ABSTRACT
The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 in blood donors in Guyana has never been estimated. We evaluated the prevalence of these viruses in blood donors by enzyme-linked immunosorbent assay and Western blotting and showed a prevalence of HTLV-1 of 1.3%; no HTLV-2 was detected. Female donors had a much higher HTLV-1 seroprevalence (3.6%) than male donors (0.7%). HTLV-1-seropositive donors tended to be slightly older than the average age for the total pool of donors. We also investigated the phylogenetic and molecular characteristics of HTLV-1 strains in Guyana and compared them with those identified in Suriname and French Guiana. Analysis of portions of the env and long terminal repeat nucleotide sequences showed that all the strains in Guyana and Suriname, like those in French Guiana, belonged to the transcontinental group of cosmopolitan subtype A. The similarities were greater between strains from Suriname and Guyana than between strains from Suriname and Guyana and those from French Guiana. Nevertheless, our results confirm that the HTLV-1 strains in all three countries have a common African origin.
Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 are members of a group of mammalian retroviruses with similar biological properties and tropisms for T lymphocytes. HTLV-1 is the causative agent of adult T-cell leukemia/lymphoma (36) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) (11). It has also been associated with a number of inflammatory diseases, including pediatric infectious dermatitis (22), uveitis (28), and some cases of arthropathy (18) and polymyositis (29). HTLV-2 may be responsible for neurological syndromes that are clinically similar to TSP/HAM (16, 31), but no hematological disorders have definitely been linked to infection with this virus (9, 17).
HTLV-1 is endemic in areas such as southern Japan, sub-Saharan Africa, the Caribbean Basin, and parts of South America (13). The overall prevalence of severe HTLV-1-associated disease is 2 to 8% among HTLV-1-infected persons, estimated to represent 15 million to 25 million individuals worldwide. HTLV-2 has been shown to be endemic in various American Indian populations (2, 3, 33, 41, 43, 44) and has also been endemic for the past 10 to 20 years among intravenous drug users in Europe and North America (32, 39, 40). In populations in which these viruses are endemic, HTLV-1 and HTLV-2 are transmitted between sexual partners and from mother to child during breast-feeding.
The viruses are also transmitted via blood by needle sharing among intravenous drug users (32), and the transmission of HTLV-1 by blood transfusion has been documented in several studies (30, 42). One report indicated that 65% of patients who received whole blood or cellular blood components from HTLV-1-seropositive donors seroconverted (19). It has also been reported that patients with histories of blood transfusion rapidly develop HTLV-1-associated diseases, such as myelopathy and uveitis, after seroconversion. Posttransfusion cases of TSP/HAM appear to be more severe and to evolve faster than nonposttransfusion cases (25, 47). Therefore, public health authorities in many countries have implemented routine screening for antibodies to HTLV-1 and HTLV-2 in blood banks. Systematic screening of all blood donated in the French overseas territories where HTLV-1 and HTLV-2 have been identified to be endemic, including French Guiana, and in the West Indian islands of Guadeloupe and Martinique began in January 1989. A study of HTLV-1-infected blood in an area where it is endemic (Guadeloupe) and an area of France (Paris) where it is not endemic concluded that screening of all donated blood components was a useful measure for preventing HTLV-1 and HTLV-2 infection by transfusion (27). In some South American countries, such as Guyana, however, there are no reliable data on the seroprevalence of HTLV-1 and HTLV-2. One report from Suriname showed a seroprevalence of 1.2% among blood donors (1). Furthermore, molecular epidemiological studies have not been conducted in these countries.
There is no defined treatment for patients infected with HTLV-1, but accurate knowledge of seroprevalence rates in various population groups may be helpful in establishing prophylactic measures to reduce the rates of viral transmission from infected individuals. The aim of this study was to evaluate the prevalence of these viruses in Guyana and to investigate the molecular characteristics of strains from different ethnic groups living in the Guyana Shelf, which comprises Suriname, Guyana, and French Guiana.
