740
Detection of Hepatitis B Virus DNA in Chronic Carriers by the Polymerase Chain Reaction C. Malay6 Lara*, M.T. Gorrifio, C. Campelo, R Lardelli, E. Sufien, R. Cisterna
The polymerase chain reaction (PCR) was used to detect hepatitis B virus D N A (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were posi. tire for H B V - D N A on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for D N A H B V on dot blot hybridisation, however D N A was detected in 30 ofthem with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected H B V - D N A in only one patient. These results indicate that while serological and conventional D N A hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients.
Hepatitis B virus infection represents a major public health problem. In addition to the mortality and morbidity caused by the acute infection, the disease becomes chronic in some patients, over 200 million people worldwide being chronic carriers of the virus (1). Prediction of development of chronic disease and carrier status is currently based on serologic and liver biopsy findings. HBeAg and HBV-DNA in serum have been claimed to be good markers of active virus replication (2), however HBV-DNA is now considered a more reliable marker of virus replication and infectivity. Thus, the presence of serum HBV-DNA has been demonstrated to correlate strongly with the production of H B V particles, while it seems to be independent of circulating antigens or antibodies (3, 4). The development of Department of Microbiologyand Immunology, University of the Basque Country, PO Box 699, Bilbao, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
the polymerase chain reaction (PCR) has allowed detection of as little as one single copy of a gene (5). Recently, this highly sensitive method has also been used for study of the pathogenesis of chronic H B V infection as well as for determination of infectivity and surveillance of H B V carriers (6-9). In the present study we used the PCR technique to detect HBV-DNA in the serum of 104 patients with chronic hepatitis B virus infection with the aim of determining the sensivity of the method in comparison with serological assays. Materials and Methods. The serum samples tested were obtained from 104 chronic carriers divided into three groups. Group A consisted of 22 subjects with active chronic disease who were positive for HBsAg, H B e A g and anti-HBc, and negative for anti-HBe. Group B consisted of 41 asymptomatic subject s who were positive for HBsAg, anti-HBc and anti-HBe, and negative for anti-HBs. Group C consisted of 41 asymptomatic subjects who were positive for anti-HBs and antiHBc, and negative for t-IBsAg. In addition serum samples from 40 healthy subjects with no serological evidence of H B V infection were tested. Serological markers of hepatitis B virus (HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc) were detected with commercial enzyme immunoassays (Abbott Laboratories, Germany) following the manufacturer's instructions. For preparation of D N A samples 200 ~1 of each serum were mixed with 200/al of lysis solution (25 mg/l proteinase K~ 0.5 % sodium dodecyl sulphate [SDS], 25 mM sodium acetate of pH 6.5, 25 mM EDTA) and incubated for 2 h at 55 °C (7). D N A was then extracted with phenolchloroform and precipitated with absolute ethanol. D N A amplification was carried out as follows. The specific primers for H B V core gene sequences used were 1763 5': GC-WTTGGGGATGG A C A T T G A C C C G T A T A A 3', and 2032 R: 5' CTG A CTA C T A A T T C C C T G G A T G C T G G G TCT 3'. These specific primers allow amplification of a highly preserved region of the core of wild-type HBV-DNA (8). The final incubation volume was 100 ~tl, containing 4 jag of DNA, 1 mM of each primer, 1.25 mM of each dNTP (dATP, dCTP, dGTP, dTFP), 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12 and 0.01% gelatin. Samples were incubated for 7 min at 98 °C in a thermal cycler (Perkin-Elmer, USA) to achieve D N A denaturation and protein inactivation, and then for 1 rain at 37 °C to allow primer
Vol. 11, 1992
741
Table lx Detection of serum HBV-DNA by PCR and dot blot hybridisation in 104 chronic HBV carriers. Serologicalstatus
Group A Group B
Group C
HBV-DNA
No. of patients
HBsAg
Anti-HBs
Anti-HBc
HBeAg
Anti-HBe
15 7
+ +
-
+ +
+ +
-
3 27 11
+ + +
-
+ + +
-
+ + +
1 22
-
+ +
+ +
18
-
+
+
annealing. Thereafter, 2.5 units of Taq polym e r a s e ( P e r k i n - E l m e r ) were added. T h e P C R conditions were 37 °C for 2 min for annealing, 70 °C for 3 min for D N A synthesis, and 92 °C for 1 min for denaturation. A f t e r 10 cycles the annealing t e m p e r a t u r e was increased to 50 °C for a n o t h e r 30 cycles. (9).
