Journal of Medical Virology 36:28-31 (1992)

Hepatitis C Virus RNA in Saliva of Patients With Posttransfusion Hepatitis and Low Efficiency of Transmission Among Spouses J i n - T o w n Wang, T e h - H o n g Wang, J i n - C h u a n Sheu, J a w - T o w n Lin, and Ding-Shinn C h e n Department of Internal Medicine (J.-T.W., T.-H.W., J.-C.S., J.-T.L.) and Graduate Institute of Clinical Medicine (D.-S.C.),National Taiwan University Hospital, Taipei, Taiwan, Republic of China In a prospective study of posttransfusion hepatitis, 14 patients w h o were diagnosed with posttransfusion hepatitis C were enrolled randomly for the study of hepatitis C virus (HCV) RNA in saliva. Saliva and serum samples were collected o n the same day. Spouses of 11 married patients were also tested for anti-C100 and HCV RNA. Paired serum and saliva samples were tested for HCV RNA by a nested polymerase chain reaction (PCR). Two primer pairs specific for the noncoding region of HCV were used for the PCR and a oligonucleotide sequence between the primers was used as the probe for Southern hybridization. Six patients were positive for HCV RNA by first round PCR amplification and an additional four patients were detected after second round PCR. All patients were negative for HCV RNA in saliva after first round PCR, while seven were positive after second round PCR amplification. All seven patients were positive for HCV RNA in paired serum samples. HCV RNA was detectable in saliva from 1 week t o 38 months after the onset of hepatitis. All spouses were negative for antLC100 and HCV RNA. We conclude that HCV RNA is present in the saliva of approximately half of patients with acute and chronic hepatitis C, and the presence of HCV RNA correlates with HCV viremia. The efficiency of HCV transmission is l o w among spouses. K E Y WORDS: anti-C100, PCR, HCV

INTRODUCTION

The only established mode of hepatitis C virus (HCV) transmission is that of percutaneous spread, including blood transfusion, shared needles among drug addicts, and perhaps accidental needle-sticks among health care workers [Alter, 19901. However, it has became apparent that many cases have no discernible route of parenteral exposure. A study of community-acquired 0 1992 WILEY-LISS, INC.

NANBH shows that approximately 50% cases had no known parenteral exposure [Alter e t al., 19821. One of the important findings of recent HCV serology is that a very high proportion of community acquired cases of NANBH are HCV related, including the true sporadic cases [Alter et al., 19891. Although the exact mode of transmissions of HCV in sporadic cases is unclear, transmission of NANBH by saliva has been demonstrated in chimpanzee experiments [Abe et al., 19871. More recently, acute hepatitis C infection after a human bite has been reported [Dusheiko et al., 19901. Using the polymerase chain reaction (PCR), we detected HCV RNA in the saliva of patients with acute hepatitis C [Wang et al., 1991al. However, only patients with acute hepatitis C were studied and the number of patients was limited. We therefore studied additional cases with acute and chronic posttransfusion hepatitis C to explore the prevalence of HCV RNA in saliva and its clinical correlation. Furthermore, a nested PCR method instead of the previous single PCR was used to improve the sensitivity. Spouses ofrthese patients were also tested to examine HCV transmission. M A T E R I A L S AND M E T H O D S In a prospective study of posttransfusion hepatitis [Wang et al., 19901, 14 patients who had a follow-up visit in August and September 1990 were enrolled randomly for the study. They had not received specific treatment before. Saliva and serum samples were collected on the same day. Spouses of 11 married patients were also tested for anti-C100 and HCV RNA 3 months later. To detect serum HCV RNA by PCR, two primer pairs from the highly conserved non-coding region of HCV were used [Wang et al., 1991aI. The outer primers were sense, 5004 5' GAC ACT CCA CCA

Accepted for publication July 11, 1991 Address reprint requests to Teh Hong Wang, Department of Internal Medicine, National Taiwan University Hospital, No. 1, Chang-Te St, Taipei, Taiwan 10016, ROC.

