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Comparison of assays for HCV RNA
Prevention of neural tube defects SIR-It has been shown that the administration of either multivitamin preparation containing folic acid (0-36 mg daily)1 or folic acid (4 mg daily)2 around the time of conception reduces the recurrence rate of neural tube defects (NTD). In the UK, a Department of Health Expert Advisory Group has recommended that, "All women should take an extra 0-4 mg folic acid prior to conception and during the early months of pregnancy". We carried out a survey at Leeds General Infirmary to determine the extent to which this advice is reaching women of child-bearing age before they conceive. 613 consecutive women making their first hospital antenatal visit between May 19, and Sept 3, 1993, were questioned. None had had a pregnancy complicated by NTD. Only 10 (1-6%) reported that folate had been prescribed or advised (in all cases by general practitioners) before conception, although 1 other patient had been advised to take more folate-rich foods. Another 4 patients who had not received advice from their doctors, but who were aware of the importance of folate in preventing NTD had increased their intake-3 by consuming more folate-rich foods and 1 by taking supplements. Thus, a total of 15 (2-4%) had increased their folate intake before conception. In 35 (5-7%) other patients folate supplements had been prescribed, but only after pregnancy had been confirmed. Neural tube closure takes place on days 26 to 27 of embryonic life. We have shown that supplements started 30 or more days after conception have no protective effect against NTD recurrence.4In the findings reported here, 97-6% of pregnant women had not increased their folate intake before this critical period. If this sample of women is representative of pregnant women throughout the UK, and perhaps elsewhere, a major programme of education for professionals and the general public is needed.
a
Figure: Southern blot analysis of complete (left) and partial (right) Xbal and Bgl digests of chromosomal DNA of V cholerae 0139 Strains from Andhra Pradesh (B02), and Calcutta (SG25).
(AP1),
Maduri
(MD012), Bangladesh
chromosomes of these organisms. For example, fragment pairs of 4-7 and 4-3 kb, or 7-2 and 6-6 kb, could arise from repetition two and three times of the pair of fragments 2-5 and 2-2 kb,
respectively. Normally, repetition of 2-5 and 1-7 kb fragments would be expected; repetition of 2-2 kb is anomalous. This apparent anomaly can be explained if it is assumed that the 2-2 kb fragment is the precursor of 1-7 kb fragment and has a less preferred site for one of the restriction enzymes used in digestion. The pattern was the same for all the strains tested. We conclude that the V cholerae 0139 strains carry a large number of copies of the toxin gene (at least 10 copies; 5 each of 2-5and 2-2) and are capable of producing very high amounts of toxin to elicit a severe pathogenic response. V cholerae 0139 strains were kindly provided by Dr G B Nair, National Institute of Cholera and Enteric Diseases, Calcutta. This work was supported by grant BT/TF/9/63/91 from the Department of Biotechnology, Government of India.
Margaret Sutcliffe, Christopher J Schorah, Alison Perry, Jennifer Wild Department of Nutrition and Dietetics, and Antenatal Clinic, United Leeds Teaching Hospitals, Leeds LS2 9NS, UK; and Department of Clinic Medicine, University of Leeds
1
2
3
4
Smithells RW, Nevin NC, Seller MJ, et al. Further experience of vitamin supplementation for prevention of neural tube defect recurrences. Lancet 1983; ii: 1027-31. MRC Vitamin Study Group. Prevention of neural tube defects: results of the Medical Research Council vitamin study. Lancet 1991; 338: 131-37. Department of Health. Folic acid and the prevention of neural tube defects: report from an expert advisory group. Heywood: DoH Health Publication Unit, 1992: 21. Sheppard S, Nevin NC, Seller MJ, et al. Neural tube defect recurrence after ’partial’ vitamin supplementation. J Med Genet 1989; 26: 326-29.
