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Armando Blondel Djiyou Djeuda�PhD student�The University of Douala, Cameroon�MIVEGEC, Université de Montpellier, CNRS, IRD, France

Development of a robust in-house HIV-1 genotyping assay for the detection of drug resistance mutations in low-level viremia samples

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Plasma

DBS

Sanger Sequencing

Point Mutation Assay

Next-Generation seq.

Nested PCR

3. Dye-terminator sequencing

Labeled cDNA

DRM report

ELISA

(Luminiscent, fluorescent, colorimetric)

Ligation

3. Labeling

q-PCR

Allele-Specific PCR

RFMP

3. Library preparation

Post-capture library

4. Parallel Sequencing

(Semi-conductor - ion Torrent, Reversible terminator - Illumina)

Emulsion PCR

(Ion Torrent)

Bridge PCR

(Illumina)

4. Detection

DRM report

DRM report

4. Sequence analysis

(Sequence editing and DRM analysis)

1. TNA/RNA extraction

2. Amplification

Technologies used for resistance testing

Background (1/2)

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Rationale and objective of the study

1000

Copies/mL

> 1 000 000

50

EACS guidelines1

WHO guidelines2

ART failure threshold

Low-level viremia (LLV)

Virological control

Virological failure

Development and evaluation of an in-house genotyping method for the detection of HIVDR mutations in patients with low- and high-level viremia using minimal input of sample specimens

Until recently, no interventions were recommended for patients with LLV, yet

  • Associated with subsequent virological failure 3,4
  • Evidence of accumulation of HIV drug resistance jeopardizing current and future treatment 5
  • Most of the commercially available and in-house tests are using the 1000 cps/mL threshold

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Background (2/2)

EACS: European AIDS Clinical Society; WHO: World Health Organisation.

1. EACS, 2021 https://www.eacsociety.org/guidelines/eacs-guidelines/; 2. WHO, 2021; 3. Chun HM, et al. Lancet Glob Health 2022; 4. Hermans LE, et al. Lancet Infect Dis 2018; 5. Djiyou AB, et al. J Antimicrob Chemother 2023

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Study cohort

280 Adolescents (10-19 years old) on ART ≥ 6 months 1

  • 90% were perinatally infected
  • Median time on ART: 9.8 years
  • ART: 52.5% on first line (mostly EFV-based) and 46.5% on 2nd line (mostly LPV-based)

​

Samples included

124 plasma samples from 86 adolescents were included

  • 89 plasma with high-level viremia (HLV, VL ≥1000 copies/mL)
  • 35 plasma with low-level viremia (LLV, VL between 200-999 copies/mL)

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Genotypic resistance testing

Attempted using a modified version of an in-house method targeting the viral PR, RT and IN regions 2

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Study procedures

Methodology (1/2)

IN: Integrase; PR: Protease; RT: Reverse Transcriptase

1. Djiyou A, et al. BMC Pediatr. 2023; 2. Tchouwa GF, et al. EClinicalMedicine. 2018

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RNA extraction (Macherey-Nagel kits)

Amplification by RT-PCR (Superscript & Platinium Taq PCR kits)

Sequencing reactions and sequencing (BigDye Terminator kit)

Subtyping analysis (Seqman, Mega & Seaview)

Identification of HIVDR mutations (Stanford Algorithm)

Step 1

Step 2

Step 3

Step 4

Step 5

Genotyping resistance testing workflow

Enrichment by ultracentrifugation

Step 0

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Methodology (2/2)

HIVDR: HIV drug resistance; RT-PCR: Reverse Transcriptase Polymerase Chain Reaction

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Optimized PCR protocol

Round 1 (Superscript III)

Final conc.

Unit volume (µL)

RNAse-free water

 

2

Tampon OS (2X)

1X

25

PR2 or INT1s (10 µM)

0,2 µM

1

TR2as or INT1as (10 µM)

0,2 µM

1

MgSO4 (5 mM)

 

8

dNTP (10 mM)

0,2 mM

1

One step RT-PCR

 

2

Final volume

 

40

RNA extracts

 

10

50°C 30 min

94°C 2 min

94°C 20s

55°C 30s

68°C 1.5min

68°C 10min

10°C ∞

40 cycles

Round 2 (Platinium II)

Final conc.

