Anis Khan

Detection of hepatitis C virus natural recombinant RF1_2k/1b strain among intravenous drug users in Uzbekistan

Aim:  A series of recent studies have indicated the presence of natural intergenotypic recombinant hepatitis C virus (HCV) strains in distinct parts of the world. The majority of the current genotyping methods are based on analysis of either 5′UTR, structural (Core/E1/E2) or non‐structural (NS5B) genomic regions of the virus.

Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan

BackgroundEnteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder.MethodsStool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases.ResultsNPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers.ConclusionThe present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.

Epidemic Spread of Hepatitis C Virus Genotype 3a and Relation to High Incidence of Hepatocellular Carcinoma in Pakistan

Studies conducted in different populations worldwide revealed an association between HCV genotype 1 and the development of hepatocellular carcinoma (HCC) than in infection with other HCV genotypes. There are reports which reveal the association of HCV genotype 3a (HCV‐3a) with hepatic steatosis and fibrosis but its relation with the development of HCC has not been investigated. In Pakistan, where the incidence of HCC is increasing, 189 patients with chronic liver disease including 82 with HCC were enrolled. HCV genotypes were determined by phylogeny in the NS5B region and the epidemic history of HCV‐3a was examined using coalescent theory based methods. HCV‐3a was the predominant genotype (81.4%) in the cohort studied, followed by 3b (9.3%), 3k (2.3%), 1a (1.5%), 1c (1.5%), 1b (0.8%), and 2a (0.8%) where 76% of HCC and 86% of non‐HCC were infected with HCV‐3a. The significant factors associated with HCC were older age (mean ± SD) 55.8 (±9.9) (P < 0.0001), and male gender (P < 0.001). HCV RNA was significantly higher in patients with HCC and chronic hepatitis than in liver cirrhosis (P < 0.0001). Molecular evolutionary analysis revealed a distinct phylogenetic cluster of HCV‐3a in Pakistan and an estimation of the effective number of HCV infections indicated the appearance of HCV‐3a in this region around 1920s and a rapid exponential growth in the 1950s. This indicates that the epidemic spread of HCV‐3a occurred earlier in Pakistan than in other countries in which this genotype has been reported. HCV‐3a which spread earlier in Pakistan may be associated with an increasing incidence of HCC. J. Med. Virol. 81:1189–1197, 2009.

Genetic variability of hepatitis C virus in South Egypt and its possible clinical implication.

Egypt is one of the countries with very high rates of hepatitis C virus (HCV) related morbidity and mortality. However, little is known about geographical and clinical differences in genetic variability of HCV in Egypt. Using direct sequencing and phylogenetic analysis of partial core/E1 and NS5B regions of the HCV genome, HCV genotype/subtype was determined in 129 HCV‐infected patients residing in three governates in south Egypt: Assuit, Sohag, and Qena. According to clinical stage of infection, patients were categorized into four groups: asymptomatic carriers, n = 16; chronic hepatitis C patients, n = 36; liver cirrhosis, n = 54; and hepatocellular carcinoma (HCC), n = 23. Genotype 4a was detected in 80.6%, whereas 1g, 4l, 4n, 4o, 4f, and 4m were identified in 7.7%, 4.7%, 3.9%, 1.6%, 0.8%, and 0.8% of cases, respectively. The prevalence of 4a differed regionally; from 88.5% (in Sohag) to 64% (in Assuit, P = 0.002). Genotypes 4l and 4n had a higher prevalence in Assuit (12.8%, 10.3%) than Sohag (0%, 0%; P ≤ 0.011). Difference in clinical features of determined genotypes/subtypes was observed; more carriers of non‐4a variants (4l and 4n, 4f, or 4m) had chronic hepatitis compared to carriers of 4a (53.3% vs. 23.1%, P = 0.025), while more patients with 4a had liver cirrhosis (45.2% vs. 13.3%, P = 0.023). Two HCV‐4o strains were isolated in this study, both from patients with HCC. In conclusion, geographical diversity of HCV was revealed in this study in southern Egypt. A further case–control study is required to confirm the trends of differential pathogenicity of HCV subtypes, indicated by this study. J. Med. Virol. 81:1015–1023, 2009.

