Molecular sequence analysis of HIV strains collected from exposed individuals during cluster investigations can provide important data in support of epidemiological evidence regarding transmission patterns. Earlier investigations of potentially related HIV infections have used genetic and phylogenetic techniques to provide data to support or refute person-to-person transmission of HIV in healthcare settings [1–3], within households , between sexual partners [5,6], and in cases of intentional infection through injection .
In April 2004, a cluster of four new HIV infections was identified among performers in the adult film industry in Los Angeles County. Epidemiological investigation, reported elsewhere , indicated that a male index case had infected three of 13 women (23% attack rate) with whom he had been employed to perform a variety of sexual acts in the production of adult films. These acts included oral, vaginal, anal, and double-anal (two penises in one woman's anus) sex. All sex acts were performed without barrier precautions (e.g. condoms). The exposures took place during a 26-day period after the male index tested HIV negative (17 March 2004) and then HIV positive (9 April 2004) using a polymerase chain reaction (PCR) DNA-based method that detects proviral DNA (Amplicor HIV-1 test; Roche Molecular Systems, Branchburg, New Jersey, USA). This test is approved for research use only and is labeled specifically not for use in diagnostic procedures, but was used in this context to reduce the window period between HIV infection and its diagnosis; PCR-based methods are estimated to reduce this window period to approximately 10 days .
The three HIV-infected female performers all tested HIV negative before working with the male index case and then HIV positive after this work (Fig. 1). The index case and two of the women reported no other sexual contacts. The sole non-occupational contact of the third woman (contact no. 3, Fig. 1) tested HIV negative 60 days after her last potential sexual exposure to HIV. The epidemiological investigation of this infection cluster concluded that the three women were infected through occupational exposure. We evaluated whether the available viral strains collected from infected individuals were related and supported this epidemiological conclusion.
Blood specimens were available from the male index case and two of his three infected female partners (contacts nos. 1 and 2, Fig. 1). Specimens from consenting individuals were collected within 3 months after the diagnosis of HIV infection before any antiretroviral exposure, and were evaluated at the Centers for Disease Control and Prevention for relatedness by RNA extraction, reverse transcriptase (RT)–PCR amplification, direct or consensus DNA sequencing, and genetic analysis. HIV RNA was extracted from blood plasma using the Qiagen UltraSens extraction kit (Qiagen Inc., Valencia, California, USA). Extracted RNA was used to amplify the C2V3C3 and gp41 regions of env[10–12] and the p17 region of gag. These regions have been used for phylogenetic comparisons of HIV strains in transmission cases because they are located in separated portions of the HIV genome, and encode proteins with different functions subject to generally independent selection pressures related to the host immune response and transmission. The Qiagen One-Step RT–PCR kit was used to amplify various amounts of template RNA. Amplification conditions were as follows: 30 min at 50°C, 15 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C (50°C for gp41 amplification), and 1 min at 72°C. Secondary amplification using 1–5 μl of primary reactions was performed using the Platinum Taq PCR Supermix (Invitrogen Corp., Carlsbad, California, USA), with the same thermocycling conditions as in the primary amplification but without the 30 min 50°C step and 15 min 95°C step. Secondary PCR amplicons were purified using QIAquick PCR purification kits (Qiagen Inc.) and were subsequently sequenced with the nested amplification primers using the BigDye Terminator version 1.1 cycle sequencing kit (Applied Biosystems, Foster City, California, USA) and an automated ABI Prism 3100 DNA sequencer (Applied Biosystems). The HIV regions sequenced included the C2V3C3 region (454 nucleotides) of env, a portion of the gp41 coding region (354 nucleotides) of env, and the p17-coding region (396 nucleotides) of gag; these regions are used commonly in phylogenetic analyses and comparisons of HIV strains in transmission cases. Sequences were edited using Sequencher version 4.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and aligned using the Se-Al Sequence Alignment Editor, version 1.0 .
