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Detection of HCV RNA in saliva of patients with hepatitis C virus infection by using a highly sensitive test
Journal of Virological Methods 101 (2002) 29 – 35 www.elsevier.com/locate/jviromet
Detection of HCV RNA in saliva of patients with hepatitis C virus infection by using a highly sensitive test M. Hermida a, M.C. Ferreiro b, S. Barral a, R. Laredo a, A. Castro c, P. Diz Dios b,* b
a Institute of Health Sciences, Juan Canalejo Hospital, La Corun˜a, Spain School of Dentistry and Medicine, Santiago de Compostela Uni6ersity, Santiago de Compostela, Spain c Department of Internal Medicine, Juan Canalejo Hospital, La Corun˜a, Spain
Received 14 June 2001; received in revised form 17 October 2001; accepted 22 October 2001
Abstract Hepatitis C virus (HCV) is transmitted primarily by direct percutaneous exposures to blood. Since HCV RNA has been found in saliva, it has been suggested that saliva might also be a source of infection. HCV RNA in saliva from plasma HCV RNA positive patients was tested by a highly sensitive PCR method. HCV RNA was detected in 32 out of 61 saliva specimens (52.4%). No correlation was found between the presence of HCV in saliva and age, sex, identified risk factors for HCV infection, time lapsed since the diagnosis, transaminases and alkaline phosphatase values and stimulated salivary flow. A statistically significant relation between plasma HCV RNA viral load and saliva HCV RNA detection was observed (PB 0.001). In conclusion, HCV RNA is often present in saliva of HCV infected patients, with plasma viral load being the only known predictable factor. Further studies on salivary HCV RNA are needed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Polymerase chain reaction; Saliva; Disease transmission
1. Introduction Hepatitis C virus (HCV) is transmitted by percutaneous exposure to infected blood. In some countries, unsafe injections and contaminated equipment used in healthcare-related procedures appear to have a predominant role in transmission, leading to epidemiological and geographical
* Corresponding author. Present address: c/Panama´ 2, 2° dcha, 36203 Vigo, Spain. Fax: + 34-981-562-226. E-mail address: [email protected] (P. Diz Dios).
differences and temporal trends (Wasley and Alter, 2000). Sexual and mother-to-child transmission have also been described, although it has been suggested that HCV infection should not be considered as a sexually transmissible infection (Kaldor et al., 2000). There remain a large number of hepatitis C carriers in whom no route of infection can be identified. Experimental infections by HCV contaminated saliva inoculation in non-human primates (Abe and Inchauspe, 1991) and HCV transmission following human bites (Dusheiko et al., 1990; Figueiredo et al., 1994) have been reported.
0166-0934/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 4 1 7 - 7
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M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
Saliva has been examined in several studies for the presence of the HCV RNA by reverse transcription polymerase chain reaction (RT-PCR) with percentages which vary between 0% (Fried et al., 1992) and 100% (Takamatsu et al., 1990). The aim of this study was to determine the prevalence of HCV RNA in saliva of plasma HCV RNA positive patients using a highly sensitive RT-PCR technique.
ployed (Navazesh and Christensen, 1982), applying a few drops of 0.1 mol/l citric acid on the anterior dorsal surface of the tongue every minute for 3 min. Saliva samples were collected in a sterile container and after measurement of volumes, were stored at − 80 °C. Saliva specimens of the 74 plasma HCV RNA positive patients were examined. Nine samples were macroscopically seen to contain blood and four samples were damaged during processing.
