Qualitative measurement of HCV-RNA by home-made PCR in chronic liver diseases compared with commercial kits

Hepatitis C is a major health problem in Egypt. As global population estimates reach 170 million infected with the hepatitis C virus [HCV], there has never been a more pressing need for sensitive, precise tests for active infection. This study aimed to develop a sensitive, precise, cost saving protocol for measuring qualitative HCV RNA by PCR. To accomplish this aim, we optimized an-already published-direct RT-PCR protocol without extracting viral nucleic acid. Neat serum [3UL] was used, denatured with phosphate-buffered saline, then nested RT-PCR was performed. Two pairs of primers [inner and outer] were used, which had been selected from the most conservative 5'- untranslated region of the HCV genome. Designing of the inner primers based on nucleotide sequence of the genotype IV which is the more prevalent among Egyptian HCV patients. Positive result showed an 237 bp-sizing band by gel electrophoresis. Sensitivity of the optimized method was estimated also, and revealed a detection limit of 460 copies/ml. This emphasizes that the optimized protocol is a good screening test in comparison with other commercially used ones. Negative cases should be confirmed by extraction method to avoid any loss of false negatives

Detection and quantification of hepatitis B virus DNA by three different methods in HBsAg positive patients

The detection and quantification of hepatitis B virus genomes appear to be the most reliable methods for monitoring HBV infection and assessing response to antiviral treatment. The aim of this study assess the performance of three HBV DNA detection and quantification assays currently used for the management of HBV infected patients. In our study. 44 HBsAg +ve samples were assayed to HBV DNA detection and quantification by three different methods [In House HBV PCR. Amplicor HBV Monitor and HBV branched DNA DNA "bDNA"]. Twenty three samples [52.27%] were HBV DNA positive by one or more of the three methods and 21 samples [47.73 3/4] were HBV DNA negative by the three methods. Twenty one samples [47.73 3/4] were HBV DNA positive by Amplicor HBV Monitor and their cops number ranged from 440 - 40x106 copies/ml. Ten samples [22.73%] were HBV DNA positive by in House HBV PCR with copy number ranged from 6400 - 40 x 106 copies/ml. Eight samples [18.18%] were HBV DNA positive by NBV branched DNA [bDNA] and their copy number ranged from I.2x106 40x106 copies/ml. Branched DNA [bDNA] versus Amplicor HBV Monitor showed that: [i] bDNA and Arnplicor HBV Monitor gave concordant results for 27 [61.63%] of 44 the samples. The assays were both positive in six samples [13.64%]. The assays were both negative in 21 samples [47.73%]. [ii] bDNA and Amplicor HBV Monitor gave discordant results in 17 cases [38.64%]: bDNA was positive and Amplicor HBV Monitor negative in two cases [4.55%]. While bDNA was negative and Amplicor HBV Monitor positive in 15 cases [34.1%], HBV-DNA load in the Monitor assay was higher in 14] NA positive samples [mean = 9.7 x 106 copies/ml] than bDNA negative samples [mean 4.1 x 104 copies ml]. In House positive [mean = 9.7 x 106 copies/ml] than bDNA negative samples [mean 4.1 x 104 copies/ml]. In House PCR and Amplicor HBV Monitor showed that [i] In House PCR and Amplicor HBV Monitor gave concordant results with 33 of the 44 samples [75.0%]. The assays were both positive with to samples [22.73%]. The assays were both negative in 23 cases [52.27%] [ii] In House PCR and Amplicor HBV Monitor gave discordant results in 11 cases [25.0%] In House PCR was negative and Amplicor HBV Monitor gave discordant results in 11 casses [26.0%] in House PCR was negative and Amplicor HBV Monitor gave discordant resutlds in 11 cases [25.0%]. In House PCR was negative and Amplicor HBV Montor positive in these ii cases. HBV-DNA load in the Monitor assay was higher in In House PCR positive samples [mean 5.88 x 106 copies/ml] than In House PCR negative samples [mean 21 x 102 copies mb. In House PCR versus bDNA showed that: [i] in House PCR and bDNA gave concordant results with 38 of the 44 samples [86.36%]. The assays were both positive in six cases [13.64%]. The assays were both negative in 32 caes [72.73%]. [ii] In House PCR and bDNA gave discordant results in six cases [13.64%] In House PCR was positive and bDNA negative with four samples [0.09%]. In House PCR was negative and bDNA positive with two samples [4.55%]. HBV-DNA load in the Monitor assay was higher in bDNA positive samples [mean x 10 copies mb than In House PCR positive samples [mean 5.88 x 102 copies mb. The monitor assasy was more sensitive than both the In House PCR and bDNA. This better sensitivity appeared to be clinically relevant [even their respective performance, these three assays should be used in complementary fashion in the management of HBV infected patients

