Cervical Infiltrate and Genital HIV
• JID 2010:202 (15 November) • 1543
M A J O R A R T I C L E
Elevated Cervical White Blood Cell Infiltrate
Is Associated with Genital HIV Detection
in a Longitudinal Cohort of Antiretroviral
Tara Randolph Henning,1Patricia Kissinger,4Nedra Lacour,1Mary Meyaski-Schluter,3Rebecca Clark,2
and Angela Martin Amedee1
and Translational Research Center, Louisiana State University Health Sciences Center and Tulane University, and
of Epidemiology, Tulane University School of Public Health and Tropical Medicine, New Orleans, Louisiana
1Microbiology, Immunology, and Parasitology and
2Medicine, Louisiana State University Health Sciences Center,
in female genital secretions is critical for intervention strategies targeting transmission and eliminating replication
of genital virus. We sought to monitor the prevalence of genital HIV shedding in antiretroviral therapy–adherent
women over time and to assess changes in the genital microenvironment.
Levels of cell-free HIV (HIV RNA) and HIV-infected cells (HIV DNA) were monitored in peripheral
blood samples and cervical and vaginal fluid samples at monthly intervals in 11 women for 1 year. Genital tract
infections and fluctuations in cervical and vaginal white blood cell counts were also evaluated at each study visit.
Plasma HIV was undetectable at the majority of study visits; when detected, it was only at low levels.
Throughout the study, genital HIV RNA and DNA were detected in each person. Combined genital HIV (RNA
and DNA) was detected at 49.2% of study visits and was associated with an elevated concentration of cervical
white blood cell infiltrate (odds ratio, 2.52 [95% confidence interval, 1.01–6.22];
associated with a clinical disorder or patient-reported symptoms.
Despite antiretroviral therapy adherence and clinically suppressed plasma viremia, HIV was in-
termittently detected in genital secretions and was associated with subclinical inflammation and cells trafficking
to the cervical mucosa.
Identification of factors associated with the presence of human immunodeficiency virus (HIV)
). Infiltrate was notP p .04
The presence of human immunodeficiency virus (HIV)
in female genital secretions presents a risk for sexual
transmission and mother-to-infanttransmissionduring
parturition [1–5]. HIV replication in the genital tract
may also increase the risk of pathogenesis because of
compartmentalized replication and evolution of virus
Received 1 March 2010; accepted 1 June 2010; electronically published 6 October
Potential conflicts of interest: none reported.
Presented in part: 2009 Keystone Symposia on Prevention of HIV/AIDS, Keystone,
Colorado, March 2009 (abstract 402).
Financial support: Louisiana State University Health Sciences Center Translational
Research Initiative and the South Louisiana Institute for Infectious Disease Research
sponsored by the Louisiana Board of Regents.
Reprints or correspondence: Dr Angela Martin Amedee, Dept of Microbiology,
Immunology, and Parasitology, LSUHSC, Box P6-1, 1901 Perdido St, New Orleans,
LA 70112 (email@example.com).
The Journal of Infectious Diseases
? 2010 by the Infectious Diseases Society of America. All rights reserved.
genotypes [6, 7]. Identification of mechanisms respon-
sible for genital HIV shedding is critical to preventing
these adverse events.
Elevated plasma HIV levels and absence of antiret-
roviral therapy (ART) have been identified as predom-
inant correlates of cervicovaginal HIV detection in sev-
eral studies [8–12]. Despite strong correlations between
plasma viremia and genital shedding, HIV is still de-
tectable in genital secretions in women with low plasma
HIV levels, and several studies have documented mea-
surable levels of cervicovaginal HIV in ART-treated co-
horts [13–17]. Additionally, differential effects of ART
on levels and incidence of genital HIV have been ob-
more effective in reducing genital HIV levels; however,
the steady-state concentration and efficacy of ART in
the female genital tract is often drug specific [16, 18,
19]. Understanding of the dynamics of genital HIV
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1544 • JID 2010:202 (15 November) • Henning et al
expression in women receiving ART is critical for developing
regimens to prevent genital HIV shedding.
Perturbations to the genital microenvironment, such as sex-
ually transmitted infections, genital ulcerations, and vulvova-
ginal candidiasis (VVC), are also correlated with genital HIV
detection [11, 20, 21]. Additionally, other researchers have as-
sociated bacterial vaginosis or the absence of Lactobacillus spe-
cies with increased incidence of genital HIV shedding [8, 22,
23]. Recent studies have also shown a correlation between local
inflammatory responses and cytokine levels associated with
genital HIV detection [13, 24–26].