To reduce the possibility of cross-contamination between samples during these analyses, RNA from each of the three blood specimens was extracted by a single laboratory scientist on different days. All subsequent amplification and sequencing of the extracted RNA was performed in duplicate by two different laboratory scientists on separate days in physically separated laboratories.
The C2V3C3, gp41, and p17 sequences from each case patient were compared with the other case patients' sequences. These sequences were then compared with equivalent sequences from HIV reference strains collected in geographical regions where the HIV transmissions occurred: California and Brazil. Reference sequences were sought by searching the HIV Sequence Database (Los Alamos National Laboratories, New Mexico, USA); the number of available reference sequences for C2V3C3, gp41, and p17 of equivalent length to the sequences from the case patients that also had adequate information on the geographical location of the collection source was limited. For these reasons, and because individuals working in the adult film industry to which the case patients were exposed often work in other US cities, we expanded the search from California and Brazil to also include sequences from any other location in the United States. We identified 17 US sequences for C2V3C3, nine Brazil sequences for V3 alone, 18 US sequences for gp41, and two US and three Brazil sequences for p17. In this database we identified no equivalent sequences from the United States that were specified as being from California. Additional comparison data were taken from unpublished sequences of HIV-1 subtype B strains previously analysed at CDC: 11 California sequences for C2V3C3, 15 non-California US sequences for gp41, and 11 California and 10 non-California US sequences for p17. All alignments were examined both visually and by computer, using MEGA, version 3.0  for differences in nucleotide sequences and to determine nucleotide distances (mean substitutions per 100 sites ± standard error).
The C2V3C3, gp41, and p17 sequences from the case patients, other identified reference sequences from HIV-1 subtype B strains, as well as representative sequences from the other eight HIV-1 subtypes were used in three separate alignments as input for phylogenetic analysis. Appropriate nucleotide substitution models were determined with Modeltest v3.6  for each of the three alignments and bootstrap (2000 replicates), neighbor-joining trees generated with PAUP* v4.0 .
Two case patient's viral isolates (the index case and contact 1, Fig. 1) were also tested for antiretroviral resistance mutations by both genotyping and phenotyping at a commercial laboratory (ViroLogic PhenoSense GT, South San Francisco, CA, USA). These specimens were collected and assayed before any antiretroviral exposure.
The three case patient HIV-1 strains were determined to be subtype B. The results of within-group and between-group comparisons are shown in Table 1 and Table 2. The three case patient isolates were identical to each other, whereas a within-group comparison of sequences from reference strains varied from 8.2 ± 0.8 to 15.5 ± 1.1 substitutions per 100 nucleotides among comparisons of all three gene regions. Between-group comparisons of the case patients' sequence group with equivalent sequences from reference strain groups showed a similar variation: 8.0 ± 1.0 to 19.6 ± 2.3 substitutions per 100 nucleotides. As expected, phylogenetic analysis indicated that the case patients' sequences clustered strongly (100% bootstrap support) with HIV-1 subtype B strains within the reference HIV subtype phylogenetic trees, but not with other strains in the subtype B trees; a representative phylogenetic tree for the C2V3C3 gene region on env is shown in Fig. 2.
There was no evidence of genotypic or phenotypic antiretroviral drug resistance in the plasma virus tested from the male index case and his female contact. For the male index case, the only differences from subtype B consensus sequence in RT were at codons Q102K, I142M, C162S, R211K, F214L, A272T, V276I and R277K. Plasma virus from the female partner had identical differences but lacked detection of the mixtures seen at codons 272 and 276. Mutational differences seen in protease included I15V, N37S, R41K and V77I, which were also identical in both individuals. All of the mutations identified reflected naturally occurring polymorphisms that are not associated with antiretroviral drug resistance.
Our analysis demonstrates that the HIV-1 strains from the three case patients represent a cluster of viruses highly related to one another, but unlike the limited available comparison strains to which the case patients could have been exposed based on geography. Two lines of evidence support a close molecular relationship between the HIV strains examined: data from the genetic sequencing performed at the Centers for Disease Control and Prevention laboratory and the genotypic and phenotypic resistance data obtained at a commercial laboratory. Although our analysis was limited to HIV strains from three of the four HIV-1 infections in the identified cluster and by the small number of reference sequences available for comparison, the molecular data support the epidemiological evidence of occupational HIV transmission.