MATERIALS AND METHODS
Population.Guyana is located in the Amazonian Forest complex, on the northeast coast of the South American continent, between Suriname, Brazil, and Venezuela, and has an area of 216,000 km2 (Fig. 1). The population of approximately 750,000 is made up of six ethnic groups: Guyanese Africans (Creoles) of mixed European and African descent; Amerindians; Marrons; and immigrants from India, China, and Portugal.
Map of Guyana, Suriname, and French Guiana, with location of Guyana Shelf in South America (inset).
Specimen collection and serological tests.Between January and May 2002, sera were collected from donors at a blood bank in Georgetown, Guyana, after informed consent had been obtained; and the ages, sexes, and ethnicities of the donors were ascertained. All the sera were screened for antibodies to HTLV-1 and HTLV-2 by two methods: a gelatin-particle agglutination assay (Serodia HTLV-1 kit; Fujirebio Inc., Tokyo, Japan), performed at the blood bank in Guyana, and an enzyme immunoassay (Cobas Core Anti-HTLV-1/2 EIA; Roche, Basel, Switzerland), performed at the Laboratory of Retroviruses at the Pasteur Institute of French Guiana. Positive or borderline-positive samples were analyzed by Western blotting (HTLV blot, version 2.3; Diagnostic Biotechnology, Singapore, Republic of Singapore, and Cambridge Biotech, Worcester, Mass.) to confirm seropositivity and to differentiate between HTLV-1 and HTLV-2 infection. Samples were considered positive for HTLV-1 when they displayed virus-specific bands corresponding to the major gag antigens p19, p24, and p53; recombinant glycoprotein gp21; and the MTA-1 peptide, an HTLV-1-specific peptide of the gp46 envelope glycoprotein. Samples were considered of indeterminate positivity when they showed only gag p19 and/or p24 and no env reactivity.
Molecular studies.DNA was extracted from uncultured peripheral blood mononuclear cells (PBMCs), and a PCR was performed with several HTLV-1-specific primers whose sequences were specific for sequences within the long terminal repeat (LTR) and env regions. Each PCR mixture contained 1.5 μg of PBMC DNA, 0.2 mmol of a deoxynucleoside triphosphate mixture (Boehringer, Mannheim, Germany) per liter, 10 μl of a 10-fold reaction buffer (Perkin-Elmer Cetus, Norwalk, Conn.), 2.5 mmol of MgCl2 per liter, 10 μmol of each oligonucleotide primer per liter, and 0.5 μl of Taq DNA polymerase (Perkin-Elmer Cetus) in a total volume of 50 μl. The PCR master mixture was prepared and the PCRs were performed by two different researchers in three physically separate rooms. Two negative PCR controls (DNA from HTLV-negative samples and water only without DNA) were used for each experiment. PCR (35 cycles) or seminested PCR was performed with genomic DNA extracted from PBMCs as described previously by Kazanji et al. (21).
Env was amplified by seminested PCR with env1 and env22 as the outer primers and env1 and env2 (522 nucleotides) as the inner primers, as described previously (23). This part of the env gene corresponds to the end of the carboxy terminus of gp46 and most of gp21. For amplification of the LTR, a nested PCR was carried out as described by Mahieux et al. (24). A first fragment of 512 bp was amplified with primers P23ACDF and LTRU5E as the outer primers, and then a second fragment of 480 bp was amplified with primers 8255 and 420LTR as the inner primers.
Purified PCR products were cloned with the pCR2.1 TOPO plasmid (Invitrogen, Carlsbad, Calif.), and positive clones were selected, extracted, purified, and sequenced with an automatic sequencing system (Euro Sequence Gene Services, Evry, France).
Phylogenetic analysis.In order to obtain more general information about the origins and the homologies of the HTLV-1 virus strains in the Guyana Shelf, PBMCs from four patients with TSP/HAM known to be infected with HTLV-1 were obtained from the University Hospital of Paramaribo, Paramaribo, Suriname. After DNA extraction, PCR amplification for the HTLV-1 env and LTR genes, cloning, and sequencing were carried out as described above.