Unit volume (µL)

RNAse-free water

 

17

Platinum™ II Hot-Start PCR Master Mix (2X)

1X

25

PR3 or INT2s (10 µM)

0,2 µM

1

TR3as or INT2as (10 µM)

0,2 µM

1

dNTP (10 mM)

0,2 mM

1

Final volume

 

45

cDNA

 

5

95°C 2 min

94°C 30s

55°C 30s

68°C 1min

68°C 10min

10°C ∞

35 cycles

124 plasma samples

  • 35 with LLV
  • 89 with HLV

Total sequences obtained

N = 115 (92.7%)

LLV Sequences obtained

N = 30 (85.7%)

  • RT: 97.1%
  • PR: 91.4%
  • IN: 85.7%

HLV Sequences obtained

N = 85 (95.5%)

  • RT: 98.9%
  • PR: 96.6%
  • IN: 95.5%

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Results

(1/4)

Samples

Negative control

Molecular Weight Marker

1070 bp

(IN)

1220 bp

(RT-PR)

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Genetic diversity of HIV strains

CRF02_AG

Subtype G

Subtype D

Subtype F2

Subtype A

Legend:

♦ Sequences obtained from our study participants

● Reference sequences

● CRF02_AG ● Subtype F2

● Subtype A ● Subtype G

● Subtype D ● Other subtypes

A

B

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Results

(2/4)

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High-level of HIV drug resistance

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Overall

Samples with LLV

Samples with HLV

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n/N tested

%

n/N tested

%

n/N tested

%

Total sequences

115/124

92.7

30/35

85.7

85/89

95.5

Overall HIVDR

91/115

79.1

25/30

83.3

66/85

77.6

NNRTI resistance

83/115

72.2

23/30

76.7

60/85

70.6

NRTI resistance

69/115

60.0

18/30

60.0

51/85

60.0

PI resistance

11/115

9.6

5/30

16.7

6/85

7.0

InSTI resistance

23/115

20.0

7/30

23.3

16/85

18.8

Dual class HIVDR

55/115

47.8

14/30

46.7

41/85

48.2

Triple-class HIVDR

18/115

15.6

5/30

16.7

13/85

15.3

Alarming rates of HIVDR found in samples with LLV (VL between 200-999 copies/mL)

  • Proportions higher than samples with HLV
  • Patients with LLV are misclassified as “non-failing” in SSA countries
  • No specific interventions required (prone to subsequent VF)

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Results

(3/4)

SSA: Sub-Saharan Africa; VF: Virological failure

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HIV drug resistance profile

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Results

(4/4)

Drug susceptibility levels in samples with LLV

Drug susceptibility levels in samples with HLV

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In summary

Our optimized in-house protocol is highly sensitive and robust for genotyping resistance testing of HIV-1 PR, RT, and IN genes using low-level viremia samples

However:

    • Requires two steps of PCR amplifications and sequencing
    • Should be adapted to the local genetic diversity
    • Should be tested using DBS samples to ensure its applicability

The efficacy of this assay in specimens with high genetic diversity and minimal input of samples proves its applicability in routine practice and highlights its clinical benefit in resource-limited countries

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Conclusion

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Thank you

Acknowledgments

  • Avelin AGHOKENG
  • Calixte Ida PENDA
  • Yoann MADEC
  • Armando DJIYOU
  • Astrid MOUKOKO
  • Christiane MEDI SIKE
  • Dallé Grase NGONDI

Research team

  • Carole EBOUMBOU
  • Rita TJOMB LOLO
  • Eric ELGUERO
  • Cécile MOINS
  • Marie VARLOTEAUX
  • Laure-Amélie de Monteynard

All the study participants

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Cameroon national health authorities

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The study sites

  • Laquintinie Hospital of Douala (Cameroon)
  • IRD Montpellier (France)

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Conference organizers