Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

8 Abstracts WePeA5717-WePeA5721

XIV International AIDS Conference

plasma were clearly distinct though phylogenetically related. In the remaining 3,
the strains appeared to be phylogenetically intermingled between the 2 sites and
included intrapatient HIV-1 recombinants in one individual. The preference of a
viral variant to use CCR5 (R5) vs. CXCR4 (X4) as a coreceptor may be predicted
by the sequence of the V3 portion of the env gene. The pattern of predicted coreceptor usage in the CVL and plasma also demonstrated compartmentalization,
with coreceptor preferences in 7 women differing in the 2 compartments. Two individuals displayed a larger proportion of X4 strains, those associated with CD4
decline, in CVL than in plasma.
Conclusion: HIV-1 sequences and predicted coreceptor usage showed striking
compartmentalization in CVL vs. plasma, suggesting that each site can serve as
a separate reservoir of replicating HIV-1 displaying different pathogenic characteristics; this variation may reflect different immune pressures in each site.
Presenting author: Kimdar Sherefa-Kemal, David Axelrod Institute, 120 New
Scotland Ave., Albany, NY 12208, United States, Tel.: [phone], Fax:
[phone], E-mail: [email protected]
WePeA571 7 Characterisation of epithelial cells from human
milk and their sensitivity to HIV strains
M. Ziccheddu, C. Serra, A. Biolchini, G. Arru, A. Dolei. Dept of Biomedical
Sciences, University of Sassari, Sassari, Italy
Background: Breast-feeding is responsible for one of the mucosal routes of HIV
transmission. Milk-borne transmission is thought to occur mainly via virus-infected
cells, since free virions are likely to be rapidly inactivated, mainly by child's saliva.
We studied the cells present in human milk, in order to clarify the role of the different cell types in mother-to-child transmission and selection of epithelial-adapted
variants, that could have a more efficient transmission to mucosal cells of the
child.
Methods: Human milk was obtained from healthy Italian mothers; Adherent cells
were kept in culture, then analysed for expression of specific antigens (by immunofluorescence) and co-receptors (by RT-PCR), and infected with T-tropic
(HIV-1 P1 and HIV-1P1-E) and M-tropic (HIV-1BaL) HIV strains. Virus binding and
replication were followed by p24, PCR and RT-PCR assays)
Results: Adherent cells were 15-25% of total milk cells. After 3-4 weeks
macrophages were no more present. The cells had epithelial morphology, were
more than 90% cytokeratin-positive, and positive for HGMF-1 e HGMF-2 (human
milk globulin fat antigen 1 e 2) and expressed both CXCR4 and CCR5 molecules.
Cultures resulted sensitive to all HIV strains used, with a preferential expression
of HIV-1P1-E, (that is an HIV variant adapted to epithelial cells) with respect to
the T-cell-derived HIV-1P1.
Conclusions: Data suggest that also the epithelial cells of human milk may become infected by HIV and constitute a possible vector of vertical transmission, in
addition to lymphocytes and monocytes. Morover the virus emerging from epithelial cells might have selective advantage for mucosal transmission.
Presenting author: Antonina Dolei, Dept.Biomedical Sciences, Sect. of Microbiology, Viale S.Pietro 43B, 1-07100 Sassari, Italy, Tel.: [phone], Fax:
[phone], E-mail: [email protected]
WePeA5718I Evaluation of a method to quantitate HIV-1 nucleic
acids in female genital tract
A. Gimeno1, J. Portilla2, J. Plazas1, J. Sanchez-PayA3, M. Ochando1,
E. Merino2, V. Boix2, C. Llopis1, J. Polache4, M.A. Pardo4, R. Scanchez2,
I. Gasc6n1. 1S. Microbiologa. Hospital General de Alicante, S. Microbiologa,
Hospital General De Alicante, Maestro Alonso 109, 03010 Alicante, Spain;
2Unidad de Enfermedades Infecciosas. Hospital General de Alicante, Alicante,
Spain; 3U. de Epidemiologfa. Hospital General de Alicante, Alicante, Spain; 4 S.
Farmacia. Hospital General de Alicante, Alicante, Spain
Objectives: To evaluate a method for sampling and processing cervico-vaginal
secretions to quantify HIV-1 nucleic acids (DNA+RNA) and HIV-1 RNA.
Methods: Cervico-vaginal secretions were aspirated directly HIV-1 positive
women. Samples were washed in 0.5 ml phosphate buffered saline (PBS). Sample weight was determined measuring PBS recipient weight before and after sample collection. Nucleic acids extraction was performed using silica columns with
QIAamp Viral RNA Kit previously evaluated in 30 plasmatic samples. HIV-1 blood
plasma viral load (BP-VL) was determined by Amplicor HIV-1 Monitor test.Vaginal
viral load (V-VL) was expressed in number of copies per gram. Reproducibility
was studied in 52-paired vaginal samples from 42 patients, obtained in an interval of no more than two days. Bland-Altman method was used to study method
reproducibility and Pearson coefficient (R) to study correlation between BP-VL
and V-VL. Number of copies are expressed in uLog.
Results: 154 cervico-vaginal samples were obtained from 61 women (median
BP-VL 3.23uLog, P25-P75: 0.00-4.64; V-VL median DNA+RNA 0.00, P25-P75:
0.00-4.48; and median RNA 0.00, P25-P75: 0.00-3.74). Method reproducibility:
mean difference between DNA+RNA copies obtained in two paired-samples was

0.1 uLog (SD+0.56) and for RNA copies was 0.10 uLog (SD~0.58). Correlation coefficient (R) between DNA+RNA copies in BP-VL and V-VL was R=0.55
(p<0.01) and for RNA copies between BP-VL and V-VL, R= 0.54 (p<0.01).
Conclusions: Aspirating secretions from genital tract and weighting sample collected can be used as a method to quantify HIV-1 viral load in female genital
tract after isolation nucleic acids in silica columns. A significant correlation exists
between BP-VL and V-VL.

