Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baron, P. Articles by Reichelderfer, P. S. Search for Related Content PubMed PubMed Citation Articles by Baron, P. Articles by Reichelderfer, P. S.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 2000, p. 3822-3824, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection and Quantitation of Human Immunodeficiency Virus Type 1 in the Female Genital Tract

Penny Baron,¹ James Bremer,² Steven S. Wasserman,³ Marek Nowicki,⁴ Barbara Driscoll,⁵ Bruce Polsky,¹ Andrea Kovacs,⁴ and Patricia S. Reichelderfer^6,*, For The Division Of Aids Treatment Research Initiative 009 Study Group

St. Luke's Hospital, New York, New York 10025¹; Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612²; University of Maryland School of Medicine, Baltimore, Maryland 21201³; University of Southern California, Los Angeles, California 90033⁴; State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203-2098⁵; and National Institute of Child Health and Human Development, Bethesda, Maryland 20892⁶

Received 22 February 2000/Returned for modification 20 April 2000/Accepted 12 June 2000

	ABSTRACT

Top Abstract Text References

Human immunodeficiency virus type 1 (HIV-1) was detected in the genital tracts of 59% of 225 women by RNA PCR and in 7% of the women by culture. In a comparison of two sampling methods, endocervical swabs were more sensitive than cervicovaginal lavage for HIV-1 RNA detection by PCR but not by culture and their sensitivity was independent of the concentration of HIV-1 RNA.

	TEXT

Top Abstract Text References

The measurement of human immunodeficiency virus type 1 (HIV-1) in the genital compartment has important implications for studies of transmission. Few studies have simultaneously assessed multiple measurement parameters, as well as sampling methods, and the various laboratory factors that may affect those measurements. HIV-1 has been cultured from the cervix and the vagina as cell-free and cell-associated virus using various sampling methods (1, 7, 10, 14, 17-19; M. A. Wainberg, R. Beaulieu, C. Tsoukas, and R. Thomas, Letter, AIDS 7:433-434, 1993). Positive-culture rates range from 11 to 71%. PCR methods have been used to quantitate HIV-1 RNA in the genital tract, including cervicovaginal lavage (CVL) (2, 16), vaginal lavage (6), vaginal (8, 9) and cervical S. Cu Uvin, A. M. Caliendo, S. E. Reinert, K. H. Mayer, T. P. Flanigan, and C. C. Carpenter, Letter, AIDS 12:826-827, 1998) swabs, and the endocervical cytobrush (6). The rates of detection ranged from 29 to 64%.

Clinical specimens were obtained from a cross-sectional study of 225 HIV-infected women, from five sites, enrolled in a substudy of the Women's Interagency HIV Study group (2). CVL was performed using a 10-ml syringe filled with sterile, nonbacteriostatic saline and aimed directly at and into the os. The pooled fluid was aspirated, and the entire volume was collected, stored on ice, and processed within 4 to 6 h. Unfractionated aliquots were stored at 70°C for RNA quantitation and were checked for the presence of blood, leukocytes, and semen. The remaining sample was centrifuged, the supernatant was collected and stored, and the pellet was resuspended in 1 ml of cell culture medium.

Two swabs for peripheral blood mononuclear cell culture and RNA determinations (Puritan Sterile Dacron Polyester Tip Applicators and Puritan Dacron Polyester, respectively) were inserted together into the cervical os and rotated 720°. The RNA swab was placed in a vial containing 4 M guanidine isothiocyanate solution stored at 70°C, and the culture swab was placed in a vial containing culture medium and stored at room temperature. Specimens were processed within 4 to 6 h. Peripheral blood mononuclear cell cultures were performed in accordance with standard procedures (5) with the addition of nystatin at 160 U/ml. A pilot study determined the optimal concentration of nystatin to address the problem of fungal contamination in genital tract cultures. RNA was quantitated using the Roche Amplicor HIV-1 Monitor test; specimens were processed using standard Roche procedures (12), and results were calculated in accordance with the manufacturer's recommendations, with a lower limit of detection of 400 copies/ml.

Only 7% of the women were culture positive in the genital tract; RNA was detectable in the genital tracts of 59% of the women. Both genital tract culture and the RNA assay were positive for 5% of the women, and one woman was positive by culture only. In sum, 64% of the women were positive by one or both methods. There was a significant association between the presence of microscopic blood and genital tract RNA. Of 74% of the women who were positive for both microscopic blood and RNA, 49% had CVL fluid collected first (Fisher's exact test, two tailed [FET], P = 0.003) and 51% had a swab collected first (FET, P = 0.02).