Biswajit Das, Ranajit Kumar Ghosh Indian Institute of Chemical
Biology, Jadavpur, Calcutta -700032, India
Charu Sharma, Navneet Vasin, Amit Ghosh Institute of Microbiol Technology, Chandigarh
1
2
3
4 5 6
Garg S, Saha PK, Ramamurthy T, et al. Nationwide prevalence of the new epidemic strain of Vibrio cholerae serogroup 0139 in India. J Infect 1993; 27: 107-08. Albert MJ, Siddiqui AK, Islam MS, et al. Large outbreak of clinical cholerae due to Vibrio cholerae non O1 in Bangladesh. Lancet 1993; 341: 704. Shimada T, Nair GB, Deb BC, Albert MJ, Sack RB, Takeda Y. Outbreaks of Vibrio cholerae non O1 in India and Bangladesh. Lancet 1993; 341: 1347. Nair GB, Takeda Y. Vibrio cholerae in disguise: a disturbing entity. World J Microbiol Biotech 1993; 9: 399-400. Mekalanos JJ. Duplication and amplification of toxin genes in Vibrio cholerae. Cell 1983; 35: 253-63. Bhattacharya U, Ghosh A, Ghosh RK. Structural organization of the cholera toxin gene and its expression in an environmental nonpathogenic strain of Vibrio cholerae. J Bioscience 1987; 11: 231-38.
1174
Comparison of assays for HCV RNA SIR-Lau and colleagues1 described a quantitative branched DNA (bDNA) assay for detection of hepatitis C virus (HCV) RNA (Quantiplex HCV-RNA, Chiron). We have compared the performance of this valuable and simple new assay with our version of HCV reverse-transcription polymerase chain reaction (HCV RT-PCR). From July, 1992, to May, 1993, 706 serum samples from patients with liver disease were tested for HCV-RNA by both bDNA assay and RT-PCR. RT-PCR was done with 5’-noncoding region nested primers. PCR products were analysed by gel electrophoresis and ethidium bromide staining. The lower limit of detection of our RT-PCR is roughly equivalent to one chimpanzee infectious dose. The HCV-bDNA assay, however, can quantify HCV-RNA with a lower limit of detection of
Table :
Comparison of bDNA assay with RT-PCR
Medical Center, San Francisco. Admission laboratory tests revealed a white blood cell count of 5-0 x 109/L, prothrombin time 16-8 s (control 12-0), serum bilirubin 236 µmol/L, alanine aminotransferase 599 IU/L, and alkaline phosphatase 180 IU/L. He was seronegative for IgM antibodies against hepatitis A, C, and D viruses, but seropostive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and IgM hepatitis B core antibody (HBc). He denied drinking. He
given supportive therapy, including lactulose and broadspectrum antibiotics. 3 days after admission, he developed pancreatitis and died. Necropsy done 1 day later showed haemorrhagic pancreatitis, cirrhosis, and submassive necrosis of the liver. Microscopical examination of the liver showed pronounced pericellular and bridging fibrosis, striking cholestasis, diffuse ballooning degeneration of hepatocytes with autolysis, and minimal inflammatory infiltrates. No acid-fast bacilli were seen. Even in autolysed specimens, immunohistochemical studies with respective antibodies detected HBsAg (figures available from The Lancet), HBcAg, and HBeAg diffusely in hepatocytes.1 HBV DNA was also seen in these cells by non-isotopic in-situ hybridisation with a digoxigenin-labelled HBV-DNA probe4(figures available from The Lancet). The presence of histological evidence of cirrhosis and IgM HBc antibody suggested that this patient had reactivation of HBV. The absence of marked inflammatory infiltrates in this patient who had such a fulminant course, and the close histological resemblance with FCH in orthotopic liver transplant patients, suggested that HBV may induce direct cytopathic damage to liver cells in HIV/HBV infection. Our report illustrates that HBV may cause liver cell damage by a direct cytopathic mechanism not only in orthotopic liver transplant recipients, but also in other immunosuppressed patients. Further studies on the mechanisms of hepatocellular damage induced by HBV in immunosuppressed patients are essential to formulate a better therapeutic strategy.
