The non-structural 5B (NS5B) gene is the target region to identify hepatitis C virus (HCV) subtypes. However, it is not always possible to amplify this region because of inherently high sequence variability. Nucleotide sequences of the non-structural 5A (NS5A) and NS5B genes and its concordance were determined from patients infected with HCV genotype 1 (HCV-1). Among the 30 HCV-1 samples, 7 (23%) were identified as subtype 1a and 23 (77%) were identified as 1b by NS5A sequencing. Sequence analysis of the NS5B showed that 13 (43%) were identified as 1a and 17 (57%) were identified as 1b. Out of the 13 samples identified as 1a by NS5B, 6 (46%) were correctly identified by NS5A. Of the 17 samples identified as 1b by NS5B, 16 (94%) were correctly identified by NS5A. The presence of glutamic acid (E) or aspartic acid (D) at position 2225 in the NS5A differentiates 1a from 1b subtypes, respectively. This study showed that the NS5A sequencing can identify HCV-1a and 1b subtypes with predictive values of 86% and 70% of cases, respectively. The overall concordance with NS5B was 73%. NS5B sequence analysis remains to be the reference method to identify HCV-1 subtypes. NS5A sequencing may be used to complement NS5B sequencing in case the NS5B gene cannot be successfully amplified.

Human ; Male ; Female ; Aged ; Middle Aged ; Adult ; ; Aspartic Acid ; Genotype ; Glutamic Acid ; Hepacivirus ; Hepatitis C ; Nucleotides ; Sequence Analysis ; Viral Nonstructural Proteins

We report a case of a 76-year old Filipino male from the province of Porac, Pampanga who presented with pain, redness, and blurring of vision of the right eye (oculus dexter, OD) on 25 February 2009. One and a half weeks prior to consultation, patient complained of right eye discharge and lid swelling. The patient consulted an ophthalmologist and was given unrecalled topical eye medications. There was no improvement from the symptoms and patient complained of worsening pain and redness in the right eye. The patient was a non-contact lens wearer and he denied any trauma to the right eye. However, he admitted to washing both eyes with tap water. Visual acuity in the right eye was counting fingers at 3 feet. Slit lamp examination showed diffuse conjunctival injection. There was note of a ring-like infiltrate in the cornea almost extending to the limbus. There was also note of significant anterior chamber cells reaction and hypopyon. Primary working impression was Acanthamoeba keratitis (AK), OD. Corneal scraping was done and sent to the St. Luke's Research and Biotechnology for detection and identification of the infectious agent. Morphological detection was done by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA. S1 (a partial 18S ribosomal DNA gene) confirmed the morphological identification (band at 461-bp). Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present from the eye of keratitis patients.

We report the case of a 76-year old Filipino male who presented with pain, redness, and blurring of vision of the right eye. Corneal scraping was done and sent to the St. Luke’s Research and Biotechnology Group for detection and identification of the infectious agent. Morphological detection was performed by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA.S1 confirmed the morphological identification. Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present in the eye of keratitis patients.