research article occult hepatitis b virus infection among...

Research ArticleOccult Hepatitis B Virus Infection among HIVPositive Patients in Nigeria

Oluyinka Oladele Opaleye1 Adeolu Sunday Oluremi1 Adetona Babatunde Atiba1

Moses Olubusuyi Adewumi2 Olatunji Victor Mabayoje3 Emmanuel Donbraye4

Olusola Ojurongbe1 and O Adekunle Olowe1

1 Department of Medical Microbiology and Parasitology Ladoke Akintola University of Technology Osogbo Nigeria2 Department of Virology College of Medicine University College Hospital University of Ibadan Ibadan Nigeria3 Department of Hematology Ladoke Akintola University of Technology Osogbo Nigeria4Department of Medical Microbiology and Parasitology College of Health Sciences Obafemi Awolowo UniversityPMB 4400 Ile-Ife Nigeria

Correspondence should be addressed to Oluyinka Oladele Opaleye yopaleyeyahoocom

Received 27 January 2014 Accepted 28 March 2014 Published 24 April 2014

Academic Editor Sukla Biswas

Copyright copy 2014 Oluyinka Oladele Opaleye et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

HIV has been known to interfere with the natural history of hepatitis B virus (HBV) infection In this study we investigatethe prevalence of occult hepatitis B virus infection (OBI) among HIV-infected individuals in Nigeria Overall 1200 archivedHIV positive samples were screened for detectable HBsAg using rapid technique in Ikole Ekiti Specialist Hospital The HBsAgnegative samples were tested for HBsAg anti-HBc and anti-HCV by ELISA Polymerase chain reaction was used for HBV DNAamplification and CD4 counts were analyzed by cytometry Nine hundred and eighty of the HIV samples were HBsAg negativeHBV DNA was detected in 21188 (112) of patients without detectable HBsAg CD4 count for the patients ranged from 2 to 2140cells120583L of blood (mean = 490 cells120583L of blood) HCV coinfection was detected only in 3188 (16) of the HIV-infected patients(119875 gt 005) Twenty-eight (292) of the 96HIV samples screened were positive for anti-HBc Averagely the HBV viral load was lt50copiesmL in the OBI samples examined by quantitative PCR The prevalence of OBI was significantly high among HIV-infectedpatients These findings highlight the significance of nucleic acid testing in HBV diagnosis in HIV patients

1 Introduction

Hepatitis B virus (HBV) is a major health problem withapproximately 400million chronically infected peopleworld-wide and 15ndash60 of the normal population in many Africancountries may be positive for one or more of the serologicalmarkers of hepatitis B virus infection [1] These chroni-cally infected patients not only are at an increased stageof developing liver cirrhosis and hepatocellular carcinomabut also serve as a potential reservoir of infection [1] Themajor structural protein of virus envelope hepatitis B surfaceantigen (HBsAg) is universally considered as a diagnosticmarker ofHBV infectionThe absence ofHBsAg in the serumand the presence of antibodies to core antigen (anti-HBc)

usually indicate resolved infection [2] Occult HBV infection(OBI) usually has a serological evidence of previous HBVinfection that has been described in a few cases [2]

HIV coinfection has been reported to modify the naturalhistory of HBV with potential consequences on morbidityand mortality [3] Data on OBI in ART untreated HIVpatients is limited from a vast number of African countrieslike Nigeria where prevalence of HBVmonoinfection modeof transmission viral genotype andmutational pattern variesconsiderably in different parts of the country No previousstudy from Nigeria on prevalence on OBI among any groupshas been carried out This is particularly important asexposure to HBV is common among HIV-infected casesbecause of shared routes of transmission [4] Notably there is

Hindawi Publishing CorporationJournal of Tropical MedicineVolume 2014 Article ID 796121 5 pageshttpdxdoiorg1011552014796121

2 Journal of Tropical Medicine

considerable variation in prevalence ofHIVHBVcoinfectionaccording to geographic regions and exposure risk [5]

Successful implementation of ART leads to immunereconstitution that can potentially result in immunemediatedliver injury in the setting of HBV coinfection Some studieshave reported an association between OBI and elevatedtransaminase [6] therefore identification of OBI is of impor-tance