was
350 000 HCV eq/mL. For samples in which results were discordant with the two assays, both assays were repeated in
duplicate or triplicate. HCV RNA was detected in 66-7% and 62-2% of samples by RT-PCR and bDNA assay, respectively (table). There was a significant correlation (p < 0°001) between the two tests. In comparison with our RT-PCR assay, the total false-positive and false-negative rates for the bDNA assay were 1-1% (8/706) and 5-5% (39/706), respectively. HCV RNA was not detected by RT-PCR in 1-8% (8/439) of samples with HCV-RNA concentrations greater than 350 000 eq/mL by bDNA, and detected by RT-PCR in 14-6%(39/267) samples with HCVRNA concentrations less than 350 000 eq/mL as measured by the bDNA assay. HCV RNA was detected by RT-PCR in 100% of 356 samples with HCV-RNA concentrations greater than 1460 000 eq/mL and in 90-4% (75/83) of samples with HCV-RNA concentrations between 350 000 and 1460 000
eq/mL. We conclude that there is a close correlation between our RT-PCR test and the HCV-bDNA assay for detection of HCV RNA. The bDNA assay has the added advantage of quantification. Overall, compared with RT-PCR, the HCVbDNA assay has a sensitivity of 91-7% and a specificity of 96-6%. Nevertheless, cases suspected of having HCV infection that are negative by the HCV-bDNA assay should be tested by RT-PCR for HCV RNA.
Xiuming Li, Maria de Medina, Silvia LaRue, Lijun Shao, Eugene R Schiff Department of Hepatology, University of Miami School of Medicine, Miami, FL 33101, USA
Jane W S
Fang, Teresa L Wright, Johnson
Y N Lau
Section of Hepatobiliary Diseases, Department of Medicine, University of Florida, Gainesvile, FL 32610, USA; and Gastroenterology Section and Department of Medicine, Departments of Veterans Affairs Medical Center, University of California, San Francisco
1 Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-04. 1
Fibrosing cholestatic hepatitis in patient with HIV and hepatitis B (HBV) is believed to cause hepatocellular damage through an immune-mediated mechanism. However, a proportion of patients with HBV recurrence after orthotopic liver transplantation develop a peculiar syndrome called fibrosing cholestatic hepatitis (FCH), which clinically and histologically is very suggestive of direct cytopathic damage by HBV associated with high expression of HBV antigens.1 It has been suggested that this may result from the proviral effect of antirejection immunosuppressive therapy, leading to high HBV antigen expression and induced hepatocellular damage as seen in the transgenic mouse model. 1-3 We report a patient with end-stage HIV and HBV infection who developed acute liver failure and had a picture compatible with FCH seen in patients after orthotopic liver transplantation. A 41-year-old man was admitted with acute liver failure in September, 1992. He was diagnosed to be seropositive for HIV with an absolute CD4 count of 36/µL (normal 445-1403) SIR-Hepatitis
B
virus
4 weeks before admission. 2 weeks before admission, he developed malaise, anorexia, and jaundice. His clinical status deteriorated steadily and he lapsed into hepatic encephalopathy 3 days before transfer to the liver unit, VA
2
3
4
Lau JYN, Davies SE, Bain VG, et al. High level expression of hepatitis B viral (HBV) antigen in fibrosing cholestatic hepatitis. Gastroenterology 1992; 102: 956-62. Chisari FV, Filippi P, Buras J, et al. Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Soc USA 1987; 84: 6909-13. Lau JYN, Bain VG, Smith HM, et al. Modulation of heptitis B viral antigen expression by immunosuppressive drugs in primary hepatocyte culture. Transplantation 1992; 53: 894-98. Lau JYN, Naoumov NV, Alexander GJM, et al. Rapid detection of HBV DNA in liver tissue by in-situ hybridisation and its combination with immunohistochemistry for simultaneous detection of hepatitis B virus (HBV) antigens. J Clin Pathol 1991; 44: 905-08.