Hybridisation
w
PCR
+
+
--
+
+
+
--
+
+
+
--
+
was allowed to proceed for 20 h at 42 °C. Positive signals were m a d e visible by an alkaline phosphatase color d e v e l o p m e n t p r o c e d u r e (Blugene, BRL, USA).
1
2
3
4
5
6
7
Ten lal of the amplified material were electrop h o r e s e d in 2 % agarose gel and stained with ethidium b r o m i d e in o r d e r to reveal D N A u n d e r U V light. PB R 328 BglI-HinfI digest (Boehringer, M a n n h e i m , G e r m a n y ) was used to m o n i t o r the size of the amplified product. D N A extraction and amplification was p e r f o r m e d at least twice in each patient. Positive and negative controls were run in parallel in every assay. In samples with deviant results D N A was re-extracted and amplified twice again. Samples which then exhibited positivity in only one of the duplicate reactions w e r e considered negative. Specific precautions were t a k e n in the P C R to prevent cross-contamination. T h e p r o c e d u r e for dot blot hybridisation was as follows. P C R products were d e n a t u r a t e d and subsequently immobilized o n t o nitrocellulose m e m b r a n e s . M e m b r a n e s were washed in 2 x SSC (1 x SSC: 0.15 M NaC1, 0.015 M sodium citrate), allowed to dry and b a k e d for 1 h at 80 °C. A f t e r incubation for 15 min in 5 x SSC, m e m b r a n e s w e r e prehybridised in 50 % (v/v) formamide/5 x SSC, 10 x D e n h a r d t ' s solution (1 x D e n h a r d t ' s solution: 0.02 % polyvinylpyrrolidone, 0.02 % Ficoll, 0.02 % bovine serum albumin), 5 m M Na2HPO4 and 0 . 1 % SDS containing denaturated salmon s p e r m D N A (250 ~g/ml) at 42 °C for 4 h. Hybridisation was p e r f o r m e d with 2 pg/ml of the biotin labelled P B H 2 0 r e c o m b i n a n t plasmid containing the H B V whole genome. Hybridisation
Figure 1' Visualization under UV light of HBV-DNA sequences in PCR amplified samples after ethidium bromide staining of agarose gels. Lanes 1 and 2: positive samples; lanes 3 and 4: negative samples; lane 5: amplified 500 bp fragment of lambda DNA run as control for the PCR technique; lane 6: non-amplified lambda DNA; lane 7: molecular weight marker PBR 328 DNA digested with
BglI-HinfI.
742
Eur. J. Clin. Microbiol. Infect. Dis.
All HBV-DNA positive samples obtained after PCR amplification were visualized directly in ethidium bromide stained agarose gels under UV illumination (Figure 1) and the results subsequently confirmed by dot blot hybridisation (Figure 2).
t-IBV-DNA detection in serum samples by dot blot hybridisation without prior amplification was carried out following the same procedure. The sensivity of the PCR assay was evaluated using serial dilutions of recombinant entire HBVD N A (PBH20) as template subjected to PCR amplification as described above. Results and Discussion. As shown in Table 1, HBV-DNA was detected by dot blot hybridisation in 15 of the 22 (68 %) subjects in group A (HBsAg, H B e A g and anti-HBc positive patients). All subjects were positive for HBVD N A in the PCR. Analysis of sera of the 41 subjects in group B (HBsAg, anti-HBc and anti-HBe positive) revealed that only three (7.3 %) were positive for HBV-DNA on dot blot hybridisation. However, 30 (73.1%) were positive in the PCR. Of the 41 subjects in group C (anti-HBs, anti-HBc positive) only one was positive for HBV-DNA on dot blot hybridisation although HBV-DNA was detected in 23 (56.1%) in the PCR. All samples from 40 healthy individuals with no serological evidence of past or present H B V infection were negative for HBV-DNA in both dot blot hybridisation and the PCR.
1
2
3
4
5
6
In this study HBV-DNA present in amounts not detected by dot blot hybridisation was easily detected after P C R amplification. While 85 of 104 serum samples from chronic H B V carriers were negative for HBV-DNA on conventional direct dot blot hybridisation, after PCR amplification HBV-DNA was demonstrated in the sera of all patients positive for H B e A g and HBsAg. Serum HBV-DNA and HBeAg have been considered markers of viral replication (2, 10). In our study, although 7 of 22 H B e A g positive patients were negative for HBV-DNA on dot blot hybridisation, the presence of H B V - D N A could be demonstrated after PCR amplification, confirming that I-IBeAg is a good marker of viral replication (3, 9, 11-13). In addition, these results also imply that all HBsAg and H B e A g positive sera are potentially infectious. On the other hand, in the group of HBsAg positive patients who also had anti-HBe, although only 3 of 41 sera were positive for HBV-DNA on
7
8
9
10
11
12
13
G
Hgure 2: Dot blot hybridisation analysisof PCR amplified HBV-DNAsequences from serum samples.