For the phylogenetic analysis, the env and LTR sequences were aligned by use of the Clustal W program and were then analyzed manually with the Ed editor of the MUST package (34). Phylogenetic relationships were reconstructed by the distance neighbor-joining method (38), and confidence levels were estimated with the distance NJBOOT program (1,000 replicates with the MUST package).
RESULTS
Serological studies for HTLV-1 and HTLV-2.Between January and May 2002, 1,035 consecutive blood donors were screened for HTLV-1 and HTLV-2. As shown in Table 1, 13 donors (1.3%) were positive for HTLV-1, as confirmed by Western blotting. Female donors had a much higher seroprevalence (3.6%) than males (0.7%). Of the 13 donors, 9 were of Guyanese African origin, 1 was Guyanese Indian, 1 was Amerindian, and 2 were of mixed origin. The median age of all the donors included in the study was 32 years (age range, 16 to years 84), and most (81%) were male. HTLV-1-seropositive donors tended to be slightly older than the average age for the total pool, with a median age of 46 years (age range, 31 to 65 years) (Table 2). Two of the blood donors were found to be seropositive by enzyme-linked immunosorbent assay and gelatin-particle agglutination tests, but Western blotting analysis showed an indeterminate serological pattern, with a unique band only against p24. No HTLV-2 was found in the donors by Western blot analysis.
Seroprevalence of HTLV-1 among regular blood donors of various ethnic groups in Guyana
Seroprevalence of HTLV-1 among male and female blood donors in various age groups
Molecular characterization.DNA was collected from six HTLV-1-infected blood donors, and portions of the env and LTR sequences were obtained (Table 3). Neither nucleotide deletions nor nucleotide insertions were observed in the 522-bp fragment of the env sequence. The six strains from Guyana were closely related (99.0 to 100% nucleotide similarity) and were similar to cosmopolitan HTLV-1 prototype strain ATK (98.2 to 98.8% similarity). The similarities of the env sequences varied from 96.1 to 96.7% with central African subtype B strain EL to 91.9 to 92.9% with strain MEL5 (Melanesian subtype) and 96.5 to 97.1% with strain PYG19 (subtype D). After alignment with the newly established sequences from Suriname, presented in Table 3, and those from French Guiana, previously reported by Talarmin et al. (46), the sequences from Guyana showed strong homology with those from Suriname (98.2 to 99.8%) and with those from French Guiana (98.0 to 99.4%). The strains from Guyana were more closely related to strains from Creoles and Amerindians (98.2 to 99.4%) in French Guiana than to strains from Noirs Marrons (98.0 to 99.2%).
Characteristics of HTLV-1-infected blood donors in Guyana and patients with TSP/HAM in Suriname
Comparison of a portion of 480 nucleotides of the LTR of six strains from Guyana showed that all were closely related, both to each other (96.9 to 99.5% nucleotide similarity) and to cosmopolitan HTLV-1 prototype strain ATK (96.4 to 96.7%). The identities of the LTR sequences of these strains varied from 93.1 to 93.4% with central African subtype B strain EL to 89.4 to 89.6% with strain MEL5 (Melanesian subtype) and 94.8 to 95.0% with strain PYG19 (subtype D). Comparative sequence analysis showed that the strains from Guyana were slightly more closely related to those from Suriname, with 95.7 to 99.2% homology, than to strains from French Guiana, with 95.5 to 99.0% homology. As with the env sequence analysis, the most widely divergent strains were those from Noirs Marrons living in French Guiana.
Phylogenetic analysis.The 522-bp fragment of the env gene was analyzed phylogenetically in 66 selected HTLV-1 sequences (24, 50) from each subtype, including the 6 new strains from Guyana, the 4 new strains from Suriname, and the 14 sequences from French Guiana published previously (46). As seen in the phylogenetic tree (Fig. 2), all the strains from Guyana belonged to the cosmopolitan subtype (HTLV-1 subtype A), and molecular clusters could be distinguished. These sequences clustered with strains from Suriname and were closely related to the strains from French Guiana derived from Amerindians and Creoles and to some strains from Brazil.