Chinese herbs and atopic dermatitis SIR-Sheehan and colleagues’ study1 of traditional Chinese herbal therapy in adult atopic dermatitis may shed light on the management of longstanding, refractory, widespread atopic dermatitis. However, it was surprising to note that details of the formulation of the ten herbs were not shown in the article. We wrote to the authors for the formulation, on the basis of the footnote in the paper: "Available from the authors for the purposes of research". The response was: "Unfortunately, I cannot give you the exact formulation and weights of each of the herbs, since the company that prepared this formulation have put in an application for a patent on this particular formulation". Since 1175
a
Figure: Southern blot analysis of complete (left) and partial (right) Xbal and Bgl digests of chromosomal DNA of V cholerae 0139 Strains from Andhra Pradesh (B02), and Calcutta (SG25).
(AP1),
Maduri
(MD012), Bangladesh
chromosomes of these organisms. For example, fragment pairs of 4-7 and 4-3 kb, or 7-2 and 6-6 kb, could arise from repetition two and three times of the pair of fragments 2-5 and 2-2 kb,
respectively. Normally, repetition of 2-5 and 1-7 kb fragments would be expected; repetition of 2-2 kb is anomalous. This apparent anomaly can be explained if it is assumed that the 2-2 kb fragment is the precursor of 1-7 kb fragment and has a less preferred site for one of the restriction enzymes used in digestion. The pattern was the same for all the strains tested. We conclude that the V cholerae 0139 strains carry a large number of copies of the toxin gene (at least 10 copies; 5 each of 2-5and 2-2) and are capable of producing very high amounts of toxin to elicit a severe pathogenic response. V cholerae 0139 strains were kindly provided by Dr G B Nair, National Institute of Cholera and Enteric Diseases, Calcutta. This work was supported by grant BT/TF/9/63/91 from the Department of Biotechnology, Government of India.
Margaret Sutcliffe, Christopher J Schorah, Alison Perry, Jennifer Wild Department of Nutrition and Dietetics, and Antenatal Clinic, United Leeds Teaching Hospitals, Leeds LS2 9NS, UK; and Department of Clinic Medicine, University of Leeds
1
2
3
4
Smithells RW, Nevin NC, Seller MJ, et al. Further experience of vitamin supplementation for prevention of neural tube defect recurrences. Lancet 1983; ii: 1027-31. MRC Vitamin Study Group. Prevention of neural tube defects: results of the Medical Research Council vitamin study. Lancet 1991; 338: 131-37. Department of Health. Folic acid and the prevention of neural tube defects: report from an expert advisory group. Heywood: DoH Health Publication Unit, 1992: 21. Sheppard S, Nevin NC, Seller MJ, et al. Neural tube defect recurrence after ’partial’ vitamin supplementation. J Med Genet 1989; 26: 326-29.
Biswajit Das, Ranajit Kumar Ghosh Indian Institute of Chemical
Biology, Jadavpur, Calcutta -700032, India
Charu Sharma, Navneet Vasin, Amit Ghosh Institute of Microbiol Technology, Chandigarh
1
2
3
4 5 6
Garg S, Saha PK, Ramamurthy T, et al. Nationwide prevalence of the new epidemic strain of Vibrio cholerae serogroup 0139 in India. J Infect 1993; 27: 107-08. Albert MJ, Siddiqui AK, Islam MS, et al. Large outbreak of clinical cholerae due to Vibrio cholerae non O1 in Bangladesh. Lancet 1993; 341: 704. Shimada T, Nair GB, Deb BC, Albert MJ, Sack RB, Takeda Y. Outbreaks of Vibrio cholerae non O1 in India and Bangladesh. Lancet 1993; 341: 1347. Nair GB, Takeda Y. Vibrio cholerae in disguise: a disturbing entity. World J Microbiol Biotech 1993; 9: 399-400. Mekalanos JJ. Duplication and amplification of toxin genes in Vibrio cholerae. Cell 1983; 35: 253-63. Bhattacharya U, Ghosh A, Ghosh RK. Structural organization of the cholera toxin gene and its expression in an environmental nonpathogenic strain of Vibrio cholerae. J Bioscience 1987; 11: 231-38.
