Objective: To describe the clinical, epidemiological and molecular evidence for transmission of HIV-1 infection from a person with unrecognized HIV infection to a family member in two unconnected families where the route of transmission could not be conclusively determined.
Design: Case studies, molecular analysis of viral strains and a clinical and laboratory investigation of risk factors for transmission.
Setting: State referral centres for HIV/AIDS in two Australian teaching hospitals.
Results: Previously unrecognized HIV-1 infection was diagnosed in two unconnected females following blood donation in different Australian cities. Initially, no source of infection was identified but subsequently HIV-1 infection was diagnosed in the sister of one case and the adult son of the other. Using nucleic acid-based methods, it was demonstrated that one index case and her sister were infected with highly homologous ‘Russian-type’ HIV-1 subtype A, and the other index case and her son were infected with highly homologous HIV-1 subtype E (CRF01_AE). Sexual history taking from the sister and the son of the respective index cases revealed prior sexual partners from geographical areas in which the corresponding subtypes are known to be prevalent. Extensive history taking, cross-validated by independent reviewers, found no evidence whatsoever that any form of sexual contact or known blood contact could explain the HIV-1 infection in the two index cases. However, there was evidence that some form of domestic contact involving unperceived blood transfer may have occurred.
Conclusion: Intra-familial transmission of HIV-1 infection should be considered when a source of HIV-1 infection cannot be determined.
From the aDepartment of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, the bDepartment of Pathology, University of Western Australia, Perth, the cDepartment of Virology, ICPMR, and Centre for Virus Research, Westmead Millennium Institute, Westmead Hospital, the dNational Centre in HIV Epidemiology and Clinical Research, and the eAlbion Street Centre, Sydney, Australia.
Correspondence to M. French, Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, GPO Box X2213, Perth 6001, Australia.
Received: 4 October 2002; revised: 7 February 2003; accepted: 19 March 2003.
HIV infection is usually transmitted by sexual, percutaneous (injecting drug use or transfusion of blood/blood products) or perinatal routes. In the vast majority of studies, household contact has not been associated with the transmission of HIV-1 infection [1–4]. However, there have been reports documenting unusual circumstances in which HIV-1 transmission has occurred [5–8], including reports of probable intrafamilial transmission [9–12]. Here we report the clinical and molecular evidence for two occurrences of transmission of HIV-1 infection from one family member to another, by a route other than sexual contact, overt blood contact or perinatal exposure. These cases are of particular interest because each source patient had unrecognized HIV-1 infection.
Case 1.1 was a previously healthy 16-year-old girl who was found to be HIV-1 antibody positive in January 1999 following a voluntary blood donation. The CD4 T-cell count was 568 × 106/l and the plasma HIV-1 RNA concentration was 517 889 copies/ml. There was no history of a primary HIV infection syndrome. All sexual contacts and her mother and father were HIV antibody negative. The only sibling was case 1.2.
Case 1.2 was the older sister of case 1.1. She was tested for HIV-1 infection in February 1999, when she was 18 years old, because her sister recalled her having had severe ‘glandular fever’ in January 1997. She was HIV-1 antibody positive. She was asymptomatic, the CD4 T-cell count was 680 × 106/l and the plasma HIV RNA concentration was 82 064 copies/ml. Testing for HIV infection had not previously been undertaken and neither stored serum nor tissues were available to test retrospectively.
She reported that 1 month prior to the January 1997 illness she had sexual intercourse for the first time in her life with a Russian man. Three subsequent sexual partners tested negative for HIV-1 antibodies but there were other partners who could not be traced.
Case 2.1 was a previously well 55-year-old woman who was found to be HIV-1 antibody positive in June 1997 after donating blood. In August 1996, she had tested negative for HIV-1 antibodies. The CD4 T-cell count was 350 × 106/l and the plasma HIV RNA concentration was 28 355 copies/ml (version 1.0 RT–PCR assay, Roche, Branchberg, NJ, USA). She had presented to her general practitioner in February 1997 with pharyngitis and cervical lymphadenopathy and was treated with antibiotics, which were ceased when she developed oral thrush. HIV testing was not done.
She had been married for 25 years and reported no other sexual partners in that time. Her husband was HIV-1 antibody negative. The couple had a teenage daughter living with them, who was also HIV-1 antibody negative. Case 2.1 also had a son in his early 30s from an earlier marriage, who had been living in Thailand. He had visited his family in December 1996 and stayed for 6 months.
