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Antimicrobial Agents and Chemotherapy, August 2009, p. 3606-3607, Vol. 53, No. 8
0066-4804/09/$08.00+0 doi:10.1128/AAC.01583-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
| LETTER TO THE EDITOR |
Lack of Antimicrobial Activity by the Antiretroviral Drug Nevirapine against Common Bacterial Pathogens 
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The extended prophylactic use of the antiretroviral drug nevirapine in breastfeeding infants of human immunodeficiency virus (HIV)-infected mothers in developing countries has been shown to reduce HIV transmission (2, 5). Nevirapine's use in this setting was also associated with a reduction in mortality in the extended nevirapine arm due primarily to a reduction in gastrointestinal and respiratory disease even among HIV-uninfected infants, suggesting possible antimicrobial activity of the drug (5). Reverse transcriptase has been shown to occur in at least two groups of bacteria: myxobacteria and Escherichia coli (3). Low levels of azidothymidine, a nucleoside analogue reverse transcriptase inhibitor, have been reported to have antimicrobial activity against a variety of certain gram-negative enteric bacteria, such as Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, and Haemophilus influenzae (1), but there have been no reports of whether nevirapine has been shown to have antimicrobial activity. The investigator brochure for nevirapine does not describe any such testing.
In order to determine whether nevirapine demonstrates antimicrobial activity in vitro, we tested manufactured nevirapine provided by Boehringer Ingelheim (maker of nevirapine). Serum concentrations in the range found physiologically in breastfeeding infants who have taken daily nevirapine prophylaxis (4 mg/kg/day) for 6 weeks or more (namely 0.25 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.0 µg/ml, and 4.0 µg/ml [4]) were tested using broth and agar dilution methods by following the manufacturer's recommendations for making the stock solutions of nevirapine. Nevirapine was initially dissolved in dimethyl sulfoxide (DMSO) to a concentration of 5% according to the manufacturer's directions and then diluted with Mueller-Hinton broth for a working concentration of 40 µg/ml of nevirapine and 0.2% DMSO. The working solution was diluted in the respective culture media to the final concentrations of nevirapine tested. The final concentrations of DMSO were no greater than 0.02% in the culture media.
For Streptococcus pneumoniae a broth macrodilution method using Mueller-Hinton broth with 3% lysed horse blood was used. For Haemophilus influenzae, a broth macrodilution method in Haemophilus test medium was used. For the remaining organisms, testing was performed using an agar dilution method. A 0.5 McFarland standard of each bacterial organism tested was inoculated in duplicate to the various test media. A total of 13 bacteria were tested (comprising eight bacterial species which commonly infect human infants—six ATCC strains [Streptococcus pneumoniae ATCC 49619, Haemophilus influenzae ATCC 4927, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Shigella sonnei ATCC 111060, and Salmonella group B ATCC 31194] and seven patient isolates [one each of Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli, Shigella flexneri group B, and Salmonella group D and two of Staphylococcus aureus]). Plates and broths were incubated at 35°C for 18 to 24 h.
In order to demonstrate that the manufactured nevirapine used in the bacterial cultures had anti-HIV activity, qualitative HIV cultures were performed with and without prophylactic physiological concentrations of nevirapine. In brief, 1 x 106 phytohemagglutinin-stimulated donor peripheral blood mononuclear cells (PBMCs) were cocultivated at 37°C in duplicate with 1 x 106 washed PBMCs from a highly viremic HIV-infected patient who was not on antiretroviral therapy (viral load of 331,008 HIV RNA copies/ml at time of collection and no history of non-nucleoside reverse transcriptase exposure). PBMCs were cultured in culture media at 37°C with 5% interleukin-2, 20% fetal bovine serum, penicillin (100 units/ml), and gentamicin (50 µg/ml) and RPMI 1640 media with L-glutamine containing 4 µg/ml, 0.25 µg/ml, or 0 µg/ml of nevirapine. Cultures were refed with 0.5 x 106 donor PBMCs at days 7 and 14, and culture supernatants were tested in Susan Fiscus's laboratory (University of North Carolina) for HIV p24 antigen using an enzyme-linked immunosorbent assay kit (Perkin Elmer, Waltham, MA). By day 14 of culture, the optical density (OD) of the signal for detection of p24 antigen was positive (1.4 and 1.6 OD; positive cutoff, 0.054 OD) in the absence of nevirapine with DMSO present, minimally positive (0.07 and 0.08 OD) in the presence of 4 µg/ml of nevirapine, and positive (1.3 and 1.8 OD) in the presence of the lowest nevirapine concentration tested (0.25 µg/ml).
After overnight incubation, MICs against these bacterial organisms exceeded achievable serum concentrations, with 4 µg/ml being the highest concentration tested. Nevirapine, which does not require metabolism to its active form, showed no activity against any of the organisms tested, which were all shown to be susceptible to various antibiotics.
It is possible that the organisms tested did not have reverse transcriptase or had a different reverse transcriptase or another essential polymerase not inhibited by physiological levels of nevirapine. Another possibility is that the cause of the higher mortality in the infants not receiving extended-dose nevirapine was due to pathogens not tested in this study.
In summary, we found no evidence that nevirapine has antimicrobial activity at the concentrations tested against organisms commonly associated with pediatric morbidity and/or mortality.
Published ahead of print on 1 June 2009. 
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[Abstract/Free Full Text] - Kumwenda, N. I., D. R. Hoover, L. M. Mofenson, M. C. Thigpen, G. Kafulafula, Q. Li, L. Mipando, K. Nkanaunena, T. Melbrahtu, M. Bulterys, M. G. Fowler, and T. E. Taha. 2008. Extended antiretroviral prophylaxis to reduce breast-milk HIV-1 transmission. N. Engl. J. Med. 359:119-129.
[Abstract/Free Full Text] - Lim, D., and W. K. Maas. 1989. MicroReview: reverse transcriptase in bacteria. Mol. Microbiol. 3:1141-1143.[CrossRef][Medline]
- Shetty, A. K., H. M. Coovadia, M. M. Mirochnick, Y. Maldonado, L. M. Mofenson, S. H. Eshleman, T. Fleming, L. Emel, K. George, D. A. Katzenstein, J. Wells, C. C. Maponga, A. Mwatha, S. A. Jones, S. S. Abdool Karim, and M. T. Bassett for the HIVNET 023 Study Team. 2003. Safety and trough concentrations of nevirapine prophylaxis given daily, twice weekly, or weekly, or weekly in breast-feeding infants from birth to 6 months. J. Acquir. Immune Defic. Syndr. 34:482-490.[CrossRef][Medline]
- Six Week Extended-Dose Nevirapine (SWEN) Study Team et al. 2008. Extended-dose nevirapine to 6 weeks of age for infants to prevent HIV transmission via breastfeeding in Ethiopia, India, and Uganda: an analysis of three randomized controlled trials. Lancet 372:300-313.[CrossRef][Medline]
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J. Brooks Jackson*
Department of Pathology Carnegie Building Room 415 600 North Wolfe Street
James Dick
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* Phone: (410) 614-4966 Fax: (410) 955-0394 E-mail: bjackso{at}jhmi.edu |
Antimicrobial Agents and Chemotherapy, August 2009, p. 3606-3607, Vol. 53, No. 8
0066-4804/09/$08.00+0 doi:10.1128/AAC.01583-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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