Most genital HIV shedding correlateshavebeenderivedfrom
cross-sectional studies evaluating a single time point in a dy-
namic disease course. Only a limited number of studies have
longitudinally evaluated genital HIV shedding, and they have
similarly associated genital HIV RNA and/or DNA detection
over time with higher plasma HIV RNA levels [14, 27, 28].
Other longitudinal studies have evaluated the effect of ART on
genital HIV detection, demonstrating a reductioninbothplasma
and genital HIV levels after initiation of therapy [15, 29]. How-
ever, limited longitudinal data are available to describe genital
HIV shedding in ART-adherent women, in whom plasma HIV
is undetectable or intermittently at a low level [30–32].
In both cross-sectional and longitudinal analyses of cohorts
with varying ART use and/or adherence, genital cellular infil-
trate has been associated with cervicovaginal HIV RNA detec-
tion [13, 33]. These analyses were derived from testing of cer-
vicovaginal lavage fluid, and it is unclear whether the cervix or
the vagina was the site of cell trafficking. Nkwanyana et al 
compared concentration and types of cervical cellular infiltrate
between HIV-1–infected women taking ART and those naive to
therapy. Infiltrate concentrations were similar in the 2 groups,
and data suggested that inflammation at the cervical mucosawas
the underlying cause . The relationship between cervical and
vaginal infiltrate and genital HIV shedding in ART-adherent
women has not been evaluated longitudinally.
Several studies have documented genital HIV shedding in
women with low levels of plasma virus [8, 16, 17]. However,
few studies have evaluated the prevalence of sheddingovertime
in an ART-adherent cohort or examined correlates of shedding
in the absence of plasma viremia. The role played byfreevirions
and infected cells in the transmission or the presence of HIV
in the genital mucosa also remains unclear. Chronically infect-
ed women adherent to ART regimens who have clinically sup-
pressed viremia present an ideal cohort to evaluate the mech-
anisms of genital HIV shedding independentof plasmaviremia.
In light of the widespread and increased use of ART, we sought
to assess the prevalence of genital HIV detection in a cohort
of ART-adherent women through monthly sampling of both
the cervix and the vagina to quantify cell-free and cell-asso-
ciated HIV levels and to identify associated changes in the
Orleans HIV clinic were enrolled and evaluated at monthly
intervals for 1 year (10 women attended 12 study visits, and 1
woman attended 10 visits). HIV-1–infected, ART-adherent,
nonpregnant women ?18 years old who had an intact cervix
were selected. Visits were not conducted during menstruation,
and overtly blood-contaminated samples were excluded from
analyses. Informed consent was obtained in accordance with
the Tulane University Health Sciences Center and Louisiana
State University Health Sciences Center Institutional Review
At each visit, patients completed questionnaires detailing de-
mographic characteristics, date of last menstruation, frequency
of vaginal sex and douching, and ART adherence. ART type and
CD4 T cell counts were obtained by medical record abstraction.
Cervical and vaginal samples were col-
lected on polyester-tipped swabs [13, 16, 34–36] during pelvic
examination by a single provider throughout the study. En-
docervical secretion samples were obtained by a 360-degree
rotation in the os, and vaginal secretion samples were collected
by rotating the swab along the vaginal vault. Afterward, a cy-
tobrush (Cooper Surgical) was inserted in the endocervical os
to collect cervical cells, placed in Roswell Park Memorial In-
stitute 1640 medium supplemented with gentamycin and am-
photericin B, and processed within 4 h of collection [25, 37].
Swab samples collected for genital HIV level analyses were
stored in 1 mL of RNAlater solution at 4?C until processed for
storage at ?80?C, as described elsewhere . Vaginalsecretion
samples were used to evaluate concomitant genital tract infec-
tion. The presence of Trichomonas vaginalis and yeast hyphae
and/or conidia were documented by the provider (wet mount).
Chlamydia trachomatis and Neisseria gonorrhoeae screenings
were conducted at 3-month intervals (GenProbe assay; Becton
Dickinson). Bacterial vaginosis diagnoses (Nugentcriteria)
were performed using Gram-stained vaginal smears. Inflam-
mation of the genital mucosa was assessed during pelvic ex-
amination on the basis of the presence or absence of erythem-
atous tissues and excessive or mucopurulent discharge.
Peripheral blood samples were collected in ethylenediamine-
tetraacetic acid–treated tubes and were fractionated by centri-
fugation. Plasma aliquots were stored at ?80?C; peripheral
blood cells (PBCs) were collected from the buffy coat layer, red
blood cells were removed by lysis, and cells were stored at
Measurement of HIV levels.
cell (HIV DNA) levels were quantified using a real-time poly-
merase chain reaction (PCR) assay targeting the conserved
HIV RNA and HIV-infected
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correlates inversely with Lactobacillus species but positively with bac-
terial vaginosis and Mycoplasma hominis. J Infect Dis 2005;191:25–32.
