HIV-1 subtype F in single and dual infections in Puerto Rico: A potential sentinel site for monitoring novel genetic HIV variants in North America.

To the Editor: Ehrlichiosis is a zoonotic disease transmitted to humans through the bite of infected ticks (1). The first recognized human ehrlichial infection, Sennetsu fever, was described in Japan in 1954 (2). The first case of human ehrlichiosis in the United States was recognized in 1986 and was reported in 1987 (3). The disease is caused by intracellular gram-negative bacteria of the Ehrlichia genus. The bacteria can be found in the monocytes and granulocytes of peripheral blood. Human monocytic ehrlichiosis is caused by E. chafeensis, and human granulocytic ehrlichiosis is caused by E. equi or E. phagocytophilia, which was first recognized in 1994 (4). Most cases occur between April and September, and the reservoirs are field animals such as rodents, deer, and dogs. The clinical spectrum of the disease is similar to that of other febrile illnesses; without adequate and timely treatment, approximately 5% of the patients die (5). In the United States, more than 400 cases of serologically confirmed E. chaffensis infection have been documented since 1996 (6). No cases have been reported in Mexico. In February 1997, we evaluated a 41-year-old male patient from Merida. The patient had been exposed to ticks during activity in a rural area 1 week before the onset of illness. Clinical manifestations included frequent hyperthermia, rash, myalgia, headache, anorexia, fatigue, and cough. Physical examination showed bilateral cervical lymphadenopathy, and a chest radio-graph showed an interstitial bilateral infiltrate. Hematic cytometry showed thrombocytopenia of 134 x 10 3 /µL and 3200 leukocytes (1440 neutrophils/µL). Hepatic transaminases were elevated, with an aspartate aminotransferase: 92 U/L (normal: 22 U/L), alanine aminotrans-ferase: 48 U/L (normal: 18 U/L), gamma-glutamyltranspeptidase: 278 U/L (normal: 28 U/L); and globulins: 4.8 g/dL with a polyclonal pattern. No antibodies against rickettsia, dengue virus, B-19 parvovirus, or HIV were detected. A serum sample gave a positive reaction by indirect immunofluorescence assay against E. chaffeensis at titers of 1:64 on week 2 and 1:128 on week 3. No infected monocytes or granulocytes were observed in peripheral blood. Remission of the clinical manifestations began on week 4 and was completed on week 6. This case indicates the existence of human ehrlichiosis in Yucatan, Mexico. Reactivity to E. chaffeensis suggests human monocytic ehrlichiosis; however, as antibody testing was not performed with E. phagocytophila or E. equi, the possibility of human granulocytic ehrlichiosis cannot be excluded. In any event, case reports indicate the need for deliberate search for …

clinical manifestations began on week 4 and was completed on week 6. This case indicates the existence of human ehrlichiosis in Yucatan, Mexico. Reactivity to E. chaffeensis suggests human monocytic ehrlichiosis; however, as antibody testing was not performed with E. phagocytophila or E. equi, the possibility of human granulocytic ehrlichiosis cannot be excluded. In any event, case reports indicate the need for deliberate search for cases. Dengue is endemic in this area of Mexico, and ehrlichiosis should be considered as a differential diagnosis.

HIV-1 Subtype F in Single and Dual Infections in Puerto Rico: A Potential Sentinel Site for Monitoring Novel Genetic HIV Variants in North America
To the Editor: Although international efforts to systematically collect, characterize, and classify HIV isolates from around the world have increased considerably, data on HIV-1 genetic variations in Puerto Rico are limited. This island (population 3.7 million) has one of the highest Letters AIDS incidence rates in the United States (53.3 cases per 100,000) (1). To evaluate the potential for a multiple subtype distribution pattern in Puerto Rico, we analyzed genetic variations between HIV-1 strains isolated from peripheral blood mononuclear cells of 63 asymptomatic HIV-infected female commercial sex workers from 12 communities. These participants were part of 290 female commercial sex workers followed in a larger cross-sectional study of risk behavior (2).
HIV-1 subtypes F (n = 4) and B (n = 44) strains were identified in persons infected with a single viral subtype with a molecular screening assay based on restriction fragment length polymorphism (RFLP) analysis and with DNA sequencing of the viral protease gene-prot (3). The remaining 15 specimens were classified by RFLP as potential dual infections. Further cloning and sequencing of prot from three of these specimens confirmed one dual infection involving subtypes F and B viruses and identified two infections caused by genetically distinct quasispecies of subtype B variants.
In further detailed pairwise analysis of HIV-1 prot genes, a small nucleotide divergence of 0.3% (0.0 to 1.1) within subtype F contrasted with a typical value of 6.3% (5.1 to 7.8) for the intrasubtype distance within subtype B prot sequences (4). The 99% similarity between prot subtype F Puerto Rican sequences suggested an epidemiologic link or a recent introduction of subtype F in Puerto Rico. Comparative sequence analysis of the C2-V3 env is useful in establishing the time that elapsed from infection on the basis of an annual nucleotide divergence of 0.5% to 1% in this region (5). Such analysis has been used to study the epidemiologic link between cases (4,6). Thus, we compared env sequences from two of five persons infected with prot subtype F strains. This analysis provided several observations. Env nucleotide divergence of 13.2% did not support a direct epidemiologic link between these strains. Furthermore, the relatively high intrasubtype diversity between env sequences suggested that evolution from a common progenitor would have taken a minimum of approximately 13 years. Phylogenetic analysis classified these two env sequences as subtype B, indicating that at least some of Puerto Rican prot subtype F viruses represent HIV-1 mosaics involving closely related prot F and significantly divergent env B sequences. Overall, discrepancy in both subtype assignment and nucleotide diversities within prot and env regions may indicate that distinct F/B mosaics circulating in Puerto Rico were likely the result of recombination between highly homogeneous subtype F of relatively recent arrival and divergent resident subtype B viruses.
HIV-1 infections with subtype F strains including B/F mosaics have been reported in Brazil (3,7). To evaluate a potential HIV-1 linkage between Brazil and Puerto Rico, a comparative phylogenetic analysis was done on subtype F viral prot sequences from these countries. This analysis documented that HIV-1 subtype F strains in Puerto Rico are distinct from both Brazilian and Romanian viruses. Furthermore, our results show that genetic analysis of prot allows tracking of subtype F viruses of different origin. Recently, by this approach, HIV-1 prot subtype F of Puerto Rican origin and F prot/B env mosaic were identified in HIV-1infected persons in New York city (8). Observation of HIV-1 subtype F strains in Puerto Rico together with the recent report describing the first cases of such infections in New York indicates the potential for further emergence of subtype F on the North American continent. The presence of a complex distribution pattern of subtype F infections in Puerto Rico has serious implications for the evaluation and development of HIV diagnostics and vaccines. Supported in part by grant G12RR-03050 (Y.Y.).
The nucleotide HIV-1 sequences obtained in this study were submitted to GenBank; their accession numbers are AF096813-AF096833.