Matrix metalloproteinase-9 [Mmp-9] circulating intercellular adhesion molecule-1 [Cicam-1], and procollagen III peptide [PIIIP] in hepatocellular carcinoma and various chronic liver diseases

A sound predictive test is lacking for the identification of cirrhotic patients at high risk of developing hepatocellular carcinoma. In the present study, plasma MMP-9, plasma clCAM-1, serum alpha-feto protein [AFP] and serum PIIIP levels were measured and evaluated in 30 patients suffered from chronic hepatitis [CH], 30 patients suffered from liver cirrhosis [LC] and 30 patients suffered from hepatocellular carcinoma [HCC], in addition to 30 normal healthy individuals as a control group using RIA method for estimation of PIIIP and ELISA methods for estimations of the AFP and clCAM-1 and MMP-9. The study showed that the mean values of plasma MMP-9, plasma clCAM-1, serum PIIIP and serum AFP levels were 44.8 ng/ml, 232.1 ng/ml, 3.4 ug/l, 3.2 ng/Ml respectively among control group, 88.61 ng/ml, 489.5 ng/ml, 11.5 ug/l, 8.7 ng/ml respectively among Chronic hepatitis patients, 96.6 ng/ml, 781.3 ng/ml, 13.9 ug/l, 26.5 ng/ml respectively among Liver cirrhosis patients and 212.1 ng/ml, 999.4 ng/ml, 26.6 ug/l, 784.6 ng/ml respectively among HCC patients. Plasma MMP-9, plasma clCAM-1, serum PIIIP and serum AFP showed statistically highly significantly increase in all patients groups [P <0.001] when compared with the healthy control group. Plasma MMP-9 showed statistically highly significant increase in HCC group when compared with CH and LC groups, while did not show any statistically significant change [P> 0.05] in CH and LC groups when compared with the control group or with each other. clCAM-1 showed statistically highly significant increase in LC and HCC groups when compared with CH group with no significant change between LC and HCC groups and lastly serum AFP and PIIIP levels showed statistically highly significantly increase in HCC group when compared with CH and LC groups. As regard HCC histopathological grading all measured parameters showed statistically nonsignificant changes in different HCC grades except MMP-9 which showed a statistically significant increase in grade III when either compared with grade I or grade II. Receiver operating characterstic curve [ROC curve] was constructed using multiple cut off points for every studied parameter and calculating the sensitivity and the specificity at each cut off point and also calculating the area under each curve. The optimum cut off point for diagnosis of HCC from CH and LC for plasma MMP-9, plasma clCAM-1, serum PIIIP, and serum AFP were 89.8 ng/ml, 905 ng/ml, 25.8 ug/L and 68 ng/ml respectively, also, the study showed that AFP was the best of the studied HCC markers as it had the biggest area under ROC curve [0.86] followed by MMP-9 [0.76], cICAM [0.715] and lastly PIIIP [0.71]

How far liver enzymes reflect the inflammatory activity in the liver of patients with chronic hepatitis C virus infection?

This study was performed to evaluate the utility of the AST/ALT ratio in patients with proven chronic hepatitis C virus infection and to distinguish patients with and without cirrhosis and to correlate the ratio with the other biochemical and histological features. This study was conducted on 51 patients with chronic hepatitis C virus, 30 of them without cirrhosis and 21 with cirrhosis according to liver biopsy. The patients were attendants of inpatients Medicine department, Liver Institute, Menoufia University. 20 healthy individual were serving as control group. They were subjected to complete clinical examination, complete liver function tests [ALT, AST, T and D- bilirubin, Albumin, ALP, GGT, PT] and platelet count. All serum samples of the patients were +ve for circulating HCV-RNA. Liver biopsy was performed to all patients to diagnose cirrhosis and histological activity index [HAI]. The AST/ALT ratio [1 was seen in 80% of cirrhotic patients and 0% of the noncirrhotic patients. Correlation of the AST/ALT ratio with the grades of HAI shows significant positive correlation with grade III [r =+0.40 p <0.01] and grade IV [r =+ 0.61 p <0.001]. While nonsignificant correlation with grade I and II [r = +0.14 p>0.05 and r =+ 0.23 p> 0.05 respectively] correlation of the platelets count with HAI show significant negative correlation with grade IV only [r = - 0.35 p< 0.01]. The 3 parameters of modified CDS score [platelets count, ALT/AST ratio, PT score] are compared in cirrhotic versus noncirrhotic patients, there were significant increase in the score of the 3 parameters and the total CDS score in cirrhotic than noncirrhotic patients. A total CDS score [8 was seen in 47.6% of cirrhotic patients and in 3.3% of noncirrhotic patients. CDS score [8 showing 96.6% specificity with positive predictive value in distinguishing patients with cirrhosis from patients without. In conclusion: AST/ALT >1 or total CDS score [8 may be useful for identifying patients of chronic HCV with cirrhosis. While patients with AST/ALT <1 or total CDS score < 7 still require histological examination of liver to identify cirrhosis

