Unusual Cluster of HIV Type 1 Dual Infections in Groningen,The Netherlands

Introduction

HIV dual infections, defined either as coinfection (infection with multiple HIV strains before seroconversion) or superinfection (infection with a second HIV strain after seroconversion), have been described as case reports since 2002, ^1,2 but overall incidences are low, at least in the United States and Western Europe. ^{3 –5} Dual infection could be the result of either serial HIV-1 infections from different partners or alternatively through infection by a multiply infected individual. The latter scenario has not been described thus far. The chance of acquiring multiple HIV-1 strains is likely to be very low. This chance is related to both exposure and risk behavior, ² and the exposure may be low in the developed world where the majority of the population, even within risk groups, is not HIV infected [see www.amsterdamcohortstudies.org: of the 532 men having sex with men (MSM) requesting HIV testing at the Municipal Health Service in Amsterdam in 2008, 481 (90%) tested HIV antibody negative]. To our knowledge patients infected with an identical mixture of virus strains have never been identified. Here, we report an unusual cluster of dual HIV-1-infected (MSM) in The Netherlands. Seven individuals were infected with the same two HIV-1 subtype B strains, which is the largest cluster of identical HIV dual infections known to date. Six others were infected with one of these two HIV-1 strains.

Materials and Methods

Results

In 2007, 14 MSM from The Netherlands filed a criminal case against three other MSM, accusing them of administering sedative drugs, sexual abuse, and deliberate subcutaneous injections with HIV-1-infected blood without consent between the end of 2005 and the beginning of 2007. For clarity, these 14 MSM will be referred to as “recipients” and the three accused men as “donors.” Twelve of the 14 recipients and three of three of the donors tested HIV-1 positive. Eight recipients and one of the alleged donors reported an acute HIV syndrome before or around the time of seroconversion in 2006–2007. Patient characteristics are summarized in Table 1. Two recipients (11 and 13) were not infected with HIV as demonstrated by a lack of anti-HIV antibodies.

Population sequencing of the HIV-1 pol gene indicated a high number of degenerate base codes (IUPAC-IUBMB codes for incompletely specified bases in nucleic acid sequences) in the RT part of the sequence for two participants, suggestive of an HIV dual infection (Table 1). ^2,9 Subsequent sequencing of env gene clones and phylogenetic analysis identified dual infections in an even higher number of study participants. In particular, a total of seven study participants, six recipients and one donor, were found to be infected with two distinct HIV-1 subtype B strains (Table 1 and Fig. 1). As an example, analysis of all V3–V4 env sequences derived from a single patient is shown for recipient 14 (Fig. 2).

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FIG. 1.

Phylogenetic analysis of HIV-1 env. HIV-1 env V3–V4 sequences were aligned, resulting in 574 aligned positions, and analyzed with the GTR + I model and the parallel version of MrBayes 3.1, modified to use the sprng library (http://sprng.cs.fsu.edu/) to generate independent streams of random numbers in the parallel processes. Five million generations were ran (average standard deviation of split frequencies = 0.006), burnin was set to 500, and the consensus tree with branch lengths and posterior probabilities ≥0.8 is shown. Contemporary subtype B reference sequences were generated from Dutch HIV-1⁺ patients sampled during 2006–2009. Sequences from donors and recipients are labeled as in Table 1. One representative clonal env sequence was used for each patient. If the patient was dually infected, a representative clonal sequence of each strain was incorporated in the analysis.

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FIG. 2.

Analysis of the env V3–V4 sequences from recipient 14. HIV-1 env V3–V4 sequences obtained from recipient 14 (indicated with 14 followed by the clone number) were aligned with reference HIV-1 subtype B sequences and analyzed with the neighbor-joining method implemented in MEGA4 using the gamma-distributed Tamura–Nei model with α = 0.38 (see the Materials and Methods section). HIV-1 subtype B strains 1 and 2 are indicated. Bootstrap values ≥90 are indicated. The scale bar indicates the genetic distance between the sequences (as calculated with the Tamura–Nei method, see the Materials and Methods section).

Counting the number of degenerate base pairs probably underestimates the incidence of early, within subtype HIV dual infection. Sequencing of an additional gene fragment improved the detection of dual infections, ¹⁰ as in two patients (4 and 12) the pol and env sequences clustered with both strains. Strain specific primers were designed that were able to amplify both strain 1 and 2 env fragments from blood plasma. This resulted in an extra double infection in subject 4, suggesting that one strain had been missed by analyzing only 16 clones. Recombination may obviously obscure the analysis of HIV-1 dual infections. In subject 12, analysis of the pol gene resulted in detection of only strain 2 sequences, while when sequencing env clones only strain 1 sequences were found.