A9: positive control; A10: negative control. Each dot corresponds to a single sample.
Vo1.11,1992
direct dot blot hybridisation, 30 ( 7 3 . 1 % ) were positive in the PCR. This finding suggests that the absence of serum H B e A g does not exclude active H B V replication. F u r t h e r m o r e , it could also imply that seroconversion from H B e A g to antiH B e does not guarantee disappearance of viral particles from the serum or loss of infectivity. Our results are in a g r e e m e n t with those reported by K a n e k o et al. (8, 14), who showed that nearly all patients positive for HBsAg, anti-HBc and antiH B e possessed circulating viral particles. Moreover, Hadziyannis et al. (15) have shown that viral replication occurs in H B e A g negative patients by demonstrating the presence of serum H B V - D N A and core antigen in hepatocyte nuclei. These findings challenge the c o m m o n belief that serum H B e A g itself is a reliable m a r k e r of viral replication. Similarly, seroconversion to anti-HBs does not seem to preclude the existence of active replication. According to our results, in 5 6 . 1 % of the H B s A g negative, anti-HBs positive subjects, H B V - D N A was detected by PCR, and in one of them also by direct dot blot hybridisation. Likewise, H B V - D N A has been detected by hybridisation in hepatic tissue from H B s A g negative, antiHBs positive patients (16), and more recently traces of viral D N A have been detected by P C R in patients with the same serological status (17). T h e r e f o r e , although the presence of anti-HBs has been so far considered a m a r k e r of past infection or previous contact with the virus, some anti-HBs positive patients may still harbor viral D N A in their hepatocytes and act as chronic carriers of HBsAg. In summary, this study demonstrates the existence of subclinical cases of H B V infection with active viral replication which is not reflected in the serological findings in these patients. Therefore, the P C R is a good technique for identification of potentially infective samples and for follow-up of chronic H B V patients and carriers. Nevertheless, although H B V - D N A positivity of a sample after P C R amplification indicates the putative infectivity of this patient and is a sufficient criterion for exclusion from organ or blood donation, P C R alone cannot define the actual infectivity o f a single patient. In fact, the quantitative measurements of H B V - D N A in serum have b e e n shown to change from day to day in H B V carriers. It is important to correlate PCR results with immunohistochemical and serological findings in order to establish a cut-off value for viremia that implies clinical significance.
743
References 1. Szmuness W, Harley EJ, lkram H, Stevens CE:
Sociodemographie aspects of epidemiology of hepatitis B. In: Vyas GN, Cohen SN~ Schmid R (ed): Viral hepatitis. Franklin Institute Press, Philadelphia, 1978, p. 297-320, 2. Scotto J, Hadchouel M, He W C, lvarl J, Tiollais P, Brechot C: Detection of hepatitis B virus DNA in
serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1982, 3: 279-284. 3. Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin J: Hepatitis B virus DNA in the sera of HBsAg
carriers: a marker of active hepatitis B replication in the liver. Hepatology 1981, 1: 386--391. 4. Brechot C, Pourcel C, Hadchouel M, Dejean A, Louise
A, Scotto J, Tiollais P: State of hepatitis B virus DNA in liver diseases. Hepatology 1982, 2, Supplement: 2734.