Occult hepatitis B in the genotype H-infected Nahuas and Huichol native Mexican population†

Mexico is considered to be a low endemic country for HBV infection. However, a high anti‐HBc against a low hepatitis B surface antigen (HBsAg) seroprevalence is the reported characteristic of native Mexicans. HBV diagnosis and genotype distribution was examined in native populations (Nahuas and Huichol, n = 306), and compared to a non‐native population (Mestizos, n = 17). Overall, 6% of the natives were positive for HBsAg and 33% had detectable anti‐HBc. HBsAg prevalence was lower in Nahuas compared to Huichols (1.4% vs. 9.4%, P < 0.002). Occult hepatitis B was detected in 14.2% (41/289) of natives, who either tested positive (5.88%, 17/289 HBsAg‐negative) or negative for anti‐HBc marker (8%, 24/289 HBsAg‐negative). Age‐adjusted anti‐HBc seroprevalence and HBsAg quantitation revealed a sub‐optimal sensitivity of conventional immunoassays. Nahuas had HBV/H and Huichol had HBV/A as the predominant genotypes followed by genotypes D, C, B, A, and D, G and H, respectively. A less variable HBV/H was characteristic in Mestizos, compared to a much variable HBV/H identified among the Nahuas. In conclusion, these findings indicate a high HBV endemicity among native Mexican groups where occult B infection is common. The different distribution of HBV genotypes among natives suggests multiple reservoirs of HBV from which these genotypes spread into the local communities. High anti‐HBc seroprevalence against a low HBsAg prevalence rate may be due to the limited sensitivity of the immunoassays for the detection of HBsAg that are available in Mexico and/or unknown immunogenetic characteristics of native Mexicans. J. Med. Virol. 82:1527–1536, 2010.

Positive Selection of Core 70Q Variant Genotype 1b Hepatitis C Virus Strains Induced by Pegylated Interferon and Ribavirin

BACKGROUND Approximately 20% of patients with hepatitis C virus (HCV) genotype 1b infection have nonresponse to the most current treatment, pegylated interferon with ribavirin. Mutations in the HCV core region were recently proposed to be associated with nonresponse. Our aim was to evaluate the viral factors associated with treatment failure. METHODS HCV variants were determined directly and after cloning in 66 HCV-1b-infected Japanese patients and in 5 urokinase-type plasminogen activator transgenic severe combined immunodeficiency mice with human hepatocytes (chimeric mice), at baseline, during treatment, and after treatment. RESULTS At baseline, glutamine at position 70 of the HCV core protein (70Q) was detected by direct sequencing in 20% of patients with virologic response and in 43.8% of patients with nonresponse. Among patients with nonresponse, who were examined during and after treatment, the prevalence of the 70Q substitution increased to 56.3%, which indicates that treatment-induced selection occurred in all patients with nonresponse who had 70Q quasispecies detectable by cloning. This observation was reinforced by the results from experimentally infected chimeric mice. Logistic regression analysis indicated that detection of 70Q quasispecies was associated with a statistically significantly increased risk of nonresponse (odds ratio, 15.1; P = .004) in the studied cohort. CONCLUSION Presence of the 70Q quasispecies at baseline was associated with an increased risk of treatment failure, as indicated by the positive selection of the 70Q clones induced by treatment with pegylated interferon with ribavirin. These results urge further investigation of the mechanisms of this association.

Novel point mutations and mutational complexes in the enhancer II, core promoter and precore regions of hepatitis B virus genotype D1 associated with hepatocellular carcinoma in Saudi Arabia

In this study, a cohort of 182 patients [55 hepatocellular carcinoma (HCC) and 127 non‐HCC] infected with hepatitis B virus (HBV) in Saudi Arabia was investigated to study the relationship between sequence variation in the enhancer II (EnhII), basal core promoter (BCP) and precore regions of HBV genotype D (HBV/D) and the risk of HCC. HBV genotypes were determined by sequencing analysis and/or enzyme‐linked immunosorbent assay. Variations in the EnhII, BCP and precore regions were compared between 107 non‐HCC and 45 HCC patients infected with HBV/D, followed by age‐matched analysis of 40 cases versus equal number of controls. Age and male gender were significantly associated with HCC (p = 0.0001 and p = 0.03, respectively). Serological markers such as aspartate aminotransferase, albumin and anti‐HBe were significantly associated with HCC (p = 0.0001 for all), whereas HBeAg positivity was associated with non‐HCC (p = 0.0001). The most prevalent HBV genotype was HBV/D (94%), followed by HBV/E (4%), HBV/A (1.6%) and HBV/C (0.5%). For HBV/D1, genomic mutations associated with HCC were T1673/G1679, G1727, C1741, C1761, A1757/T1764/G1766, T1773, T1773/G1775 and C1909. Age‐ and gender‐adjusted stepwise logistic regression analysis indicated that mutations G1727 [odds ratio (OR) = 18.3; 95% confidence interval (CI) = 2.8–118.4; p = 0.002], A1757/T1764/G1766 (OR = 4.7; 95% CI = 1.3–17.2; p = 0.01) and T1773 (OR = 14.06; 95% CI = 2.3–84.8; p = 0.004) are independent predictors of HCC development. These results implicate novel individual and combination patterns of mutations in the X/precore region of HBV/D1 as predictors of HCC. Risk stratification based on these mutation complexes would be useful in determining high‐risk patients and improving diagnostic and treatment strategies for HBV/D1.

