Triple HIV-1 Infection with Group O and Group M of Different Clades in a Single Cameroonian AIDS Patient

To the Editor: Coinfection or superinfection of more than one viral genotype (genetic population) is indicated by the coexistence of multiple human immunodeficiency virus (HIV) strains belonging to different phylogenetic lineages in a single person (1,2). Recently, there has been well-documented evidence of double infection with HIV-1 and HIV-2 (3), and with different clades of HIV-1 (4) as well as recombinational events between viruses belonging to different clades of HIV-1 (5) or HIV-2 (6). Here, we present evidence of triple HIV-1 infection with a highly divergent group O virus and group M viruses of clades A and D. The patient studied was a 23-year-old single Cameroonian woman diagnosed with AIDS. Her blood samples were collected twice at an interval of 12 months in 1994 and 1995. Sera were screened by particle agglutination (PA) test (Serodia-HIV, Fujirebio, Tokyo, Japan) and examined by Western blotting (WB) assay (New Lavblot HIV-1 and HIV-2; Sanofi Diagnostics Pasteur, Marnes la Coquette, France). Both specimens were dual reactive for HIV-1 and HIV-2 by PA and WB. The sera were then discriminated using a peptide-based assay (Peptilav HIV-1 and HIV-2; Sanofi Diagnostics Pasteur) and judged as HIV-1 positive. The viral strains obtained in 1994 and 1995 were designated as CMR61 and CMR709, respectively. A part of the env gene (C2, V3, and C3 regions, 390 bp) was amplified by nested polymerase chain reaction from peripheral blood mononuclear cells, and their sequences were analyzed. Phylogenetic analysis of the env region revealed the presence of three different HIV-1 strains belonging to HIV-1 group O and HIV-1 clades A and D in both CMR61 and CMR709 (Fig. 1). Clades A and D were determined using the same primer pair for detecting group M viruses in the env region. HIV-1 group O was also detected using a different primer pair in the corresponding env position designed specially for group O viruses. Thus, the coexistence of three different sequences in the env region clearly testifies the first case of triple HIV-1 infection. Since the patient had no history of blood transfusion, we conclude that she underwent a superinfection with three HIV-1 strains, although a possibility of simultaneous infection cannot be excluded. Further studies would provide useful information concerning the host's immune response against highly divergent superinfecting viruses. *Jun Takehisa; †Léopold Zekeng; *Tomoyuki Miura; *Eiji Ido; *Masahiro Yamashita; *†Innocent Mboudjeka; ‡Lutz G. Gürtler; *Masanori Hayami; †Lazare Kaptue †Laboratory of Pathogenic Virus, Institute for Virus Research; Kyoto University;Sakyo-ku, Kyoto, Japan †Laboratoire d'Hémtologie et d'Immunologie; Centre Hospitalier Universitaire; Yaoundé, Cameroon ‡Max von Pettenkofer Institute; University of Munich; Munich, GermanyFIG. 1: . A phylogenetic tree of HIV-1 env sequences. DNA sequences (approximately 390 bp) were aligned by the ODEN program package of the National Institute of Genetics in Japan. Nucleotide substitutions were estimated by the six-parameter method, and a phylogenetic tree was constructed by the neighbor-joining method, reliability being estimated by 100 bootstrap replications. GenBank accession numbers of respective nucleotide sequences relating to this study are as follows: U58148, CMR61-A: U58149, CMR61-D; U58150, CMR709-A; U58151, CMR709-D; U58152 AND U58153, CMR61-O; U58154-U58156, CMR709-O; U58157, CMR61-A (pol); U58158, CMR61-O (pol); U58159, CMR709-O (pol).