Molecular diagnosis and phylogenetic analysis of human papillomavirus type-16 from suspected patients in Pakistan.

BACKGROUND: Human Papillomavirus (HPV) is well known pathogen that can cause benign and malignant tumors in humans, yet there is very little information regarding HPV types prevalent in Pakistan. METHODS: A total of 92 cervical secretions were collected from suspected married female patients and used for DNA isolation using a novel isolation method. The samples were tested through Polymerase Chain Reaction (PCR) using already reported primers MY09/MY11, GP5/GP6, GP5+/GP6+, CP65/CP70, CP66/CP69 and SPF1/SPF2 and with those developed in this study including HRT1 and HRT2 primer sets for typing HPV types and HACTB primer set for human beta actin gene as internal positive control. Sequencing and phylogenetic analyses were performed for two isolates to determine circulating HPV types. RESULTS: PCR with HRT1 and HRT2 indicated 2 (2.17 %) patients were positive for HPV type- 16 while 1 (1.08 %) with HPV type 18. Sequencing and phylogenetic analysis of isolates confirmed HPV type-16 in genus alpha 9 which have 99 % homology with already reported HPV from Japan and Costa Rica. CONCLUSION: This is the first report of HPV type-16 genus alpha 9 in Pakistan and the reported assay and sequence data will serve as valuable tools in further epidemiological studies for HPV surveillance to improve public health, especially of females in Pakistan.

Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Punjab Province in Pakistan and a phylogenetic analysis.

BACKGROUND: Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. In Pakistan, more than 10 million people are living with HCV. Very little is known about the genotype distribution in Punjab Province, the largest province of Pakistan. Pretreatment genotype identification is very important, as different HCV genotypes respond differently to interferon therapy. METHODS: In this study we performed HCV genotyping for 1537 HCV-infected patients from different districts of Punjab Province, Pakistan. Sequencing of partial HCV NS5B sequences from 14 samples belonging to genotypes 3 and 1 was also done. A sequence comparison was made of our reported sequences with those reported in the National Center for Biotechnology Information (NCBI), and a phylogenetic tree was constructed. RESULTS: Our study showed that the most frequent HCV genotype was 3a (in 88.1% of infected individuals), followed by 1a (3.5%), 3b (3.0%), 1b (0.8%), and 2a (1.0%). A mixed genotype infection was found in 3.6% of infected individuals, with 0.3% living with 1a + 1b co-infection, 3.1% with 3a+3b, and 0.2% suffering from 3a+1b co-infection. The sequence comparison showed that HCV NS5B motif B residues G283, T287, and N291 were highly conserved in both genotype 1 and genotype 3 sequences, while the motif B residue T286 was mutated to proline in all the genotype 3 sequences. The GDD motif, which forms the catalytic pocket and binding site for the divalent cations, was highly conserved in all the reported sequences. The phylogenetic tree suggests clustering of genotype 1 sequences with sequences from the USA, UK, Germany, and France, while genotype 3 sequences are clustered with sequences from Japan and the UK. CONCLUSIONS: The major prevalent genotype in Punjab Province of Pakistan was genotype 3a, followed by genotype 1a, and only 3.6% of infected individuals had a mixed genotype infection. Sequencing of the HCV NS5B gene suggested that the active site residues were highly conserved in all the reported sequences. Our sequences, which are clustered with sequences from the USA, UK, France, and Japan, show the diversity in origin of the different genotypes prevalent in Pakistan.

Analytical variables influencing the HCV RNA determination by TaqMan real-time PCR in routine clinical laboratory practice.

Hepatitis C virus (HCV) quantification is used as a prognostic marker for treatment success. In a routine clinical laboratory some infinitesimal sample handling factors can contribute to variability and loss of precision in HCV quantification. This may include blood collection tubes, blood drawing procedure, sample processing and storage temperatures. In current study blood was collected in tubes with different anticoagulant type (spray vs. liquid), group 1, blood was drawn with possible suck of methylated spirit through needle (experimental group) while avoiding the methylated spirit suck (control group) group 2, plasma separation was delayed from 0 to 60 min for group 3, plasma storage at different temperatures group 4. All samples were analyzed using Corbett research real time PCR system using AJ Roboscreen Kit. Mean viral load difference between spray vs. liquid was found 3.6 × 10(5) IU/ml (p < 0.001). Methylated spirit inhibited the viral load quantification with a value of 4.8 × 10(5) IU/ml (p < 0.001). Mean viral load difference was found 1.2 × 10(5) IU/ml (p < 0.05). Delay in centrifugation from 0 to 60 min and plasma placement at 25 °C for 15 min before freezing had no effect (p = 0.5996). Plasma storage temperature at -80 and -20 °C did not affect significantly on RNA levels (p > 0.05). In conclusion blood collection tubes and procedures can be a key factor in variability of results, that might affect the treatment response decision.

Predictors of response to antiviral therapy in patients with chronic hepatitis C from Pakistani population.

BACKGROUND: Hepatitis C virus (HCV) constitutes a major public health issue around the world, especially in developing countries like Pakistan. In this study, we assessed outcome of interferon (INF) treatment in chronic hepatitis C patients categorized by gender, age, and viral load. METHODS: In this study, 750 HCV positive patients with genotype 3 were selected, out of which 616 completed the entire treatment. Their personal history, pre-treatment HCV RNA and serum alanine transaminase (ALT) was quantified. Patients were treated with combination therapy of INF-α 2b three million units (thrice a week) plus ribavirin (1000 - 1200 mg per day) for 24 weeks. After 24 weeks their HCV RNA and serum ALT level was quantified. RESULTS: Out of the 616 patients, 391 (63.5%) responded to therapeutic regimen (INF-α 2b plus ribavirin). Among the responders, 27.1% were men and 36.4% were women. Best treatment response was observed in patients having low viral load < 8 × 10(5) IU/ml and age ≤ 40 years than patients having low viral load and age > 40 years (73.2% vs. 60.3%, P = 0.05). CONCLUSIONS: Better response to IFN-α 2b plus ribavirin was observed in patients with lower viral RNA and younger age. It suggests that all patients considered for treatment should have quantification of serum HCV RNA level. The result can be used to counsel patients on the likelihood of response and may influence the patient's decision on treatment.