2. Materials and methods
2.3. HCV RNA detection
2.1. Population
HCV RNA detection was carried out in the remaining 61 saliva samples using a nested RTPCR technique. This technique was applied amplifying a fragment of 251 bp from the 5%UTR region of the HCV, as described previously (Roy et al., 1995). Stored samples of saliva were centrifuged (3500 rpm, 5 min). HCV RNA was extracted from 200 ml of supernatant and 200 ml of the cell fraction, with the addition of TRIZOL (Life Technologies, Barcelona, Spain), according to the manufacturer’s instructions. The RNA was resuspended in 10 ml of diethyl pirocarbonate (DEPC)-treated water. Synthesis of cDNA was undertaken by reverse transcription from 10 ml of RNA extracted at 38 °C for 30 min and posteriorly 95 °C for 5 min, in 20 ml solution containing 10 mM/l Tris –HCl (pH 8.4), 50 mM/l KCl, 3 mM/l MgCl2, 10 mM DTT, 2.5 mM/l of the primers 209 and 939 (Life Technologies), 200 U of Moloney Murine Leukaemia Virus (MMLV) (Life Technologies), 20 U of Rnasin (Life Technologies) and 0.5 mM dNTPs. PCR was carried out in a 50 ml mixture containing 20 ml of cDNA, 10 mM/l Tris –HCl (pH 8.4), 50 mM/l KCl, 1.5 mM/l MgCl2, 200 mM dNTPs and 1 U Taq Pol (Life Technologies). Amplification occurred over 30 cycles (94 °C for 30 s; 53 °C for 45 s; 72 °C for 60 s). PCR reaction product (1 ml) was reamplified using the same cycling program in a mixture containing 10 mM/l Tris – HCl (pH 8.4), 50 mM/l KCl, 1.5 mM/l MgCl2, 200 mM dNTPs, 1 U Taq Pol (Life Technologies) and 1 mM/l of the primers 211 and
The study population included 74 patients with hepatitis C virus infection, diagnosed in the Internal Medicine Service of the Juan Canalejo Hospital (La Corun˜ a, Spain). The selection of patients was carried out according to the following inclusion criteria: presence of HCV RNA in plasma confirmed by nested reverse transcription polymerase chain reaction (RT-PCR); absence of other concomitant liver diseases; negative HIV test; no prior interferon and/or ribavirine treatment; subjects stated that they were neither habitual alcohol consumers nor active intravenous drug users. The following data were recorded from their medical records: age, sex, identified risk factors for HCV infection and time lapsed since the diagnosis. Plasma HCV RNA levels determined by the PCR system (Amplicor HCV Monitor, Roche Molecular Systems, Barcelona, Spain), aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) values from the last month before enrollment in the study, were also recorded.
2.2. Sali6a collection Stimulated whole saliva was collected between 10:00 and 12:00 h by a single research worker. Subjects refrained from eating, drinking, smoking and oral hygiene procedures for 90 min before salivary collection. The spitting method was em-
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
940 (Life Technologies). Amplified cDNA was detected by 1.5% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the technique was determined by studying the products of the PCR at serial plasma dilutions (100, 10, 1, 0.1, 0.01 and 0.001%) in saliva. Blood came from an HCV infected patient with viral load of 1× 106 copies/ml. The saliva was obtained from a healthy HCV negative volunteer. The detection limit of this technique was ten HCV RNA copies/ml (Fig. 1). A Hem-Check-1 kit based in monoclonal antibodies (Menarini Diagnostics, Barcelona, Spain) was used in order to search for the presence of hemoglobin (hidden blood). The presence of HCV RNA in saliva was examined in relation to the demographical, clinical and analytical variables applying the two-tailed Student’s t-test, the 2-test and logistic regression analysis. Informed consent from patients was obtained.
31
3. Results The study group comprised of 54 males (73%) and 20 females (27%) with a mean age of 40.59 14.6 years (range 20–71 years). Risk factors were identified in 55.8% of patients. Injection drug use was the main mode of transmission (45.9%), followed by blood transfusion (8.1%). The mean duration of HCV infection was 11.49 8 years, although this was only determined in 23 cases. The mean time lapsed from the diagnosis of hepatitis C virus infection was 2.99 2.4 years (range 1–10 years). The mean values of the liver enzymes were: AST= 60.49 42.7 IU/l (range 5–200); ALT = 106.49 96.8 IU/l (range 11–447); GGT= 55.09 60.5 IU/l (range 9–387); ALP=191.49 86.7 IU/l (range 74– 488). The average viral load detected in the study group was 582,1209 383,258 copies/ml (range 790–1,599,000 copies/ml). HCV RNA was found in 32 out of 61 saliva specimens (52.4%). No correlations were found between HCV detection in saliva and age, sex,
Fig. 1. Agarose gel electrophoresis of amplified HCV RNA at serial plasma dilutions from an HCV infected patient (1 × 106 copies/ml) in saliva from a healthy HCV negative volunteer, using a nested reverse transcription polymerase chain reaction. Lane 1: plasma positive control. Lane 2: plasma from an HCV infected patient with viral load =1 × 106 copies/ml. Lane 3: negative saliva control. Lane 4: 10% plasma dilution in saliva (1 × 105 copies/ml). Lane 5: 1% plasma dilution in saliva (1 ×104 copies/ml.) Lane 6: 0.1% plasma dilution in saliva (1 × 103 copies/ml). Lane 7: 0.01% plasma dilution in saliva (1 ×102 copies/ml). Lane 8: 0.001% plasma dilution in saliva (1 ×101 copies/ml). Lane 9: negative saliva control. Lane 10: molecular weight control.