1174
Comparison of assays for HCV RNA SIR-Lau and colleagues1 described a quantitative branched DNA (bDNA) assay for detection of hepatitis C virus (HCV) RNA (Quantiplex HCV-RNA, Chiron). We have compared the performance of this valuable and simple new assay with our version of HCV reverse-transcription polymerase chain reaction (HCV RT-PCR). From July, 1992, to May, 1993, 706 serum samples from patients with liver disease were tested for HCV-RNA by both bDNA assay and RT-PCR. RT-PCR was done with 5’-noncoding region nested primers. PCR products were analysed by gel electrophoresis and ethidium bromide staining. The lower limit of detection of our RT-PCR is roughly equivalent to one chimpanzee infectious dose. The HCV-bDNA assay, however, can quantify HCV-RNA with a lower limit of detection of
Table :
Comparison of bDNA assay with RT-PCR
Medical Center, San Francisco. Admission laboratory tests revealed a white blood cell count of 5-0 x 109/L, prothrombin time 16-8 s (control 12-0), serum bilirubin 236 µmol/L, alanine aminotransferase 599 IU/L, and alkaline phosphatase 180 IU/L. He was seronegative for IgM antibodies against hepatitis A, C, and D viruses, but seropostive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and IgM hepatitis B core antibody (HBc). He denied drinking. He
given supportive therapy, including lactulose and broadspectrum antibiotics. 3 days after admission, he developed pancreatitis and died. Necropsy done 1 day later showed haemorrhagic pancreatitis, cirrhosis, and submassive necrosis of the liver. Microscopical examination of the liver showed pronounced pericellular and bridging fibrosis, striking cholestasis, diffuse ballooning degeneration of hepatocytes with autolysis, and minimal inflammatory infiltrates. No acid-fast bacilli were seen. Even in autolysed specimens, immunohistochemical studies with respective antibodies detected HBsAg (figures available from The Lancet), HBcAg, and HBeAg diffusely in hepatocytes.1 HBV DNA was also seen in these cells by non-isotopic in-situ hybridisation with a digoxigenin-labelled HBV-DNA probe4(figures available from The Lancet). The presence of histological evidence of cirrhosis and IgM HBc antibody suggested that this patient had reactivation of HBV. The absence of marked inflammatory infiltrates in this patient who had such a fulminant course, and the close histological resemblance with FCH in orthotopic liver transplant patients, suggested that HBV may induce direct cytopathic damage to liver cells in HIV/HBV infection. Our report illustrates that HBV may cause liver cell damage by a direct cytopathic mechanism not only in orthotopic liver transplant recipients, but also in other immunosuppressed patients. Further studies on the mechanisms of hepatocellular damage induced by HBV in immunosuppressed patients are essential to formulate a better therapeutic strategy.
