Hepatitis C Virus Genetic Diversity and Drug Mutational Analysis in Cameroon: 1992 to 2013

Judith Ndongo Torimiro, Laure Arlette Tchapda, Maurice Boda, Jude Saber Bimela, Henry Namme Luma, Oudou Njoya

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Introduction

• Globally, an estimated 71 million people have chronic hepatitis C virus infection (WHO, July 2019)

• Hepatitis C is caused by Hepatitis C virus (HCV), a flavivirus, blood-borne

• HCV Infection is a growing public health concern globally

• There is currently no effective vaccine against hepatitis C

• Cameroon shows a broad range of prevalence depending on diagnostic test/algorithm and study target

• Antiviral agents can cure more than 95% of persons with hepatitis C infection

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HCV Virology

- HCV infection is a highly dynamic process with a viral half-life of only a few hours

- Production and clearance of an estimated 1012 virions per day in a given individual (Neumann et al, 1998)

- Globally, 7 HCV genotypes (1 to 7) and 67 subtypes have been classified

- Cameroon:

* great genetic variability with Genotypes 1, 2 and 4 being prevalent (Torimiro et al, 2016; Galani et al,

2016; Tagnouokam-Ngoupo et al, 2019).

* two unclassified variants isolated from Cameroon (Li et al, 2013)

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Genetic Diversity of HBV, HIV and HCV

Figure 1: Phylogenetic analysis of HBV, HIV and HCV(Almudena Torres-Cornejo, Georg M. Lauer, 2017)

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Figure 2: Proteins encoded by the HCV genomeStructural proteins (core (C), E1 and E2)Non structural (NS2-5) replicative proteins

NS5B- highly conserved sequence- central component of the HCV replicase- important for viral maturation and replication- a major target for antiviral intervention

HCV Genome

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But what do we know about the level of susceptibility of thesedrugs against circulating HCV strains in Cameroon?

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Current All-Oral Therapies Highly Effective, Simple, Well Tolerated

IFN6 Mos

PegIFN/RBV 12 Mos

IFN12 Mos

IFN/RBV12 Mos

PegIFN12 Mos

2001

1998

2011

StandardInterferon

(IFN)

Ribavirin(RBV)

Peginterferon(pegIFN)

1991

PegIFN/RBV +DAA

IFN/RBV6 Mos

6

16

34

4239

55

70+

0

20

40

60

80

100

DAA + RBV ±PegIFN

90+

2013

All-OralDAA±

RBV

Current95+

All-Oral Therapy

References in sliFigure 3: Hepatitis Oral therapies

Direct-Acting Antivirals

(DAAs)

Slide credit: clinicaloptions.com 7

First Direct-acting Agent (DAA) introduced in Cameroon

SOFOSBUVIR (NS5B protein, polymerase inhibitor, interrupts viral

replication, pan genotypic anti-HCV activity)

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Rationale of study

• Sofosbuvir (polymerase inhibitor) was incorporated into the standard of care in Cameroon in 2016

• No record susceptibility reported in Cameroon

• Lessons learnt from the HIV molecular epidemiology: late development of sequence database

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Methods

• Ethical review by the Cameroon National Ethics Committee

• HCV treatment-naïve individuals (1992 to 2013)

• HCV NS5B sequences obtained from patients (Douala and Yaoundé, Cameroon) and Los Alamos National Library HCV Database (Total of 252)

• HCV Core sequences = 45 (from LANL)

• HCV Envelope sequences = 112 (from LANL)

• PCR, Sanger sequencing using the 3130XL DNA analyzer

• Geno2Pheno tool version 0.92 for identification of mutations associated to resistance to SOFOSBUVIR

• Genotyping by phylogeny

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Results

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HCV mutations associated to resistance to Sofosbuvir

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1. Drug resistance analysis

S282T

0.4% : Torimiro et al ( 2016)

0%: Costantino et al (2015)