32
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
Fig. 2. Relationship between plasma HCV RNA titers and saliva HCV RNA detection (PB 0.001). Mean plasma viral load in patients without HCV in saliva = 372,7299 302,053 copies/ml. Mean plasma viral load in patients with HCV in saliva = 784,292 9 345,859 copies/ml.
identified risk factors for HCV infection, time lapsed since the diagnosis, AST, ALT, GGT and ALP values. Hemoglobin was detected in 14 out of 61 saliva samples (22.9%), but its presence was not a predictor of HCV RNA in saliva. A statistically significant relationship between plasma HCV RNA viral load and saliva HCV RNA detection was observed (P B 0.001) (Fig. 2)
4. Discussion All patients included in the present study were infected with HCV confirmed by RT-PCR. Al-
though the most recent enzyme-linked immunoassays for HCV infection have increased sensitivity and specificity, these assays still have relatively high false–positive rates among low-risk populations. Because some infected persons fail to generate an immune response to HCV, reactivity by RT-PCR is considered to be the definitive proof of hepatitis C infection (Robbins et al., 2000). It has been indicated that the presence of HCV RNA in saliva correlates with HCV viremia (Wang et al., 1992; Numata et al., 1993; Mariette et al., 1995; Caldwell et al., 1996; Fabris et al., 1999). Variations of plasma HCV RNA levels have been related to coinfection with other viral
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
agents such as HBV (Sagnelli et al., 2000) and HIV (Hisada et al., 2000), self reported alcohol consumption (Degos, 1999) and antiviral therapy (Barbaro et al., 2000). In consequence, these conditions have been considered exclusion criteria. In our study, salivary HCV RNA prevalence was 52.4%, this figure being similar to those reported by other researchers (Roy et al., 1996, 1998; Fabris et al., 1999). Nonetheless, studies published on this topic in recent years show great variability (Table 1). Low prevalence results may indicate a quantity of HCV RNA in saliva below the level of PCR sensitivity (Roy et al., 1995; Fabris et al., 1999). We have applied a highly sensitive technique with a detection limit of ten HCV RNA copies/ml. Although handling and storage of saliva specimens are technically complex and could effect the stability of viral RNA (Roy et al., 1999), collection and processing of
33
saliva were carried out as described previously by others (Fabris et al., 1999). In HCV infected patients, transaminase values have been found to be higher in those with HCV RNA than in those without detectable HCV RNA in the saliva (Sugimura et al., 1995). Our results did not reveal any relation between HCV presence in saliva and enzyme levels and/or duration of disease, which was in agreement with other reports (Wang et al., 1992; Fabris et al., 1999). In the present study, as it has been suggested by others (Wang et al., 1992; Numata et al., 1993; Mariette et al., 1995; Caldwell et al., 1996; Fabris et al., 1999), the best predictive factor for HCV detection in saliva was a high level of plasma HCV RNA. There are several reasons why HCV RNA may be present in the saliva of patients with detectable virus in the plasma. Contamination with blood
Table 1 Recent studies on prevalence of HCV RNA in saliva of plasma HCV RNA positive patients Author, year
No. of patients
Type of patients
HCV diagnostic method
HCV RNA prevalence in saliva
(Mariette et al., 1995) (Chen et al., 1995) (Sugimura et al., 1995) (Roy et al., 1995) (Tang et al., 1996) (Jorgensen et al., 1996) (Caldwell et al., 1996) (Roy et al., 1996)
28
Plasma HCV+ (13 HIV+)
PCR
17 (61%)
23
Plasma HVC+ (11 HIV+)
PCR
4 (17%)
76
Anti-HCV+
RIBA
27 (36%)
14 16
Plasma HCV+ blood donors Plasma HCV+
RIBA PCR PCR
9 (64%) 5 (31%)
16
Plasma HCV+Sicca syndrome
RIBA PCR
13 (83%)
33
PCR
5 (15%)
PCR
10 (48%)
11
Plasma HCV+ (21 liver transplantated) Plasma HCV+hemophiliacs (6 HIV+) Plasma HCV+women postpartum
PCR
4 (36%)
10
Plasma HCV+hemodialysed
PCR
3 (30%)
20
Plasma HCV+CAH or cirrhosis
PCR
3 (15%)
33 39
Plasma HCV+IVDUs (19 HIV+) Plasma HCV+
PCR PCR
19 (58%) 22 (56%)
61
Plasma HCV+
PCR
32 (52%)
(Kage et al., 1997) (Ustundag et al., 1997) (Taliani et al., 1997) (Roy et al., 1998) (Fabris et al., 1999) Present series
21
HCV, hepatitis C virus; RIBA, recombinant immunoblot assay; PCR, polymerase chain reaction; IVDU, intravenous drug user.