Case 2.2 was the son of case 2.1. He returned to live in Australia in 1998. Hepatitis C virus (HCV) infection was diagnosed in November 1998 during investigation of arthritis. Antibodies to HIV-1 were subsequently detected. Apart from a 4-year-history of plaque-type psoriasis, he had been well. The CD4 T-cell count was 10 × 106/l and the plasma HIV RNA concentration was 40 475 copies/ml (version 1.0 RT–PCR assay). He reported occasional unprotected sex while living in Southeast Asia during the 1990s but no sexual partners since returning to Australia.
DNA samples were extracted from buffy coat fractions of each patient's blood and processed at different times. HIV-1 env fragments (spanning the V1–V5 or C2–V5 regions) were amplified from the DNA samples using a nested PCR, as previously described . In addition, codons 20–240 of the RT gene from Cases 1.1 and 1.2 were amplified in a nested PCR, also at separate times.
Heteroduplex mobility assay for HIV-1 subtyping and analysis of HIV-1 quasispecies diversity
HIV-1 subtyping was undertaken on env fragments using a heteroduplex mobility assay (HMA) and reference fragments of env from subtypes A to G . Shannon entropy was used to measure the degree of diversity of the bands on a scale of 0 to 1, where 0 indicates the lowest and 1 indicates the highest diversity .
Cloning and sequencing of env and RT fragments
V1–V5 env fragments generated by PCR were cloned into a pCR2.1 vector (Invitrogen, Carlsbad, California, USA). gp120 and codons 20 to 240 of RT were sequenced using Big Dye Terminator Cycle Sequencing Kits on an Applied Biosystems 373 Sequencer with XL upgrades (PE Biosystems, Foster City, California, USA).
Env gene sequences (C2–V5) from each patient were aligned with env sequences of other HIV-1 subtypes obtained from the Los Alamos HIV database (A: RW20-U08794, SF170-M66533; B: TH14-U08801, SF2-K02007, JRFL-U63632; C: IN747-X65638, MW 959-U08453, BR25-U08797; D: UG21-U27399, CRF 01_AE, TH253-U51189; F: BZ163-L22085; G: RU 131-U30312, pBLV21-7-U09664) including sequences from Russian and Ukrainian injecting drug users (IDU) (RU1175-U93611, UA1127-U93665 and UA970063-AF082486) and a subtype O sequence (MVP5180-L20571). Alignments were edited manually and gap stripped prior to analysis. A Neighbour-Joining tree was constructed using DNADIST, NEIGHBOR and SEQBOOT programs from the PHYLIP package . One hundred bootstrap analyses were performed. Sequence homology was determined by a Bestfit analysis and genetic distance was determined from a distance matrix generated using the Kimura 2 square parameter.
Assessment of exposure history
Following the initial investigations into the possible routes of transmission by the treating clinicians, an external review was undertaken of the available evidence. Cases 1.1, 1.2 and 2.1 agreed to be interviewed in detail. Interviews were undertaken by a female clinician with extensive experience in Sexual Health and HIV Medicine, and by an HIV epidemiologist. Case 2.2 declined to be interviewed, but agreed to answer questions in writing. A structured questionnaire covering all potential routes of transmission was administered separately to each person being interviewed, and the medical records of all cases were reviewed. The parents of cases 1.1 and 1.2 were also interviewed extensively.
HIV-1 subtype and quasispecies diversity
The HMA demonstrated that the HIV-1 env sequences isolated from Cases 1.1 and 1.2 were subtype A and that those isolated from Cases 2.1 and 2.2 were subtype E (CRF01_AE ). Analysis of quasispecies diversity by HMA gave Shannon entropy values of 0.7466 and 0.8428 for Cases 1.1 and 1.2 respectively, and 0.7326 and 0.8428 for cases 2.1 and 2.2 respectively. These findings indicated a lower degree of quasispecies complexity in both index cases, suggesting more recently acquired HIV infection.
