Concomitant poliovirus infection during an outbreak of hepatitis A

Concomitant Maurizio

Divizia*l,

Poliovirus

Infection During Hepatitis A

an Outbreak

Rosanna Gabrieli’, Domenica Denial, Vito Rusciol, Degener* and August0 Pans1

of

Anna Marta

‘Departnwnt qf Public Health, Faculty of Medicine, Tar Veqata University. Rome, Italy; ‘Department of Developlne&al Biology, Faculty of Akdicine. La Sqienza University, Rome, Italy

Cellular

and

Aim of the SAL&: The present study was designed to evaluate the possible co-infection, with other enteric viruses, during an outbreak of hepatitis A (HA). LMnt~~rinZ and Mrthods: Forty-two stool samples and sera were collected during an outbreak of hepatitis A. Sera were analysed by the Abbott test for IgG-IgM anti-HAV antibodies. Stool samples were used to identify the presence of enteric viruses. HAV genome was identified by a RT-PCR test, other enteric viruses were identified, after cell passage and seroneutralization test on BGM cells, by RT-PCR and RFLP assay. Resrdls: The samples were obtained from 2 7 employees of an industrial plant, nine household contaicts and six nonemployee controls. The attack rate was 12.5X, whereas the overall prevalence was 63%. In the employee group, 12 out of 27 stool samples were positive for the presence of HAV by reverse transcriptase polymerase chair realction (RT-PCR). All the other samples (30) were negative. Five samples from employees. three from household contacts and one from non-employees were also found positive for enteroviruses. These viruses were classified by seroneutralization as poliovirus and RFLP assay as Sabin poliovirus type 1. Four samples were positive both for HAV and poliovirus. Conrhsions: This study confirms co-infection with different enteric viruses may occur and also emphasizes the wide circulation of HA\’ and the existence of silent infection with poliovirus. 0 1999 The British Infection Society

Introduction Hepatitis A (HA) is a worldwide disease normally causing mild to severe illness. It is occasionally fatal. In recent years in Italy’ and in the European industrialized countries’ there has been a significant reduction of the incidence which dropped from 10x100 000 in 1985 to 5x100 000 in 1994.3,1 At the same time it was observed that a clear shift occurred in the age of first viral contact in the population, which moved from childhood to older children. A large portion of the adult population is now susceptible to infection. The causative agent of hepatitis A is a virus resistant to inactivation by natural5 and artificial disinfectants” which in addition survives longer than other enteric viruses. In the environment these characteristics could explain the continued circulation of the virus.‘.” Both these situations, the trend of the first contact towards adult age and the higher resistance of the virus in

‘.Address all correspondence to: Dr Maurizio Divizia. 1Jniv. of Tor Vergata, Faculty of Medicine, Dept. of Public Health. Hygiene Institute. Via di tar Vergata, 135. 00133.Rome: Italy. Accepted for publication 29 July 1999.

the environment, could explain the repeated outbreaks of hepatitis A from northern to southern Italy.9-‘3 In this paper we report the results obtained on 42 stool samples examined for the presence of hepatitis A and other enteric viruses.

Materials and Methods Smyle nnalysis All the sera were analysed for IgG and IgM anti-HAV using the Abbott test. All the tests were performed according to the manufacturer’s instructions. Stool samples were collected and immediately frozen. The stool extracts were prepared in a 10% solution in sterile phosphate buffered saline (PBS). An aliquot (0.5 g) of stool was diluted in 5 ml of sterile PBS. After vigorously mixing, the suspension was centrifuged at 6000 rpm/20 min/4”C. The supernatant was added to Polyethylene-gl,ycol 6000 at a final concentration of 10%. After overnight shaking, the sample was centrifuged at 10 000 g/45 min/4”C. The pellet was re-suspended in 400 ~1 plus 400 ,ul of sterile PBS. The stool suspension was distributed in aliquots and frozen until used.’ 0 1999

The British

Infection

Society

228

ivimia et ab Inftclion

of

cell

cultures

Buffalo Green Monkey cells (BGlM) were seeded in 2 5 cm2 and the infectioc was performed on the second day. An aliquo! of the stool suspension (l/5 volume) was extracted twice with chloroform. The sample was mixed for 15 min at room temperature with 30% chloroform. then centrifuged at 3500 rpm/lO min. A pool of antibiotics was added to the supernatant (1 hi37”C) just prior to virus infection.

Neutralization of enteric isolated viruses was performed according to the manufacturer’s instruction, with a panel of antiserum obtained from the Rijksinstituut. Bilthoven (The Netherlands).

