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High Frequency of Genotype D and Spontaneous HepatitisB Virus Genomic Mutations Among Haitians in a Multiethnic

North American Population

Claudia A. Couto, MD, PhD,* Cynthia Levy, MD,w Carol J. Morris, MD,wMary Hill, MT, ASCP,wMaria de Medina, MSPH,w Mark R. Sanborn, PhD,z Gavin A. Cloherty, PhD,z

Eugene R. Schi!, MD,w and Paul Martin, MDw

Goals/Background: The importance of hepatitis B virus (HBV)genotype and mutations has been increasingly recognized. Weaimed to determine HBV genotype, precore (PC), and basal corepromoter region (BCP) mutations in a HBV multiethnic SouthFlorida population.

Study: Samples from 213 patients were tested for HBV-DNA usingAbbott RealTime HBV IUO assay, and for mutations usingINNO-LiPA assay.

Results: Patients were predominantly male (67%); 61 (31%) wereAfrican American, 60 (28%) Hispanic, 37 (17%) Haitian, 27 (19%)white non-Hispanic, and 14 (6.6%) Asian. Genotype A was foundin 101 (69%), D in 25 (17%), F in 9 (6%), G in 7 (5%), C and E in6 (4%) each, B in 4 (3%), and H in 2 (1%) patients. Mixed gen-otypes were detected in 11 patients. Genotype A was more preva-lent in all ethnicities except for Asian. Among hepatitis B e antigen(HBeAg)-negative patients (59%), BCP, PC, and combined BCP/PC mutations were found in 30 (37.5%), 13 (16.3%), and 14(17.5%), respectively. Genotype D was associated with higherfrequency of HBeAg-negative status [18/24 (75%) vs. 62/121 (51%)P=0.03] and mutations [16/19 (84%) vs. 40/67 (60%) P=0.04]compared with others. Genotype A was negatively associated withmutations [26/31 (84%) vs. 30/55 (55%), P=0.009]. PC mutationswere more common in genotype D (14/19, 73%) compared withgenotype A (7/54, 13%, P<0.0001). One-hundred percent and79% of Asians and Haitians had spontaneous mutations, respec-tively. All Haitians with genotype D had PC mutations and 3(50%) had BCP/PC.

Conclusions: The present study shows that HBeAg-negative statusand spontaneous mutations were more common with genotype D;the presence of genotype D in Haitians was always associated withspontaneous mutations.

Key Words: ethnicity, genotype, hepatitis B, mutations, Haitians

(J Clin Gastroenterol 2012;00:000–000)

Approximately 350 million people worldwide are chron-ically infected with the hepatitis B virus (HBV), with

600,000 person-deaths annually due to complications ofcirrhosis and hepatocellular carcinoma (HCC).1

To date, 9 genotypes of HBV, A to H, J, have beenrecognized and have distinct geographic variability. Geno-type A is widely distributed in Europe, North America,India, Sub-Saharan Africa, and Australia. Genotypes Band C are prevalent in Southeast Asia. In addition, geno-type C is also found in infected Pacific islanders. GenotypeD occurs mainly in the Mediterranean Basin and MiddleEast. Genotype E is found in West and South Africa andgenotype F is found mainly in Central and South America.Genotype G has been reported in France and in the UnitedStates, and genotype H in Central America.2–6 Recently,genotype J has been identified in Japan.7

Outcomes of chronic HBV infection are heterogenous.Geographical di!erences in the natural history of chronicHBV infections are largely determined by molecular char-acteristics of the prevailing HBV genotypes, subgenotypes,and their selected mutations.8–10 Structural and functionalfeatures of specific HBV genotype can influence theseverity, course and likelihood of complications, and hep-atitis B e antigen (HBeAg) seroconversion.11–14 Sponta-neous mutations in the HBV genome occur frequently inpatients with chronic HBV in the precore (PC) and basalcore promoter region (BCP).15 These mutations, in additionto specific HBV genotype, have been implicated in diseaseprogression and risk of HCC development.11–16 Fur-thermore, specific HBV genotype has been shown to influ-ence the outcome of therapy in di!erent populations.16–18

The reported prevalence of chronic HBV infection inthe United States (0.3% to 0.5%) is likely to be an under-estimate, as it does not reflect the impact of widespreadimmigration from areas of the world where HBV infectionis hyperendemic.19,20 CDC recommendations for HBVtesting vary according to HBV prevalence in the country oforigin. Recent recommendations include testing children ofimmigrants of Asian/Pacific ancestry.21–23 The aim of thecurrent study was to determine specific HBV genotype, PC,BCP, and drug resistance mutations in a multiethnicAmerican population of chronic hepatitis B patients.

