Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR.

Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.

RNA extraction and RT-PCR procedures adapted for the detection of enterovirus sequences from frozen and paraffin-embedded formalin-fixed spinal cord samples.

Methods for detecting enterovirus RNA in both paraffin-embedded, formalin-fixed and frozen spinal cord sections from amyotrophic lateral sclerosis (ALS) patients were established. A proteinase K digestion following the deparaffinization procedure was required for the fixed spinal cord sections, whereas only one step of crushing in phosphate buffered saline was necessary for the frozen samples prior to the extraction of the RNA. With an optimized reverse transcription and PCR procedure, enterovirus RNA could be detected from frozen and fixed archival spinal cord samples.

Molecular analysis of iduronate -2- sulfatase gene in Tunisian patients with mucopolysaccharidosis type II.

UNLABELLED: Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is X-linked recessive lysosomal storage disorder resulting from the defective activity of the enzyme iduronate-2-sulfatase (IDS). Hunter disease can vary from mild to severe, depending on the level of enzyme deficiency. We report the IDS mutation and polymorphisms causing the Hunter syndrome in patients from one family in Tunisia PATIENTS AND METHODS: A preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS II probands. The IDS mutation and polymorphisms were determined on these probands and their family members by amplifying and sequencing each of the exons and intron-exon junctions of IDS gene. RESULTS: The studied probands were homoallelic for p.R88P mutation. In addition, three known polymorphisms/sequence variants: IVS3-16 (c.419-16 delT), T214M (c.641C > T), T146T (c.438 C > T), IVS5-87(c.709-87G > A) and one previously unknown: IVS7+38(c.1006+38T > C were identified in the MPS II patients. These are the first Tunisian MPS II patients to be genotyped. CONCLUSION: The identification of these mutation and polymorphisms and their genotype-phenotype correlation should facilitate prenatal diagnosis and counseling for MPS II in Tunisia, where a very high rate of consanguinity exists.

Mucopolysaccharidosis type I: molecular characteristics of two novel alpha-L-iduronidase mutations in Tunisian patients.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an autosomal storage disease resulting from defective activity of the enzyme α-L-iduronidase (IDUA). This glycosidase is involved in the degradation of heparan sulfate and dermatan sulfate. MPS I has severe and milder phenotypic subtypes. AIM OF STUDY: This study was carried out on six newly collected MPS I patients recruited from many regions of Tunisia. PATIENTS AND METHODS: Mutational analysis of the IDUA gene in unrelated MPS I families was performed by sequencing the exons and intron-exon junctions of IDUA gene. RESULTS: Two novel IDUA mutations, p.L530fs (1587_1588 insGC) in exon 11 and p.F177S in exon 5 and two previously reported mutations p.P533R and p.Y581X were detected. The patient in family 1 who has the Hurler phenotype was homozygous for the previously described nonsense mutation p.Y581X.The patient in family 2 who also has the Hurler phenotype was homozygous for the novel missense mutation p.F177S. The three patients in families 3, 5 and 6 were homozygous for the p.P533R mutation. The patient in family 4 was homozygous for the novel small insertion 1587_1588 insGC. In addition, eighteen known and one unknown IDUA polymorphisms were identified. CONCLUSION: The identification of these mutations should facilitate prenatal diagnosis and counseling for MPS I in Tunisia.