was
350 000 HCV eq/mL. For samples in which results were discordant with the two assays, both assays were repeated in
duplicate or triplicate. HCV RNA was detected in 66-7% and 62-2% of samples by RT-PCR and bDNA assay, respectively (table). There was a significant correlation (p < 0°001) between the two tests. In comparison with our RT-PCR assay, the total false-positive and false-negative rates for the bDNA assay were 1-1% (8/706) and 5-5% (39/706), respectively. HCV RNA was not detected by RT-PCR in 1-8% (8/439) of samples with HCV-RNA concentrations greater than 350 000 eq/mL by bDNA, and detected by RT-PCR in 14-6%(39/267) samples with HCVRNA concentrations less than 350 000 eq/mL as measured by the bDNA assay. HCV RNA was detected by RT-PCR in 100% of 356 samples with HCV-RNA concentrations greater than 1460 000 eq/mL and in 90-4% (75/83) of samples with HCV-RNA concentrations between 350 000 and 1460 000
eq/mL. We conclude that there is a close correlation between our RT-PCR test and the HCV-bDNA assay for detection of HCV RNA. The bDNA assay has the added advantage of quantification. Overall, compared with RT-PCR, the HCVbDNA assay has a sensitivity of 91-7% and a specificity of 96-6%. Nevertheless, cases suspected of having HCV infection that are negative by the HCV-bDNA assay should be tested by RT-PCR for HCV RNA.
Xiuming Li, Maria de Medina, Silvia LaRue, Lijun Shao, Eugene R Schiff Department of Hepatology, University of Miami School of Medicine, Miami, FL 33101, USA
Jane W S
Fang, Teresa L Wright, Johnson
Y N Lau
Section of Hepatobiliary Diseases, Department of Medicine, University of Florida, Gainesvile, FL 32610, USA; and Gastroenterology Section and Department of Medicine, Departments of Veterans Affairs Medical Center, University of California, San Francisco
1 Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-04. 1
Fibrosing cholestatic hepatitis in patient with HIV and hepatitis B (HBV) is believed to cause hepatocellular damage through an immune-mediated mechanism. However, a proportion of patients with HBV recurrence after orthotopic liver transplantation develop a peculiar syndrome called fibrosing cholestatic hepatitis (FCH), which clinically and histologically is very suggestive of direct cytopathic damage by HBV associated with high expression of HBV antigens.1 It has been suggested that this may result from the proviral effect of antirejection immunosuppressive therapy, leading to high HBV antigen expression and induced hepatocellular damage as seen in the transgenic mouse model. 1-3 We report a patient with end-stage HIV and HBV infection who developed acute liver failure and had a picture compatible with FCH seen in patients after orthotopic liver transplantation. A 41-year-old man was admitted with acute liver failure in September, 1992. He was diagnosed to be seropositive for HIV with an absolute CD4 count of 36/µL (normal 445-1403) SIR-Hepatitis
B
virus
4 weeks before admission. 2 weeks before admission, he developed malaise, anorexia, and jaundice. His clinical status deteriorated steadily and he lapsed into hepatic encephalopathy 3 days before transfer to the liver unit, VA
2
3
4
Lau JYN, Davies SE, Bain VG, et al. High level expression of hepatitis B viral (HBV) antigen in fibrosing cholestatic hepatitis. Gastroenterology 1992; 102: 956-62. Chisari FV, Filippi P, Buras J, et al. Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Soc USA 1987; 84: 6909-13. Lau JYN, Bain VG, Smith HM, et al. Modulation of heptitis B viral antigen expression by immunosuppressive drugs in primary hepatocyte culture. Transplantation 1992; 53: 894-98. Lau JYN, Naoumov NV, Alexander GJM, et al. Rapid detection of HBV DNA in liver tissue by in-situ hybridisation and its combination with immunohistochemistry for simultaneous detection of hepatitis B virus (HBV) antigens. J Clin Pathol 1991; 44: 905-08.
Chinese herbs and atopic dermatitis SIR-Sheehan and colleagues’ study1 of traditional Chinese herbal therapy in adult atopic dermatitis may shed light on the management of longstanding, refractory, widespread atopic dermatitis. However, it was surprising to note that details of the formulation of the ten herbs were not shown in the article. We wrote to the authors for the formulation, on the basis of the footnote in the paper: "Available from the authors for the purposes of research". The response was: "Unfortunately, I cannot give you the exact formulation and weights of each of the herbs, since the company that prepared this formulation have put in an application for a patent on this particular formulation". Since 1175