5. Saiki R, Scharf S, Faloona F, Muilis K, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of beta-
globin genomie sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985, 230: 1350-1353. 6. Larznl D, Guigue F, Sninsky J J: Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. Journal of Virological Methods 1988, 20: 227-237. 7. Thiers V, Nakajima E, Kremsdorf D, Mack D, Schellekens H, Driss F, Goudea A, Wands J, Sninsky J, Tioilais P, Brechot C: Transmission of hepatitis B from hepatitis B-seronegative subjects. Lancet 1988, ii:
1273-1276. 8. Kaneko S, Miller R, Feinstone S, Unuora M, Kobayashi K, Hatori N, Ruscell RH: Detection of
serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proceedings of the National Academy of Science of the USA 1989, 86: 312-316. 9. Sumasaki R, Motz M, Wolf H, Heining J, Jilg W, Deinhardt F: Detection of hepatitis B virus in serum using
amplification of viral DNA by means of the polymerase chain reaction. Virology 1989, 27: 304-308. 10. Lok ASF, Hadziyannis SJ, Weller IVD, Montjardino J, Karayiannis P, Montana L, Thomas HC: Contribu-
tion of low level HBV replication to continuing inflammatory activity with anti-HBe positive chronic hepatitis B virus infection. Out 1984, 25: 1283-1287. 11. Berninger M, Hammer M, Hoyer B, Gerin GL: An assay for the detection of the DNA genome of hepatitis B virus in serum. Journal of Medical Virology 1982, 9: 57--68. 12. Chen DS, Lay MY, Lee SC, Yang PM, Sheu JC, Sung JL: Serum HBsAg, HBeAg, anti-HBe and hepatitis B viral DNA in asymptomatic carriers in Taiwan. Journal of Medical Virology 1986, 19: 87-94. 13. Baker BL, Di Bisceglie AM, Kaneko S, Miller R, Feinstone SM, Waggoner JG, Hoofnagle JG: Deter-
mination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology 1991, 13: 632-636. 14. Kaneko S, Miller RH, Di Bisceglie AM, Feinstone SM, Hoofnagle H J, Purcell RH: Detection of hepatitis
B virus DNA in serum by polymerase chain reaction. Application for clinical diagnosis. Oastroenterology 1990, 99: 799-804.
744
15. Hadziyannis SJ, Lieberman H, Karvountzis G, Shafritz D: Analysis of liver disease, nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983, 3: 656--662. 16. Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S,
France D, Bismuth H, Trepo C, Benhamou JP, Wands J, lsselbacher K, Tiollais P, Berthelot P: Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. New England Journal of Medicine 1985, 312: 270-276. 17. Shafritz DA, Lieberman HM, Isselbacher K, Wands
J: Monoclonal radioimmunoassayfor hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes. Proceedings of the National Academy of Science of the USA 1982, 79: 5675-5679.
Choroiditis and Meningitis in Experimental Murine Infection with Listeria monocytogenes N. Prats 2, V. Briones 1, M.M. Blanco 1, J. Altimira 2, J.A. R a m o s 2, L. D o m f n g u e z 1, A. M a r c o 2.
In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously with Listeria monocytogenes. Systemic infection developed, as shown by the isolation ofListeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningifts were the most relevant lesions. Inflammatory lesions were associated with the presence of Llsteria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of Listeria monoeytogenes within
1Departamento de Patologfa Animal I, Faeultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. 2Departamento de Patolog[a y Producciones Animales (Anatomfa Patol6gica), Facultad de Veterinaria Universidad Aut6noma de Barcelona, 08193 (Bellaterra) Barcelona, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.
There are two main forms of nervous system involvement in listeriosis: encephalitis, which is the predominant form in small ruminants (1), and meningitis, which is the most frequent form of listeriosis in humans (2). In listeric encephalitis in small ruminants, involvement of the medulla oblongata and pens is a consequence of neurotropic ascension of Listeria monocytogenes via the trigeminal nerve (1), whereas in cases of meningitis in humans and other species, spread of the microorganism to the central nervous system (CNS) is assumed to be hematogenous. Nervous system involvement in murine listeriosis has also been reported (3), but a clear-cut distinction between encephalitis and meningitis has not been established in this experimental model. Moreover, most of the studies on listeriosis in mice report no changes in the CNS (4-6). In this paper we report on an experimental model of CNS infection in mice infected subcutaneously with Listeria monocytogenes, which is similar to the meningitic form in humans. Materials and Methods. Listeria monocytogenes (type strain, serovar 1/2a, SLCC 2371/NCTC 7973) was grown in 250 ml of brain heart infusion broth (BHI; Difco, USA) at 37 °C for 22 h, centrifuged and resuspended in 20 ml of sterile 0.9 % saline. This suspension (2 x 101° cfu/ml) was diluted to obtain the desired inoculum. Twenty-seven 6-week-old female Swiss CD1 listeria-resistant mice (Interfauna, Spain) were inoculated subcutaneously with 1 x 109 cfu/ml of Listeria monocytogenes. This dose represents approximately 1.3 x LD50 for this route and for this strain of mice (7). Three animals were sacrificed daily from day 1 to day 9 post-inoculation by inhalation of ethyl ether. The mice to be sacrificed were randomly selected. Necropsy was performed under aseptic conditions. Samples of the liver and spleen were weighed and homogenated in 0.9 % saline, and serial dilutions plated in duplicate on BHI agar for the counting of Listeria monocytogenes. The whole brain, and samples from the liver and spleen were removed, fixed in 10 % formol saline and embedded in paraffin according to routine histological procedures. Sections were stained with hematoxylin and eosin (HE) or processed for immunocytochemistry by the peroxidase anti-
Detection of Hepatitis B Virus DNA in Chronic Carriers by the Polymerase Chain Reaction C. Malay6 Lara*, M.T. Gorrifio, C. Campelo, R Lardelli, E. Sufien, R. Cisterna
The polymerase chain reaction (PCR) was used to detect hepatitis B virus D N A (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were posi. tire for H B V - D N A on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for D N A H B V on dot blot hybridisation, however D N A was detected in 30 ofthem with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected H B V - D N A in only one patient. These results indicate that while serological and conventional D N A hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients.