2 Materials and Methods

Of the 1200 HIV-infected patients enrolled in the HAARTClinic of the Specialist Hospital Ikole Ekiti State Nigeriafrom October 2012 to April 2013 we identified 980 HBsAgnegative patients (ART-naıve subjects) Among them 188were selected for the study by a simple random methodInformed consent was obtained from the patients and theinstitutional committee approved the study protocol Thesera were stored at minus20∘C until tested Ethical clearancewas obtained from the Ethical Committee of the Ikole EkitiSpecialist Hospital

21 Serological Testing All samples were tested for HBsAganti-HBs anti-HBc anti-HCV and anti-HIV using ELISA(DRG Diagnostics Marburg Germany) All anti-HBc posi-tive samples were retested for HBsAg as well as for anti-HBcand only repeat positive samples were included in the study

22 DNA Extraction DNA was extracted from all the serumsamples using QIAampDNABloodMini kit (Qiagen GmbHHilden Germany) following themanufacturersrsquo instructionsBriefly samples (200120583L) were incubated with protease andlysis buffer After incubation there were two washing stepsand the nucleic acids were eluted in a volume of 50 120583L ofelution buffer The eluted DNA was stored at minus20∘C untiltested

23 Hepatitis B Virus Specific Nested PCR The presence ofHBV DNA was examined in all samples using a routinediagnostic PCR Primer pairs were designed from the highlyconserved overlapping regions of the S and P genes of theHBV genome A nested PCR was performed outer primerpairs were HBPr134 (sense) 51015840-TGCTGCTATGCCTCA-TCTTC-31015840 and HBPr135 (antisense) 51015840-CAGAGACAAAA-GAAAATTGG-31015840 and the inner primer pairs were HBPr75(sense) 51015840-CAAGGTTATGTTGCCCGTTTGTCC-31015840 andHBPr94 (antisense) 51015840-GGTATAAAGGGACTCACGATG-31015840 PCR amplifications were carried out in 25 120583L reactionvolumes with 5 ng of genomic DNA 10x PCR buffer (20mMTris-HCl pH84 50mMKClQiagen) 2mMof dNTPs 50 ngof each primer and 1U Ampli Taq gold DNA polymerase(Applied Biosystems) on a PTC 200 cycler (Peltier Thermalcycler Watertown Massachusetts USA) Thermal cyclingparameters were initial denaturation at 94∘C for 2minfollowed by 35 cycles of 30 sec at 94∘C denaturation 30 secat 52∘C annealing temperature and 45 sec at 72∘C extensionfollowed by a final extension of 5min at 72∘CThermal cyclingparameters remained the same as in the first PCR round

except for the number of cycles that is increased to 40 cyclesof amplification Each PCR product (5120583L) was analysed byelectrophoresis in 2 agarose gels A positive control (HBVplasmid DNA) and a negative control of the master mix onlywere integrated to each run to validate the PCR products thatyielded a 340 bp fragment

3 Quantification of HBV DNA

Quantification of HBV DNA was performed with quantita-tive real-time PCRusing a previously described procedure [7]in a GeneAmp 7300 sequence analyzer (Applied BiosystemsPerkin-Elmer Foster City CA) HBV-plasmid DNA wasused to generate a standard curve following a serial 10-folddilution

4 Statistical Analysis

Mean age and all the numerical data were analysed usingStudentrsquos 119905-test The chi-square test and Fisherrsquos exact testwere used to compare categorical data For the purpose of ourstudy 119875 value le 005 was considered statistically significant

5 Results

The demographic biochemical and virological parametersof the study group are summarized in Table 1 The mean agewas 35 (range 3ndash67) years The majority (45) had multiplesexual partners and 25 of the subjects had a history ofconcomitant alcohol use HCV coinfection was found in 296(21) Overall 2996 (292) of patients were reactive foranti-HBc an indication of prior exposure to HBV DNAand majority 68 (75) of the patients were female (Table 2)Thus in the total study population 21188 (112) of patientswere identified as OBI and 625 of the OBI patients hadCD4 count less than 200 cellsmm3 Averagely the HBV viralload was lt50 copiesmL in the OBI samples examined byquantitative PCR

