Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

772 Abstracts 41148-41152

12th World AIDS Conference

Methods: Twenty three HIV infected babies were recruited from the MRC
transmission study; 17 were HIV-1 of whom six died before the age of one year,
and six were infected with HIV-2. Perinatal mother-to-child HIV transmission was
diagnosed if the baby was positive by PCR before 9 months of age, and postnatally if negative by PCR before 9 months of age but positive at 18 months.
Proviral DNA load in PBMC was quantitated by PCR using HIV-1 and HIV-2
specific primers and external controls.
Results: (Table)
Geometric mean (~ SD) of proviral DNA copies/10 PBMCs (log 10)
2 month 9 month 18 month
HIV-1 infected babies (n = 17)
Perinatal
Died (n = 6) 2.962 ~ 1.343a 2.375 ~ 0.046
Survived (n = 5) 1.171 ~ 1.209a 2.207 ~ 1.741 1.501 ~ 1.124b
Postnatal (n = 5) 0.301 1.977 ~ 0.919b
Unclassified (n = 1) N.D. 4.0 N.D.
HIV-2 infected babies (n = 6)
Perinatal (n = 4) 2.554 ~ 0.409C 2.107 ~ 0.535d
Postnatal (n = 2) 0.300 ~ 0.000c 1.004 ~ 0.320d
ap < 0.05; bp < 0.1; Cp = 0.002; dp = 0.084; Not done
Conclusions: No significant difference in DNA viral load between HIV-1 and
HIV-2 infected babies. A high DNA viral load was strongly associated with poor
survival within the first year of life in babies infected perinatally with HIV-1 (p <
0.05). Therefore, an early assessment of DNA viral load in children infected with
HIV might be of use in estimating prognosis, and in management.
41148 Viral load and clinical manifestations in patients with HIV
infection
Maia Butsashvili, T.K. Shartava, O.V. Chubinishvili. Georgian AIDS & Clin.
Immun. Center 16, Al Kazbegi Av. Tbilisi 380060, Georgia
Objectives: To study the relationship between viral load and clinical manifestations in patients with HIV infection.
Design: Cross-sectional study
Methods: 34 adult patients with confirmed HIV infection by Western Blot and
qualitative PCR methods were included in the study. HIV viral load was measured by quantitative PCR method. Diagnostics of opportunistic infections was
performed by clinical and laboratory (Culture, ELISA and PCR) investigations.
Three groups of patients were separated according to the level of viral load: 1st
group - 11 patients with low level of viral load (<10 000 copies/ml); 2nd group -
13 patients with medium level of viral load (10,000-100,000 copies/ml); 3rd group
- 10 patients with high level of viral load (>100,000 copies/ml).
Results: The frequency of clinical manifestations was as follows:

41150 I A high input PCR method (Mega-PCR) for the detection of
HIV-1 DNA
Jurg Boni1, C. Shah2, M. Fleep3, R. Luthy3, J. Schupbach2. 1Univ Zurich, Natl.
Center F Retroviruses, Gloriastrasse 30, 8028 Zurich; 2Swiss National Center F
Retroviruses, 3Infectious Diseases, University Hospital, Zurich, Switzerland
Objectives: To establish an internally controlled high input PCR method for the
detection of HIV-1 provial DNA and to evaluate the method in a model system of
pooled blood samples.
Methods: EDTA blood from HIV-1-infected individuals was obtained with informed consent. Buffy coats were obtained via the local blood transfusion center.
Pooling of blood samples was imitated by mixing 0.3 ml of a patient's blood into
buffy coat containing 50 Mio. white blood cells. DNA was extracted by proteinase
K/phenol extraction after Ficoll hypaque purification of PBMC and 93% of the extracted DNA was used for Mega-PCR analysis. DNA was hybridized to two biotin
labeled probes directed to each strand of HIV-1 proviral DNA and located outside
the amplified sequence. Hybrids were then bound to paramagnetic strepavidin
beads and unbound DNA removed by washing. Bound HIV-DNA was identified
by a first round of duplex PCR of two gag regions followed by second round
amplification with only one primer pair and ELISA detection of the amplicon.
Efficient provirus DNA selection and amplification were controlled for by inclusion
of two mimic plasmids at low copy numbers. Negative buffy coat samples were
included during each step of the analysis.
Results: 100% (106/106) of the specimens from HIV infected individuals were
positive by a PCR protocol using 1 tig DNA and the same primers as in the
Mega-PCR. Amplification of 1 /gg DNA from pooled samples resulted in 64%
(68/106) double positive, 13% (14/106) single positive, and 23% (24/106) double
negative reactions. Average input in the Mega-PCR was 260 ~ 84 ig DNA
with an estimated of 1.1 ~ 0.6% patient DNA. 96% (102/106) samples scored
positive and 3% (3/106) negative in the mega-PCR. 1% (1/106) failed to select
the mimic plasmid. Dilution experiments showed that the detection limit of the
Mega-PCR was at about 5 copies/reaction. Further analysis of the three false
negative cases indicated that very low copy numbers but not sequence variations
were responsible for the negative results. Among the negative buffy coat controls
0% (0/107) were false positive and 4% (4/107) exhibited a failure of the internal
reaction controls.
Conclusion: Mega-PCR is a highly sensitive and specific PCR method which
lowers the detection limit to about 0.01 HIV-1 DNA copies//zg of applied DNA
and has a wide field of potential applications, including the detection of other rare
microrganisms.
411511 A multiplex nested PCR assay for the detection of six
clinically important herpesvirus genomes
Stuart Kirk, R.S. Tedder, M.R. Howard. Dept. of Virology, UCLMS, 46 Cleveland
St., London, UK
Background: Rapid and accurate detection of herpesvirus infections in the immunocompromised individual is vital for effective patient management. Recently
the assay of choice for such analysis has been the polymerase chain reaction,
however large-scale use of this assay is expensive and time-consuming.
Methods: To overcome the problems inherent in testing clinical samples for a
number of herpesvirus genomes by individual nested PCR (nPCR) amplification,
we have developed a multiplex PCR assay with an enzyme-linked oligonucleotide
detection system (mPCR-ELONA). This assay allows simultaneous detection of
HSV-1, HSV-2, VZV, EBV, CMV and KSHV DNA.
Results: The multiplex assay was found to have the same detection sensitivity, of a theoretical single target genome as our established nPCR's reactions.
Analysis of a panel of 192 clinical samples by both individual and multiplex PCR
showed a 98% concordance. Concurrent analysis for all six herpesvirus genomes
in the multiplex assay has been found to provide a saving of approximately 60%
in terms of reagent cost and set up time.
Conclusion: The mPCR-ELONA assay has proved to be as effective a technique for detection of herpesvirus genomes as our previously established individual nPCR's. The assay allows rapid screening for six clinically significant
herpesviruses on a single sample volume. Such a multiplex assay has significant
advantages for both diagnostic and research virology in comparison to individual
genome detection techniques.
41152 Sputum induction and ligase chain reaction as adjuncts
to the diagnosis of sputum negative pulmonary
tuberculosis
Majid S. Twahir1, D.W. Muhindi2, Gathua3, F.S. Rana2, J.J. Bwayo2. 1PO. Box
19978, Nairobi; 2University of Nairobi; Nairobi3 Ministry of Health, Nairobi, Kenya
Background: The world health organisation (WHO) has recently identified as a
global crisis. Case rates of tuberculosis (TB) have soared since the HIV pandemic. Diagnosis of Pulmonary tuberculosis (PTB) has traditionally been based

