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  12th International HIV Drug Resistance Workshop
June 10-14, 2003, Los Cabos, Mexico
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Super-Infection and Co-Infection
Reported by Jules Levin
  There were two abstracts on super-infection and co-infection presented orally at this morning's sessions. Co-infection is when a person gets infected with two different strains of HIV at once, this is not to be confused with HCV/HIV co-infection. Super-infection is when a person gets infected by one strain of HIV and after time gets infected with a second and different strain of HIV. There is much discussion about this subject in the community and at this conference these two presentations caused quite a bit of discussion. How often does super-infection occur? We don't know because this question has not been studied. There have been several research reports identifying a small number of individuals with super-infection, but there has no been a concerted effort to conduct research to discover the prevalence. There appeared to be broad agreement at this conference that super-infection occurs and presents a health problem to individuals as well as a public health concern, and should receive further research attention to ascertain the prevalence, better document it's occurrence, and understand it better.
There are several concerns if a person gets super-infection with two different strains of HIV, which apparently can occur sexually or through IVDU: (1) one virus may be a more virulent or potent virus than the other and this may make a person sicker and develop HIV-related symptoms; (2) one of the viruses may be a drug resistant virus and may prevent the patient from responding to HIV-treatment; (3) viral load may increase and CD4 count might decline after becoming infected with a second virus. We don't know how often co-infection or super-infection occurs.
Sarah Palmer (HIV Drug Resistance Program, NCI, NIH, Frederick, Md) presented her study, which is reported below, and discussed one particular patient who is a patient of Eric Daar at UCLA. He is a 32 yr old homosexual male, and discussed in more detail below. He was infected with a multi-drug resistant virus (MDR) initially and later it appears super-infected with a wild-type virus. Subsequent to initial HIV-infection the patient reported an unprotected sexual encounter after which his viral load increased. He has now been on HAART for 1 month and responding well to therapy. Palmer is concerned that the MDR will emerge and he will fail therapy.
In the second abstract, discussed in more detail below, Luc Perrin, a noted HIV researcher for years, reported on his study of IVDUs and found 5% (n=3) in the study had co-infection and several had super-infection. 58 individuals with primary HIV-infection or recently infected were studied and 19% had drug resistant virus. The average viral load was about 100,000 copies/ml. The prevalence of co-infection and super-infection in IVDUs or transmitted sexually is not known and could be greater than Perrin found (5%) in this study. Perrin suggests IVDUs are at greater risk for co-infection and super-infection since sharing dirty needles permits direct insertion of another's virus into the recipient's blood. In order for HIV to infect another by sexual means the virus must pass through the mucosal system and other barriers.
Perrin made particular note of two long-term non-progressors who after they were super-infected experienced symptoms of HIV. One was aviremic (no viral load) for 5 years in the absence of HIV treatment and after a second strain of HIV was identified (super-infection) experienced acute retroviral syndrome (fever, fatigue, multiple lymphadenopathy) with viral load of 1 million. The second patient was aviremic for 3 years in the absence of HIV treatment then after a second strain of HIV was identified (super-infection) developed multiple lymphadenopathies, viremia at around 100,000 copies/ml and a drop in CD4 count from 1225 to 674. Perrin concluded that co-infection and super-infection are relatively frequent among IVDUs. Both strains persist for years in co-infected patients. He found one strain tended to replace the other. He stated that the successful control of the first strain is not associated with a better response or resistance to the super-infection.
There was quite a lot of discussion by participants at the microphone after the presentations. Almost all agreed the studies were adequately done to be convincing that super-infection did occur. One dissenter was Bob Grant who insisted that Palmer's testing was not conclusive. A number of other observers expressed they felt her test methodoloy was convincing and her conclusions compelling. I spent quite a bit of time talking with Perrin, Palmer, and researchers at the workshop immediately after the presentations. Every researcher I spoke with is convinced that super-infection occurs and perhaps much more often than we think. The problem is that we have not studied super-infection. We don't know how often this can occur, the prevalence, and we don't understand why some individuals are co-infected or super-infected. Some individuals may have innate host protection, and if we could study this we might understand more about HIV and the host immune system. This type of research might help develop therapy and protect individuals from super-infection. I think it's important to study super-infection. We have no idea of it's prevalence. We don't a firm understanding of response to HAART if a person has super-infection. Of course we don't know if viral non-response or failure is due to super-infection or co-infection.
Aditional NATAP coverage and analysis from this conference and on this subject is forthcoming from NATAP writers including Andrew Zolopa, MD, Stanford University. All NATAP reports are archived at the NATAP website: http://www.natap.org
Population genetics in HIV-1 super-infection
BACKGROUND: In recent reports of HIV-1 superinfection, both patients had received antiretroviral therapy for primary infection. In the present case, the patient was a homosexual man who had primary infection with a multidrug-resistant HIV-1 (Daar et al. 9th CROI). Within 2 months, viraemia declined to 1000 copies/ml without antiretroviral therapy. Approximately 4 months later, viraemia increased to 10000 copies/ml and showed no drug resistance by standard genotype analysis. We used two different analytical approaches to characterize the populations of virus and determine their origin.
