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HIV-1 co/super-infection in intravenous drug users
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AIDS: Volume 18(10) 2 July 2004
Yerly, Sabinea; Jost, Stéphaniea; Monnat, Martineb; Telenti, Amalioc; Cavassini, Matthiasc; Chave, Jean-Philipped; Kaiser, Laurenta; Burgisser, Philippee; Perrin, Luca; and the Swiss HIV Cohort Study
From the aLaboratory of Virology and AIDS Center, Geneva University Hospital, the bCenter Saint-Martin, Lausanne, the cAIDS Center, Lausanne University Hospital, the dLa Source Hospital, Lausanne and the eDivision of Immunology, Lausanne University Hospital, Switzerland.
Received: 26 December 2003; revised: 5 March 2004; accepted: 29 March 2004.
Author's conclude: "…..Overall the data presented [in this study] indicate that the frequency of co- and super-infection is relatively high in IVDUs, and that some individuals who are able to control a first infection very efficiently are still susceptible to infection by a second strain. This not only has obvious consequences for the development of vaccines based on single HIV-1 strain but also indicates that all HIV-1 infected persons should adhere strictly to the guidelines aiming at the prevention of HIV-1 transmission….."
Abstract
Background: The frequency of HIV-1 co/super-infection is unknown despite their implications for public health and vaccine development. This issue was addressed during an epidemic of both CRF11 and B subtype among intravenous drug users (IVDUs).
Methods: Bulk sequencing of reverse transcriptase, protease and C2V3 regions and subtype-specific nested polymerase chain reaction (PCR) in plasma and proviral DNA were performed using baseline and follow-up samples collected in recently infected IVDUs between 1998-2002 and in IVDUs with chronic infection living in the same area and presenting an unexpected rise of viremia (> 1 log10).
Results: In 58 recently infected patients, three B/CRF-11 co-infections, 25 B, 28 CRF-11 and two other subtypes were detected at baseline. In the three co-infected patients, both CRF-11 and B were detected in plasma and proviral DNA and persisted during follow-up. B- and CFR-11-specific PCR performed on follow-up samples of 40 of 58 recently infected patients (median follow-up, 14.5 months) revealed a transient B super-infection in a patient initially infected by CRF-11. Five of 156 chronic IVDUs (total follow-up: 346 years) had an unexpected rise of viremia. In two of them, aviremic without treatment for years after an initial B infection, a symptomatic CRF-11 super-infection occurred and was associated with high viral load and a fall of CD4 cell count.
Conclusions: In recently infected IVDUs, co-infection B/CRF-11 is relatively frequent (5%). In chronically infected IVDUs super-infection may be transient and may occur in patients controlling efficiently HIV infection by the initial strain.
Author's Discussion
We estimated the frequency of HIV-1 co and super-infection in IVDUs actively using cocaine and heroin in an area where both B and CRF-11 subtypes are present in IVDUs. It is well appreciated that cocaine usage is associated with a compulsive comportment and may increase the risk of unsafe practices such as sharing of the injection material. Within this population we show that co-infection is relatively frequent and that super-infection is not a rare event. All the detected co and super-infection occurred between CRF-11 and B subtype. Selection of patients for the detection of super-infection in chronically infected patients was based on an unexpected rise of viremia. As shown for patient LS 650, transient super-infection can occur without viremia change; thus we might have missed some cases. Consequently, the frequency of super-infection was only assessed strictu sensu in the 40 patients with follow-up but not in the 156 IVDUs with chronic infection for whom the selection was based on 1 log10 increase of viremia.
In the present investigation we have taken advantage of the recent spreading of a non-B circulating recombinant form in the western part of Switzerland, an area where B subtype used to be the only circulating subtype in IVDUs. We developed a subtype-specific RT-PCR of high sensitivity and specificity resulting in the possibility to detect less than 0.1% of the heterologous strain in patients with 10 000 HIV-1 RNA copies/ml and a much lower percentage in those with higher viremia. This RT-PCR also had the advantage over a cloning approach, of a larger capacity.