The finding that the case patients' viruses shared identical nucleotide sequences at the evaluated gene loci, in one case 3 months after transmission, is unusual but not unexpected. The male index case transmitted HIV during the primary acute phase of his infection, the women were infected by the index case within a few days of one another, and blood specimens for molecular and microbiological analyses were collected before exposure to selective drug pressure. Investigations of other well-characterized HIV infection clusters have also found identical sequences in strains collected 3 months apart from individuals with epidemiologically related infections [18–20]. The high rate of spontaneous mutation of the virus can produce a variety of quasispecies within a single infected individual. The methods used in this and other similar investigations as well as in clinical HIV medicine detect the gene sequences predominating in the population of circulating virions; these methods are widely accepted for identifying mutations. The development of fixed mutations in the population of virions leading to detectable genetic divergence is slow compared with the rate of change within individual virus particles as they reproduce. Quantifying the number of quasispecies as a means of documenting mutational variation is labor-intensive, costly, and generally considered impractical.
The 23% attack rate in this cluster of infections was considerably higher than the most conservatively estimated risk of less than 0.5% for a single act of receptive anal or receptive vaginal sex with an HIV-infected partner [21,22]. The HIV transmission risk for the presently investigated cluster was greater for at least three reasons. First, the sexual contact involved in adult film production can be prolonged and traumatic, increasing the opportunity for infection to occur; notably in the case of these infections there was a substantially increased risk of trauma to the anorectum (i.e. double-anal penetration). Second, most estimates of transmission risk are ‘per coital act’ calculations. The risk for adult film workers is increased by their multiple coital and other exposures over a short period; the attack rate for this cluster represents a cumulative rather than per-act risk. Finally, the transmissibility of HIV is greatest within the first months after infection during and near the time of seroconversion. Epidemiological studies in Africa have observed a 12-fold higher risk per coital act for heterosexual transmission within the first 5 months after initial infection . Mathematical models using US data estimate that the higher loads of HIV in semen observed during seroconversion increase the risk eight to 10-fold that a man acutely infected with HIV and free of other sexually transmitted diseases (STD) will transmit the virus to his female partner, infecting 7–24% of susceptible female partners during the first 2 months after infection .
Routine frequent HIV testing can identify individuals during the acute primary phase of HIV infection when they are most infectious to others. Such testing should be encouraged, particularly among individuals at a substantially increased risk of exposure to HIV, such as adult film workers. HIV testing should be coordinated with STD screening when there is any risk of co-infection, especially since STD infections increase the risk of both HIV transmission and acquisition. Routine testing is important for early diagnosis and referral for care. However, as this cluster of occupational HIV infections illustrates, testing alone, even by the most sensitive available approaches, is an inadequate prevention strategy. Condoms and other barrier methods should be used consistently during filming to reduce occupational exposure to infected fluids and prevent the transmission of HIV and STD between workers.
The nucleotide sequence data for the C2V3C3 region and partial gp41 of env and the p17-coding region of gag were deposited in the GenBank nucleotide sequence database (accession numbers DQ148506–DQ148514).
The authors would like to thank Dr Walid Heneine for useful comments on protease–RT (polymerase) sequences. They are especially grateful to Dr Terry Chorba for his support, insight, and continued guidance.
Sponsorship: For authors employed by governmental agencies, data were collected and analysed as part of each author's routine duties supported by public funds. E.S.D. is supported by National Institutes of Health grant AI43638.
Portions of the findings and conclusions in this report have been officially disseminated in the Morbidity and Mortality Weekly Report (United States Government, Department of Health and Human Services, Centers for Disease Control and Prevention). The present report has not been formally disseminated by the Centers for Disease Control and Prevention and should not be construed to represent any agency determination or policy.
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