34
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
could be a contributing factor, but positive hemoglobin samples were independent of HCV RNA results (Fabris et al., 1999). Peripheral blood mononuclear cells (PBMCs) are another possible source for transferring HCV to saliva (Roy et al., 1995; Fabris et al., 1999), but the presence of HCV RNA in PBMCs and in saliva is not correlated closely (Young et al., 1993). HCV could also enter saliva through the gingival crevicular fluid, as has been shown in other viral diseases (Maticic et al., 2000), but HCV RNA in saliva has been found even in edentulous patients (Roy et al., 1998). The issue of intraglandular replication of HCV remains controversial. Recent studies employing assays optimized for identifying replicative intermediates of HCV, found HCV quasispecies in different tissue samples compatible with independent replication at extrahepatic sites (Laskus et al., 2000), which could include major salivary glands. The results of this study suggest that HCV RNA is present in saliva in a high proportion of HCV infected patients. Some issues, such as the mechanism by which the virus enters the saliva, its clinical significance and the possible HCV transmission through exposure to this body fluid, remain unclear. However, HCV RNA detection in saliva may be useful for monitoring antiviral treatment (Roy et al., 1995).
References Abe, K., Inchauspe, G., 1991. Transmission of hepatitis C by saliva. Lancet 337, 248. Barbaro, G., Di Lorenzo, G., Soldini, M., Giancaspro, G., Pellicelli, A., Grisorio, B., Barbarini, G., 2000. Evaluation of long-term efficacy of interferon alpha-2b and ribavirin in combination in naive patients with chronic hepatitis C: an Italian multicentre experience. J. Hepatol. 33, 448 –455. Caldwell, S.H., Sue, M., Bowden, J.H., Dickson, R.C., Driscoll, C.J., Yeaton, P., Stevenson, W.C., Ishitani, M.B., McCulloug, C.S., Pruett, T.L., Lovell, M.A., 1996. Hepatitis C virus in body fluids after liver transplantation. Liver Transpl. Surg. 2, 124 –129. Chen, M., Yun, Z.B., Salberg, M., Schvarcz, R., Bergquist, I., Berglund, H.B., Sonnerborg, A., 1995. Detection of hepatitis C virus RNA in the cell fraction of saliva before and after oral surgery. J. Med. Virol. 45, 223 –226. Degos, F., 1999. Hepatitis C and alcohol. J. Hepatol. 31 (Suppl. 1), 113 – 118.
Dusheiko, G.M., Smith, M., Scheuer, P.J., 1990. Hepatitis C virus transmitted by human bite. Lancet 336, 503 – 504. Fabris, P., Infantolino, D., Biasin, M.R., Marchelle, G., Venza, E., Terribile Wiel Marin, V., Benedetti, P., Tossitti, G., Manfrin, V., del Lalla, F., 1999. High prevalence of HCV RNA in the saliva fraction of patients with chronic hepatitis C but no evidence of HCV transmission among sexual partners. Infection 27, 86 – 91. Figueiredo, J.F., Borges, A.S., Martinez, R., Martinelli, A.L., Villanova, M.G., Covas, D.T., Passas, A.D., 1994. Transmission of hepatitis C virus but not human immunodeficiency virus type1 by a human bite. Clin. Infect. Dis. 19, 546 – 547. Fried, M.W., Shindo, M., Fong, T.L., Fox, P.C., Hoofnagle, J.H., Bisceglie, M., 1992. Absence of hepatitis C viral RNA from saliva and semen of patients with chronic hepatitis C. Gastroenterology 102, 1306 – 1308. Hisada, M., O’Brien, T.R., Rosenberg, P.S., Goedert, J.J., 2000. Virus load and risk of heterosexual transmission of human immunodeficiency virus and hepatitis C virus by men with hemophilia. The multicenter hemophilia cohort study. J. Infect. Dis. 18, 1475 – 1478. Jorgensen, C., Legouffe, M.C., Perney, P., Coste, J., Tissot, B., Segarra, C., Bologna, C., Bourrat, L., Combe, B., Blanc, F., Sany, J., 1996. Sicca syndrome associated with HCV virus infection. Arthritis Rheum. 39, 1166 – 1171. Kage, M., Ogasawara, S., Kosai, K., Nakashima, E., Shimamatsu, K., Kojiro, M., Kimura, A., Fujisawa, T., Matsukuma, Y., Ito, Y., Kondo, S., Kawano, K., Sata, M., 1997. Hepatitis C virus RNA present in saliva but absent in breast-milk of the hepatitis C carrier mother. J. Gastroenterol. Hepatol. 12, 518 – 521. Kaldor, J.M., Dore, G.J., Correll, P.K., 2000. Public health challenges in hepatitis C virus infection. J. Gastroenterol. Hepatol. 15, S83 – 90. Laskus, T., Radkowski, M., Wang, L.F., Nowicki, M., Rakela, J., 2000. Uneven distribution of hepatitis C virus quasispecies in tissues from subjects with end-stage liver disease: confounding effect of viral adsorption and mounting evidence for the presence of low-level extrahepatic replication. J. Virol. 74, 1014 – 1017. Mariette, X., Loiseau, P., Morinet, F., 1995. Hepatitis C virus in saliva. Ann. Intern. Med. 122, 556. Maticic, M., Poljak, M., Kramar, B., Tomazic, J., Vidmar, L., Zakotnik, B., Skaleric, U., 2000. Proviral HIV-DNA in gingival crevicular fluid of HIV-1-infected patients in various stages of HIV disease. J. Dent. Res. 79, 1496 – 1501. Navazesh, M., Christensen, C.M., 1982. A comparison of whole mouth resting and stimulated salivary measurement procedures. J. Dent. Res. 61, 1158 – 1162. Numata, N., Ohori, H., Hayakawa, Y., Saitoi, Y., Tsunoda, A., Kanno, A., 1993. Demonstration of hepatitis C virus genome in saliva and urine of patients with type C hepatitis: usefulness of the single polymerase chain reaction method for detection of the HCV genome. J. Med. Virol. 41, 120 – 128.