Hepatitis B virus infection represents a major public health problem. In addition to the mortality and morbidity caused by the acute infection, the disease becomes chronic in some patients, over 200 million people worldwide being chronic carriers of the virus (1). Prediction of development of chronic disease and carrier status is currently based on serologic and liver biopsy findings. HBeAg and HBV-DNA in serum have been claimed to be good markers of active virus replication (2), however HBV-DNA is now considered a more reliable marker of virus replication and infectivity. Thus, the presence of serum HBV-DNA has been demonstrated to correlate strongly with the production of H B V particles, while it seems to be independent of circulating antigens or antibodies (3, 4). The development of Department of Microbiologyand Immunology, University of the Basque Country, PO Box 699, Bilbao, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
the polymerase chain reaction (PCR) has allowed detection of as little as one single copy of a gene (5). Recently, this highly sensitive method has also been used for study of the pathogenesis of chronic H B V infection as well as for determination of infectivity and surveillance of H B V carriers (6-9). In the present study we used the PCR technique to detect HBV-DNA in the serum of 104 patients with chronic hepatitis B virus infection with the aim of determining the sensivity of the method in comparison with serological assays. Materials and Methods. The serum samples tested were obtained from 104 chronic carriers divided into three groups. Group A consisted of 22 subjects with active chronic disease who were positive for HBsAg, H B e A g and anti-HBc, and negative for anti-HBe. Group B consisted of 41 asymptomatic subject s who were positive for HBsAg, anti-HBc and anti-HBe, and negative for anti-HBs. Group C consisted of 41 asymptomatic subjects who were positive for anti-HBs and antiHBc, and negative for t-IBsAg. In addition serum samples from 40 healthy subjects with no serological evidence of H B V infection were tested. Serological markers of hepatitis B virus (HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc) were detected with commercial enzyme immunoassays (Abbott Laboratories, Germany) following the manufacturer's instructions. For preparation of D N A samples 200 ~1 of each serum were mixed with 200/al of lysis solution (25 mg/l proteinase K~ 0.5 % sodium dodecyl sulphate [SDS], 25 mM sodium acetate of pH 6.5, 25 mM EDTA) and incubated for 2 h at 55 °C (7). D N A was then extracted with phenolchloroform and precipitated with absolute ethanol. D N A amplification was carried out as follows. The specific primers for H B V core gene sequences used were 1763 5': GC-WTTGGGGATGG A C A T T G A C C C G T A T A A 3', and 2032 R: 5' CTG A CTA C T A A T T C C C T G G A T G C T G G G TCT 3'. These specific primers allow amplification of a highly preserved region of the core of wild-type HBV-DNA (8). The final incubation volume was 100 ~tl, containing 4 jag of DNA, 1 mM of each primer, 1.25 mM of each dNTP (dATP, dCTP, dGTP, dTFP), 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12 and 0.01% gelatin. Samples were incubated for 7 min at 98 °C in a thermal cycler (Perkin-Elmer, USA) to achieve D N A denaturation and protein inactivation, and then for 1 rain at 37 °C to allow primer
Vol. 11, 1992
741
Table lx Detection of serum HBV-DNA by PCR and dot blot hybridisation in 104 chronic HBV carriers. Serologicalstatus
Group A Group B
Group C
HBV-DNA
No. of patients
HBsAg
Anti-HBs
Anti-HBc
HBeAg
Anti-HBe
15 7
+ +
-
+ +
+ +
-
3 27 11
+ + +
-
+ + +
-
+ + +
1 22
-
+ +
+ +
18
-
+
+
annealing. Thereafter, 2.5 units of Taq polym e r a s e ( P e r k i n - E l m e r ) were added. T h e P C R conditions were 37 °C for 2 min for annealing, 70 °C for 3 min for D N A synthesis, and 92 °C for 1 min for denaturation. A f t e r 10 cycles the annealing t e m p e r a t u r e was increased to 50 °C for a n o t h e r 30 cycles. (9).