PATIENTS AND METHODSSerum/plasma samples were obtained from 213

chronic HBV patients seen at our South Eastern US referralcenter, which serves a large immigrant population. Sampleswere unlinked and deidentified being collected sequentiallyfrom patients previously tested for HBV-DNA by Roche

Received for publication January 17, 2012; accepted July 3, 2012.From the *Department of Internal Medicine and Alfa Gastro-

enterology Institute, School of Medicine, Federal University ofMinas Gerais, Belo Horizonte, MG, Brazil; wDivision of Hepatol-ogy, Center for Liver Diseases, University of Miami, Leonard M.Miller School of Medicine, Miami, FL; and zAbbott Molecular,DesPlaines, IL.

Supported in part by Abbott Molecular, Grant number UM 20080863.M.d.M.: participated in the Advisory Board meeting October 28, 2010;

M.R.S.: Abbott Molecular employee and shareholder; G.A.C.:Abbott Molecular employee and shareholder; P.M.: Abbott con-sultant. The remaining authors declare that they have nothing todisclose.

Reprints: Paul Martin, MD, Division of Hepatology, Center for LiverDiseases, University of Miami Leonard M. Miller School of Med-icine, 1500 NW, 12th Avenue, suite 1101, Miami, FL 33136 (e-mail:[email protected]).

Copyright r 2012 by Lippincott Williams & Wilkins

ORIGINAL ARTICLE

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mailto:[email protected]


Cobas TaqMan HBV assay in our laboratory betweenDecember 2005 and October 2009.

HBV-DNA viral load was measured by the AbbottReal Time HBV IUO assay m2000 sp/rt system accordingto the manufacturers’ instructions. Patient samples werefurther subdivided into HBeAg-positive and HBeAg-neg-ative and tested for specific genotype, PC, and BCP muta-tions by INNO-LiPA assay. In addition, samples wereevaluated for drug resistance with Abbott HBV Sequenc-ing. HBeAg were tested by ELISA (ETI-EBK Plus andETI-AB-EBK Plus Assay; DiaSorin, Stillwater, MN).

Sex, ethnicity, HBeAg status, genotype, PC and BCPmutations, and drug resistant mutations were analyzed.

HBV Genotyping, PC and BCP MutationsHBV genotyping was performed by line-probe assay

(INNO-LiPA HBV Genotyping Assay; Innogenetics NV,Ghent, Belgium), designed to identify HBV genotypes A toH by detection of type-specific sequences in the HBV pol-ymerase gene domain B to C. The amplification productwas used for hybridization to LiPA strips, as described inthe manufacturers’ instructions. Results were interpretedaccording to these instructions, which indicate that ifmultiple genotype-specific lines are observed for one gen-otype together with a single positive line for a secondgenotype, only the genotype corresponding to the multiplepositive lines is reported. The single exception to this rule isgenotype G, whose reactivity is only due to the last line onthe strip. The genotypes were characterized in 147 patientsbut in 38 cases genotyping was not possible due to technicallimitations (viral load was too low in 28 and reading wasequivocal or invalid in 8). HBV PC and BCP mutationswere detected with INNO-LiPA HBV PreCore (Innoge-netics NV). This test simultaneously detects HBV wild-typemotif and mutations in the BCP and PC region of the HBVgenome. This line probe assay is designed to identify

nucleotide polymorphism at no1762 and nt1764 in the BCPand at codon 28 in the PC region of the HBV.

Quantification of HBV-DNAHBV-DNA viral loads were measured by comparing

the Abbott RealTime HBV IUO assay with m2000 sp/rtsystem and Roche Cobas TaqMan HBV assay with HighPure extraction, and processed according to the manu-facturers’ instructions. The detection limit of the AbbottRealTime HBV IUO assay is 10 IU/mL. Results lost duringextraction or deemed invalid were excluded from theanalysis.