Serum levels of AST andALTwere higher among patientswith OBI in comparison to anti-HBc positive HBV DNAnegative individuals but the difference failed to reach stan-dard significance (119875 = 013 and 119875 = 007) respectivelyThe comparison of different demographic biochemical andvirological factors between HBV DNA positive and negativecases was illustrated in Table 3 The distribution of the studyparticipants as per the 1993 Revised Classification System forHIV Infection and Expanded Surveillance Case Definitionfor AIDS among Adolescents and Adults was as shown inTable 4 Figure 1 shows the 07 agarose gel picture showinga 340 base pairs amplicon

6 Discussion

Thepresent study represents a comprehensive cross-sectionalanalysis of prevalence of OBI in an ART naıve HIV positivecohort comprising various risk groupsMost previous studieslooking at the clinical effects of OBI in HIV include a largenumber of patients on anti-HBV drugs as a component of

Journal of Tropical Medicine 3

Table 1 Evaluation of overall demographic biochemical and virological parameters of the study populations 119875 values by 119905-test

Characteristicunder analysis

Overall status in thestudy population

Anti-HBc status in the study population (119899 = 96)Positive119899 = 28 (292)

Negative119899 = 68 (708) 119875 value

Male 48 (50) 18 (643) 30 (441) 005Female 48 (50) 10 (357) 38 (559) 005Mean age (range) 35 (3ndash67) 34 (19ndash67) 34 (3ndash55) 0009Alcohol addiction 24 (25) 17 (607) 34 (50) 041Sexual promiscuity 45 (50) 11 (393) 14 (206) 070CD4 lt200 cellsmm3 23 (25) 13 (461) 14 (206) 002ALT gt40 IUL 27 (281) 12 (423) 15 (221) 011AST gt30 IUL 46 (50) 17 (607) 34 (50) 014Anti-HCV positive 2 (21) 1 (38) 1 (14) 024Anti-HBs positive 9 (94) 9 (321) 2 (29) 00001

Table 2 Evaluation of overall demographic biochemical and virological parameters of anti-HBc positive samples 119875 values calculated byFisher test and 119905-test

CharacteristicOBI (anti-HBcAg +ve) (119899 828)

HBV DNA +ve (119899 = 8)Anti-HBc +ve

HBV DNA ndashve (119899 = 20)Anti-HBc +ve 119875 value

Male 2 (25) 6 (30) 047Female 6 (75) 14 (70) 047Mean age (range) 345 (12ndash67) 34 (3ndash55) 048Alcohol addiction 3 (345) 6 (30) 100Sexual promiscuity 3 (375) 13 (65) 015CD4 lt200 cellsmm3 5 (625) 7 (233) 047ALT gt40 IUL 6 (75) 8 (267) 013AST gt30 IUL 7 (875) 12 (60) 007Anti-HCV positive 1 (13) 1 (5) 053

Table 3 Comparison of different demographic biochemical and virological factors between HBV DNA positive and HBV DNA negative

Characteristics HBV DNA negative (119899 = 167) HBV DNA positive (119899 = 27)Male 48 (287) 7 (26)Female 119 (713) 14 (512)Mean age (range) 35 (12ndash67) 34 (3ndash55)Alcohol addiction 41 (246) 11 (407)Sexual promiscuity 50 (30) 21 (778)CD4 (mean) 410 215ALT gt40 IUL 412 58AST gt30 IUL 398 726

Table 4 The distribution of the Human Immunodeficiency Virus (HIV) infected in study participants as per Centers for Disease Controlclassification for HIV-infected adults and adolescents with the mean CD4 lymphocyte count in each category (WHO 2009)

Category No of patients Mean CD4 count (per mm3)1 T cells gt500 cellsmm3 69 5062 T cell 200ndash499 cellsmm3 81 3543 T cells lt200 cellsmm3 38 143


1 2 3 4 6 minusC +C 27 30 37 41 L

1000

340

100

Figure 1 PCR for detection of HBV DNA in HBsAg negativepatients Representative agarose gel electrophoresis of PCR prod-ucts Lanes 6 and 27 were positive for HBVDNA Lanes 1 2 3 437 and 42 were negative for HBVDNA +C positive control minusCnegative control and L molecular weight size marker