Hybridisation
w
PCR
+
+
--
+
+
+
--
+
+
+
--
+
was allowed to proceed for 20 h at 42 °C. Positive signals were m a d e visible by an alkaline phosphatase color d e v e l o p m e n t p r o c e d u r e (Blugene, BRL, USA).
1
2
3
4
5
6
7
Ten lal of the amplified material were electrop h o r e s e d in 2 % agarose gel and stained with ethidium b r o m i d e in o r d e r to reveal D N A u n d e r U V light. PB R 328 BglI-HinfI digest (Boehringer, M a n n h e i m , G e r m a n y ) was used to m o n i t o r the size of the amplified product. D N A extraction and amplification was p e r f o r m e d at least twice in each patient. Positive and negative controls were run in parallel in every assay. In samples with deviant results D N A was re-extracted and amplified twice again. Samples which then exhibited positivity in only one of the duplicate reactions w e r e considered negative. Specific precautions were t a k e n in the P C R to prevent cross-contamination. T h e p r o c e d u r e for dot blot hybridisation was as follows. P C R products were d e n a t u r a t e d and subsequently immobilized o n t o nitrocellulose m e m b r a n e s . M e m b r a n e s were washed in 2 x SSC (1 x SSC: 0.15 M NaC1, 0.015 M sodium citrate), allowed to dry and b a k e d for 1 h at 80 °C. A f t e r incubation for 15 min in 5 x SSC, m e m b r a n e s w e r e prehybridised in 50 % (v/v) formamide/5 x SSC, 10 x D e n h a r d t ' s solution (1 x D e n h a r d t ' s solution: 0.02 % polyvinylpyrrolidone, 0.02 % Ficoll, 0.02 % bovine serum albumin), 5 m M Na2HPO4 and 0 . 1 % SDS containing denaturated salmon s p e r m D N A (250 ~g/ml) at 42 °C for 4 h. Hybridisation was p e r f o r m e d with 2 pg/ml of the biotin labelled P B H 2 0 r e c o m b i n a n t plasmid containing the H B V whole genome. Hybridisation
Figure 1' Visualization under UV light of HBV-DNA sequences in PCR amplified samples after ethidium bromide staining of agarose gels. Lanes 1 and 2: positive samples; lanes 3 and 4: negative samples; lane 5: amplified 500 bp fragment of lambda DNA run as control for the PCR technique; lane 6: non-amplified lambda DNA; lane 7: molecular weight marker PBR 328 DNA digested with
BglI-HinfI.
742
Eur. J. Clin. Microbiol. Infect. Dis.
All HBV-DNA positive samples obtained after PCR amplification were visualized directly in ethidium bromide stained agarose gels under UV illumination (Figure 1) and the results subsequently confirmed by dot blot hybridisation (Figure 2).
t-IBV-DNA detection in serum samples by dot blot hybridisation without prior amplification was carried out following the same procedure. The sensivity of the PCR assay was evaluated using serial dilutions of recombinant entire HBVD N A (PBH20) as template subjected to PCR amplification as described above. Results and Discussion. As shown in Table 1, HBV-DNA was detected by dot blot hybridisation in 15 of the 22 (68 %) subjects in group A (HBsAg, H B e A g and anti-HBc positive patients). All subjects were positive for HBVD N A in the PCR. Analysis of sera of the 41 subjects in group B (HBsAg, anti-HBc and anti-HBe positive) revealed that only three (7.3 %) were positive for HBV-DNA on dot blot hybridisation. However, 30 (73.1%) were positive in the PCR. Of the 41 subjects in group C (anti-HBs, anti-HBc positive) only one was positive for HBV-DNA on dot blot hybridisation although HBV-DNA was detected in 23 (56.1%) in the PCR. All samples from 40 healthy individuals with no serological evidence of past or present H B V infection were negative for HBV-DNA in both dot blot hybridisation and the PCR.