Statistical AnalysisJMP and SAS (SAS Institute, Cary, NC) were used for

statistical analyses. Baseline characteristics were summar-ized using descriptive statistics. w2 and t tests were used forstatistical comparison as appropriate. A 2-tailed P-value<0.05 was considered statistically significant.

RESULTS

Patient CharacteristicsThe sample population consisted of 213 patients with

chronic HBV infection, 41% HBeAg-positive, 59% HBeAg-negative, and 3 undetermined. Patients were predominantlymale [143 (67%)]: 61 (31%) were African American, 60(28%) Hispanic, 37 (17%) Haitian, 27 (19%) white non-Hispanics, and 14 (6.6%) Asian (Fig. 1). Ethnicity was notrecorded for 14 patients. HBV-DNA was detectable byAbbott Assay in 199 (93.4%): 115 of 125 (92%) HBeAg-positive and 82 of 85 (96%) HBeAg-negative. The mean viralload was higher for HBeAg-positive patients (4.8±2.4 logIU/mL) compared with HBeAg-negative (2.9±1.6 log IU/mL), P<0.0001. HBe status and viral load were not sig-nificantly di!erent across ethnicities.

FIGURE 1. A, HBV-studied population distribution according to ethnicity (N=199). B, Miami population distribution according toethnicity (N=2,457,044), US Census Bureau, 2005 to 2009. HBV indicates hepatitis B virus.

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HBV Genotyping and EthnicityHBV genotype distribution was as follows: genotype A

was found in 101 (69%), D in 25 (17%), F in 9 (6%), G in 7(5%), C and E in 6 (4%) each, B in 4 (3%), and H in 2 (1%)patients. Besides, in 11 patients, >2 genotypes weredetected. Table 1 shows genotype distribution of 147patients according to ethnic group, eAg status, and pres-ence or absence of spontaneous mutations. Overall geno-types A and D comprised 86% of the study population.Genotype D was the only genotype associated with HBestatus. A higher frequency of HBeAg-negative status [18/24(75%) vs. 62/121 (51%), P=0.03] was found comparedwith other genotypes.

Table 2 shows distribution of spontaneous mutationsamong patients who are HBeAg-negative and who havegenotypes A and/or D, according to ethnicity. AfricanAmericans most frequently had genotype A (83%), andonly rarely had D (9%). Genotypes A and D were also themost common in Hispanics (66% and 18%, respectively)and in non-Hispanic whites (77% and 33%, respectively).In Haitians, genotype A (65%), genotype D (28%), andgenotype E (10%) were more frequent. Genotypes B and Gwere also found among Haitians. Asians represented only6.6% of total population and most frequently had genotypeC (45%) and B (27%), as shown in Tables 1 and 2.

Spontaneous Mutations (PC and BCP)and Ethnicity

Fifty-seven out of 75 (76%) HBeAg-negative patientstested positive for spontaneous mutations. BCP mutationswere found in 30 (37.5%) whereas PC mutations werepresent in 13 (16.3%), and combined BCP/PC in 14(17.5%), as shown in Table 1. Genotype D was associatedwith a greater frequency of spontaneous mutations com-pared with other genotypes [16/19 (84%) vs. 40/67 (60%),P=0.04]. In contrast, genotype A was negatively asso-ciated with spontaneous mutations [26/31 (84%) vs. 30/55(55%), P=0.009]. When analyzed separately, PC muta-tions were also more commonly found with genotype Dcompared with genotype A [14/19 (73%) vs. 7/54 (13%),P<0.0001]. Table 2 shows the percentage of PC (33%) andBCP (52%) mutations in HBeAg-negative patientsaccording to ethnicity. Asians and Haitians had the highestprevalence of spontaneous mutations: 100% and 79%,respectively. All Haitian patients with genotype D had PCmutations and 3 (50%) presented both BCP/PC.

Drug resistance mutations were found in 18 patients,of whom only 3 were HBeAg-negative. All 3 HBeAg-neg-ative patients with drug resistance mutations also hadspontaneous mutations. Table 3 shows the sequencingreport, genotype, eAg status, and viral load for all patientswith drug resistance mutations.