ART This study describes the risk factors associated withOBI frequently of anti-HBc positivity and its possible valuesas a serological marker for identifying HIV-infected patientswho benefit from HBV DNA assay We found the prevalenceof occult HBV to be 112 among a random selected group ofHIV-infected patientsThe prevalence of OBI inHIV positiveindividuals varies worldwide between 0 and 90 dependingon the geographic regions risk factors and the exposureinvolved [6]

In the present study the prevalence of anti-HBc (29228 of 96) and OBI (286 8 of 28) among the ART naıveHIV positive cohort was higher compared to previous reporton blood donors from studies done in areas of Indiaareas which reported 213 OBI among the HBsAg negativeanti-HBe positive donors [8] Within Nigeria HBV andHCV coinfection among HCV-infected patients have beenreported sporadically from different regions [9] Most ofthe previous studies on HBVHIV co-infection are aimed atdetecting HBV prevalence in the HIV population are basedon HBsAg positivity (prevalence 99 to 11) but reports onOBI are scarce A previous study on intravenous drug usersin northeastern India detected a prevalence of 159

Among the OBI cases the rate of anti-HBs was lower(28 25) which may be due to the fact that HIV-infectedpatients are prone to lose anti-HBs immunity at a higherfrequency than the general population [10] Previous reportssuggested that the lower HBV replication was associated withmilder hepatic damage [11] Among the subjects with OBIelevated ALT or AST was found among 75 (6 of 8) and notsignificant However our study is cross-sectional thereforeevaluation of long term clinical significance of OBI should bebetter addressed by follow-up studies

The low level of viral load obtained in this study buttressthe findings in another study that showed that showed thatalmost all OBI cases are infected with replication incompe-tentHBV revealing a strong suppression of overall replicationactivity and gene expression thereby resulting in a significantreduced viral load [12]

HIV patients are screened for concomitant chronic hep-atitis B using HBsAg ELISA and it is not considered cost-effective to perform HBV DNA testing for all HIV patientsin our resource-poor setting Our study tried to identifypossible clinical and serological markers which could guide

DNA testing in these patients OBI is reported to be commonamong HCV infection but we found its prevalence to be lowamong our study group However anti-HCV was not testedamong the other HIV positive samples attending the Special-ist Hospital Ikole Ekiti State Nigeria Furthermore noneof the risk factors were found to be statistically significantmarkers ofOBI and cannot be used as an independentmarkerfor identifying patients who should benefit from HBV DNAestimation

However as one third of the anti-HBc positive neg-ative patients were positive for HBV DNA (8 of 28) itis recommended that HIV positive patients with HBsAgnegativeanti-HBc positive patterns should be tested for thepresence of HBV DNA irrespective of their anti-HBs statusNevirapine is commonly included in the first line ARTregimens at most treatment centers in Nigeria Our studyidentified only 21 subjects with OBI and all the sampleswere collected from a single center indicating that resultsmight differ in setting with significant different demographiccharacteristics Thus in future multicenter study involvinglarge sample size should be taken up

7 Conclusion

A main implication of the presently viable data is thereforefurther emphasizing the need for efficient HBV vaccinationprograms Overall the present study highlights the need forscreening HBV before the initiation of any HAART contain-ing anti-HBV regimens in HBVHIV coinfected patients Itnecessitates the use of NAT for effective laboratory diagnosisof occult HBV infections in HIV positive patients especiallyin developing countries where these assays are not widelyavailable

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] M Torbenson and D L Thomas ldquoOccult hepatitis Brdquo TheLancet Infectious Diseases vol 2 no 8 pp 479ndash486 2002

[2] C Brechot V Thiers D Kremsdorf B Nalpas S Pol and PPaterlini-Brechot ldquoPersistent hepatitis B virus infection in sub-jects without hepatitis B surface antigen clinically significant orpurely lsquooccultrsquordquo Hepatology vol 34 no 1 pp 194ndash203 2001

[3] M T Perez-Rodriquez B Sopenia M Crespo et al ldquoClinicalsignificance of ldquoanti-HBc alonerdquo in human immunodeficiencyvirus positive patientsrdquo World Journal of Gastroenterology vol15 no 10 pp 1237ndash1241 2009

