Occult HCV infection has been defined as the presence of HCV RNA in liver and/or lymphoid cells despite undetectable HCV-RNA in the serum in HCV-infected patients who have a spontaneous clearance or antiviral treatment response. The current study was primarily aimed at determining the prevalence of occult HCV in predominantly 3(a/b)-infected HCV patients and secondly to investigate if PBMC positivity can be seen as an indicative marker for later recurrence of viral particles in the serum. Our cohort was infected with the subtype of occult HCV defined by the presence of HCV RNA in PBMCs of patients with treatment-induced HCV RNA clearance from serum (antiHCV- positive, serum HCV RNA-negative). It should also be mentioned that the sample collection was random for the genotype selection, but the cohort turned out to be 100% genotype 3, which has already been reported as the predominant type of HCV in Pakistan, especially in the Potohar region [11, 12]. This also reinforces our own previous observation of genotype 3 as the most prevalent genotype in the region; therefore our conclusions are more relevant for HCV genotype 3. Our study also shows a 70% success rate for the sustained response achieved through interferon alpha/ribavirin combined therapy as reported previously  for genotype 3.
Liver biopsy samples are considered the gold standard for occult HCV diagnosis; but due to the invasive nature of this method, especially in the absence of clinical manifestations, reliable alternative methods of investigation have also been developed . HCV RNA has been reported in PBMC as well as ultracentrifuged serum samples in a high percentage of patients (70%) and is considered a reliable method for occult detection as repeat liver biopsy samples are not usually available in clinical practice . We therefore, relied on the HCV detection in lymphocytes for our method of investigation. According to the current definition of occult as serum-clear, treated patients, we considered the presence of positive PBMCs in the end-of-treatment specimens as evidence of occult HCV. Our results showed viral genomic HCV present in the PBMCs of 15.8% of 3 (a/b) chronically infected HCV patients at EOT. The follow up studies, however, showed that the HCV particles that are present in the PBMC of the patients at the end of treatment are ultimately cleared out by 6-12 months after EOT at the SVR stage. We did not find any association of delayed clearance with any of the baseline parameters listed in Table 1 as previously reported [14, 15]. The SVR stage results therefore lead us to conclude that the 15.8% EOT viral presence reflects a difference in the viral kinetics in various compartments rather than a true case of occult HCV. It is also worth mentioning that there has been a recent report on the study of occult virus in the region; but the cohort consisted of non-treated cryptogenic virus-infected patients who were antiHCV-negative and showed increased ALT (alanine amino transferase) and AST (aspartate amino transferase) levels . These results may reflect a true case of occult HCV. Our results also indicate the likelihood that the immune response remains active even after the completion of the treatment regimen and that it helps in clearing of the residual virions that might remain detectable in other compartments.
It could be argued that lack of detection of HCV in the PBMC of our cohort at SVR is due to low sensitivity of our test (50 IU/ml), which may be too low to detect the presence of very small amounts of occult viral particles; but in our study plan we followed the same cohort of patients who initially (EOT) showed the presence of occult virus in their PBMC with the same sensitivity level. It is also worth mentioning that one of the 'occult' samples, although still weakly positive at 6 months post EOT, was observed to gradually clear the residual viremia at one year post-treatment. However, we could not exclude the possibility of viral persistence in the hepatic cells in our cohort of patients as none of them presented any indication for a liver biopsy test, such as raised ALTs at the EOT. These patients will be periodically followed up and liver biopsies carried out if needed. According to our current findings we conclude that clinicians may be confident of the clearance of the virus as indicated by the absence of viral RNA in the serum at the EOT especially in the case of genotype 3 infection. A previous study on five geno 3 patients also showed clearance of virus after 56 months of treatment .
The question remaining is why the virus is cleared later from the PBMC than the serum (15.8% cases). This cannot be explained clearly due to the incomplete understanding of the HCV infection and evasion processes. It can be propounded, however, that HCV has developed a number of evasion mechanisms, infection of PBMCs being one of those where the virus can avoid the immune defense system, while hepatocytes remain the actual target. We tried to find an association of baseline viral load and other clinical parameters with final clearance of virus, since some researchers have shown an association of occult presence with high cholesterol and triglyceride levels [14, 15]. Statistical tests were applied to determine if there was an association with any of the base line parameters given in Table 1, but no such evidence was found. It may also be that the virus developed some new quasi-species in PBMC that showed delayed response to the antiviral therapy but we could not confirm that speculation.
The outcome of therapy involves an interplay of host and viral factors in their specific environments, which ultimately determines the immune response at both the innate and humoral response levels of the host. It is difficult, therefore, at this stage to determine the effect of clinical and biochemical parameters as markers for disease outcome. It would be worthwhile to study the known host and viral genetic factors that might contribute to the immune response in the patients and provide a better understanding of the observed treatment responses. The existence of occult HCV as a reservoir of the virus, that can become active has remained controversial since its first report in 2004 . There have been recent negative reports in other genotypes also [18–22]. Therefore, the picture is still unclear. Further detailed, long-term studies are needed before clinicians can be confident that the serum based tests are accurate.