Phylogenetic analyses indicate an atypical nurse-to-patient transmission of human immunodeficiency virus type 1

Abstract

A human immunodeficiency virus (HIV)-negative patient with no risk factor experienced HIV type 1 (HIV-1) primary infection 4 weeks after being hospitalized for surgery. Among the medical staff, only two night shift nurses were identified as HIV-1 seropositive. No exposure to blood was evidenced. To test the hypothesis of a possible nurse-to-patient transmission, phylogenetic analyses were conducted using two HIV-1 genomic regions (pol reverse transcriptase [RT] and env C2C4), each compared with reference strains and large local control sets (57 RT and 41 C2C4 local controls). Extensive analyses using multiple methodologies allowed us to test the robustness of phylogeny inference and to assess transmission hypotheses. Results allow us to unambiguously exclude one HIV-positive nurse and strongly suggest the other HIV-positive nurse as the source of infection of the patient.

FIG. 1

Chronology of PBMC and plasma samplings for the two nurses and the patient. Rectangles represent periods of hospitalization; vertical plain and dashed arrows indicate sampling of lymphocytes and plasma, respectively. Nurse 1 and nurse 2 had been in contact, between May 28 and June 8, 1996, each during two nights (indicated by asterisks), with the patient, who had a negative HIV-1 serology and no detectable viral RNA at the time of her hospitalization. Nurse 1 had been aware of his HIV-1 seropositivity for 5 years, and HIV-1 sequences were characterized from lymphocytes (N1) obtained on February 19, 1997, i.e., after the virological investigation was requested. Nurse 2's HIV serological status was unknown until she was diagnosed with HIV-1 and HCV infection during a hospitalization that started on June 20, 1996. The first nurse 2 HIV sequence (N2) used for molecular epidemiology analyses was from lymphocytes obtained on May 7, 1997, 1 month after introduction of antiretroviral treatment. Retrospectively, we also characterized viral sequences from cryopreserved plasma samples (JV44 and JV27) obtained during nurse 2's hospitalization. The second hospitalization (July to August 1996) of the patient was for HIV-1 primary infection. Plasma (JV32 and JV48) and lymphocyte (P) samples were obtained from the patient for viral sequence amplification. HIV-1 viral load, when available, is indicated (logarithm of the number of copies per milliliter) above corresponding sampling pointers.

FIG. 2

MP and ML trees among P, N1, N2, and reference sequences (RT). (a) Strict consensus of the MP trees under no weighting of substitution types. BV under exclusion and inclusion of group O sequences are indicated above and below the branches, respectively. Including the group O sequences collapsed the branch indicated as dashed and rooted the tree (with a BV value of 53) as indicated by the arrow. N1 and P are separated by three branches (thick line) supported by BV > 95. Sequence labels correspond to reference strain names from the LANL website (see text). (b) Strict consensus of the 3 ML trees (−ln L = 2062.54867). Estimation of Ti/Tv ratio, proportion of invariable sites, and gamma shape parameter for variable sites are 4.09, 0.32, and 0.64, respectively. When group O sequences are included, the branching pattern is unchanged but the tree is rooted as indicated by the arrow.

FIG. 3

NJ trees among P, N1, N2, and reference sequences for RT (a) and C2C4 (b). For each subtype (A to H), BV (in percent) are given for 10⁴ NJ/10⁴ MP replicates. Arrows indicate branches separating P from N1 with BV > 94%. Scale is in percent expected substitution per position. Note the difference of scale between RT and C2C4. Reference sequences used for RT were the same as in Fig. 2, with the addition of nine sequences from subtypes A, F, G, H (see Materials and Methods) and the exclusion of ibng (see text for details). The accession numbers of the 29 reference sequences (subtypes A to H) used for C2C4 are given in the text.

FIG. 4

Inference of phylogenetic relationships among RT sequences for P, N1, N2, and local controls. All local control sequences were obtained from plasma; P, N1, and N2 were obtained from lymphocytes from the patient, nurse 1, and nurse 2, respectively. Scale is in percent expected substitution per position. (a) NJ tree for the full set of 59 local control sequences (RO). Relevant BV (10⁴ replicates) are indicated. Letters in bold indicate subtypes. (b) ML tree (−ln L = 1913.91065) for the subset including the 15 local controls closest to [P + N2]. BV > 50% (ML, 1,000 replicates) are indicated.

FIG. 5

Phylogenetic relationships among C2C4 sequences from the patient, the two nurses, and multiple local controls. (a) Strict consensus among 382 MP trees (tree length = 1,191). BV obtained under unweighted MP (10³ replicates) and under NJ (10⁴ replicates) are indicated above and below the branches, respectively. na, not applicable (the group was not found in the NJ bootstrap consensus tree). (b) ML phylogram (−ln L = 2662.67099) among patient, nurse 2, and a reduced set of local control sequences (i.e., redundant sequences, indicated by asterisks, were excluded from these analyses; see text for details). BV obtained under ML are indicated on the branches. Plasma viral sequences from the patient (JV32 and JV48) and nurse 2 (JV27 and JV44) were processed blind during the analyses.

FIG. 6

ML phylogram of the [patient + nurse 2] clade. ML BV are indicated above the branches. The branching pattern of the MP bootstrap consensus tree is identical to that shown here, and corresponding MP BV are indicated below the branches. The full V4 loop sequence is indicated for each sample. Duplication of an FNSTW motif (boxed) was observed only in JV32 and JV48 (patient plasma viral sequences) as well as in the unrelated reference sequence LAI. Horizontal brackets indicate amino acid positions sensitive to alignment parameters (see Materials and Methods) when all local control C2C4 sequences are included. These positions have been excluded from phylogeny inference analyses. Note that the position indicated by an asterisk adds support to the grouping of P with JV32 and JV48.