Homology and phylogenetic analyses of env and RT sequences
Comparison of env gene sequences (990 nucleotides) from Cases 1.1 and 1.2 (registered in Genbank as AUS-2 and AUS-1 respectively) demonstrated 97% homology and a pairwise distance of 0.0168. The env sequences from Cases 2.1 and 2.2 demonstrated > 96% homology and a pairwise distance of 0.015. For comparison, pairwise distances for 4950 random subtype B sequences from Genbank were 0.03–0.37 . Comparison of env sequences from cases 1.1 and 1.2 with env sequences from Genbank demonstrated that they were more similar to sequences from subtype A than other HIV-1 subtypes, particularly subtype A sequences from Russian IDU. Phylogenetic analysis confirmed this, as sequences from cases 1.1 and 1.2 clustered with those from Russian and Ukranian IDU (Fig. 1). A FASTA and BLASTN homology search using the RT sequence (599 nucleotides) from case 1.1 showed greater similarity to the RT sequence of case 1.2 than to any of 1091 sequences from other patients in the same geographical area (data not shown).
Route of transmission of HIV-1 infection between family members
The interviews revealed no evidence that cases 1.1 and 1.2 or 2.1 and 2.2 had had any form of sexual contact with each other or had a sexual partner in common. In addition, there was no evidence of acquisition or transmission of HIV-1 infection by injecting drugs, transfusion of blood or blood products, exposure to blood or bodily fluids, or medical or dental procedures. Cases 1.1 and 1.2 shared a bathroom and sometimes used the same razor to shave off body hair. Cases 2.1 and 2.2 reported no sharing of razors or toothbrushes. However, case 2.2 had active psoriasis in late 1996 and case 2.1 had applied topical therapy to the skin lesions. She did not wear gloves to do this and reported that the lesions had been dry and did not bleed. She did not have dermatitis on her hands.
We have documented two unconnected cases of HIV-1 infection in females who had a family member that was subsequently shown to also have HIV-1 infection. Molecular epidemiology studies demonstrated that the family members were highly likely to have been the source of the infection. Thus, cases 1.1 and 1.2 were infected with ‘Russian-type’ HIV-1 subtype A, which has not previously been identified in Australian patients , and cases 2.1 and 2.2 were infected with HIV-1 subtype E (CRF01_AE), which is very uncommon in Australia . Furthermore, sequencing of the env gene of proviral DNA from each case demonstrated very close homology of sequences from cases 1.1 and 1.2 on the one hand, and cases 2.1 and 2.2 on the other. In addition, sequencing of the RT gene from case 1.1 demonstrated a greater degree of homology with the RT sequence from case 1.2 than any of over 1000 sequences from patients in the same geographical area.
Cases 1.2 and 2.2 appear to have been the sources of the HIV-1 infection because they gave a history of sexual partners from regions of the world in which their particular HIV-1 subtype is common. It is also possible that case 2.2 had an undisclosed history of injecting drug use because he also had HCV infection. The greater HIV-1 quasispecies diversity in cases 1.2 and 2.2 also provided evidence that they had been infected before their respective index cases. A source of HIV-1 infection, other than the infected family member, was not identified for either index case by extensive interviews and by contact tracing of sexual contacts of case 1.2 or dental contacts of case 2.2.
No route of transmission from either source case to the corresponding index case was conclusively identified. The only potential route of transmission for case 1.1 was the sharing of razors for shaving of body hair. Sharing of razors has been reported to be a possible source of HIV infection  and HCV infection . Case 2.1 appears to have had primary HIV infection shortly after living in the same house as case 2.2. At this time, case 2.2 had active psoriasis and his mother had assisted in the application of therapy to the lesions. This contact is the only potential route of transmission that was elicited by the interviews.
There have been previous reports of pairs of family members infected with HIV-1 species that were clearly of the same origin, between whom no route of transmission could be definitively established [5–7,9–12]. However, these reports have usually involved at least one child. The transmission of HIV-1 infection between adults in the absence of identified risk factors has been reported rarely [9,10] and only once  with the shared HIV-1 species confirmed by DNA sequencing. The source patient in these reports had AIDS complicated by mucocutaneous lesions which could have provided a route for HIV transmission. In contrast, both of the source patients reported here were unaware of their HIV-1 infection until after the HIV-1 infection was diagnosed in the corresponding index case. These therefore appear to be the first reports of intra-familial transmission of HIV-1 infection between adults involving a source patient with unrecognized infection.
The total reliance on self-reported history in HIV surveillance means that there can never be absolute certainty in determining routes of HIV transmission. The residual doubt about the cases presented in this paper can easily be used to throw into question the conclusions reached by the investigation. A more constructive interpretation is that unsuspected transmission via non-sexual routes can occur on rare occasions among adults, but that the overwhelming majority of HIV transmission is due to sexual contact or overt blood contact. These two cases of apparent domestic transmission do not alter in any way the public health advice that has been promulgated for over a decade and a half about HIV transmission and the means of its avoidance.