Table 1 shows the lack of agreement between positive swab and CVL samples. This lack of concordance was significant for RNA detection but not for culture, presumably because of the small number of positives. The effect of specimen collection order on the detection of HIV by either culture or RNA assay was assessed by alternating collection methods. When CVL was performed first (n = 102), the rate of swab positivity for both culture and the RNA assay increased from 2.6 to 4.1% and from 47.3 to 51.8%, respectively. Similarly, when swabs were taken first (n = 123) the rate of CVL positivity increased from 3.1 to 5.3% for culture and from 22.0 to 52.4% for the HIV-1 RNA assay. A similar quantitative effect on HIV-1 RNA levels was seen. None of the effects reached significance (FET or Wilcoxon's test).

View this table: [in this window] [in a new window]   TABLE 1.   Lack of agreement between positive swab and CVL samples

The standard female genital tract sampling method has been swabbing or scraping for culture (1, 10, 18, 19; Wainberg et al., Letter) or CVL for RNA assay (2, 6, 16). Although virus could be cultured equally well from swab and CVL specimens, the ability to detect RNA and absolute levels of RNA were highest for swab specimens (Fig. 1), no other studies have made a direct comparison. The approximately 10-fold dilution factor associated with CVL may, in part, explain the lower detection rate and level of RNA for CVL. Lavage cell pellets were used for culture, and there was no difference between the ability to culture virus from CVL samples or swab samples, also suggesting that those RNA differences are due to dilution. The median difference in RNA level was approximately 0.5 log higher for swab specimens and was independent of the overall level of virus in the genital tract.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Differences between log10 estimated RNA concentrations from swab collections and CVL collections plotted against the log-transformed mean of the two estimates. A positive difference indicates that the swab collection RNA copy number was higher than the CVL collection RNA copy number.

We confirmed the findings for women on antiretroviral therapy that have been shown previously for women not on antiretroviral therapy that multiple samplings are necessary to rule out the absence of infectious virus (10; A. Kovacs, P. Reichelderfer, and D. Wright for the DATRI 009 Study Group, Proc. 12th World AIDS Conf., abstr. 23488, p. 439, 1998). The level of infectious virus detected by culture is lower in the female genital tract than in the male genital tract (4). This may be, in part, a biologic explanation for the lower rate of HIV-1 transmission from females to males. No studies have simultaneously compared swab and CVL samples for HIV-1 RNA. We found that the percentage of RNA-positive samples was greater for swabs than for CVL, although some women had detectable RNA in CVL fluid but not in swab specimens. This lack of agreement between sampling methods again suggests that multiple sampling may be as important as the sampling method used. Other longitudinal studies using additional specimen collection methods have confirmed the necessity of multiple sampling (P. S. Reichelderfer, R. Coombs, D. Wright, D. Burns, and A. Kovacs, for The WHS 001 Study Group, Proc. 38th Intersci. Conf. Antimicrob. Agents Chemother. abstr. I-251, p. 443, 1998).

In summary, we have shown that it is possible to detect HIV-1 in the genital tracts of most infected women by using a combination of assays and sampling methods. In this study, RNA was best measured by endocervical swab. Regardless of the sampling method used, multiple samples appear to be needed to rule out the absence of HIV-1 in the genital tract.

	ACKNOWLEDGMENTS

We thank the following for their contributions: S. Lewis, V. Goveia, A. Soloviov, J. Bykoski, and K. Balfe of Westat, Rockville, Md.; H. Minkoff of the State University of New York Health Science Center, Brooklyn; M. Young of Georgetown University Medical Center, Washington, D.C.; R. Greenblatt of the University of California at San Francisco; M. Cohen of Cook County Hospital, Chicago, Ill.; K. Liu of St. Luke's-Roosevelt Hospital Center, New York, N.Y.; B. Meyers of Quest Diagnostics, Baltimore, Md.; and Eric Peterson of the University of Washington, Seattle.

This study was supported by the Division of AIDS Treatment Research Initiative, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Md. (contract N01-A1-15123); the Program Support Center, U.S. Department of Health and Human Services (contract 282-98-0015, task order 21); and the Women's Interagency HIV Study, which is funded by NIAID with supplemental funding from the National Cancer Institute, the National Institute of Child Health and Human Development (NICHD), the National Institute of Drug Abuse, the National Institute of Dental Research, the Agency for Health Care Policy and Research, and the Centers for Disease Control and Prevention (contracts U01-AI-35004, U01-AI-31834, U01-AI-34994, AI-34989, U01-HD-32632 [NICHD], U01-AI-34993, and U01-AI-42590).

