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Highly sensitive method uncovers high levels of resistance in mothers given single-dose nevirapine
Chris Gadd & Michael Carter, Friday, February 25, 2005
A sophisticated laboratory test has demonstrated that single-dose nevirapine given to mothers can lead to a higher incidence of resistance than detectable by standard assays, according to three similar studies presented on Thursday at the Twelfth Annual Retrovirus Conference in Boston.

All three studies assessed the prevalence of nevirapine resistance mutations in HIV-1-positive African women with subtype C virus who were given single-dose nevirapine during labour to prevent transmission of HIV to their babies. Although the clinical relevance of the high level of resistance is still unclear, this suggests that nevirapine resistance mutations can arise in the majority of these women, possibly jeapordising future treatment options.

Standard assays to test for resistant virus are unable to detect mutations that comprise less than 20% of the viral population. Accordingly, investigators have developed a method called real-time polymerase chain reaction (RT-PCR), which detects prevalence of specific mutations down to around 0.1%. This method allows researchers to make copies of a particular gene in the test tube, and monitor the amount being produced in real time by labelling the DNA with fluorescent markers.

Researchers from South Africa and the United States, led by Jeffrey Johnson of the Centers for Disease Control and Prevention, used RT-PCR to detect for the commonest resistance mutation seen with nevirapine, K103N, to reassess the emergence of drug resistance in 50 women who received single dose nevirapine. Blood samples were obtained before the drug was administered during labour and a second sample was collected between six and 36 weeks after delivery.

Using the real-time analysis, the investigators found that none of the pre-nevirapine samples had detectable K103N. The investigators’ assay also successfully identified the ten post-treatment samples in which conventional tests had found the K103N mutation to be present.

In addition, the real-time assay detected the K103N mutation in a further 17 (43%) of post-treatment samples not found in the conventional analysis. This gave a total incidence of 65% of women with resistance, an increase of 63% on previous estimates. A similar pattern was seen for the Y181C mutation.

Sequencing was carried out on five samples, and the K103N mutation was confirmed in each case. The prevalence of K103N virus was between 1 and 11% of the total HIV population, much too low be detected by a conventional assay.

This finding was confirmed in a second study presented by Sarah Palmer from the National Cancer Institute at Frederick. The investigators, from the United States and South Africa, obtained plasma samples from 17 women and used an RT-PCR assay to detect the K103N and Y181C mutations.

Using standard genotypic testing, blood samples from the women were tested for the presence of NNRTI-resistance. The investigators then retested the samples from all 17 women using the RT-PCR assay, finding one or both mutations in seven (88%) of the eight women previously found to have neither mutation at twelve months after delivery. The median frequency of mutant HIV particles ranged from 1 to 63% in these women, with a median of 3%.

When they extrapolated their results to the full population of women treated with single-dose nevirapine, the investigators calculated that up to 69% of women could have NNRTI resistance mutations in the first four months after delivery, declining to at least 32% in months 5 to 9 and at least 22% in months 11 to 14.

Finally, Shayne Loubser from the National Institute for Communicable Disease, Johannesburg, presented data from a third similar study assessing the frequency of K103N in women given single-dose nevirapine. While standard assays revealed the mutation in nine (50%) of 18 women at six weeks after delivery, this increased to 16 women (89%) using the RT-PCR method.

Encouragingly, this last study involved longitudinal follow-up of 16 women, finding that at one year after delivery nevirapine resistance was seen in HIV samples isolated from 25% of the women, a comparable value to the extrapolation by Sarah Palmer's study. However, DNA from the women’s blood cells did not show any evidence of nevirapine resistance, despite use of the more sensitive assay. This suggests that archiving of the nevirapine-resistant mutation may be a rare event.

Despite these findings, RT-PCR is an expensive and time-consuming laboratory test. It is unlikely to become a routine tool for use in monitoring resistance in the clinic in the near future, and will probably remain confined to use in scientific research for the foreseeable future.

Reference

Johnson J et al. Resistance emerges in the majority of women provided intrapartum single-dose nevirapine. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 100, 2005.

Palmer S et al. Persistence of NNRTI-resistant variants after single-dose nevirapine in HIV-1 subtype C infected women. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 101, 2005.

Loubser S et al. Sensitive real-time PCR quantification of 103N resistance mutants following single-dose treatment with nevirapine. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 102, 2005.