[4] C LThio ldquoHepatitis B in the human immunodeficiency virus-infected patient epidemiology natural history and treatmentrdquoSeminars in Liver Disease vol 23 no 2 pp 125ndash136 2003

[5] N J Shire S D Rouster N Rajicic and K E Sherman ldquoOcculthepatitis B in HIV-infected patientsrdquo Journal of AcquiredImmune Deficiency Syndromes vol 36 no 3 pp 869ndash875 2004

[6] M Nunez P Rios M Perez-Olmeda and V Soriano ldquoLack ofldquooccultrdquo hepatitis B virus infection in HIV-infected patientsrdquo


Agency for International Development vol 16 no 15 pp 2099ndash2101 2002

[7] P Bhattacharya P K Chandra S Datta et al ldquoSignificantincrease in HBV HCV HIV and syphilis infections amongblood donors in West Bengal Eastern India 2004-2005exploratory screening reveals high frequency of occult HBVinfectionrdquoWorld Journal of Gastroenterology vol 13 no 27 pp3730ndash3733 2007

[8] N Kumarasamy S Solomon T P Flanigan R Hemalatha SP Thyagarajan and K H Mayer ldquoNatural history of humanimmunodeficiency virus disease in southern Indiardquo ClinicalInfectious Diseases vol 36 no 1 pp 79ndash85 2003

[9] S S Solomon A K Srikrishnan S H Mehta et al ldquoHighprevalence of HIV HIVhepatitis C virus coinfection and riskbehaviors among injection drug users in Chennai India a causefor concernrdquo Journal of Acquired ImmuneDeficiency Syndromesvol 49 no 3 pp 327ndash332 2008

[10] L Piroth C Binquet M Vergne et al ldquoThe evolution ofhepatitis B virus serological patterns and the clinical relevanceof isolated antibodies to hepatitis B core antigen inHIV infectedpatientsrdquo Journal of Hepatology vol 36 no 5 pp 681ndash686 2002

[11] A Knoll A Hartmann H Hamoshi K Weislmaier and WJilg ldquoSerological pattern ldquoanti-HBc alonerdquo characterization of552 individuals and clinical significancerdquo World Journal ofGastroenterology vol 12 no 8 pp 1255ndash1260 2006

[12] G Raimondo J-P Allain M R Brunetto et al ldquoStatementsfrom the Taormina expert meeting on occult hepatitis B virusinfectionrdquo Journal of Hepatology vol 49 no 4 pp 652ndash6572008

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


considerable variation in prevalence ofHIVHBVcoinfectionaccording to geographic regions and exposure risk [5]

Successful implementation of ART leads to immunereconstitution that can potentially result in immunemediatedliver injury in the setting of HBV coinfection Some studieshave reported an association between OBI and elevatedtransaminase [6] therefore identification of OBI is of impor-tance

2 Materials and Methods

Of the 1200 HIV-infected patients enrolled in the HAARTClinic of the Specialist Hospital Ikole Ekiti State Nigeriafrom October 2012 to April 2013 we identified 980 HBsAgnegative patients (ART-naıve subjects) Among them 188were selected for the study by a simple random methodInformed consent was obtained from the patients and theinstitutional committee approved the study protocol Thesera were stored at minus20∘C until tested Ethical clearancewas obtained from the Ethical Committee of the Ikole EkitiSpecialist Hospital

21 Serological Testing All samples were tested for HBsAganti-HBs anti-HBc anti-HCV and anti-HIV using ELISA(DRG Diagnostics Marburg Germany) All anti-HBc posi-tive samples were retested for HBsAg as well as for anti-HBcand only repeat positive samples were included in the study

22 DNA Extraction DNA was extracted from all the serumsamples using QIAampDNABloodMini kit (Qiagen GmbHHilden Germany) following themanufacturersrsquo instructionsBriefly samples (200120583L) were incubated with protease andlysis buffer After incubation there were two washing stepsand the nucleic acids were eluted in a volume of 50 120583L ofelution buffer The eluted DNA was stored at minus20∘C untiltested