Env phylogenetic tree constructed by the neighbor-joining method with a fragment of 522 bp encompassing the end of the carboxy terminus of the gene for gp46 and most of the gene for gp21 in 66 HTLV-1 strains, including 6 new strains from Guyana (with the prefix Gya, in bold), 4 new strains from Suriname (with the prefix Sur, in bold), and 14 previously published strains from French Guiana (F. Guiana; with the prefixes AM, CR, and NM, in bold). Strain PTM3 (an STLV-1 strain) was used to root the tree. The numbers along the ancestral segments indicate the robustness of each node, as estimated from 1,000 bootstrap samplings of the data. The geographical origins of the strains other than those from Guyana, Suriname, and French Guiana reported in the cosmopolitan HTLV-1 subtype A are as follows, from top to bottom: PT12, PT3, and PT5, Brazil; PT1 and PT18, Caribbean; Abel2, Reunion Island; PH121, Senegal; CH, Caribbean; ARGMF, Argentina; TSP1, MEL, ATK, H5, OUL, AKR, AKE, MAP, BAT, and MT2, Japanese group; HS35, Caribbean; SIE and SIK, Ivory Coast; PH122, Senegal; and PH333, Mauritania. The sequences in the phylogenetic tree are reported elsewhere (12, 13, 23, 24, 46, 49, 50).
Comparison of the total LTR sequences of the six strains from Guyana and those of the selected HTLV-1 sequences for each subtype confirmed the results of the env gene analysis: four main genomic clades were identified in the phylogenetic tree (Fig. 3). The first clade corresponds to the cosmopolitan group (HTLV-1 subtype A, transcontinental subgroup) and includes strains from Guyana (strains Gya468, Gya542, Gya572, Gya813, Gya1280, and Gya1515), Suriname (Sur230, Sur229, SurHM22, and Sur1597), and Creoles (strain CAM) and Amerindians (strain NAR) from French Guiana, whereas the strain from the Noirs Marrons (strain NM1626) in French Guiana clusters with a strain originating from West Africa (strain HS35) (12). The second clade corresponds to the Central African cluster (HTLV-1 subtype B); and the third clade corresponds to two pygmy strains (strains H23 and PYG19), which form a distinct molecular cluster (subtype D) (24). The fourth clade corresponds to the more distant Asian HTLV-1 and includes the MEL5 strain (Solomon Islands). Strain PTM3 (a simian T-cell lymphotropic virus type 1 [STLV-1] strain) from Sulawesi was used as the outgroup (48).
LTR phylogenetic tree constructed by the neighbor-joining method with 47 HTLV-1 strains, including 6 new strains from Guyana (with the prefix Gya, in bold), 4 new strains from Suriname (with the prefix Sur, in bold), and 3 previously published strains from French Guiana (CAM, NAR, and NM1626, in bold) for a portion of 480 bp from the LTR. Strain PTM3 (STLV-I strain) was used to root the tree. The numbers along the ancestral segments indicate the robustness of each node, as estimated from 1,000 bootstrap samplings of the data. The geographical origins of the strains other than those from Guyana, Suriname, and French Guiana reported in the cosmopolitan HTLV-1 subtype A are as follows, from top to bottom: BOI, French Caribbean; TSP1, Japan; Cr1, Caribbean; AFS911, South Africa; FCR, MASU, MAQS, AMA, and JCP, Brazil; CMC, Taiwan; CH, Caribbean; MT2-LB, Japan; P73RM, United States; YS, MT4-LB, HKN, H5, HCT, ATK, and ATM, Japanese group; and HS35, Caribbean. The sequences in the LTR phylogenetic tree are reported elsewhere (12, 13, 23, 24, 46, 49, 50).
DISCUSSION
We present here the results of serological and molecular studies showing that Guyana is a region where HTLV-1 infection is endemic. Serological studies of samples from 1,035 blood donors showed that this virus might infect 1.3% of this population. This seroprevalence rate is comparable to those in areas of the Caribbean where the rates of endemicity are high, such as Jamaica (2.1% in 1987) (26), Trinidad and Tobago (1.5% in 1991) (6), Suriname (1.2% in 1995) (1), French Guiana (2.0% in 1985) (8), and Brazil (1.4% in 1994) (7). In some Caribbean countries, such as French overseas territories, however, the prevalence of HTLV-1 infection among blood donors has been reduced significantly by systematic screening of all donated blood, the prevalences being 0.33% in Guadeloupe and 0.4% in Martinique in 1999 (5, 37).