N316C

11.22%43%: Costantino et al (2015)

Primary mutation

Polymorphism

KEY

Figure 4a: RAM to SOF Figure 4b: Common

mutation in HCV NS5B

SA

N009

4f

AY

74

31

48

2

4f A

Y685025

0

4f A

Y685025(2

)

0

4f A

Y632208

0

4f A

Y632200(2

)

1

4f A

Y632200(3

)

2

4f A

Y743148(2

)3

4f A

Y632200

7

SA

N00

5C

024

4f A

Y68

5012

4f A

Y63

2196

4f A

Y26

5434

(2)

SAN01

3

4f A

Y26

5434

72012

10

1

SAN00

8

C012

4m F

J462433

4o KY608668

C0234f A

Y265441

40C0064 AY743090

39 4a EF694486

84a FJ872311

4c GU088166

4c L29614

4c FJ462436

2741

355

22

400

1

4f AY632211(2)

4f AY632211

19

4f AY632211(3)

50

4 JQ3184294f AY6322094f AY265440(2)4f EU3921754f AY265440

4f AY685013(2)4f AY685013

626

3

2

6

19

15

0

4f AY632207

0

SAN010C021

19

0

4f AY743056

4f AY685023

4f AY632198

11

19

10

4f AY685021

74

C013

C010

1e AY685019

9

1e A

Y265450

5

1e A

Y685051

0

1e A

Y632083

2

1e A

Y632083(2

)

3

1e A

Y265450(2

)

18

1e A

Y632099

1e A

Y632098

SA

N014

SA

N003

43

29

18

17

1e

AY

68

50

49

16

C019

1h

AJ29

12

79

1h

KC

24

81

99

1h A

Y632134

585878

12

1a E

U781787

1a E

U256096

70

1l K

C248193(2

)1l K

C248193 21

1l K

C248193(3

) 58

SA

N002

16

1l K

Y608618

1l A

Y632109

4

1l A

Y63

2116 0

1l K

C96

0812

0

1l A

Y68

5046

1

1l K

C24

8197

0

1l K

C24

8197

(2)

0

C01

6

25

C017SAN011

C0021l AY632113(2)

1l AY632125

1l AY6321151l AY6321241l AY6321131l AY257091 6

1001 5 44 46

3

47

12

31

64

972 AY265423C014

2 JN642974

12

2 AY265436

3

2 JF735112

0

2 AY632186

0

2 AY632178

0

C011

3

SAN004

10

2 AY632177

18

2 AY265437

2 AY257079

2 AY632168

2 AY632163

2 AY632161

2 AY265451(2)

2 AY685022

2 AY265451

2 AY265444(2)

2 AY265444

55SA

N007

C025

46SA

N001

2 A

Y257099

2 A

Y632158

2 A

Y632157

2 A

Y685048

4112310

31

0

0

0

0004044

Figure 5: Phylogenetic relationship of HCV NS5B Genotypes and sub types

Genotype 2Genotype 4

Genotype 1

HCV NS5B Genetic Diversity

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• Genotypes 1 (16), 2 (17) and 4 (21) were identified at 29.6%, 31.5% and 38.9% rate, respectively

Genotype 129.6%

Genotype 231.5%

Genotype 438.9%

Génotype 1 Génotype 2 Génotype 4

Prevalence of HCV NS5B Genotypes

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Figure 6: HCV Genotypes

HCV Core and Envelope Diversity

Figure 7a: HCV Core (C) diversity Figure 7b: HCV envelope (E) Diversity

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Conclusion

• Sofosbuvir resistant variants are not common in treatment-naive individuals in Cameroon

• HCV Genotypes 1, 2 and 4 co-circulate in this population

Recommendation

Routine surveillance of resistance-associated mutations (RAMs) in treatment-naive population useful for

developing national diagnostic and treatment guidelines for hepatitis C.

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Thank you for your kind attention!

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