The polymerase chain reaction (PCR) for HAV was performed according to Jansen ELa1.14but using the one-tube PCR test. The tubes were coated with monoclonal antibody 7e7 (n/lab 7e7) kindly obtained from B. Flehmig and the reverse transcription was performed in 20 rntir Tris pH 8.8, 75 m~~II(Cl.2.5 m;\IMgC12, 0.25 mnleach dNTP (Promega) and 100 pmol each of negative (HAV -4008, 5’-GAACAATATCTCTTAACCA-3’) and positive oligonucleotide (HAV +3636. 5’- ATGCTTGGATTGTCTGGAGT-3’) and two units of reverse transcriptase MuMLV (Promega). The final volume of the reverse transcription was 90 ~1. After 1 h at 42°C. the PCR was performed, followirig the addition of 2 U of Taq polymerase (Perkin Elmer), and by subjecting the volume to 30 automated cycles of denaturation at 95W25 s, to annealing al 42W10 s and elongation at 7OWl. min. The final volume of the reaction was 100 ,ul. To identify the enteric viruses isolated during the outbreak, after proteinase I< digestion (Boeringer) and pheno!-chlorophorm-isoamylic alcohol extraction on cellular lysate, the reverse transcriptase reaction was performed at 42Wl h. The cDNA was amplified by 30 cycles of 10 s denaturation (95”C), 1. min annealing (45°C) and 1 min eiongation (70°C). To increase the final amplified product, the genomic RNA of some samples was extracted by the antigen-capture method, as used for HAV.‘” The well of the PCR tube was coated in a mixture of the antibody used for the serological identification of the isolated poliovirus. and the PCR performed as for the HAV-PCR. but using the above temperature cycles. The inner-outer primers were: 5’-GAATTCCATGTCAAATCTAGA-3’ and 5’-TTTGTGTCAGCGTGTGTAATGA-3’.15

The reverse-transcribed PCR-amplified fragments of about 480 nt were anaiysed by restriction er_zyme digestion:‘j,‘” IO ~1 out of 100 ~1 DNA solution obtained by PCR were digested with 6 IJ Dde:. 5 U Hae III, or 5 U Hpa II (Promega enzymes) in a final volume of 20 1-11 with the appropriate buffer. After incubation at 3i”C for 2 11,the DNA fragments were applied to a 3% agarose gel containing 1. I;g/ml ethidium bromide. At the end of electrophoresis the gels were examined under UV light, al: which time the restriction pattern of the sample was similar to one of the reference strains; the similarity was confirmed by parallel close migration.

The epidemiological data were reported ai the ‘Viral Hepatitis and Liver Disease’ congress held in Rome in 1996.” HAV-PCR analysis of the stoo! extracts showed that 12 out of 2 7 from employees (44.4%) were positive but none ir, the other two groups (Table I). The mean period of the stool collection was 12.7 days after the onset of the disease (range 2-45 days). Attempt at HAV cultivation was not successful, but nine samples (five from the industrial employees, three from household contacts and one from non-employees) were positive for the presence of other enteric viruses. These viruses were classified by serum neutralization as type 1 poliovirus. On the whole, the positivity for po!iOkus isolation was 18.5% for the industrial employees. 33.3% for the household contacts and 16.6% for the non-employees. In four cases (14.8%) the presence of both BAV and Doliovirus was demonstrated. Two employees were co-wins: one, from an HAV/poliovirus-positive household. was poliovirus positive HAV negative. The other was negative for both viruses. Two other poliovir:us-positive household contacts were relatives of two of the employees: one of the employees was HAV and poliovirus negative. while the other was HAV positive. The amplified genome of the isolated poliovirxses shcwed a specific and characteristic band of about 480 at on agarose gel electrophoresis. In the first analysis using the RFLP assay, all the positive samples appeared to belong to the type 1 poliovirus Sabin strain (PVl./Sabin) (Fig.1). Another aliquot from each sample was digested with the restriction enzymes in parallel with the DNA amplified from PVl./Sabin. As shown in Fig.2, all the fragments had a restriction profile identical !o PVl/Sabin. After phenol extraction in two poliovirus-positive samples, the PCR produced only a weak specific amplified

Double Table 1.

Positive

samples

for hepatitis

Stool samples from: Employees Household Others Total

A and/or

II 27 9 6 42

contacts

Enteric

Virus Infection

229

poliovirus.