Although HTLV-2 infection is endemic in many populations in South America, especially in Brazil, this virus was not detected in our study of blood donors in Guyana. The rate of infection with this virus may therefore be very low in this population, although a seroepidemiological survey will be necessary to confirm these observations.
The majority of the blood donors were male; however, female donors had a much higher seroprevalence of HTLV-1 than males, perhaps due to preferential sexual transmission from male to female. Furthermore, HTLV-1-seropositive donors tended to be slightly older than the average age for the total pool of donors. Although the number of infected individuals was low, a similar pattern has been observed in previous studies (20, 45).
Molecular epidemiological studies have shown few nucleotide changes in HTLV-1 strains that are specific for the geographical origin of the patient. Our molecular studies showed that all the strains from Guyana belonged to the transcontinental group in cosmopolitan subtype A. Furthermore, we found sequences from Suriname, French Guiana, Brazil, the Caribbean, and Africa in the env and LTR phylogenetic trees and in the cluster formed by the sequences from Guyana. These results clearly show the common origin of these strains. The finding that some strains from Guyana were more closely related to strains from Suriname than to other strains, such as strains from French Guiana, might be explained geographically, as there is more exchange between Suriname and Guyana than between Suriname or Guyana and French Guiana.
Our findings confirm the results of other groups (14) and also of demonstrations that in South America HTLV-1 is found mainly in populations of African ancestry (4, 12). In our study, most infected individuals whose samples were in the blood bank were Guyanese Africans; only one Amerindian had HTLV-1 infection. Many studies of the seroprevalence of HTLV-1 in South America have shown that the prevalence of HTLV-1 is much higher among peoples of African origin than among Amerindians (15). It has been suggested that the Amerindian populations were infected through contact with slaves of African origin (13, 49). In a previous study (46), it was also shown that Amerindians in French Guiana were infected through contact with Creoles.
In that study (46), it was also shown that the Noirs Marrons strains constitute a separate cluster in the transcontinental subgroup, belonging to the West African subgroup and being different from the Creole and Amerindian clusters. A high seroprevalence of HTLV-1 (8.0%) and a high incidence of cases of adult T-cell leukemia/lymphoma were found among the Noirs Marrons in French Guiana, an isolated population descended from slaves who escaped from Suriname in the 18th century (10, 35). Curiously, in our sequence comparison and phylogenetic analysis, none of the strains from Guyana or Suriname was closely related to the strains from Noirs Marrons. In French Guiana, interethnic mixing is very rare, especially between Noirs Marrons and the other ethnic groups, and the high seroprevalence of HTLV-1 in this population is probably due to its isolation and to high rates of mother-to-child and sexual transmission (35).
In conclusion, our study demonstrates clearly that blood donors in Guyana have a high prevalence of HTLV-1 infection. Therefore, preventive measures to decrease the spread and transmission of this human retrovirus are warranted. These measures could include (i) systematic screening of blood donors for HTLV-1, which has been effected in French Guiana since 1990; (ii) systematic screening of pregnant women and counseling about the risk for HTLV-1 transmission associated with prolonged breast-feeding; and (iii) prevention of sexual transmission of HTLV-1 by educational programs emphasizing the importance of using condoms.
ACKNOWLEDGMENTS
We are grateful to Antoine Gessain for critical reading of the manuscript.
We also thank the Ministry of Research (Programme de Recherche Fondamentale en Microbiologie des Maladies Infectieuses et Parasitaires) and the Association pour la Recherche contre le Cancer (ARC grants 7590 and 5700 [to M.K.]) for financial support.
FOOTNOTES
- Received 6 October 2003.
- Returned for modification 19 November 2003.
- Accepted 13 January 2004.
- Copyright © 2004 American Society for Microbiology