1
2
3
4
5
6
In this study HBV-DNA present in amounts not detected by dot blot hybridisation was easily detected after P C R amplification. While 85 of 104 serum samples from chronic H B V carriers were negative for HBV-DNA on conventional direct dot blot hybridisation, after PCR amplification HBV-DNA was demonstrated in the sera of all patients positive for H B e A g and HBsAg. Serum HBV-DNA and HBeAg have been considered markers of viral replication (2, 10). In our study, although 7 of 22 H B e A g positive patients were negative for HBV-DNA on dot blot hybridisation, the presence of H B V - D N A could be demonstrated after PCR amplification, confirming that I-IBeAg is a good marker of viral replication (3, 9, 11-13). In addition, these results also imply that all HBsAg and H B e A g positive sera are potentially infectious. On the other hand, in the group of HBsAg positive patients who also had anti-HBe, although only 3 of 41 sera were positive for HBV-DNA on
7
8
9
10
11
12
13
G
Hgure 2: Dot blot hybridisation analysisof PCR amplified HBV-DNAsequences from serum samples.
A9: positive control; A10: negative control. Each dot corresponds to a single sample.
Vo1.11,1992
direct dot blot hybridisation, 30 ( 7 3 . 1 % ) were positive in the PCR. This finding suggests that the absence of serum H B e A g does not exclude active H B V replication. F u r t h e r m o r e , it could also imply that seroconversion from H B e A g to antiH B e does not guarantee disappearance of viral particles from the serum or loss of infectivity. Our results are in a g r e e m e n t with those reported by K a n e k o et al. (8, 14), who showed that nearly all patients positive for HBsAg, anti-HBc and antiH B e possessed circulating viral particles. Moreover, Hadziyannis et al. (15) have shown that viral replication occurs in H B e A g negative patients by demonstrating the presence of serum H B V - D N A and core antigen in hepatocyte nuclei. These findings challenge the c o m m o n belief that serum H B e A g itself is a reliable m a r k e r of viral replication. Similarly, seroconversion to anti-HBs does not seem to preclude the existence of active replication. According to our results, in 5 6 . 1 % of the H B s A g negative, anti-HBs positive subjects, H B V - D N A was detected by PCR, and in one of them also by direct dot blot hybridisation. Likewise, H B V - D N A has been detected by hybridisation in hepatic tissue from H B s A g negative, antiHBs positive patients (16), and more recently traces of viral D N A have been detected by P C R in patients with the same serological status (17). T h e r e f o r e , although the presence of anti-HBs has been so far considered a m a r k e r of past infection or previous contact with the virus, some anti-HBs positive patients may still harbor viral D N A in their hepatocytes and act as chronic carriers of HBsAg. In summary, this study demonstrates the existence of subclinical cases of H B V infection with active viral replication which is not reflected in the serological findings in these patients. Therefore, the P C R is a good technique for identification of potentially infective samples and for follow-up of chronic H B V patients and carriers. Nevertheless, although H B V - D N A positivity of a sample after P C R amplification indicates the putative infectivity of this patient and is a sufficient criterion for exclusion from organ or blood donation, P C R alone cannot define the actual infectivity o f a single patient. In fact, the quantitative measurements of H B V - D N A in serum have b e e n shown to change from day to day in H B V carriers. It is important to correlate PCR results with immunohistochemical and serological findings in order to establish a cut-off value for viremia that implies clinical significance.
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serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1982, 3: 279-284. 3. Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin J: Hepatitis B virus DNA in the sera of HBsAg
carriers: a marker of active hepatitis B replication in the liver. Hepatology 1981, 1: 386--391. 4. Brechot C, Pourcel C, Hadchouel M, Dejean A, Louise
A, Scotto J, Tiollais P: State of hepatitis B virus DNA in liver diseases. Hepatology 1982, 2, Supplement: 2734.