The frequency of 5% of co-infection observed in this study is a minimal estimate since we have not explored the frequency of co-infection with two different B or CRF-11 strains. The high occurrence of co-infection and super-infection detected is in line with the observation of an ever increasing diversity of circulating HIV-1 strains which is only partially reflected in the current subtypes and circulating recombinant forms nomenclature. Owing to the relatively high frequency of co/super-infection detected in our population of IVDUs and to the fact that they are living in the same geographical area, one may wonder whether these patients have been infected with the same B and/or CRF-11 strain during the same event. This was excluded for B strains since B-specific amplicons did not cluster upon phylogenetic analysis. The same analysis for CRF-11 sequences suggest that two of the super-infected patients were infected by the same infected patients since the patient's history excluded transmission of the infection from one to the other. Finally, the time frame supports separate events occurring over a 2-year period with two events in early 2000, three in late 2000, and one in early 2002.
In terms of pathogenicity, it is not surprising that two HIV-1 strains would establish and replicate in an individual infected concomitantly by two strains or within weeks since specific immune responses are not fully expressed. In the three co-infected patients both strains persisted during the observation period varying from 14 months to more than 2 years suggesting that the B and CRF-11 strains had similar growth kinetics and/or that HIV-1-specific immune responses control HIV-1 replication of the two strains to a similar degree. The efficacy of the immune responses appears to be low in two of the three co-infected IVDUs who had viremia levels > 100 000 copies/ml 6 months after the infection. The low number of events precludes generalization on whether infection with multiple strains has an impact on the magnitude of viral replication. However, a recent contribution indicate that co- and super-infection are associated with more rapid progression.
The biological and clinical history of two of the three super-infected patients is evocative of that of long-term non-progressors since they exhibited control of viremia and had normal CD4 cell counts for several years in the absence of antiretroviral therapy. The current consensus is that these patients have raised specific immune responses that are able to control viral replication since the other possibility, namely control associated with particular host genetic polymorphism(s) involved in protection is probably excluded in view of the outcome of the second infection. At the time of super-infection, both patients were symptomatic and had high viremia levels that remained higher than 100 000 HIV-1 RNA copies 6 months later. This evolution is in contrast to the milder course of experimentally induced second infection in macaques immunized with attenuated simian immunodeficiency virus strain(s) and later on challenged with another strain. It is however similar to what has been observed in other patients with super-infection. More direct evidence of the low efficacy of cross protection mediated by specific cell-mediated responses derives from another case report. This last patient with an initial and a super-infection due to B subtypes had immune responses directed against multiples HIV-1 T-cell epitopes shared by both the initial and the second B strain before super-infection.
There are a wide variety of likely outcomes following exposure to a second viral strain. Indeed, the first patient with a super-infection in the current study had only a transient expression of the second strain in plasma and at a much lower level than the initial strain. This patient, in contrast to the other super-infected patients, had high viremia for years including the time of super-infection. Thus, transient expression of a second viral strain may occur frequently in viremic patients and be unnoticed. The search for super-infection in our study was based on a sensitive technique but was limited by the number of samples tested, especially for the population of chronically infected patients whose selection was driven by an unexpected rise of viremia. The detection of a transient infection achieved with a very sensitive method modulates the conclusions derived from the other reported cases of super-infection including those reported in the present investigation. Indeed, if super-infection is more frequent than expected and usually transient, that means that for most of the time a second virus can not establish a productive infection probably as a consequence of cross-protective immune responses or due to unfavorable growth kinetics. A recent study in IVDUs in an area where HIV-1 B subtype is predominant fails to detect superinfection in 37 IVDUs; this may indicate that cross-protection is usually subtype dependent.
Assessment of cell-mediated responses in the three patients reported here, which could potentially provide clues on this issue, was prevented by the unavailability of PBMCs in sufficient amount.
Overall the data presented indicate that the frequency of co- and super-infection is relatively high in IVDUs, and that some individuals who are able to control a first infection very efficiently are still susceptible to infection by a second strain. This not only has obvious consequences for the development of vaccines based on single HIV-1 strain but also indicates that all HIV-1 infected persons should adhere strictly to the guidelines aiming at the prevention of HIV-1 transmission.