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35 Robbins, D.J., Pasupuleti, V., Cuan, J., Chiang, C.S., 2000. Reverse transcriptase PCR quantitation of hepatitis C virus. Clin. Lab. Sci. 13, 23 –30. Roy, K.M., Bagg, J., Bird, G.L.A., Spence, E., Follett, E.A., Mills, P.R., Lau, R.L., 1995. Serological and salivary markers compared with biochemical markers for monitoring interferon treatment for hepatitis C virus infection. J. Med. Virol. 47, 429 – 434. Roy, K.M., Bagg, J., Follett, E.A., Brewer, A., Lowe, G.D., 1996. Hepatitis C virus in saliva of haemophiliac patients attending an oral surgery unit. Br. J. Oral Maxillofac. Surg. 34, 162 – 165. Roy, K.M., Bagg, J., McCarron, B., Good, T., Cameron, S., Pithie, A., 1998. Predominance of HCV type 2a in saliva from intravenous drug users. J. Med. Virol. 54, 271 –275. Roy, K.M., Bagg, J., McCarron, B., 1999. The effect of saliva specimen collection, handling and storage protocols on hepatitis C virus (HCV) RNA detection by PCR. Oral Dis. 5, 123 – 127. Sagnelli, E., Coppola, N., Scolastico, C., Filippini, P., Santantonio, T., Stroffolini, T., Piccinio, F., 2000. Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C and delta viruses in patients with chronic hepatitis. Hepatology 32, 1106 – 1110. Sugimura, H., Yamamoto, H., Watabiki, H., Ogawa, H., Harada, H., Saito, I., Miyamura, T., Inoue, M., Tajima, K., Kino, I., 1995. Correlation of detectability of hepatitis C virus genome in saliva of elderly Japanese symptomatic
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HCV carriers with their hepatic function. Infection 23, 258 – 262. Takamatsu, K., Koyanagi, Y., Okita, K., Yamamoto, N., 1990. Hepatitis C virus in saliva. Lancet 336, 1515. Taliani, G., Celestino, D., Badolato, M.C., Pennica, A., Bozza, A., Poliandri, G., Riccieri, V., Benfari, G., Sebastiani, A., De Bac, C., Quaranta, G., Aceti, A., 1997. Hepatitis C virus infection of salivary gland epithelial cells. J. Hepatol. 26, 1200 – 1206. Tang, Z., Yang, D., Hao, L., Tang, Z., Huang, Y., Wang, S., 1996. Detection and significance of HCV RNA in saliva, seminal fluid and vaginal discharge in patients with hepatitis C. J. Tongji Med. Univ. 16, 11 – 13. Ustundag, Y., Hizel, N., Boyacioglu, S., Akalin, E., 1997. Detection of hepatitis GB virus-C and HCV genomes in the saliva of patients undergoing maintenance haemodyalisis. Nephrol. Dial. Transplant. 12, 2807. Wang, J.T., Wang, T.H., Sheu, J.C., Lin, J.T., Chen, D.S., 1992. Hepatitis C virus RNA in saliva of patients with posttransfusion hepatitis and low efficiency of transmission among spouses. J. Med. Virol. 36, 28 – 31. Wasley, A., Alter, M.J., 2000. Epidemiology of hepatitis C: geographic differences and temporal trends. Semin. Liver Dis. 20, 1 – 16. Young, K.C., Chang, T.T., Liou, T.C., Wu, H.L., 1993. Detection of hepatitis C virus RNA in peripheral blood mononuclear cells and in saliva. J. Med. Virol. 41, 55 – 60.