Evaluation of inhouse HBV DMA PCR, and its comparison with amplicor HBV PCR [Roche] and quantiplex HBV bDNA [Chiron] in detection of hepatitis b virus infection

To evaluate the diagnostic efficacy of inhouse HBV DNA PCR detected either by ELISA or agarose gel electrophoresis techniques this study was designed. 60 patients having either HbsAg antigen or HBc IgG or both were selected from the outpatients medical clinic and inpatient medical section of the National liver institute, Minoufiya University. Inhouse HBV DNA PCR detected either by ELISA or agarose gel electrophoresis techniques were done for those patients, and the results were expressed in comparison with the results of Amplicor HBV PCR [Roche], and quantiplex HBV bDNA [Chiron]. Out of sixty patients who were serologically positive for either HbsAg, anti- HBc IgG or both, the inhouse HBV DNA PCR detected by ELISA was positive in 11 patients [18.33%], while inhouse HBV DNA PCR detected by EB and gel electrophoresis was positive in 9 patients [15%], also, quantiplex HBV bDNA [Chiron] was positive in 8 [17.78%] out of 45 patients only and amplicor HBV DNA PCR [Roche] was positive in 28 [46.67%] out of 60 patients. Also, the study revealed that the sensitivity of inhouse HBV DNA PCR detected by ELISA was 39.28%, with 100% specificity and 71.67% diagnostic accuracy, while inhouse HBV DNA PCR detected by agarose gel electrophoresis after ethidium bromide staining showed 32.14% sensitivity, 100% specificity and 68.33% diagnostic accuracy also, Also, quantiplex HBV bDNA [Chiron] showed 28.57% sensitivity, 91.66% specificity and 62.22% diagnostic accuracy when compared with the positive results obtained with amplicor HBV PCR [Roche]. So, due to its low sensitivity in comparison with amplicor HBV DNA PCR [Roche] we can not rely on inhouse PCR detected either by ELISA or agarose gel electrophoresis after ethidium bromide staining in diagnosis of HBV infection, further optimization of inhouse PCR is recommended for good yield and better diagnosis

Development of home-made PCR technique for HCV-RNA detection

Hepatitis C virus is a single stranded RNA virus; it is closely related to flavi and pesti-viruses. It is 50-60 nm in size and contains 3011 amino acids and 9033 nucleotides. Virus particles have not been visualized by immune-electron microscopy. HCV-antibody by EIA technique is considered as a good screening test for HCV infection, but PCR is mandatory for therapy. Amplification of HCV-RNA is a difficult procedure due to low copy number of the virus, so nested PCR is recommended to have good sensitivity and specificity. Commercial kit as Roche Amplicor and others are available in the market. Their results are satisfactory especially Roche but they are expensive for performing a research on large number of patients or even for routine work. The aim of this study was the comparison between Amplicor Roche PCR as a gold standard and Homemade [PCR] to evaluate accuracy and cost efficiency of Home made RT-PCR. Several trials were made to optimize the different conditions of the homemade PCR until we reached the best conditions to perform it. Home made PCR showed 97.5% sensitivity and 93.5% specificity compared to Roche as a gold standard. Finally we conclude that Homemade [POR] is accurate, sensitive, specific and cost efficient compared to Amplicor Roche PCR as a gold standard. However, Homemade PCR need very good technical experience and excellent reagents to keep its sensitivity and specificity

value of branched DNA hybridization assay [chiron] in patients with chronic HBV infection in comparison with HBeAg and HBV DNA-PCR