Introduction
Co-infection refers to the acquisition of two or more HIV-1 strains at the time of acute infection or in the following weeks, whereas super-infection refers to infection with a new HIV-1 strain in an individual with an established HIV-1 infection. Co-infection and super-infection have been reported in a handful of HIV-1 infected individuals. There are no data available on the frequency of these events. However, the characterization of new circulating recombinant forms (CRFs) and/or of unique recombinant forms in increasing numbers over recent years suggests that co-infection and super-infection are occurring relatively frequently. One of the difficulties in assessing the frequency of these events derives from the fact that most studies are performed in patients infected with HIV-1 B subtype in whom it is relatively difficult to distinguish a new B strain from variants derived from the original strain.
Over recent years in Western Europe an increase in the prevalence of HIV-1 non-B infections has been reported and nowadays up to 35% of recent infections in Switzerland are due to non-B subtypes. Up to 1998, European intravenous drug users (IVDUs) were infected only by the B subtype. Since then, the A subtype has spread in Eastern European IVDUs and more recently CRF-03 emerged in Russia, probably following local recombination between A and B subtypes. More recently, geographically localized epidemics of non-B subtypes in IVDUs have been characterized in Finland, Spain and Switzerland. In Switzerland, the non-B epidemic is due to CRF-11, a circulating recombinant form whose genome is a mosaic of A, G, E and J subtypes. CRF-11 is detected only in IVDUs living in the western part of the country.
For this investigation, we took advantage of the ongoing spread of both CRF-11 and B subtype in IVDUs living in the same geographical area to assess the frequency of both co-infection and super-infection.
Methods
Patient population and sample selection
For the evaluation of co-infection frequency, all IVDUs with documented HIV-1 recent infection between January 1998 and December 2002 in the Western part of Switzerland, namely the Geneva and Lausanne AIDS Centers of the Swiss HIV Cohort Study (SHCS) were included. Recent infection refers either to acute infection or to the documentation of an HIV-1 antibody negative test less than 1 year before the collection of an antibody positive sample. The first available plasma and/or peripheral blood mononuclear cell (PBMC) sample was analyzed for the evaluation of the frequency of co-infection.The frequency of super-infection was assessed in both patients with recent infection (samples collected > 6 months after the baseline sample) and in IVDUs with chronic infection. The selection of IVDUs with chronic infection was based on a documented infection for more than 2 years, a residence in the same geographical area as recently infected IVDUs and an HIV-1 viremia increase of more than 1 log10 unrelated to treatment change. The selection was derived from the database of the SHCS and from all IVDUs patients followed at the Geneva and Lausanne AIDS Centers not included in the SHCS.
Results
Frequency of co-infection in newly infected IVDUs
More than two-thirds of the 58 recently infected IVDU study participants consume cocaine and heroin. Twelve patients were tested at the time of acute HIV-1 infection, whereas 46 had a negative antibody test less than 12 months before collection of the sample(s) analyzed. The median HIV-1 viremia was 4.9 log10 RNA copies/ml (range, 3.0-7.4), a similar proportion of patients were infected with B and CRF-11 subtypes.
Phylogenetic analysis based on reverse transcriptase sequences from 55 patients (sequences from the three co-infected patients are not included) revealed a high bootstrap value (975/1000) for the large cluster of patients infected with CRF-11. Due to the recent spreading of CRF-11 in IVDUs population, the genetic diversity of CRF-11 sequences was lower than that of B sequences. Six clusters of two to four individuals were observed for B subtype and four of them were confirmed by contact tracing. Similar results were obtained using trees based on protease and C2-V3 sequences (data not shown). Twenty-five sequences from IVDUs patients with recent infection living in the eastern and northern part of Switzerland were all of B subtype (data not shown).
Using subtype-specific PCR, three of 58 (5%) IVDUs were found to be co-infected with B subtype and CRF-11. Both subtypes were detected in amplicons derived from PBMC collected at the same time. Follow-up samples were available from these three patients and persistence of both strains was observed in all plasma tested. Sequences of the specific PCR amplicons were determined in the three co-infected patients, and in 10 IVDUs infected with B and CRF-11 respectively. Phylogenetic analysis derived from these sequences showed that the co-infected patients were infected with different B strains, the phylogenetic tree derived from sequences of the CRF-11 amplicons was not conclusive due to the low heterogeneity of CRF-11 strains (data not shown). The histories of the three co-infected patients are given here.