HAV positive

Polio positive

II and ?/o

II and ?/n

,I and %

12 (44.4) 0 0 12 (28.6)

5 3 1 9

4 (13.8) 0 0 4 (9.5)

A

(18.5) (33.3) (16.6) (21.4)

HAV-Polio

posrlive

B

c

MO123456123456123456

Figure 1. RFLI’ of polioviruses reference strains (Sahin and wild type). Hpa II (panel C). l\/l=molecular weight marker: O=undigested poliovirus: and wild, 3 and 6=Poliovirus type 3 Sahin and wild.

A Ml23456

B 7M123456

Figure 2. Comparison between Poliovirus type with Hae III (panel B) and with Hpa II (panel corresponding enzyme indicated in the panel.

PCR products digested with DdeI (panel A), with Hae 111 (panel B) and with 1 and L=Poliovirus type 1 Sabin and wild: 3 and 4=Poliovirus type 2 Sabin

C 7M1234567

1 Sahin and the different isolates from stool samples. I’CR products digested with Ddel (panel C). l-2=undigested and digested Poliovirus 1 &bin: 3%7=different isolates digested with

band. In these two cases the RNA was extracted by the antigen-capture method. as used for HAV, and a specific band was identified by RFLP as Sabin 1 poliovirus.

Discussion The epidemiological pattern of HA disease has been substantially modified in recent years, leaving many people unprotected for HAV4 In this outbreak. the mean age of HAV cases was 41 (the attack rate was 12.5% whereas the overall prevalence was 6 3 %).

A), the

In Italy the principal risk factor, accounting for more than 50% of all HAV cases, is the consumption of uncooked mussels.10~18.The origin of HAV virus spread is well defined: among catering staff 1.8 out of 22 were positive for anti-HAV Ig; the other four were negative. All efforts to isolate HAV on cell culture were negative. This could result from progressive inactivation of the virus in the stool or from the presence of other enteric viruses (e.g. poliovirus which can interfere with the replication of HAV). Hepatitis A virus has a slow replication cycle on cell” and the first passage, starting from a stool extract or an environmental sample. can take more than

230

ivizia et aL

30-45 days. In a previous study1 only four out of 16 ELISA-positive samples were HAV positive in cell cultures and this result was evident after several blind passages. In the first isolation from highly polluted river water the infection took more than 30-40 days.lO The presence of other enteric viruses, besides poliovirus, was not investigated. The contemporaneous presence of two viruses in ihe intestinal tract, whilst documental. is not common. The source of poliovirus in these cases is unknown but since the incubation period of HAV is 30 days (range 19-41) a common source (water or food) was not suspected. It probably results from transmission from vaccinated children to adults. The presence of poliovirus in a significant proportion (18.50/o, 16.6% and 3 3.3%. respectively) of the three groups demonstrates the ‘silent’ intrafamilg circulation of this virus. In 1964 mass vaccination with Sabin strain vaccine was introduced in Itaiy and the incidence of poliomyelitis dropped from 3000 cases in the pre-vaccination era to only seven cases in 198 i-1 985. Two other cases were recorded in 1987-1988 but. starting from 1989. no case has been notified to the Health AuthorityL1 Since vaccination, Italy has become classified by the WHO as a ‘poiio-free’ area although reintroduction of wild type polioviruses f~-om highly endemic areas is always possible as confirmed by the two outbreaks (1978 and 1.992/‘93) in the NetherlandP among members of the Orthodox religious community refusing vaccinations. Severai studies have confirmed a significant number of polio seronegative subjects in Italy, reaching 12% in type 3 poliovirus. with the lowest level of immunity in the age range 10-l 9 years.‘?-14 This diminution of antibodies could be due to a reduction of antigenic stimuli caused by the limited circulation of the vaccine strain poliovirus in the environment. The increase in the antibody level in the next age range could be explained by the intra-family dissemination of poliovirus after the children’s vaccination. Our data seem to confirm this hypothesis. This study underlines that HAV is still present in Italy, and the necessity to continue poliovirus surveillance, as suggested by the WHO, in order to maintain the level of protection and to avoid reintroduction of wild strains to Italy.‘j

hIele A, Cialdea 4

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14

15

If

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eference 1 De Filippis I? Divizia M. Mele A. Adamo 13, Pana A. Detection of hepalitis A virus in the stools of healthy people from endemic area. Eur J Epidemial 198 7; 3: 172-l i5. 2 Gag NJ, Morgan-Capner P. Wright J, Farrington CP, Miller E. Age-specific antibody prevalence to hepatitis A jn England: implications for disease control. E1kkwliol b&t 1994: 113: 11 3-I 20.

