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Clinical Chemistry 52: 1258-1266, 2006. First published April 20, 2006; 10.1373/clinchem.2006.066498
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(Clinical Chemistry. 2006;52:1258-1266.)
© 2006 American Association for Clinical Chemistry, Inc.

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

Christian Drosten1,a, Marcus Panning1, Jan Felix Drexler1,2, Florian Hänsel1, Celia Pedroso2, Jane Yeats3, Luciano Kleber de Souza Luna1, Matthew Samuel4, Britta Liedigk1, Ute Lippert1, Martin Stürmer5, Hans Wilhelm Doerr5, Carlos Brites2 and Wolfgang Preiser5,1

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

aAddress correspondence to this author at: Bernhard Nocht Institute for Tropical Medicine, Clinical Virology, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany. Fax 49-42818-378; 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.




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