Patient LS1 693 was a 32-year-old female consuming cocaine and heroin who presented with an acute retroviral syndrome with 7.4 log10 HIV-1 RNA copies/ml and 528 x 106 CD4 cells/l (first sample analyzed). After 9 months of triple therapy viremia was still 3 log10 and treatment was interrupted due to poor adherence. For the next 2 years viremia remained at more than 6 log10 and the CD4 cell count declined to 195 x 106 cells/l .
Patient LS0 761 was a 36-year-old acutely infected female consuming cocaine and heroin with 5.6 log10 HIV-1 RNA copies/ml and 259 x 106 CD4 cells/l at presentation (first sample analyzed). Viremia fluctuated between 5 and 6 log10 for the next 2 years and her last CD4 cell count was 201 ¥ 106 CD4 cells/l in the absence of treatment.
Patient LS0 766 was a 29-year-old female consuming cocaine and heroin. She had a negative HIV-1 antibody test 10 months before she presented with a viremia of 4.5 log10 HIV-1 RNA copies/ml and CD4 count of 928 x 106 cells/l (first sample analyzed). Fourteen months later, her viremia was 4.2 log10 and her CD4 cell count was 850 x 106 cells/l in the absence of treatment.
Frequency of super-infection
The frequency of super-infection was assessed in both follow-up samples collected in 40 of 58 recently infected IVDUs and in five of 156 IVDUs with established infection with an HIV-1 viremia increase of more than 1 log10 not related to treatment changes.
Follow-up samples were available for 40 of 55 patients with recent infection with a median follow-up of 14.5 months (range, 6-60). Super-infection was detected in a single 41-year- old female patient (LS 650), cocaine user, initially infected with CRF-11 (viremia, 5.11 log10 HIV-1 RNA copies/ml). This patient was not treated. In a plasma collected 24 months after acute infection (viremia, 5.12 log10 HIV-1 RNA copies/ml) both CRF-11 and B subtype were detected. In this sample, based on limiting dilutions, the concentration of CRF-11 was 1000-fold higher than that of the B strain (detection of B-specific amplicons up to a 10 dilution versus detection of CRF-11-specific amplicons up to a 10 000 dilution, data not shown). Sequencing of subtype-specific PCR confirmed the presence of both CRF-11 and B strains in this plasma sample (data not shown). In the next plasma sample collected 6 months later, only CRF-11-specific amplicons were detectable. Analysis of all samples was repeated twice in separate experiments to exclude a potential contamination. This patient had recurrent episodes of bacterial endocarditis, no symptoms were associated with the transient super-infection, and CD4 count and viremia were stable.
Five IVDUs with chronic infection out of 156 IVDUs followed for a total of 346 years presented an unexpected 1 log10 increase in viremia not associated with treatment change. In two of them, previously infected with B subtype, a super-infection with CRF-11 occurred. Both patients had a similar clinical history; they were able to control the viral replication of the first B subtype infection in the absence of treatment for years before the super-infection. No plasma was available before the super-infection, thus the strain-specific PCR was performed on proviral DNA.
The first patient was a 34-year-old Caucasian female (LS 172), using cocaine and heroin, infected with B subtype, she had undetectable viremia and stable CD4 cell count > 500 x 106 cells/l for 5 years in the absence of treatment. She then presented with an acute retroviral syndrome (fever, fatigue, multiple adenopathies) with viremia at around 6 log10 HIV-1 RNA copies/ml and a drop of CD4 cell count. The strain-specific PCR revealed the presence of CRF-11 strain in the absence of B subtype in plasma. Both B and CRF-11 strains were detected in proviral DNA after super-infection.
The second patient was a 40-year-old African male (LS 163), using cocaine and heroin, infected with B subtype, with undetectable viremia for 3 years in the absence of treatment. He presented with fatigue and the physical examination revealed multiple adenopathies not present 6 months before at the previous medical visit. His viremia was 5 log10 HIV-1 RNA copies/ml, and there was a drop of the CD4 count from 1225 to 674 x 106 CD4 cells/l. At this time the strain-specific PCR revealed the presence of CRF-11 in the absence of B strain in plasma. CRF-11 strains from the two patients were clustering in phylogenetic analyses suggesting that they were infected by the same unidentified person but not during the same event since the symptomatology associated with the super-infection was observed 4 months apart. Analysis of samples from these two patients were performed on two different experiments and were repeated twice with identical results.
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