The optimal hepatitis B virus [DNA] quantitative assay for clinical use remains to be determined. Information on the virus load and the replicative activity of I-IBV is of importance in the management of patients with chronic HBV infection. We evaluate the branched-DNA [bDNA] assay [Quantiplex: Chiron Corp.] in-patients with chronic HBV infection in comparison with HBeAg and in-house HBV DNA-PCR. Serum samples from 53 hepatitis B surface antigen [HBsAg]-positive patients and 20 HBsAg-negative controls were assayed. According to presence or absence of HBeAg, the patients were divided into two groups: [1] First group consists of 9 HBsAg-positive and HBeAg-positive patients, [2] Second group consists of 44 HBsAg-positive and HBeAg-negative patients. Our results showed that the in-house HBV DNA-PCR was positive 77.78% [7 patients] and 15.91% [13 patients] in the first and second groups respectively. The branched DNA was positive [above the detection level] 55.56% [5 patients] and 9.1% [4 patients] in the first and second groups respectively. All the positive patients for HBV DNA by bDNA assay were positive also by in-house HBV DNA-PCR [9 patients]. Only 5 patients were positive for HBV DNA by in-house HBV DNA-PCR and at the same were negative by bDNA [below the detection level]. The rest of patients [39 Patients] were negative for HBV DNA by in-house DNA-PCP and bDNA assays. These results showed that PCR is a more sensitive method for detecting HBV DNA in serum than bDNA assay

value of PCR in the diagnosis of HCV infection in chronic liver disease patients

Hepatitis C virus is a single stranded RNA virus. HCV strains could be grouped into at least 12 genotypes. Diagnosis of HCV infection was made in chronic liver disease patients and the presence of HCV-RNA in the circulation was correlated with the severity of the liver disease in anti- HCV positive chronic liver diseased patients using the polymerase chain reaction [PCR] technique in 159 cases and 50 controls. The prevalence of anti-HCV among the chronic liver disease patients was 83.6% and 38% in the control group. The PCR results were 72.3% in the patient group and 26% in the control group. The percentage of RNA positive cases in the RIBA positive samples was 87.4%. HCV-RNA was present in 89.7% of the cirrhotic patients. ALT level correlated with anti-HCV positivity and there was a significant difference between the patient and control regarding ALT level, 51.6% of the cases in the patient group had elevated ALT and 84.1% of the cases who had elevated ALT were HCV-RNA positive. We concluded that there was a high prevalence of HCV infection among chronic liver disease patients and HCV-RNA was found to correlate with the liver pathology, symptom free HCV infected patients exist and some have normal liver function tests, despite the presence of liver affection which might be severe

Hepatic disorders among workers exposed to cotton dust containing pesticide residues

One hundred exposed and fifty control workers in a cotton ginning factory were chosen for this study. All were subjected to a special questionnaire including personal data together with clinical examination. The investigations involved detrmination of liver function parameters, detection of hepatitis markers [B and C], haemagglutination test for schistosomiasis, urine and stool analysis and liver sonography. Environmental study was done for estimation of concentration and pesticide residue contents of cotton dust collected from the factory atmosphere. This study was carried out twice; before and after [1991 - 1992] and before [1992 - 1993] ginning seasons. Subgrouping of studied workers was done according to the presence or absence of schistosomiasis and / or hepatitis markers, or according to sonographic pattern of the liver. The results of environmental study showed a concentration of respirable cotton dust in the atmosphere of different departments higher than the recommended threshold limit value in this industry. In cotton dust, the median measurements of different pesticide residues [organochlorine, organophosphorus, and carbamate] were below their threshold limit values. The study demonstrated a significant increase in SGPT, SGOT, SAP and gamma-GT among exposed than control groups at the end of the ginning season. Other liver functions including total and direct bilirubin, serum albumin and total serum protein showed no significant abnormal changes. Significant increase in SGPT, SGOT, SAP and gamma-GT were found among exposed subgroups when the comparison was done between pre and post seasons in each, or between different subgroups. Subgroup IV [free from schistosomiasis and / or hepatitis] and subgroup A [free from cirrhosis and periportal fibrosis] showed the significantly lowest mean values. The seasonal rest was associated with return to basic pre seasonal values of liver parameters. Moreover, there was a trend of increase in mean values of SGPT, SGOT, SAP and gamma-GT in exposed workers as the duration of exposure or the concentration of pesticide residues increased

Serum cholinesterase changes in workers exposed to organophosphorus and carbamate residues in cotton dust