L. Epatite A: nuori sl-iluppi per una malattia antica. 1994: 4: 184-I 90. Mele A, Slroffolini T, Pasquini l? SEIEVA: Integrated epidemiologicai spstem for acute viral hepatitis. Report 1985-1994. Rapporti Istisan 199613. Biziagos E, Passagot J. Crance JU. Deloince R. Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water. !Q+ Cnvirm >Iicrobiol 198X; 54: 2705-2710. Grabow WOK. Gauss-Muller T! Prozesky OW Deinhardt I? Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals. &II j?:iwiron Xlicrobiol 1983; 46: 619-624. Divizia M, Morace G. Gabrieli R, Pisani G. Pan2 A. Application of the PCR technique lo the detection of hepatitis A virus in the environment. l&l Sci Techid 1993: 27: 223-225. Horace G, Pisani G. Divizia M. Pana A. Detection of hepatilis A virus in concentrated river water by polymerase chain reaction. Zh1 Hyn 1993: 193: 521-527. Divizia M. Gnesivo C, Bonapasta RA, i\/lorace G. Pisani 6. Pan8 A. Virus isolation and identification in an outbreak of hepatitis A: epidemiological investigation. I&t Sri ‘l’ecilnoi 1993; 27: 199-205. Yvlele A, Rastelli MG. Gill ON. Recurrent epidemic hepatitis A associated with consumption of raw shellfish. probably controlled through public health measures. Ain J l$id~~lliol 1989; 130: 340-546. Mele A, Catapano R, Cialdea L, Piscanc A, Mladen AS. Kresevic L. Outbreak of hepatitis A in Tricste, Italy. J Plih He&h &kf 1994; 16:242&244. Sagliocca I,, Mele A, Gill ON. Cappetta 6. A village outbreak of hepatitis A: acquaintance network and inapparent pre-school transmission compared. J Epidemic 1988; 0: 470-472. Slroffolini T, Biagini TA! Lorenzoni L, Palazzesi Gl? Divizia M, Frongillo R. An outbreak of hepatitis A in young adults in central Ital~~.GurJ~~idei~~iol1990; 6: 156-159. Jansen R\k7, Siegel G, Lemon SM. Molecular epidemiology of human hepatitis A virus defined by antigen-capture polymerase chain reaction method. Piuc2iTnt/AcdSci US/l 1990: 87: 2867-2871. Vonsorer A. Handsher R, Newman 21, Guillot S, Balanant J, Rudich H. Mendelson E, Swartz T, Crainic R. Molecular epidemiology of type 1 poliovirus isolated in Israel and defined by restriction fi-agment length polymorphism assay.] I&t Dis 1993: 167: 199-203. Purione M. Guillol S. Olelea D. Ralanant J, Candrea A. Crainic R. Poliovirus with natural recombinant genomes isolated from vaccineassociated paralytic poliomyelilis. Vii-d 1993: 196: 199-208. Bonanni I? Norman A. Flehmig 13. I,o Monaco M, Orione I,, Raffo AM. Crovari P. A common-source outbreak of hepatitis A in italy: casecontrol study and molecular characterization of isolates (poster A41). IX International Symposium on Viral Hepaptitis. April 2 l-25. I 9 9 6 Rome, Italy Giusti G, Galanti B. Gaela GB. Acute viral hepatitis in Italy: an analysis of 8604 cases admitted lo 53 clinical centers in 1982. Artn MedizlTrr~nen 1984; 3: 13-38. Flehmig B. Hepatitis A virus in cell culture. II. Growth characteristics of hepatitis A virus in Frhlc-4/R cells. r\/red ;Iflicrobiol 1rnn~~r~~I 1981; 170: 73-8 1. Flehmig E, Weckeire A. lsolierung von infektiosem hepatitis A virus aus klarschlamm. Zbl Wyo 1988: 24: 43-45. WHO. Expanded programme on immunisation (EPI). Poliomyelitis in 1993. Weekly l$idmiologicni Record 1994; 69: 169-1 76. CDC. Update: poliomyelitis outbreak. Netherlands. LMLMWR 1992: 41: 917-919. Bellelli E, Bracchi U. Tanzi ML. Benagli G, Montanarini G. Poliomyelitis immunity status at different intervals from vaccination. Eur JIm~~~unol 19S6: 2: 197-204. Reali D. Carducci A. Ruschi MA. Serum antibody to polioviruses in a Tuscan population, Italy EwJl$id~~iliol 1990; 6: 309-3 12. Squarcione S, Germinario C, Iandolo E. Lo Caputo S. Bwgamini I?. Profeta MD, Gredo D. Quart0 M. Barbuti A. Seroimmunily to poliomyelitis in an Albanian immigranl population. Vcmine 1992; 10: 853-856. Igicm

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