Detection of HCV RNA in saliva of patients with hepatitis C virus infection by using a highly sensitive test M. Hermida a, M.C. Ferreiro b, S. Barral a, R. Laredo a, A. Castro c, P. Diz Dios b,* b
a Institute of Health Sciences, Juan Canalejo Hospital, La Corun˜a, Spain School of Dentistry and Medicine, Santiago de Compostela Uni6ersity, Santiago de Compostela, Spain c Department of Internal Medicine, Juan Canalejo Hospital, La Corun˜a, Spain
Received 14 June 2001; received in revised form 17 October 2001; accepted 22 October 2001
Abstract Hepatitis C virus (HCV) is transmitted primarily by direct percutaneous exposures to blood. Since HCV RNA has been found in saliva, it has been suggested that saliva might also be a source of infection. HCV RNA in saliva from plasma HCV RNA positive patients was tested by a highly sensitive PCR method. HCV RNA was detected in 32 out of 61 saliva specimens (52.4%). No correlation was found between the presence of HCV in saliva and age, sex, identified risk factors for HCV infection, time lapsed since the diagnosis, transaminases and alkaline phosphatase values and stimulated salivary flow. A statistically significant relation between plasma HCV RNA viral load and saliva HCV RNA detection was observed (PB 0.001). In conclusion, HCV RNA is often present in saliva of HCV infected patients, with plasma viral load being the only known predictable factor. Further studies on salivary HCV RNA are needed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Polymerase chain reaction; Saliva; Disease transmission
1. Introduction Hepatitis C virus (HCV) is transmitted by percutaneous exposure to infected blood. In some countries, unsafe injections and contaminated equipment used in healthcare-related procedures appear to have a predominant role in transmission, leading to epidemiological and geographical
* Corresponding author. Present address: c/Panama´ 2, 2° dcha, 36203 Vigo, Spain. Fax: + 34-981-562-226. E-mail address: [email protected] (P. Diz Dios).
differences and temporal trends (Wasley and Alter, 2000). Sexual and mother-to-child transmission have also been described, although it has been suggested that HCV infection should not be considered as a sexually transmissible infection (Kaldor et al., 2000). There remain a large number of hepatitis C carriers in whom no route of infection can be identified. Experimental infections by HCV contaminated saliva inoculation in non-human primates (Abe and Inchauspe, 1991) and HCV transmission following human bites (Dusheiko et al., 1990; Figueiredo et al., 1994) have been reported.
0166-0934/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 4 1 7 - 7
30
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
Saliva has been examined in several studies for the presence of the HCV RNA by reverse transcription polymerase chain reaction (RT-PCR) with percentages which vary between 0% (Fried et al., 1992) and 100% (Takamatsu et al., 1990). The aim of this study was to determine the prevalence of HCV RNA in saliva of plasma HCV RNA positive patients using a highly sensitive RT-PCR technique.
ployed (Navazesh and Christensen, 1982), applying a few drops of 0.1 mol/l citric acid on the anterior dorsal surface of the tongue every minute for 3 min. Saliva samples were collected in a sterile container and after measurement of volumes, were stored at − 80 °C. Saliva specimens of the 74 plasma HCV RNA positive patients were examined. Nine samples were macroscopically seen to contain blood and four samples were damaged during processing.
2. Materials and methods
2.3. HCV RNA detection
2.1. Population
HCV RNA detection was carried out in the remaining 61 saliva samples using a nested RTPCR technique. This technique was applied amplifying a fragment of 251 bp from the 5%UTR region of the HCV, as described previously (Roy et al., 1995). Stored samples of saliva were centrifuged (3500 rpm, 5 min). HCV RNA was extracted from 200 ml of supernatant and 200 ml of the cell fraction, with the addition of TRIZOL (Life Technologies, Barcelona, Spain), according to the manufacturer’s instructions. The RNA was resuspended in 10 ml of diethyl pirocarbonate (DEPC)-treated water. Synthesis of cDNA was undertaken by reverse transcription from 10 ml of RNA extracted at 38 °C for 30 min and posteriorly 95 °C for 5 min, in 20 ml solution containing 10 mM/l Tris –HCl (pH 8.4), 50 mM/l KCl, 3 mM/l MgCl2, 10 mM DTT, 2.5 mM/l of the primers 209 and 939 (Life Technologies), 200 U of Moloney Murine Leukaemia Virus (MMLV) (Life Technologies), 20 U of Rnasin (Life Technologies) and 0.5 mM dNTPs. PCR was carried out in a 50 ml mixture containing 20 ml of cDNA, 10 mM/l Tris –HCl (pH 8.4), 50 mM/l KCl, 1.5 mM/l MgCl2, 200 mM dNTPs and 1 U Taq Pol (Life Technologies). Amplification occurred over 30 cycles (94 °C for 30 s; 53 °C for 45 s; 72 °C for 60 s). PCR reaction product (1 ml) was reamplified using the same cycling program in a mixture containing 10 mM/l Tris – HCl (pH 8.4), 50 mM/l KCl, 1.5 mM/l MgCl2, 200 mM dNTPs, 1 U Taq Pol (Life Technologies) and 1 mM/l of the primers 211 and
The study population included 74 patients with hepatitis C virus infection, diagnosed in the Internal Medicine Service of the Juan Canalejo Hospital (La Corun˜ a, Spain). The selection of patients was carried out according to the following inclusion criteria: presence of HCV RNA in plasma confirmed by nested reverse transcription polymerase chain reaction (RT-PCR); absence of other concomitant liver diseases; negative HIV test; no prior interferon and/or ribavirine treatment; subjects stated that they were neither habitual alcohol consumers nor active intravenous drug users. The following data were recorded from their medical records: age, sex, identified risk factors for HCV infection and time lapsed since the diagnosis. Plasma HCV RNA levels determined by the PCR system (Amplicor HCV Monitor, Roche Molecular Systems, Barcelona, Spain), aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) values from the last month before enrollment in the study, were also recorded.
