Received on January 6, 2006
Accepted on April 4, 2006

Molecular Diagnostics and Genetics

Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5' Long Terminal Repeat Domain

¹ Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
² Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, and Servico de Infectologia, Hospital Universitario Prof. Edgard Santos, Salvador, Brazil
³ Servico de Infectologia, Hospital Universitario Prof. Edgard Santos, Salvador, Brazil
⁴ Department of Medical Virology, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa
⁵ Department of Experimental Medicine, Tamil Nadu Medical University, Madras, India
⁶ Institute of Medical Virology, Johann Wolfgang Goethe University, Frankfurt/M., Germany
⁷ Institute of Medical Virology, Johann Wolfgang Goethe University, Frankfurt/M., Germany, and Current affiliation: Discipline of Medical Virology, University of Stellenbosch, Stellenbosch, South Africa

^* To whom correspondence should be addressed. E-mail: drosten{at}bni-hamburg.de.

Background: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes.

Methods: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127).

Results: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (>95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%-51%. The quantification range was 50-10 000 000 IU/mL. Viral loads for subtypes A-D, F-J, AE, and AG yielded mean differences of 0.31 log₁₀ compared with Amplicor in the 10³-10⁴ IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) (P <0.001 for all).

Conclusions: This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.