DISCUSSIONOur study identified genotypes A and D as the most

frequent in this ethnically diverse South Florida populationalthough 8 HBV genotypes were identified. Genotype Awas the most frequent genotype in African Americans(83%), white non-Hispanics (77%), Hispanics (66%), andHaitians (65%), but not in Asians (9%). Genotype D, incontrast, was rare among African Americans but was foundin approximately 20% of other ethnic groups. Importantly,we found genotype D to be more frequently associated withHBeAg-negative status and PC mutations than genotype A. T

ABLE

1.

Gen

otypeDistributionAccordingto

Ethnic

Group,eA

gStatus,

andSp

ontaneo

usMutations

EthnicGroup

Spo

ntan

eous

Mutations

HBV

Genotyp

eN

AA

Hispa

nic

Haitian

sWhite

Asian

HBeA

g-nega

tive

BCP

PC

BCP/P

CTotal

A91

3422

179

144

200

525

D19

16

64

214

15

713

Mixed

11—

——

——

7—

——

—A,D

41

01

20

30

20

2A,D,F

10

10

00

11

00

1A,D,G

10

01

00

0—

——

—A,F

20

20

00

22

00

2A,G

20

00

20

0—

——

—F,H

10

10

00

10

10

1B

40

01

03

31

20

3C

61

00

05

32

00

2E

63

03

00

51

31

5F

50

30

10

21

01

2G

42

20

00

10

00

0H

10

10

00

10

00

0Total

147

4238

2918

1180

3013

1457

AA

indicates

African

American

;BCP,basal

core

promoterregion;HBeA

g,hepatitisBean

tigen;HBV,hepatitisBvirus;PC,precore.

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Further, all Haitians with genotype D had detectable PCmutations.

Our study population was mainly composed of AfricanAmericans (31%) and Hispanics (30%). Thirty-seven patients(17%) were Haitian (Fig. 1A). Compared with Miami pop-ulation data from the US Census Bureau, we had a greaterrepresentation of African Americans and a smaller pro-portion of Hispanics in our HBV population (Fig. 1B), whichmay suggest di"culty in access to health care. This study,corroborating previous concerns, would also suggest a

potentially large number of HBV-infected persons in theUnited States, who are undiagnosed and could benefit from aculturally targeted integrated program for chronic hepatitis Bas proposed by Gish and colleagues.20,24

It has been previously reported that the prevalence ofHBV genotypes varies in di!erent regions of the UnitedStates.21 Our findings are in agreement with those reportedby the HBV Epidemiology Study Group with genotype Abeing the most common genotype in a Southern US pop-ulation.21 Likewise, those investigators also found that

TABLE 2. Distribution of HbeAg-negative, Spontaneous Mutations, and Genotypes A and D According to Ethnicity

EthnicityHBeAg-negative PC BCP

GenotypeA

% MutationsGenotype A

GenotypeD

% MutationsGenotype D Total

Asians 7 (64%) 4 (57%) 4 (57%) 1 (9%) NA 2 (19%) NA 100%AfricanAmericans

15 (36%) 4 (27%) 7 (47%) 35 (83%) 53.3% 2 (5%) 67% 56%

Haitians 19 (66%) 9 (43%) 11 (53%) 19 (65%) 47% 8 (28%) 100% 79%Hispanics 25 (66%) 4 (16%) 14 (56%) 25 (66%) 62.5% 7 (18%) 83.3% 64%Whites 9 (53%) 5 (45%) 5 (45%) 13 (77%) 71% 6 (33%) 100% 78%Total 75 26 (33%) 41 (52%) 93 (67%) — 25 (18%) — 67.5%P 0.043 0.13 0.96 0.002 — 0.04 — —

P-value <0.05 means that a di!erence exists across ethnicities.BCP indicates basal core promoter region; HBeAg, hepatitis B e antigen; PC, precore.