2.2. Sali6a collection Stimulated whole saliva was collected between 10:00 and 12:00 h by a single research worker. Subjects refrained from eating, drinking, smoking and oral hygiene procedures for 90 min before salivary collection. The spitting method was em-
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
940 (Life Technologies). Amplified cDNA was detected by 1.5% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the technique was determined by studying the products of the PCR at serial plasma dilutions (100, 10, 1, 0.1, 0.01 and 0.001%) in saliva. Blood came from an HCV infected patient with viral load of 1× 106 copies/ml. The saliva was obtained from a healthy HCV negative volunteer. The detection limit of this technique was ten HCV RNA copies/ml (Fig. 1). A Hem-Check-1 kit based in monoclonal antibodies (Menarini Diagnostics, Barcelona, Spain) was used in order to search for the presence of hemoglobin (hidden blood). The presence of HCV RNA in saliva was examined in relation to the demographical, clinical and analytical variables applying the two-tailed Student’s t-test, the 2-test and logistic regression analysis. Informed consent from patients was obtained.
31
3. Results The study group comprised of 54 males (73%) and 20 females (27%) with a mean age of 40.59 14.6 years (range 20–71 years). Risk factors were identified in 55.8% of patients. Injection drug use was the main mode of transmission (45.9%), followed by blood transfusion (8.1%). The mean duration of HCV infection was 11.49 8 years, although this was only determined in 23 cases. The mean time lapsed from the diagnosis of hepatitis C virus infection was 2.99 2.4 years (range 1–10 years). The mean values of the liver enzymes were: AST= 60.49 42.7 IU/l (range 5–200); ALT = 106.49 96.8 IU/l (range 11–447); GGT= 55.09 60.5 IU/l (range 9–387); ALP=191.49 86.7 IU/l (range 74– 488). The average viral load detected in the study group was 582,1209 383,258 copies/ml (range 790–1,599,000 copies/ml). HCV RNA was found in 32 out of 61 saliva specimens (52.4%). No correlations were found between HCV detection in saliva and age, sex,
Fig. 1. Agarose gel electrophoresis of amplified HCV RNA at serial plasma dilutions from an HCV infected patient (1 × 106 copies/ml) in saliva from a healthy HCV negative volunteer, using a nested reverse transcription polymerase chain reaction. Lane 1: plasma positive control. Lane 2: plasma from an HCV infected patient with viral load =1 × 106 copies/ml. Lane 3: negative saliva control. Lane 4: 10% plasma dilution in saliva (1 × 105 copies/ml). Lane 5: 1% plasma dilution in saliva (1 ×104 copies/ml.) Lane 6: 0.1% plasma dilution in saliva (1 × 103 copies/ml). Lane 7: 0.01% plasma dilution in saliva (1 ×102 copies/ml). Lane 8: 0.001% plasma dilution in saliva (1 ×101 copies/ml). Lane 9: negative saliva control. Lane 10: molecular weight control.
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M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
Fig. 2. Relationship between plasma HCV RNA titers and saliva HCV RNA detection (PB 0.001). Mean plasma viral load in patients without HCV in saliva = 372,7299 302,053 copies/ml. Mean plasma viral load in patients with HCV in saliva = 784,292 9 345,859 copies/ml.