METHODS: Plasma samples were obtained from the patient at 1, 5, 13 and 17 months after the onset of acute retroviral syndrome. DNA sequences from about 20 individual viral genomes were obtained for each sample using single genome RT-PCR sequence (SGS) analysis of the p6 region of gag, protease and the first 900 nucleotides of reverse transcriptase (RT). For minority species quantification, a target region containing mutant or wild-type sequence at position 103 was amplified, diluted and used as template for a round of allele-specific PCR, which was performed using either non-discriminating primers to quantify the total virus population or mutant discriminating primers specific for 103N.
RESULTS: Phylogenetic analysis revealed two genetically distinct virus populations (6% difference; both subtype B) at different times after infection. SGS analysis showed a nearly monomorphic multidrug-resistant viral population 1 month after infection (0.025% sequence diversity) with all sequences containing drug resistance mutations, including the 69SS insertion and K103N. Allele-specific PCR detected wild-type 103K virus at levels indistinguishable from assay background. Analysis of subsequent samples (5, 13 and 17 months after infection) showed wild-type virus with increasing genetic diversity over time. The wild-type virus present in the 5-month sample was nearly homogeneous by SGS (0.007% sequence diversity), indicating recent infection with wild-type virus. In the same sample, allele-specific PCR revealed a substantial population of virus with 103N (~20%). The wild-type virus popula-tion became more varied in the 13- and 17-month samples, showing 0.062 and 0.18% sequence diversity, respectively, while the K103N mutant virus declined to barely detectable levels (0.1%). There was no evidence of recombination (both viruses in one cell) between the wild-type and multidrug-resistant populations.
CONCLUSIONS: Our results indicate that this patient was initially infected with a multidrug-resistant virus, and was subsequently super-infected with a wild-type virus, which dominated the replicating virus population, leaving only a very small proportion of resistant virus (0.1% or less). Longitudinal samples showed the diversification of the wild-type super-infecting virus from a monomorphic population to a more heterogeneous one by 1-1.5 years after infection. The diversification of the super-infecting wild-type virus occurred slowly by de novo mutation rather than recombination with the initially infecting virus.
Co- and super-infection: persistent replication of both HIV-1 strains?
L Perrin, Laboratory of Virology Geneva University, Switzerland
BACKGROUND: In co-infected individuals two or more HIV-1 strains established close to the time of PHI (primary HIV infection, aka acute HIV infection), whereas in super-infection a second strain established several months to years after PHI. Does this lead over time to differential replication of the strains?
PATIENTS AND METHODS: Three intravenous drug users (IVDUs) co-infected with B subtype and CRF-11, two IVDUs initially infected with B subtype and later on infected with CRF-11. Population sequencing of reverse transcriptase, protease and gag p24, subtype-specific nested PCR with a first generic PCR (amplification of a 1397 bp gag/pol fragment -1872-3251 in ref to HXB2-) followed by a nested B and CRF-11- specific PCR. Population sequencing of the B and CRF-11 amplicons.
RESULTS: Using subtype-specific PCR and limiting dilutions with constant amount of the heterologous subtype (250000 HIV-1 RNA copies) and decreasing amount of the homologous subtype (1000-10 HIV-1 RNA copies), the detection limit was <10 copies of the homologous subtype in 250000 copies of the heterologous subtype, and there was no amplification of the heterologous subtype in presence of 250000 copies. In the three co-infected patients, both CRF-11 and B sub-types were detected in plasma and proviral DNA analysed over a follow-up of 14, 20 and 24 months, respectively. Two of the three patients had viraemia >400000 copies/ml during the follow-up. A genetic tree based on sequences of the B-specific amplicons of 10 IVDUs infected with B subtypes and of the three co-infected patients indicated that two of three co-infected patients were infected with different B strains. The two super-infected patients, initially infected by a B subtype, can be referred as LTNPs since they control their viraemia without treatment to <50 copies/ml and had <500 CD4/mm 3 for, respectively, 3 and 5 years before becoming infected with CRF-11. In these two patients, super-infection with CRF-11 was associated with high viraemia, steep drop in CD4 and an acute retroviral syndrome (ARS). CFR-11 was the only detectable subtype in the plasma at the time of super-infection and later on in follow-up samples. Both sub-types were detectable in proviral DNA after the super-infection and during the follow-up. Genetic trees performed on B and CRF-11 amplicons indicate that the patients were initially infected with different B strains, whereas results for CRF-11 were inconclusive due to the high degree of homogeneity of sequences of the CRF-11 amplicons for both the super-infected IVDUs and other IVDUs infected with CRF-11 only.
CONCLUSIONS: In co-infected patients both HIV-1 subtypes persist during the follow-up, whereas in super-infected patients, despite massive immune activation associated with the ARS at the time of super-infection, only the second strain was detectable in plasma.