The application of sequence analysis to the detection of

3.12.1 Objectives

• To apply the study of sequence homology in the precore and surface genes to the investigation of clustered out breaks of hepatitis B infection.

3.12.2 Introduction

Perinatal and sexual transmission of HBV between anti-HBe positive individuals was demonstrated in sections 3.9 and 3.10. The regions of the HBV genome chosen for analysis in these studies were sufficiently well conserved to follow HBV transmission. The area of the surface genome analysed encompasses the highly conserved A determinant and the less conserved regions immediately upstream of it Lauder et al,. (1993). Similarly the area around the precore region contains both highly conserved and more variable regions.

These regions show sufficient divergence between subtypes and between individuals of the same subtype to allow confirmation of the source of infection in patients studied. The possibility of applying this type of analysis to the study of transmission events generally was therefore investigated.

The assay of HBsAg serological subtypes within HBV outbreaks is generally used to determine possible routes of transmission. However, this method is of limited use in such situations as the subtype alone may not distinguish the patients under investigation from HBV infected individuals in the general population.

An advantage of direct sequencing of amplified HBV DNA over routinely applied serological analysis is the ability to distinguish between subtypes w/r as well as d/y. In some cases this may represent a viral population in which d and y subtypes (Ashton- Rickardt and Murray, 1989) and w and r (Kanagawa et a /.,1992) may be exhibited by the

same virus. In cases where serological subtyping is inconclusive, evidence for

during surgical procedures and between patients through shared equipment (Oren et

a/., 1989).

In the following study HBV DNA sequences from 15 samples were analysed to determine which, if any, were linked to an epidemiologically identified clustered outbreak of HBV infection. Samples were coded before analysis so that the study was carried out under blinded conditions. This allowed us to asses the possibility of applying the method to situations where epidemiological data was not available.

3.12.3 P a tie n t population

Fifteen samples from 15 patients were analysed. These patients had been selected to include nine HBsAg positive, anti-HBe positive patients and six HBsAg positive, HBeAg positive patients. The group included five patients who had all been involved in an HBV outbreak.

The first patient (patient 1) (figure 3.12(i)) had breast cancer and received blood from the implicated donor. She underwent a bilateral oophorectomy and died of metastatic disease. There were no specimens available from this patient who did not have a documented hepatitis.

Two health care workers (HCW 1 and HCW 2) were involved in the clinical management of this patient. HCW 1 was the surgeon who operated on patient 1. He developed an acute symptomatic HBV infection within two months of performing the operation and recovered fully. HCW 2 was the nurse involved in the care of the patient. This included procedures involving the drainage of pleural effusion and laying out of the body. Within two months HCW 2 developed an acute HBV infection and died of fulminant hepatitis.

The surgeon operated on Patient 2 whilst incubating HBV. Patient 2 developed an acute HBV infection and recovered.

Patient 3 was a 75 year old woman who underwent an orthopaedic procedure during which she received a blood transfusion from the implicated donor. She developed an acute HBV infection two months later and died from fulminant hepatitis.

Figure 3.12(1)

Relationship between individuals involved in an

hepatitis B outbreak

Blood donor

(Donor)

Donation A

Donation B

Patient 1

(No documented

HBV infection)

Patient 3

(Fatal, fulminant

hepatitis)

HCW 1

HCW 2

Surgeon

Nurse

(Resolved acute (Fatal, fulminant

HBV infection)

hepatitis)

Patient 2

(Resolved acute HBV

infection)

3.12.4 Methods

HBsAg detection was performed by EIA (Wellcozyme; Murex Diagnostics Ltd, UK.) and quantification carried out by reverse passive haemagglutination assay. Subtypes were determined by RIA and HBeAg/anti-HBe were assayed according to methods described by Tedder et a/.,(1981).

Antibody to HCV was assayed by ELISA (Ortho HCV ELISA Test System, Third generation, Raritan, new Jersey, USA and Murex anti-HCV) Markers for HDV and HAV infections were tested for by ELISA (Murex Diagnostics Ltd, UK.).

HBV DNA was amplified from 20 jA of serum as described in section 2.6. The X/precore/core region and a determinant of the surface region were amplified as previously described .

Diiect sequencing of both regions was carried out as described in section 2.10. The samples were coded and assayed by PCR, surface gene sequencing being completed before the code was broken. Codon 28 of the precore region was analysed by PMA as described in section 2.11.

The Kimura two-parameter method was used to analyse variance between the outbreak samples compared with that between control samples. The Fitch-Margoleish method was used to construct an unrooted phylogenetic tree.

3.13.5 Results

HBV DNA was amplified from 14 o f 15 samples tested, with primers to the surface and precore regions.

The surface region had one silent nucleotide substitution in codon 138 (TGC to TGT) in five samples which were all of the same subtype (ayw). This group had a significantly less variation between themselves than was present between the other nine samples assayed (p <0.05)(Figure 3.12(ii)). This was also the case when a similar comparison was made between this group and seven unrelated control samples of the same subtype.

control samples helped to confirm this as a related group of samples. Thus, it was possible to conclude that these individuals formed a discrete group. The remaining nine samples were all highly conserved but none were identical. In all cases the inferred subtype by sequencing corresponded with the results of serological subtyping.