We thank A. McDonald of The National Centre in HIV Epidemiology and Clinical Research (NCHECR) for national surveillance information, the clinicians involved in the care of the cases reported and the patients and their relatives for assistance given in undertaking this study.
Sponsorship: Supported by The Commonwealth Department of Health and Ageing.
1. Gershon RR, Vlahov D, Nelson KE. The risk of transmission of HIV-1 through non-percutaneous, non-sexual modes - a review. AIDS
2. Berthier A, Chameret S, Fauchet R, Fonlupt J, Genetet N, Gueguen M, et al
. Transmissibility of human immunodeficiency virus type 1 in haemophiliac and non-haemophiliac children living in a private school in France. Lancet
3. Fischl MA, Dickinson GM, Scott GB, Klimas N, Fletcher MA, Parks W. Evaluation of heterosexual partners, children and household contacts of adults with AIDS. JAMA
4. Romano N, de Crecenzo L, Lupo G, Torregrossa MV, Alesi DR, Portera M, et al
. Main routes of transmission of human immunodeficiency virus (HIV) infection in a family setting in Palermo, Italy. Am J Epidemiol
5. Wahn V, Kramer HH, Voit T, Bruster HT, Scrampical B, Scheid A. Horizontal transmission of HIV infection between two siblings. Lancet
6. Fitzgibbon JE, Gaur S, Frenkel LD, Laraque F, Edlin BR, Dubin DT. Transmission from one child to another of human immunodeficiency virus type 1 with a zidovudine resistance mutation. N Engl J Med
7. Blank S, Simonds RJ, Weisfuse I, Rudnick J, Chiasson MA, Thomas P. Possible nosocomial transmission of HIV. Lancet
8. Gilbart VL, Raeside F, Evans BG, Mortimer JY, Arnold C, Gill ON, et al
. Unusual HIV transmissions through blood contact: analysis of cases reported in the United Kingdom to December 1997. Commun Dis Publ Health
9. CDC. Human immunodeficiency virus transmission in household settings – United States. MMWR
10. Belec L, Mohamed AS, Muller-Trutwin MC, Gilquin J, Gutmann L, Safar M, et al
. Genetically related human immunodeficiency virus type 1 in three adults of a family with no identified risk factor for intrafamilial transmission. J Virol
11. Salvatori F, De Martino M, Galli L, Vierucci A, Chieco-Bianchi L, De Rossi A. Horizontal transmission of human immunodeficiency virus type 1 from father to child. AIDS Res Hum Retroviruses
12. Orth H, Engelbrecht S, Cotton MF, Robson BA, Smith T-L, Schaaf HS, et al
. HIV transmission between two siblings in Africa. AIDS
13. Delwart EL, Herring B, Rodrigo AG, Mullins J. Genetic subtyping of human immunodeficiency virus using a heteroduplex mobility assay. PCR Methods and Applications
14. Delwart EL, Pan H, Sheppard HW, Wolpert D, Neumann AU, Korb Mullins JI. Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS. J Virol
15. Felsenstein J. PHYLIP (Phylogeny Inference Package) Version 3.5c
. Seattle, WA: University of Washington; 1993.
16. Robertson DL, Anderson JP, Bradac JA, Carr JK, Foley B, Funkhouser RK, et al
. HIV-1 nomenclature proposal. Science
17. Li F, Nickle DC, Shankarappa R, He X, He H, Jensen M, et al
. HIV-1 evolutionary dynamics in a dually infected HIV-1 patient. Ninth Conference on Retroviruses and Opportunistic Infections
. Seattle, February 2002 [abstract 356-M].
18. Herring BL, Saksena NK, Cunningham AL, Dwyer DE. Molecular surveillance of HIV-1 env subtypes in Australia. Keystone Symposium. HIV Pathogenesis and Treatment.
Park City, Utah, March 1998 [abstract 4090].
19. Centers for Disease Control and Prevention. HIV transmission between two adolescent brothers with hemophilia. MMWR
20. Tumminelli F, Marcellin P, Rizzo S, Barbera S,Corvino G, Furia P, et al
. Shaving as a potential source of hepatitis C virus infection. Lancet