5. Saiki R, Scharf S, Faloona F, Muilis K, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of beta-
globin genomie sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985, 230: 1350-1353. 6. Larznl D, Guigue F, Sninsky J J: Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. Journal of Virological Methods 1988, 20: 227-237. 7. Thiers V, Nakajima E, Kremsdorf D, Mack D, Schellekens H, Driss F, Goudea A, Wands J, Sninsky J, Tioilais P, Brechot C: Transmission of hepatitis B from hepatitis B-seronegative subjects. Lancet 1988, ii:
1273-1276. 8. Kaneko S, Miller R, Feinstone S, Unuora M, Kobayashi K, Hatori N, Ruscell RH: Detection of
serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proceedings of the National Academy of Science of the USA 1989, 86: 312-316. 9. Sumasaki R, Motz M, Wolf H, Heining J, Jilg W, Deinhardt F: Detection of hepatitis B virus in serum using
amplification of viral DNA by means of the polymerase chain reaction. Virology 1989, 27: 304-308. 10. Lok ASF, Hadziyannis SJ, Weller IVD, Montjardino J, Karayiannis P, Montana L, Thomas HC: Contribu-
tion of low level HBV replication to continuing inflammatory activity with anti-HBe positive chronic hepatitis B virus infection. Out 1984, 25: 1283-1287. 11. Berninger M, Hammer M, Hoyer B, Gerin GL: An assay for the detection of the DNA genome of hepatitis B virus in serum. Journal of Medical Virology 1982, 9: 57--68. 12. Chen DS, Lay MY, Lee SC, Yang PM, Sheu JC, Sung JL: Serum HBsAg, HBeAg, anti-HBe and hepatitis B viral DNA in asymptomatic carriers in Taiwan. Journal of Medical Virology 1986, 19: 87-94. 13. Baker BL, Di Bisceglie AM, Kaneko S, Miller R, Feinstone SM, Waggoner JG, Hoofnagle JG: Deter-
mination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology 1991, 13: 632-636. 14. Kaneko S, Miller RH, Di Bisceglie AM, Feinstone SM, Hoofnagle H J, Purcell RH: Detection of hepatitis
B virus DNA in serum by polymerase chain reaction. Application for clinical diagnosis. Oastroenterology 1990, 99: 799-804.
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15. Hadziyannis SJ, Lieberman H, Karvountzis G, Shafritz D: Analysis of liver disease, nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983, 3: 656--662. 16. Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S,
France D, Bismuth H, Trepo C, Benhamou JP, Wands J, lsselbacher K, Tiollais P, Berthelot P: Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. New England Journal of Medicine 1985, 312: 270-276. 17. Shafritz DA, Lieberman HM, Isselbacher K, Wands
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Choroiditis and Meningitis in Experimental Murine Infection with Listeria monocytogenes N. Prats 2, V. Briones 1, M.M. Blanco 1, J. Altimira 2, J.A. R a m o s 2, L. D o m f n g u e z 1, A. M a r c o 2.
In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously with Listeria monocytogenes. Systemic infection developed, as shown by the isolation ofListeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningifts were the most relevant lesions. Inflammatory lesions were associated with the presence of Llsteria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of Listeria monoeytogenes within
1Departamento de Patologfa Animal I, Faeultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. 2Departamento de Patolog[a y Producciones Animales (Anatomfa Patol6gica), Facultad de Veterinaria Universidad Aut6noma de Barcelona, 08193 (Bellaterra) Barcelona, Spain.
Eur. J. Clin. Microbiol. Infect. Dis.
mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.
There are two main forms of nervous system involvement in listeriosis: encephalitis, which is the predominant form in small ruminants (1), and meningitis, which is the most frequent form of listeriosis in humans (2). In listeric encephalitis in small ruminants, involvement of the medulla oblongata and pens is a consequence of neurotropic ascension of Listeria monocytogenes via the trigeminal nerve (1), whereas in cases of meningitis in humans and other species, spread of the microorganism to the central nervous system (CNS) is assumed to be hematogenous. Nervous system involvement in murine listeriosis has also been reported (3), but a clear-cut distinction between encephalitis and meningitis has not been established in this experimental model. Moreover, most of the studies on listeriosis in mice report no changes in the CNS (4-6). In this paper we report on an experimental model of CNS infection in mice infected subcutaneously with Listeria monocytogenes, which is similar to the meningitic form in humans. Materials and Methods. Listeria monocytogenes (type strain, serovar 1/2a, SLCC 2371/NCTC 7973) was grown in 250 ml of brain heart infusion broth (BHI; Difco, USA) at 37 °C for 22 h, centrifuged and resuspended in 20 ml of sterile 0.9 % saline. This suspension (2 x 101° cfu/ml) was diluted to obtain the desired inoculum. Twenty-seven 6-week-old female Swiss CD1 listeria-resistant mice (Interfauna, Spain) were inoculated subcutaneously with 1 x 109 cfu/ml of Listeria monocytogenes. This dose represents approximately 1.3 x LD50 for this route and for this strain of mice (7). Three animals were sacrificed daily from day 1 to day 9 post-inoculation by inhalation of ethyl ether. The mice to be sacrificed were randomly selected. Necropsy was performed under aseptic conditions. Samples of the liver and spleen were weighed and homogenated in 0.9 % saline, and serial dilutions plated in duplicate on BHI agar for the counting of Listeria monocytogenes. The whole brain, and samples from the liver and spleen were removed, fixed in 10 % formol saline and embedded in paraffin according to routine histological procedures. Sections were stained with hematoxylin and eosin (HE) or processed for immunocytochemistry by the peroxidase anti-