One hundred exposed and fifty control workers in a cotton ginning factory were chosen for this study. All were subjected to a special questionnaire. The investigations involved determination of serum cholinesterase activity, detection of hepatitis markers [B and C], haemagglutination test for schistosomiasis, urine and stool analysis and liver sonography. Environmental study was done for estimation of pesticide [or-ganophosphorus and carbamate] residue contents of cotton dust collected from the factory atmosphere. This study was carried out twice: before and after [1991-1992] and before [1992-1993] ginning seasons. The results of environmental study showed that the median measurements of or-ganophosphorus and carbamate pesticide residues in the atmospheric dust were below their threshold limit values. The study demonstrated a non-significant lower level of serum cholinesterase activity in exposed workers before the beginning of work season than controls. A significantly lower level of serum cholinesterase activity was observed at the end of the ginning season among the exposed workers than controls, in subgroups of exposed workers with schistosomiasis, hepatitis markers and liver cirrhosis than other subgroups free from these diseases and in subgroups exposed to higher level of or-ganophospborus and carbamate residues. There was a trend of decrease in serum cholinesterase activity with the increase in duration of exposure

Studies on alcoholism. III alterations of nucleic acids, enzyme profile and sex hormones in rat testes of growing rats due to chronic alcohol ingestion

This paper discusses the chronic effects of ethyl alcohol [20% in drinking water] on some biochemical and physiological parameters in testes of growing albino rats. This study lasted for 20 weeks of daily treatment with the test article. Following are the results obtained: 1- Chronic alcohol consumption has resulted in reduced testicular weight particularly 9 weeks post intoxication and hereafter [expressed in g/100g body weight]. 2. Nucleic acids were found to decrease significantly due to alcohol intake especially RNA which was shown to be more sensitive and decreased earlier than DNA. 3. Similar results were also obtained concerning total protein content. 4. With the exception of nuclease enzymes [RNase and DNase] which elevated in response to alcohol abuse, the remaining enzymes assayed in this study such as alcohol dehydrogenase [ADH], lactate dehydrogenase [LDH], alkaline phosphatase [ALP] and aspartate aminotransferase [AST] showed lower values than control levels but with different magnitudes of decreases. 5. Sex hormones were found to be changed differently. Testosterone levels were found to be reduced in both serum and testis whereas estradiol elevated substantially in response to alcohol intake. 6. Alcohol consumption has resulted in decreased level of zinc in the testis. The foregoing changes appeared to be, at least partly, behind the deleterious effects that ethanol exerts on male reproductive organs especially the testis

Effect of Ramadan fasting on lipid metabolism in pregnant women

In this study, serum lipid in pre-Ramadan and Ramadan blood samples of 75 pregnant women were determined. The results showed that, the serum levels of total lipids, cholesterol, triglycerides were reduced while the free fatty acids were increased up to the maximum normal levels under the effect of Ramadan Fasting. So in this pattern of short-term fasting during Ramadan, the reduction in triglycerides and total lipids may protect the pregnant women from acute pancreatitis, and conceptus continues to grow. Also, pregnant women with normal lipid metabolism can keep Ramadan without any dangerous consequences to them

Biochemical features of scanty milk supply

Insufficient and/or thin watery breast milk secretion is a very common concept among mothers. This study was carried out in order to evaluate how far this claim holds true. The different constituents of breast milk including milk protein, fat, lactose, sodium and potassium, were within normal. The growth of these babies was also within normal limits and they kept growing satisfactorily for the first 6 months

Evaluation of the optical density determination of the amniotic fluid in assessing fetal lung maturity

Respiratory distress syndrome [R.D.S.] is the commonest respiratory disorder in the premature infant which accounts for a high incidence of neonatal death. R.D.S. is due to a deficiency of surface active material [surfactant] in the neonatal lung. This surfactant was only reliably assessed by measuring the Lecithin/sphingomyelin ratio [L/S] in the amniotic fluid. Amniotic fluid optical density [O.D.] at 650 nm was reported to be as reliable as L/S in assessing fetal Lung maturity. Fifteen pregnant women of the high risk group between 28 and 34 weeks were studied. L/S ratio and optical density at 650 nm of the amniotic fluid were estimated on all patients before and 48 hrs after 4 intra-muscular injections of betamethazone [6 mg every 12 hrs]. Six patients were delivered within 2 -6 days after the injection, one baby died of R.D.S. and the others survived. The remaining 9 patients passed safely through their pregnancies and delivered at term. Amniotic fluids with O.D. at 650 nm of 0.15 were found to have an L/S ratio over 2 while readings <0.05 corresponded to L/S ratio of about 1.3 and readings between 0.05 and 0.15 corresponded to L/S ration of 1.5. Betamethazone injections raised the O.D. [as well as the L/S ratio] from immature pre treatment values to mature post treatment values in 12 out of the 15 cases studied. O.D. readings at 650 nm is a simple, easy, fast and as reliable as the L/S ratio in deciding fetal lung maturity