Once the code was broken it was revealed that the five individuals with the novel mutation had all been involved in the clustered outbreak of HBV described above. Precore sequencing revealed further mutations common to the group, codon 126 of the X gene (isoleucine to leucine), codon 28 of the precore region (tryptophan to a stop codon), and codon 29 of the precore region glycine to arginine. PMA analysis of codon 28 revealed a homogeneous mutant population in these samples. Serological analysis of HBeAg/anti-HBe using standard techniques did not reveal HBeAg in any samples. Modification of the assay did show a minor amount of HBeAg in the serum of HCW 1.

There was some sequence variation within the group in both regions studied. In the surface region (Appendix 7) this was restricted to published variants. The outbreak samples were compared with control samples of the same subtype as it was important to be able to distinguish groups from other samples of the same subtype (Appendix 7). A similar comparison of the X and precore regions was made between the outbreak samples and ayw control samples (Appendix 7).

Figure 3.12(ii) Unrooted pliylogeiieiic analysis of

cluster and control samples

# 1 1

Patient

Patient 3'

HCW1'

ICW2

Donor

Donor, HCW 1, HCW 2,

’ Patient 2 and Patient 3 are as described in

figure 3.12(i)

3.12.6 Discussion of results

Five samples from a clustered outbreak of HBV infection were identified in a blind study from nine other non-related samples by sequence analysis of the 3’ end of the X gene, the precore region and the a determinant of the surface gene.

The outbreak samples shared the same HBsAg subtype (ayw) and also contained a novel silent m otif in the surface gene. Three codon changes in the X precore region determined amino acid substitutions at codon 126 (isoleucine-leucine), codon 28 (tryptophan to stop codon) and codon 29 (glycine to arginine). The non-related samples did not have any conserved novel changes.

HBV DNA sequence analysis has been applied to many cases of transmission and as in our study most have described a limited amount of sequence variation between related samples. The presence of multiple sequences (section 3.9, Kaneko and Miller, 1989) in some individuals which are not apparent after direct sequence analysis may partly explain this. One variant may become dominant and therefore apparent only after transmission.

HBV has been shown to be generally stable on transmission. In common with other groups (Lin et a/.,(1990) our study has shown that passage of the virus over three transmission generations did not increase sequence variation in the regions of the genome that were studied.

HBV DNA sequencing of the precore and core genes has been used to exclude patients from epidemiologically defined outbreaks (Oren et a/., 1989, Liang et a /.,1991).

These reports describe the epidemiological and molecular investigations respectively of a nosocomial outbreak of hepatitis B in six patients, five o f whom developed a fulminant hepatitis. The proposed mode of transmission was via a multiple-dose vial of heparin and normal saline flush solution. The index case was, as in our study, an HBsAg positive, anti-HBe positive carrier. Precore HBV DNA was analysed by sequencing. The source plus five patients were nearly identical to one another and contained unique mutations in that region. The sequence in the sixth patient did not have these mutations. In addition Patient 6 had 60 nucleotide differences in the 822 bp HBV DNA sequence spanning the

only excluded from the out break by HBV DNA sequencing.

An alternative approach is the analysis of short hypervariable sequences (Lin etal.y 1991). A 100 nucleotide sequence between the core gene and the pre-S region was amplified. Sequences were highly conserved between children and their HBV infected parents. Furthermore, all infected siblings in nine families also had identical HBV sequences which

were not homologous with any recognised genotypes. However, as in our study

differences were detected between individuals presumed to have been infected from the same source. This may reflect the relatively high mutation rate of HBV (1-5 x 10 (Orito

et a/., 1989) which is comparable with that of RNA viruses.

In another study, the same group (Lin et a/., 1990 ) showed that the less variable S region was completely conserved within HBV infected members of Chinese families. This emphasises the influence that different rates of mutation throughout regions of the HBV genome may have on genotypic analyses. This must be controlled for when this method is used to identify HBV outbreaks.

We have tried to address this by analysing part of three different regions (X, precore and surface) which have different levels of conservation.

We have shown that subtype analysis by sequencing can be used to augment serological subtyping. An alternative approach using multiple primer sets to identify all four HBsAg subtypes in cases where serological subtyping had failed was described by Echevarria et

a/.,(1994). The main disadvantage of this method is that it would not allow the detection of a cluster among other samples of the same subtype.

The relationship of the precore mutation to disease severity has been discussed elsewhere. In our study the same mutation was associated with four different clinical pattems;chronic carriage (Donor), symptomatic acute infection (HCW 1) and fatal acute fulminant hepatitis. The two patients that died had apparently little in common, one was an elderly patient and the other a 22 year old nurse.

A possible dose effect was considered. However, Patient 3 had become infected via transfusion and HCW 2 through her care of Patient 2. Patient 1 had also received a blood

donation but did not develop a symptomatic hepatitis.

In this study we have shown that the analysis of short conserved regions of the genome can be used to support epidemiological and serological evidence in the investigation of transmission events.