	FOOTNOTES

^* Corresponding author. Mailing address: National Institute of Child Health and Human Development, National Institutes of Health, 6100 Executive Blvd., Room 8B13D, Bethesda, MD 20852. Phone: (301) 435-6991. Fax: (301) 480-1972. E-mail: pr20f{at}nih.gov.

This study group also includes D. Burns, M. Cohen, S. Beckner, J. Cohn, P. Miotti, R. Greenblatt, S. Wahl, M. Redford, L. Corey, M. Young, K. Weber, A. Landay, and B. Weiser.

	REFERENCES

Top Abstract Text References

1.	Archibald, D. W., D. J. Witt, D. E. Craven, M. W. Vogt, M. S. Hirsch, and M. Essex. 1987. Antibodies to human immunodeficiency virus in cervical secretions from women at risk for AIDS. J. Infect. Dis. 156:240-241[Medline].
2.	Barkan, S. E., S. L. Melnick, S. Preston-Martin, K. Weber, L. A. Kalish, P. Miotti, M. Young, R. Greenblatt, H. Sacks, and J. Feldman. 1998. The Women's Interagency HIV Study. Epidemiology 9:117-125[CrossRef][Medline].
3.	Clemetson, D. B., G. B. Moss, D. M. Willerford, M. Hensel, W. Emonyi, K. K. Holmes, F. Plummer, J. Ndinya-Achola, P. L. Roberts, S. Hillier, and J. Kreiss. 1993. Detection of HIV DNA in cervical and vaginal secretions: prevalence and correlates among women in Nairobi. JAMA 269:2860-2864[Abstract/Free Full Text].
4.	Coombs, R. W., C. E. Speck, J. P. Hughes, W. Lee, R. Sampoleo, S. O. Ross, J. Dragavon, G. Peterson, T. M. Hooton, A. C. Collier, and L. Corey. 1998. Association between culturable human immunodeficiency virus type 1 (HIV-1) in semen and HIV-1 RNA levels in semen and blood: evidence for compartmentalization of HIV-1 between semen and blood. J. Infect. Dis. 177:320-330[Medline].
5.	Division of AIDS, National Institute of Allergy and Infectious Diseases. 1997. DIADS virology manual for HIV laboratories, NIH-97-3828. U.S. Department of Health and Human Services, Washington, D.C.
6.	Hart, C. E., J. L. Lennox, M. Pratt-Palmore, T. C. Wright, R. F. Schinazi, T. Evans-Strickfaden, T. J. Bush, C. Schnell, L. J. Conley, K. A. Clancy, and T. V. Ellerbrock. 1999. Correlation of human immunodeficiency virus type 1 RNA levels in blood and the female genital tract. J. Infect. Dis. 179:871-882[CrossRef][Medline].
7.	Henin, Y., L. Mandelbrot, R. Henrion, R. Pradinaud, J. P. Couland, and L. Montagnier. 1993. Virus excretion in the cervicovaginal secretions of pregnant and nonpregnant HIV-infected women. J. Acquir. Immune Defic. Syndr. 6:72-75.
8.	Iversen, A. K., L. Fugger, J. Eugen-Olsen, U. Balslev, T. Jensen, S. Wahl, J. Gerstoft, J. I. Mullins, and P. Skinhoj. 1998. Cervical human immunodeficiency virus type 1 shedding is associated with genital beta-chemokine secretion. J. Infect. Dis. 178:1334-1342[CrossRef][Medline].
9.	Iversen, A. K., A. R. Larsen, T. Jensen, L. Fugger, U. Balslev, S. Wahl, J. Gerstoft, J. I. Mullins, and P. Skinhoj. 1998. Distinct determinants of human immunodeficiency virus type 1 RNA and DNA loads in vaginal and cervical secretions. J. Infect. Dis. 177:1214-1220[Medline].
10.	Kreiss, J. K., R. Coombs, F. Plummer, K. K. Holmes, B. Nikora, W. Cameron, E. Ngugi, J. O. Ndinya Achola, and L. Corey. 1989. Isolation of human immunodeficiency virus from genital ulcers in Nairobi prostitutes. J. Infect. Dis. 160:380-384[Medline].
11.	Loussert-Ajaka, I., L. Mandelbrot, M. C. Delmas, H. Bastian, J. L. Benifla, I. Farfara, I. de Vincenzi, S. Matheron, F. Simon, and F. Brun-Vezinet. 1997. HIV-1 detection in cervicovaginal secretions during pregnancy. AIDS 11:1575-1581[CrossRef][Medline].
12.	Mulder, J., N. McKinney, C. Christopherson, J. Sninsky, L. Greenfield, and S. Kwok. 1994. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection. J. Clin. Microbiol. 32:292-300[Abstract/Free Full Text].
13.	Nuovo, G. J., A. Forde, P. MacConnell, and R. Fahrenwald. 1993. In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. Am. J. Pathol. 143:40-48[Abstract].
14.	Pomerantz, R. J., S. M. de la Monte, S. P. Donegan, T. R. Rota, M. W. Vogt, D. E. Craven, and M. S. Hirsch. 1988. Human immunodeficiency virus (HIV) infection of the uterine cervix. Ann. Intern. Med. 108:321-327.
15.	Rasheed, S. 1998. Infectivity and dynamics of HIV type 1 replication in the blood and reproductive tract of HIV type 1-infected women. AIDS Res. Hum. Retroviruses 14(Suppl. 1):S105-S118.
16.	Rasheed, S., Z. Li, D. Xu, and A. Kovacs. 1996. Presence of cell-free human immunodeficiency virus in cervicovaginal secretions is independent of viral load in the blood of human immunodeficiency virus-infected women. Am. J. Obstet. Gynecol. 175:122-129[CrossRef][Medline].
17.	Vogt, M. W., D. J. Witt, D. E. Craven, R. Byington, D. F. Crawford, M. S. Hutchinson, R. T. Schooley, and M. S. Hirsch. 1987. Isolation patterns of the human immunodeficiency virus from cervical secretions during the menstrual cycle of women at risk for the acquired immunodeficiency syndrome. Ann. Intern. Med. 106:380-382.
18.	Vogt, M. W., D. J. Witt, D. E. Craven, R. Byington, D. F. Crawford, R. T. Schooley, and M. S. Hirsch. 1986. Isolation of HTLV-III/LAV from cervical secretions of women at risk for AIDS. Lancet i:525-527.
19.	Wofsy, C. B., J. B. Cohen, L. B. Hauer, N. S. Padian, B. A. Michaelis, L. A. Evans, and J. A. Levy. 1986. Isolation of AIDS-associated retrovirus from genital secretions of women with antibodies to the virus. Lancet i:527-529.