24. Ghanem KG, Shah N, Klein RS, et al. Influence of sex hormones, HIV
status, and concomitant sexually transmitted infection on cervicovag-
inal inflammation. J Infect Dis 2005;191:358–366.
25. Gumbi PP, Nkwanyana NN, Bere A, et al. Impact of mucosal inflam-
mation on cervical human immunodeficiency virus (HIV-1)-specific
CD8 T-cell responses in the female genital tract during chronic HIV
infection. J Virol 2008;82:8529–8536.
26. Nkwanyana NN, Gumbi PP, Roberts L, et al. Impact of human im-
and yield of cervical mononuclear cells in the female genital tract.
27. Cu-Uvin S, Snyder B, Harwell JI, et al. Association between paired
plasma and cervicovaginal lavage fluid HIV-1 RNA levels during 36
months. J Acquir Immune Defic Syndr 2006;42:584–587.
28. Iversen AK, Attermann J, Gerstoft J, et al. Longitudinal and cross-
sectional studies of HIV-1 RNA and DNA loadsinbloodandthefemale
genital tract. Eur J Obstet Gynecol Reprod Biol 2004;117:227–235.
29. Nagot N, Ouedraogo A, Weiss HA, et al. Longitudinal effect following
initiation of highly active antiretroviral therapy on plasma andcervico-
vaginal HIV-1 RNA among women in Burkina Faso. Sex Transm Infect
30. DeMasi RA, Graham NM, Tolson JM, et al. Correlation between self-
reported adherence to highly active antiretroviral therapy (HAART)
and virologic outcome. Adv Ther 2001;18:163–173.
31. Nachega JB, Stein DM, Lehman DA, et al. Adherence to antiretroviral
therapy in HIV-infected adults in Soweto, South Africa.AIDSResHum
32. Spire B, Carrieri P, Sopha P, et al. Adherence to antiretroviral therapy
in patients enrolled in a comprehensive care program in Cambodia: a
24-month follow-up assessment. Antivir Ther 2008;13:697–703.
33. Anderson BL, Wang CC, Delong AK, et al. Genital tract leukocytes and
shedding of genital HIV type 1 RNA. Clin Infect Dis 2008;47:1216–1221.
34. Benki S, Mostad SB, Richardson BA, et al. Cyclic shedding of HIV-1
RNA in cervical secretions during the menstrual cycle. J Infect Dis 2004;
35. Henning TR, Lacour N, Amedee AM. Efficient methodologies for sen-
sitive HIV-1 RNA quantitation from plasma and vaginal secretions. J
Clin Virol 2009;46:309–313.
36. Kissinger P, Amedee A, Clark RA, et al. Trichomonas vaginalis treat-
ment reduces vaginal HIV-1 shedding. Sex Transm Dis 2009;36:11–16.
37. Quayle AJ, Kourtis AP, Cu-Uvin S, et al. T-lymphocyte profile and
total and virus-specific immunoglobulin concentrations in the cervix
of HIV-1-infected women. J Acquir Immune Defic Syndr2007;44:292–
38. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial
vaginosis is improved by a standardized method of gram stain inter-
pretation. J Clin Microbiol 1991;29:297–301.
39. Endtz AW. A rapid staining method for differentiating granulocytes
from “germinal cells” in Papanicolaou-stained semen. Acta Cytol 1974;
40. Donder GG, Vereecken A, Bosmans E, et al. Definition of a type of
abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic
vaginitis. Bjog 2002;109:34–43.
41. Sobel JD. Nontrichomonal purulent vaginitis: clinical approach. Curr
Infect Dis Rep 2000;2:501–505.
42. Sheth PM, Kovacs C, Kemal KS, et al. Persistent HIV RNA shedding
in semen despite effective antiretroviral therapy. AIDS 2009;23:2050–
43. Money DM, Arikan YY, Remple V, et al. Genital tract and plasma
human immunodeficiency virus viral load throughout the menstrual
cycle in women who are infected with ovulatory human immunode-
ficiency virus. Am J Obstet Gynecol 2003;188:122–128.
44. Reichelderfer PS, Coombs RW, Wright DJ, et al; WHS 001 Study Team.
Effect of menstrual cycle on HIV-1 levels in the peripheral blood and
genital tract. AIDS 2000;14:2101–2107.
45. Quayle AJ, Shah M, Cu-Uvin S, et al. Implications of blood contam-
ination for assessment of local cellular immunity in the endocervix.
AIDS Res Hum Retroviruses 2004;20:543–546.
by guest on October 29, 2015