HCV RNA in Saliva TAG ATC ACT CCC CTG TGA; antisense, J296R 5' CAC TCG CAA GCA CCC TAT CAG GCA GTA CCA [Okamoto et al., 19901. RNA from serum or saliva was extracted as described previously [Garson et al., 19901. Briefly, 100 pl of serum or saliva was incubated with 200 pg/ml proteinase K, containing 0.2 M tris-HC1 (pH 7.5),25 mM EDTA, 0.3 M NaC1, and 2% sodium dodecyl sulfate (SDS) a t 37°C for 40 min. After phenol/ chloroform extraction and ethanol precipitation, the pellet was dissolved in water. RNA from 20 r~.lserum was reverse transcribed in 20 p1 mixture containing 0.6 pM of the outer antisense primer, 50 mM tris-HC1 (pH 7.5), 75 mM KC1, 3 mM MgCl,, 10 mM dithiothreitol, 0.5 mM of each dNTP, 20 units of RNAsin (Bethesda Research Laboratories, Gaithersburg, MD), and 15 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) at 37°C for 90 min. First round PCR was carried out in a 50 p1 mixture containing 10 mM tris-HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgCl,, 0.01% gelatin, 2.5 units recombinant Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk), 200 pM each dNTP, 0.6 pM of each outer primer and 5 p1 of the cDNA mixture. Thirty-five cycles of denaturation for 1 min at 95"C, annealing for 1 min at 46°C and extension for 2 min at 72°C were employed as described previously [Wang et al., 1991aI. Two 30-nucleotide sequences from the 5' non-coding region were used as the inner primers (,sense5021: 5' CAC TCC CCT GTG AGG AAC TAC TGT CTT CAC, anti-sense J270R: 5' ACC ACA CGG CCT TTC GCG ACC CAA CAC TAC) [Okamoto et al., 19901. One microliter of the reaction mixture was transferred to the round 2 reaction mixture containing 0.6 pM of each inner primer and the same buffer in first round PCR. A fast temperature cycling was carried out as described before to improve sensitivity [Wang et al., 1991bI. Briefly, 40 cycles of 96°C for 30 sec, 56°C for 15 sec, and 74°C for 30 sec were done; the temperature was maintained at 74°C for 10 min at the end of the last cycle. Samples with 2-2 x lo3 copies of cloned A HCV cDNA fragment amplified by the outer primers were cloned and serially diluted. Samples with 2-2 x lo3 copies of cloned HCV cDNA fragment were used for sensitivity assay; saliva and serum samples from healthy persons and reagents without DNA were used as negative controls in PCR. Ten microliters of the products were analyzed by electrophoresis on a 2% agarose gel after ethidium bromide staining and were transferred to a nylon membrane (Oncor, Gaithersburg, MD), prehybridized with a rapid hybridization buffer (Amersham, Bukinghamshire, UK) for 1hr at 65"C, and hybridized with a y3'P labelled oligo-nucleotide probe (51315' CTG CGG AAC CGG TGA GTA CAC CGG AAT TGC) [Okamoto et al., 19901for 2 h at 65°C. The membrane was washed in 6 x SSC for 10 min at 40°C twice. Autoradiography was carried out by a double intensified screen for 72 hr. The saliva samples were tested for the presence of blood by a multistix strip (Hemoccult, Miles, Elkhart).

29

RESULTS One of the 14 patients was negative for anti-C100 in both pre- and posttransfusion serum samples. One was already positive for anti-HCV before transfusion, while the remaining 12 seroconverted to anti-C100 (Table I). All the patients were positive for HCV RNA by PCR in the acute sera and were therefore diagnosed as hepatitis C. Nine patients developed chronic hepatitis, two recovered after the acute attack, and three were followed for less than 6 months. The time of sample collection ranged from 1 week to 38 months after the onset of hepatitis. Serum ALT activities ranged from 13 to 719 IU/L (normal

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Fig. 1. A. PCR products on gel electrophoresis. Lanes 1 4 : First round PCRproducts of positive controls with 2,000,200,20,or 2 copies of HCV cDNA, respectively. Lane 5 A DNA-Hind HI/+ x 174 DNAHinc 11. Lanes 69 Second round PCR products of positive controls with 2,000, 200, 20, or 2 copies of HCV cDNA, respectively. Arrowhead: 293 base pairs. Arrow: 250 base pairs. B: Southern blot and autoradiography of above PCR products. Arrowhead 293 base pairs. Arrow: 250 base pairs.

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has also been recorded in patients with liver cirrhosis and hepatocellular carcinoma [Takamatsu et al., 1990 I. Although difficulty in standardizing PCR efficiency precluded quantitation of HCV genomes, the levels of HCV RNA contained in the saliva were low compared with those found in the serum as shown by absence in all saliva samples on gel electrophoresis after first PCR amplification. Because the presence of HCV RNA in the saliva may occur in the absence of occult blood in the saliva, apart from contamination of blood in saliva, the mechanism of appearance of HCV in saliva is unclear. Perhaps there is transudation of fluid containing virus from the general circulation into various body fluids rather than active virus replication a t the site of secretion. This might explain the low efficiency of non-parenteral HCV transmission as shown by our study in the spouses as well a s previous reports [Esteban et al., 1989; Everhart et al., 1990; Stevens, in press]. It is concluded that HCV RNA is present in the saliva of approximately half of patients with acute and chronic hepatitis C, and the titer of virus is low in saliva compared to that in serum. Although the transmission efficiency is low among spouses, these results provide a biologic basis for transmission of hepatitis C

HCV RNA in Saliva

+

Fig. 2. A PCR products on gel electrophoresis. Lane 0 x 174 DNNHae 111. Lanes 1,2: First round PCR products of serum and saliva from a healthy person. Lanes 3 4 First round PCR products of serum and saliva from a patient with posttransfusion hepatitis C. Lanes 5-8: Second round PCR products of sample in lanes 1 4 , respectively. Arrowhead: 293 base pairs. Arrow: 250 base pairs, B: Southern blot and autoradiography of above PCR products. Arrowhead: 293 base pairs. Arrow: 250 base pairs.

via contaminated saliva, and call for precautions in dental and upper gastrointestinal endoscopic practices.