Presenting author: Adelina Gimeno, S. Microbiologfa, Hospital General De Alicante, Maestro Alonso 109, 03010 Alicante, Spain, Tel.: [phone], Fax:
[phone], E-mail: gimenolin @ gva.es
WePeA5719I HIV-1 provirus in the peripheral CD4+ T
lymphocytes from the HIV-1 infected patients
under highly active antiretroviral therapy
T. Kaneda, T. Hagiwara, J. Hattori, M. Utsumi. Department of Clinical Research,
Nagoya National Hospital, Department of Clinical Research, Nagoya National
Hospital, 4-1-1 Sannomaru Naka-ku, Naogya, 460-0001, Aichi, Japan
Background: Using a PNA-probed in situ hybridization method (PNA-ISH), we
detected the HIV-1 provirus in peripheral CD4+ T lymphocytes from HIV-1 infected
individuals under highly active antiretroviral therapy (HAART), and determined its
positivity.
Methods: Samples: 62 samples of CD4+ T lymphocytes were purified from HIV1 infected individuals. The viral loads of 30 samples were <50 copies/ml, and
that of 32 were between 1.lx102 and 2.1x105 copies/ml. Fixed smear specimens
were incubated with a FITC-labeled PNA probe. After hybrid formation, an HRPlabeled anti-FITC antibody was reacted, and the signal was amplified by the catalyzed signal amplification method. We evaluated the HIV-1 provirus by detecting
the fluorescent signal of Alexa488.
Results: Positive signals were observed as dots, and one or two were present in
the cell nucleus. HIV-1 provirus positivity in the CD4+ T lymphocytes in the total
cases was 0.3-7.9% (average 2.7%). In the samples with VL>50 copies/ml, the
rate was 0.3-7.9% (average 2.7%), and that with VL<50 copies/mi was 0.8-6.1%
(average 2.7%). Even in successful cases of HAART, CD4+ T lymphocytes preserving the provirus still remain. HIV-1 provirus positivity had no correlation with
either the viral load or the CD4+ T lymphocyte count. HIV-1 provirus positivity in
3 HAART-receiving patients was measured 3 times in a year, and the tendency to
progressively decrease was observed.
Conclusion: The PNA-ISH method for detecting HIV-1 provirus is useful to estimate the efficacy of HAART in HIV-1 infected patients.
Presenting author: Tsuguhiro Kaneda, Department of Clinical Research,
Nagoya National Hospital, 4-1-1 Sannomaru Naka-ku, Naogya, 460-0001, Aichi,
Japan, Tel.: [phone] (ext.2770), Fax: [phone], E-mail: kanedat
@ nagoya.hosp.go.jp
WePeA5720 Detection and quantification of HIV-1 provirus by
real-time PCR and PNA-ISH
K. Wada, H. Nagai, T. Hagiwara, N. Hotta, M. Utsumi, T. Kaneda. Department of
Clinical Research, Nagoya National Hospital, Department of Clinical Research,
Nagoya National Hospital, 4-1-1 Sannomaru Naka-ku 460-0001 Nagoya, Japan
Background: HIV-1 proviral DNA persists in CD4+ cells in peripheral blood and
lymphoid tissue even in patients with a suppressed plasma RNA level. Therefore,
a sensitive method for quantifying total HIV-1 DNA in infected cells is required for
monitoring highly active antiretroviral therapy
Methods: The PCR primers and Taqman type probe were designed in the conserved regions of the HIV-1 gag gene. Total DNA was extracted from purified
CD4+ T lymphocytes, and then HIV-1 DNA in the DNA extract was quantified using Roche LightCycler. HIV-1 provirus positively was simultaneously determined
by peptide nucleic acid-probed in situ hybridization (PNA-ISH). Twenty-nine patients receiving HAART and 5 naiive patin e ents were enrolled in this study.
Results: The minimum level of detectable HIV-1 DNA was 5 copies(c)/assay. In
13 HAART-receiving patients with viral load (VL)>50 c/m, the levels of HIV-1
DNA ranged from 1,000 to 62,000 c/106 cells except 2 cases which were below the detectable level. The results agreed with those obtained by PNA-ISH. In
HAART-receiving patients with VL<50 c/ml, 11 of 16 cases were lower than the
detectable level.
Conclusion: High HIV-1 DNA levels found in naive patients mainly reflect the existence of unintegrated HIV-, suggesting active reverse transcription in cells. The
quantification methods of total HIV-1 DNA established in this study will contribute
to therapy-monitoring during HAART.
Presenting author: Kaoru Wada, Department of Clinical Research, Nagoya National Hospital, 4-i-i Sannomaru Naka-ku 460-0001 Nagoya, Japan, Tel.: [phone] ext.2770, Fax: [phone], E-mail: [email protected].
JP
WePeA5721] Response of HIV RNA carrying CD4+ and CD8+ T
lymphocytes in HIV infected patients during
HAART using ultrasensitive fluorescence in situ
hybridisation (UFISH)
A. Rubbert, A. Woehrmann, D. Passon, A. Juete, B. Salzberger,
G. Faetkenheuer. Med Dep I, University of Cologne, Med Klinik I,

Joseph-Stelzmann Str 9, 50924 Cologne, Germany, Germany
Background: CD8+ T lymphocytes in HIV infected patients have recently been
shown to harbor HIV RNA. Little is known about how this viral reservoir within
CD8+ lymphocytes responds to HAART therapy
Methods: PBMC were obtained from 25 HIV infected patients before and after initiation of HAART at 3, 6 and 12 months and cells were assessed by immunophe