An explanation could be that a recombinant virus was generated that outgrew the parental strains. Alternatively, patient 12 could already have been infected with a recombinant virus. Strain 2 env sequences were not amplified from subject 12 using strain-specific primers, indeed suggesting the presence of only a recombinant form. One recipient (8) and one donor (1) were infected with HIV-1 subtype B strains unrelated to each other or to the virus variants of any of the other subjects (Fig. 1). Subject 8 was already HIV positive far before the 2006–2007 period.

The relatively high number of degenerate base codes seen in donor 1 is probably related to an advanced disease state, as this individual presented at his first HIV test in 2003 with low CD4⁺ T cell counts that warranted immediate start of antiretroviral therapy. Donor 3, who first tested HIV-1 antibody positive in April 2005, was dually infected with the two subtype B strains, named strain 1 and 2 (Fig. 1). Donor 2, who seroconverted in May 2006, was infected with only one of these strains, strain 1 (Fig. 1). The three donors were involved in an unsafe sexual relationship with each other. Donor 1 has been effectively treated with antiretroviral therapy since 2003 with a plasma viral load <50 copies/ml and is thus unlikely to transmit virus. In addition, he was found to be infected with a subtype B strain unrelated to those found in the other donors or recipients.

A total of six recipients were infected with the same two strains as donor 3, whereas three recipients were infected with strain 1 only and two others only with strain 2 (Table 1). Recipient 5 harbored a triple HIV-1 infection with the two strains first identified in donor 3 and an unrelated subtype B strain. Donor 3 represents most likely the viral source for the double infections in the six recipients, as he tested HIV positive in 2005, well before the other persons did.

Discussion

We report an unusual cluster of identical HIV-1 dual infections in seven recently infected MSM from the city of Groningen in The Netherlands. In addition, another six men were infected with either one of the strains found in the dual infections. It remains possible that some of these are also dual infected, but that the second virus is replicating at a reduced level, thus escaping detection. The recipients knew the three alleged donors, who organized both private meetings and sex parties at their home. At these parties, HIV-1 could not only have been transmitted sexually, but the donors may also have injected their own blood subcutaneously into the recipients on several occasions.

Direct blood injections could explain the extremely high number of HIV-1 dual infections seen among the recipients, given the fact that one of the donors was dually infected with two HIV-1 strains early on. Simultaneous transmission of these strains by subcutaneous injection would be a much more efficient way to transmit both viruses than sexual transmission, which is characterized by the transmission of a single infectious unit in over 75% of cases as determined for heterosexual transmission of subtype B and subtype C HIV-1 variants. ^11,12 Indeed, multivariant transmission is common in intravenous drug users compared to heterosexuals. ¹³ Donor 2, who became infected with strain 1 only, most likely derived from donor 3, denied the use of needles in his relationship with donor 3, suggesting that the single virus strain was transmitted sexually.

In the recipient patients, only five individuals (45%) were infected with a single strain (possibly the result of a single transmitted virus) while six other men (55%) were infected with at least two infectious viruses as documented by their dual infection. Because most samples were analyzed relatively early after acute infection, these cases likely represent coinfections and not superinfections. In the single infected patients, a second strain could have been missed because only 16 env clones were sequenced. With a confidence level of 95%, sequencing 16 clones detects a minority population that comprises at least 17% of the viral population. In the blood plasma sample of donor 3, strain 2 comprised the majority of the clones that were sequenced (38/42 = 90%) versus 4/42 clones (10%) for strain 1. A similar pattern was seen in four dually infected recipients, where strain 2 clones were overrepresented (77–93% of env clones). In contrast, strain 1 was the major HIV variant in recipient 14, where 62% of env clones belonged to this strain. In the single infected recipients, strain 1 was seen in three individuals and strain 2 was seen in two individuals. In the two individuals with a single strain 2 infection, the possibility that strain 1 was erroneously missed during amplification was tested with a strain-specific PCR amplification. The additional experiments did not reveal strain 1 sequences in these individuals, suggesting that they are infected exclusively with strain 2.

To our knowledge, an outbreak of epidemiologically linked HIV-1 dual infections has never been reported before. It would be of interest to monitor disease progression in the individuals described here, as there has been no systematic research on the effect of HIV dual infection compared to single infection on disease progression. In this specific transmission cluster, the impact of host factors could also be examined, as viral variation is limited. On the other hand, unique recombinant viruses may evolve in individual patients, which would also be an important subject for future study.