23 Hepatitis B Virus Specific Nested PCR The presence ofHBV DNA was examined in all samples using a routinediagnostic PCR Primer pairs were designed from the highlyconserved overlapping regions of the S and P genes of theHBV genome A nested PCR was performed outer primerpairs were HBPr134 (sense) 51015840-TGCTGCTATGCCTCA-TCTTC-31015840 and HBPr135 (antisense) 51015840-CAGAGACAAAA-GAAAATTGG-31015840 and the inner primer pairs were HBPr75(sense) 51015840-CAAGGTTATGTTGCCCGTTTGTCC-31015840 andHBPr94 (antisense) 51015840-GGTATAAAGGGACTCACGATG-31015840 PCR amplifications were carried out in 25 120583L reactionvolumes with 5 ng of genomic DNA 10x PCR buffer (20mMTris-HCl pH84 50mMKClQiagen) 2mMof dNTPs 50 ngof each primer and 1U Ampli Taq gold DNA polymerase(Applied Biosystems) on a PTC 200 cycler (Peltier Thermalcycler Watertown Massachusetts USA) Thermal cyclingparameters were initial denaturation at 94∘C for 2minfollowed by 35 cycles of 30 sec at 94∘C denaturation 30 secat 52∘C annealing temperature and 45 sec at 72∘C extensionfollowed by a final extension of 5min at 72∘CThermal cyclingparameters remained the same as in the first PCR round

except for the number of cycles that is increased to 40 cyclesof amplification Each PCR product (5120583L) was analysed byelectrophoresis in 2 agarose gels A positive control (HBVplasmid DNA) and a negative control of the master mix onlywere integrated to each run to validate the PCR products thatyielded a 340 bp fragment

3 Quantification of HBV DNA

Quantification of HBV DNA was performed with quantita-tive real-time PCRusing a previously described procedure [7]in a GeneAmp 7300 sequence analyzer (Applied BiosystemsPerkin-Elmer Foster City CA) HBV-plasmid DNA wasused to generate a standard curve following a serial 10-folddilution

4 Statistical Analysis

Mean age and all the numerical data were analysed usingStudentrsquos 119905-test The chi-square test and Fisherrsquos exact testwere used to compare categorical data For the purpose of ourstudy 119875 value le 005 was considered statistically significant

5 Results

The demographic biochemical and virological parametersof the study group are summarized in Table 1 The mean agewas 35 (range 3ndash67) years The majority (45) had multiplesexual partners and 25 of the subjects had a history ofconcomitant alcohol use HCV coinfection was found in 296(21) Overall 2996 (292) of patients were reactive foranti-HBc an indication of prior exposure to HBV DNAand majority 68 (75) of the patients were female (Table 2)Thus in the total study population 21188 (112) of patientswere identified as OBI and 625 of the OBI patients hadCD4 count less than 200 cellsmm3 Averagely the HBV viralload was lt50 copiesmL in the OBI samples examined byquantitative PCR

Serum levels of AST andALTwere higher among patientswith OBI in comparison to anti-HBc positive HBV DNAnegative individuals but the difference failed to reach stan-dard significance (119875 = 013 and 119875 = 007) respectivelyThe comparison of different demographic biochemical andvirological factors between HBV DNA positive and negativecases was illustrated in Table 3 The distribution of the studyparticipants as per the 1993 Revised Classification System forHIV Infection and Expanded Surveillance Case Definitionfor AIDS among Adolescents and Adults was as shown inTable 4 Figure 1 shows the 07 agarose gel picture showinga 340 base pairs amplicon

6 Discussion

Thepresent study represents a comprehensive cross-sectionalanalysis of prevalence of OBI in an ART naıve HIV positivecohort comprising various risk groupsMost previous studieslooking at the clinical effects of OBI in HIV include a largenumber of patients on anti-HBV drugs as a component of






Negative119899 = 68 (708) 119875 value












1 2 3 4 6 minusC +C 27 30 37 41 L

1000

340

100









7 Conclusion




References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Negative119899 = 68 (708) 119875 value












1 2 3 4 6 minusC +C 27 30 37 41 L

1000

340

100









7 Conclusion




References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 3 4 6 minusC +C 27 30 37 41 L

1000

340

100









7 Conclusion




References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article occult hepatitis b virus infection among...

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