ACKNOWLEDGMENTS This study was supported by grants from the Department of Health, Executive Yuan, Republic of China. The authors thank Ms. Yi-Ying Chen, Ms. Shiu-Hui Liu, and Ms. Sy-Ming Lin for their excellent assistance. REFERENCES Abe K, Kurata T, Sugitani M, Oda T (1987): Experimental transmission of non-A, non-B hepatitis by saliva. Journal of Infectious Diseases 155:1078-1079.

Alter H J (1990): The hepatitis C virus and its relationship to the clinical spectrum of NANB hepatitis. Journal of Gastroenterology and Hepatology 1:78~-94s. Alter MJ, Coleman PJ, Alexander WJ, Kramer E, Miller J K , Mandel E, Hadler SC, Mardolis HS (1989): Importance of heterosexual activity in the transmission of hepatitis B and non-A, non-B hepatitis. Journal of the American Medical Association 262:12011205. Alter MJ, Gerety J , Smallwood LA, Sampliner RE, Tabor E, Deinhardt F, Frosner G, Matanoski GM (1982): Sporadic non-A, non-B hepatitis: frequency and epidemiology in an urban U.S. population. Journal of Infectious Diseases 145:886-893. Chen DS, Kuo G, Sung JL, Lai MY, Sheu JC, Chen PJ, Yang PM, Hsu HM, Chang MH, Chen CJ, Hahn LC, Choo QL, Wang TH, Houghton M (1990): Hepatitis C virus infection in a n area hyperendemic for hepatitis B and chronic liver disease: The Taiwan experience. Journal of Infectious Diseases 1622317-822. Davison F, Alexander GJM, Trowbridge R, Fagan EA, Williams R (1987):Detection of hepatitis B virus DNA in spermatozoa, urine, saliva and leucocytes, of chronic HBsAg carriers. Journal of Hepatology 4:37-44. Dusheiko GM, Smith M, Scheuer PJ (19901: Hepatitis C virus transmitted by human bite. Lancet 336:503-504. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, Roget M, Vargas V, Genesca J , Buti M, Guardia J, Houghton M, Choo QL, Kuo G (1989):Hepatitis C virus antibodies among risk groups in Spain. Lancet 2994-296. Everhart J E , Bisceglie AM, Murray LM, Alter HJ, Melpolder J J , Kuo G, Hoofnagle J H (1990): Risk for non-A, non-B (type C) hepatitis through sexual or household contact with chronic carriers. Annals of Internal Medicine 1123546555. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JAJ, Contreras M, Aloysius S (1990):Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 335:1419-1422. Karayiannis P, Novick DM, Lok ASF, Fowler MJF, Monjardino J, Thomas HC (1985): Hepatitis B virus DNA in saliva, urine, and seminal fluid of carriers of hepatitis B e antigen. British Medical Journal [Clinical Research] 290:1853-1855. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, Miyakawa Y, Mayumi M (1990): The 5‘-terminal sequence of the hepatitis C virus genome. Japanese Journal of Experimental Medicine 60:167-177. Stevens C (in press): Perinatal and sexual transmission of HCV. In Hollinger FB (eds): “The 1990 international Symposium on Viral Hepatitis and Liver Disease.” New York: Liss, Inc. Sung JL, Chen DS (1983):Hepatitis B surface antigen in saliva, urine and ascites. Hepatogastroenterology 30:183-186. Takamatsu K, Koyanagi Y, Okita K, Yamamoto N (1990): Hepatitis C virus RNA in saliva. Lancet 336:1515. Wang JT, Wang TH, Lin JT, Sheu JC, Lin SM, Chen DS (1991a): Hepatitis C virus RNA in saliva of patients with posttransfusion hepatitis C infection. Lancet 337:48. Wang JT, Wang TH, Lin JT, Sheu JC, Sung JL, Chen DS (1990): Hepatitis C virus in a prospective study of posttransfusion non-A, non-B hepatitis in Taiwan. Journal of Medical Virology 32:83-86. Wang JT, Wang TH, Sheu JC, Shih LN, Lin JT, Chen DS (1991b): Detection of hepatitis B virus DNA by polymerase chain reaction in plasma of volunteer blood donors negative for hepatitis B surface antigen. Journal of Infectious Diseases 163:397-399.

Hepatitis C virus RNA in saliva of patients with posttransfusion hepatitis and low efficiency of transmission among spouses.

In a prospective study of posttransfusion hepatitis, 14 patients who were diagnosed with posttransfusion hepatitis C were enrolled randomly for the st...
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