on examination of Ziehl Neelsen (ZN) stained sputum smears, and where necessary TB cultures. In patients with dual infection, PTB and HIV, the clinical and
radiological features maybe protean and atypical. Due to the pathogenesis of
tuberculosis in HIV infected patients, most are unable to expectorate sputum and
hence there is no specimen to examine. In the few instances when this group
of patients with dual infection can produce sputum the bacilli pick up rate using
ZN staining is low. TB cultures traditionally take long and may delay diagnosis
thereby impacting on spread, and therefore incidence, of PTB.

Group Pneumo- Herpes-simplex Cyto- CNS Oral and
(number cystosis & Varicella- megalo- manifes- esoof & zoster virus viral tations phageal
patients) infections infection candidiasis
1 (n=11) 1 4 0 0 2
2(n=13) 7 6 1 1 7
3 (n=10) 7 7 3 4 0

Wasting Tuber- Mycobacsyn- culo- terium
drome sis avium
complex
0 1 0
2 3 0
3 3 1

Conclusion: The correlation between the level of viral load and frequency of
various clinical manifestations of HIV infection has been observed.
41149 Importance of CMV load as a risk factor for CMV disease
Kirsten Schliefer1, B. Kupfer2, J.K. Rockstroh1, R. Kaiser2, M. Addo1, B. Matz2,
U. Spengler1. 1Department of Internal Medicine; 2Department of Microbiology -
University of Bonn, Sigmund Freud Str. 25, Bonn, Germany
Objective: Early diagnosis of imminent CMV infection in HIV-infected patients is
important for initiating primary CMV prophylaxis or early preemptive therapy.
Methods: To identify an useful strategy for this situation, we monitored CMVspecific IgG and IgM antibodies, CMV antigen, quantitative CMV-DNA (Hybrid
Capture System, HCS), NucliSens pp 67 CMV-mRNA, shell vial assay and virus
isolation from urine (VI) in monthly samples from 41 HIV-infected patients with
AIDS or CD4 T-cell counts below 100 x 106/1 over a mean of 4 months (range
1-7).
Results: 83% (34/41) had CMV IgG at baseline and 2/41 patients (5%) also
had CMV-IgM antibodies. VI was possible in 24% (10/41) and the shell vial
assay was positive in 20% (8/41). 17% (7/41) displayed CMV-DNA (HCS) >1500
copies/ml (range 1645-37000 copies/ml). In 4 of the patients with CMV-DNA
>1500 copies/ml CMV-antigen became positive during follow up, and in two of
them CMV-mRNA could be found at the same time. In these two patients a
relapse of CMV-retinitis occurred. The third patient received preemptive CMVtherapy (intravenous ganciclovir for 21 d) which could prevent CMV-disease. With
therapy HCS, antigen and VI disappeared. The fourth patient has not developed
CMV-disease so far, probably because highly active antiretroviral therapy was
introduced.
Conclusion: Positive VI, shell vial assay and IgM antibodies are apparently not
associated with subsequent CMV-reactivation. In contrast, elevated CMV-DNAlevels and the presence of mRNA may be useful predictors of imminent CMV-disease.