---

Journal of Clinical Microbiology, October 2000, p. 3822-3824, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Sherlock, C. H., Lott, P. M., Money, D. M., Merrick, L., Arikan, Y., Remple, V. P., Craib, K., Burdge, D. R. (2006). Use of Sno Strip Filter-Paper Wicks for Collection of Genital-Tract Samples Allows Reproducible Determination of Human Immunodeficiency Virus Type 1 (HIV-1) RNA Viral Load with a Commercial HIV-1 Viral Load Assay.. J. Clin. Microbiol. 44: 1115-1119 [Abstract] [Full Text]  
• Hartmann, S. U., Berlin, C. M., Howett, M. K. (2006). Alternative Modified Infant-Feeding Practices to Prevent Postnatal Transmission of Human Immunodeficiency Virus Type 1 Through Breast Milk: Past, Present, and Future. J Hum Lact 22: 75-88 [Abstract]  
• Ndjoyi-Mbiguino, A., Ozouaki, F., Legoff, J., Mbopi-Keou, F.-X., Si-Mohamed, A., Onas, I. N., Avoune, E., Belec, L. (2003). Comparison of Washing and Swabbing Procedures for Collecting Genital Fluids To Assess Cervicovaginal Shedding of Herpes Simplex Virus Type 2 DNA. J. Clin. Microbiol. 41: 2662-2664 [Abstract] [Full Text]  
• Andreoletti, L., Gresenguet, G., Chomont, N., Matta, M., Quiniou, Y., Si-Mohamed, A., Belec, L. (2003). Comparison of Washing and Swabbing Procedures for Collecting Genital Fluids To Assess Shedding of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Asymptomatic HIV-1-Infected Women. J. Clin. Microbiol. 41: 449-452 [Abstract] [Full Text]  
• Si-Mohamed, A., Andreoletti, L., Colombet, I., Carreno, M.-P., Lopez, G., Chatelier, G., Kazatchkine, M. D., Belec, L. (2001). Quantitation of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Cell-Free Cervicovaginal Secretions: Comparison of Reverse Transcription-PCR Amplification (AMPLICOR HIV-1 MONITOR 1.5) with Enhanced-Sensitivity Branched-DNA Assay (Quantiplex 3.0). J. Clin. Microbiol. 39: 2055-2059 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baron, P. Articles by Reichelderfer, P. S. Search for Related Content PubMed PubMed Citation Articles by Baron, P. Articles by Reichelderfer, P. S.