Performance of two Real-Time RT-PCR assays for quantitation of hepatitis C virus RNA: evaluation on HCV genotypes 1-4.

Accuracy for monitoring of the concentration of hepatitis C virus (HCV) RNA represents a major challenge throughout the management of patients with chronic hepatitis C. To investigate the genotype‐independent efficiency and the accuracy of two real‐time detection reverse transcription‐polymerase chain reaction (RT‐PCR) assays; the Cobas Ampliprep/Cobas TaqMan (CAP/CTM); and the Abbott RealTime HCV (ART), a total of 184 samples with different HCV subtypes were examined; 1b (n = 58), 2a (n = 39), 2b (n = 26), 3a (n = 20), and 4 (n = 41). A robust linear correlation was observed between the two assays applied to genotypes 1b, 2a, 2b, and 3a [the correlation coefficient (R) ranged from 0.99 to 0.98], but not to genotype 4 specimens (R = 0.78). A significant difference in measurements of HCV RNA using CAP/CTM and ART in serum samples with genotypes 1b and 4 was observed (0.72, −0.53 log IU/ml, P < 0.0001, 0.01, respectively). A robust correlation was observed between the HCV core antigen and HCV RNA values by either of the HCV RNA quantitation assays applied to all genotypes with exception of genotype 4, for which R was higher with ART (R = 0.95) than with CAP/CTM (R = 0.80). The lower limit of detection of CAP/CTM and ART were 41.4 and 28.5 IU/ml using the WHO standards, respectively. In conclusion, two RT‐PCR assays had a high efficiency and accuracy for quantitation of HCV RNA of genotypes 2a, 2b, and 3a, but the mean values of HCV RNA differed for genotype 1b and 4. J. Med. Virol. 82:1878–1888, 2010.

Multiple intra-familial transmission patterns of hepatitis B virus genotype D in north-eastern Egypt†

The transmission rate of intra‐familial hepatitis B virus (HBV) and mode of transmission were investigated in north eastern Egypt. HBV infection was investigated serologically and confirmed by molecular evolutionary analysis in family members (N = 230) of 55 chronic hepatitis B carriers (index cases). Hepatitis B surface antigen (HBsAg) and hepatitis B core antibody (anti‐HBc) prevalence was 12.2% and 23% among family members, respectively. HBsAg carriers were prevalent in the age groups; <10 (16.2%) and 21–30 years (23.3%). The prevalence of HBsAg was significantly higher in the family members of females (19.2%) than males (8.6%) index cases (P = 0.031). HBsAg and anti‐HBc seropositive rates were higher significantly in the offspring of females (23%, 29.8%) than those of the males index cases (4.3%, 9.8%) (P = 0.001, 0.003), as well as higher in the offspring of an infected mother (26.5, 31.8%) than those of an infected father (4.7%, 10.5%) (P = 0.0006, 0.009). No significant difference was found in HBsAg seropositive rates between vaccinated (10.6%) and unvaccinated family members (14.8%). Phylogenetic analysis of the preS2 and S regions of HBV genome showed that the HBV isolates were of subgenotype D1 in nine index cases and 14 family members. HBV familial transmission was confirmed in five of six families with three transmission patterns; maternal, paternal, and sexual. It is concluded that multiple intra‐familial transmission routes of HBV genotype D were determined; including maternal, paternal and horizontal. Universal HBV vaccination should be modified by including the first dose at birth with (HBIG) administration to the newborn of mothers infected with HBV. J. Med. Virol. 84:587–595, 2012.

An outbreak of acute hemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant in Pakistan

Acute hemorrhagic conjunctivitis (AHC) is a self-limiting viral infection of the eyes but having epidemic potential. In winter 2004-2005, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Islamabad, Pakistan. The etiological agent was confirmed as coxsackievirus A24 variant (CA24v) by virus isolation and sequencing of a part of the VP1/VP3 gene. Phylogenetic analysis in VP1 region showed that Pakistan isolates has closest matches both in Asia and Europe while in VP1/VP3 region they were more closely related to Chinese strains, suggesting their common source in Asia which is constantly evolving to cause AHC outbreaks in susceptible hosts in different parts of the world.