identified risk factors for HCV infection, time lapsed since the diagnosis, AST, ALT, GGT and ALP values. Hemoglobin was detected in 14 out of 61 saliva samples (22.9%), but its presence was not a predictor of HCV RNA in saliva. A statistically significant relationship between plasma HCV RNA viral load and saliva HCV RNA detection was observed (P B 0.001) (Fig. 2)
4. Discussion All patients included in the present study were infected with HCV confirmed by RT-PCR. Al-
though the most recent enzyme-linked immunoassays for HCV infection have increased sensitivity and specificity, these assays still have relatively high false–positive rates among low-risk populations. Because some infected persons fail to generate an immune response to HCV, reactivity by RT-PCR is considered to be the definitive proof of hepatitis C infection (Robbins et al., 2000). It has been indicated that the presence of HCV RNA in saliva correlates with HCV viremia (Wang et al., 1992; Numata et al., 1993; Mariette et al., 1995; Caldwell et al., 1996; Fabris et al., 1999). Variations of plasma HCV RNA levels have been related to coinfection with other viral
M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
agents such as HBV (Sagnelli et al., 2000) and HIV (Hisada et al., 2000), self reported alcohol consumption (Degos, 1999) and antiviral therapy (Barbaro et al., 2000). In consequence, these conditions have been considered exclusion criteria. In our study, salivary HCV RNA prevalence was 52.4%, this figure being similar to those reported by other researchers (Roy et al., 1996, 1998; Fabris et al., 1999). Nonetheless, studies published on this topic in recent years show great variability (Table 1). Low prevalence results may indicate a quantity of HCV RNA in saliva below the level of PCR sensitivity (Roy et al., 1995; Fabris et al., 1999). We have applied a highly sensitive technique with a detection limit of ten HCV RNA copies/ml. Although handling and storage of saliva specimens are technically complex and could effect the stability of viral RNA (Roy et al., 1999), collection and processing of
33
saliva were carried out as described previously by others (Fabris et al., 1999). In HCV infected patients, transaminase values have been found to be higher in those with HCV RNA than in those without detectable HCV RNA in the saliva (Sugimura et al., 1995). Our results did not reveal any relation between HCV presence in saliva and enzyme levels and/or duration of disease, which was in agreement with other reports (Wang et al., 1992; Fabris et al., 1999). In the present study, as it has been suggested by others (Wang et al., 1992; Numata et al., 1993; Mariette et al., 1995; Caldwell et al., 1996; Fabris et al., 1999), the best predictive factor for HCV detection in saliva was a high level of plasma HCV RNA. There are several reasons why HCV RNA may be present in the saliva of patients with detectable virus in the plasma. Contamination with blood
Table 1 Recent studies on prevalence of HCV RNA in saliva of plasma HCV RNA positive patients Author, year
No. of patients
Type of patients
HCV diagnostic method
HCV RNA prevalence in saliva
(Mariette et al., 1995) (Chen et al., 1995) (Sugimura et al., 1995) (Roy et al., 1995) (Tang et al., 1996) (Jorgensen et al., 1996) (Caldwell et al., 1996) (Roy et al., 1996)
28
Plasma HCV+ (13 HIV+)
PCR
17 (61%)
23
Plasma HVC+ (11 HIV+)
PCR
4 (17%)
76
Anti-HCV+
RIBA
27 (36%)
14 16
Plasma HCV+ blood donors Plasma HCV+
RIBA PCR PCR
9 (64%) 5 (31%)
16
Plasma HCV+Sicca syndrome
RIBA PCR
13 (83%)
33
PCR
5 (15%)
PCR
10 (48%)
11
Plasma HCV+ (21 liver transplantated) Plasma HCV+hemophiliacs (6 HIV+) Plasma HCV+women postpartum
PCR
4 (36%)
10
Plasma HCV+hemodialysed
PCR
3 (30%)
20
Plasma HCV+CAH or cirrhosis
PCR
3 (15%)
33 39
Plasma HCV+IVDUs (19 HIV+) Plasma HCV+
PCR PCR
19 (58%) 22 (56%)
61
Plasma HCV+
PCR
32 (52%)
(Kage et al., 1997) (Ustundag et al., 1997) (Taliani et al., 1997) (Roy et al., 1998) (Fabris et al., 1999) Present series
21
HCV, hepatitis C virus; RIBA, recombinant immunoblot assay; PCR, polymerase chain reaction; IVDU, intravenous drug user.
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M. Hermida et al. / Journal of Virological Methods 101 (2002) 29–35
could be a contributing factor, but positive hemoglobin samples were independent of HCV RNA results (Fabris et al., 1999). Peripheral blood mononuclear cells (PBMCs) are another possible source for transferring HCV to saliva (Roy et al., 1995; Fabris et al., 1999), but the presence of HCV RNA in PBMCs and in saliva is not correlated closely (Young et al., 1993). HCV could also enter saliva through the gingival crevicular fluid, as has been shown in other viral diseases (Maticic et al., 2000), but HCV RNA in saliva has been found even in edentulous patients (Roy et al., 1998). The issue of intraglandular replication of HCV remains controversial. Recent studies employing assays optimized for identifying replicative intermediates of HCV, found HCV quasispecies in different tissue samples compatible with independent replication at extrahepatic sites (Laskus et al., 2000), which could include major salivary glands. The results of this study suggest that HCV RNA is present in saliva in a high proportion of HCV infected patients. Some issues, such as the mechanism by which the virus enters the saliva, its clinical significance and the possible HCV transmission through exposure to this body fluid, remain unclear. However, HCV RNA detection in saliva may be useful for monitoring antiviral treatment (Roy et al., 1995).
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