonal antibodies (Pan-Enterovirus Blend, Light Diagnostics, USA). Enteroviral RNA in the samples was detected by a commercial RT-PCR kit (Enterovirus Consensus Kit, Argene, France). Sixty-one (92.4%) of 66 samples from patients with suspected viral CNS infection were found to be negative for enterovirus both with RT-PCR and shell vial cell culture methods. Three samples (4.5%) were positive by shell vial culture method. In one CSF sample that was culture positive, RT-PCR was also positive. However, the remaining two culture positive samples yielded negative result by RT-PCR. Intermediate results with RT-PCR were obtained in two samples (3%) that were identified as negative by cell culture. Two of the three positive samples in cell culture were identified as echovirus, however, the remaining sample could not be identified due to small sample amount. As a result, the commercial assay was found non-practical and labor intensive, giving indeterminant results in some cases and missing two culture positive samples. Since it didn’t have an advantage over the cell culture method used, it was found inappropriate for routine diagnosis in our laboratory. On the other hand, it has been known that nucleic acid amplification tests (NAT) have markedly improved the diagnosis of enterovirus infections by increasing the sensitivity compared with cell culture methods. An alternative NAT method should be evaluated in parallel with cell culture method especially in CSF samples of children with suspected viral central nervous system infections.
Açıklama
Anahtar Kelimeler
Mikrobiyoloji
Kaynak
Mikrobiyoloji Bülteni
WoS Q Değeri
Scopus Q Değeri
Cilt
45
Sayı
3