TABLE 3. Drug resistance Mutations According eAg Status and Hepatitis B Virus (HBV) Genotypes

eAgstatus

AbbottGenotype

Abbott Real Time ViralLoad (log IU/mL)

Abbott HBV Sequencing RUO Drugresistance mutations Abbott HBV Sequencing RUO Report

+ A 7.92 80 and 180 and 204 Resistant lamivudine, telbivudine; reducedsensitivity entecavir


+ A 7.96 180 and 204 Resistant lamivudine, telbivudine; reducedsensitivity entecavir



+ A 4.79 181 Reduced sensitivity: telbivudine, adefovir,and tenofovir

+ A 3.74 180 and 204 Reduced sensitivity: telbivudine, adefovirand tenofovir







+ C 3.20 181 Reduced sensitivity: telbivudine, adefovir,and tenofovir

+ F 5.37 173 and 180 and 204 Resistant lamivudine, telbivudine; reducedsensitivity entecavir

" A 2.91 180 and 204 Reduced sensitivity: telbivudine, adefovir,and tenofovir

" D 5.13 236 Resistant lamivudine, telbivudine; reducedsensitivity entecavir

" E 2.89 173 and 180 and 204 Resistant lamivudine, telbivudine; reducedsensitivity entecavir




patients infected with HBV genotype D were more likely tobe HBeAg-negative and to harbor spontaneous mutations.Finally, the frequency of PC (33%) and BCP (52%)mutations found in our HBeAg-negative patients is verysimilar to the high prevalence of mutations found by Chuet al.25 However, unlike the HBV Epidemiology StudyGroup,21 we found that our population had a higher propor-tion of genotype D and a smaller proportion of genotypes Band C. In addition, African Americans most frequently hadgenotype A (83%), and also genotype E (7%), D (5%), G(5%), and C (2%). Overall, genotypes B and C were not asprevalent as previously reported by Chu and colleagues,being found in only 7% of patients. The higher frequency ofgenotype D, in addition to the lower frequency of geno-types B and C at our center is likely related to di!erences inethnic composition of our patient population (Fig. 1) andthe influx of immigrants from Caribbean, Central andSouth American countries with a high prevalence of HBV.Screening for HBV has been strongly advised for patientswho originated from countries with high prevalence ofHBV.23

We verified a very high frequency of HBV mutationsin Asians and also in Haitians. All Haitian patients withgenotype D presented PC mutations. A prior study fromHaiti, a country with a high prevalence of HBsAg (5%),where >90% of the population is of African ancestry,identified HBV genotypes as follows: genotype A (71.5%),D (22.4%), and E (6.1%).26 Our study confirms a similarproportion of genotype D HBV infection (28%) and furthercharacterizes these as being mostly HBeAg-negative anduniversally associated with PC mutations. This is the firsttime this association is described in the Haitian population.

In contrast to the study by Chu and colleagues, whoexcluded previously treated patients, our study includedconsecutive patients with chronic HBV despite previoustreatment, providing better representation of our pop-ulation. However, lack of information regarding treatmentlimited our analysis of resistance mutations.

It is well known that HBV genotype, viral load, andspecific viral mutations are associated with the hetero-genous disease progression in hepatitis B infection. Forinstance, acute infection with genotypes A and D results inhigher rates of chronicity than genotypes B and C.27,28 Inaddition, patients with genotypes C and D have lower ratesof spontaneous HBeAg seroconversion and also delayedseroconversion compared with genotype A and B.13 Fur-ther, genotype D has a higher prevalence of BCP andPC mutations than genotype A, which has been implicatedin disease progression11,29 and risk of HCC develop-ment.6,26,30–35 Finally, patients with HBV genotype A or Bhave better responses to interferon-based therapy thangenotypes C and D.16–18 These observations suggestimportant pathogenic di!erences between HBV genotypesthat may contribute to more severe liver disease, includingcirrhosis and HCC with genotypes C and D HBV infec-tion.13,30,31,36,37 The association of genotype D HBV withHBeAg-negative status and PC mutations in the presentstudy suggests that genotype D HBV in the United Statesmay show similar molecular characteristics to that genotypeprevailing in European and African countries.

In conclusion, our findings add to the current datapointing toward a highly significant impact of ethnicity inchronic HBV. Our results imply that the epidemiology ofHBV infection will continue to change over time as a resultof immigration patterns. Testing for HBV genotype and

mutations can help practicing physicians identify those atrisk for disease progression and determine optimal antiviralregimen.

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