HIV-1 diversity in Romania

Objectives: To evaluate the prevalence and the dynamics of HIV-1 subtypes in Romanian adults and children, and to investigate the origins of the nosocomial epidemic.

Design: A total of 1000 serum and plasma samples, from adults (n = 579) and children (n = 421) who were diagnosed as being HIV-1-infected during 1990-1997 in 39 of the 41 Romanian districts, were serotyped. Viral DNA was isolated from blood samples of 84 patients and the viruses were genotyped.

Methods: Serotyping was performed with a peptide subtype-specific enzyme immunoassay (SSEIA), based on in vitro competition for antibody binding between the representative V3 peptides of the different clades (A-F). Proviral HIV-1 DNA was genotyped by heteroduplex mobility assay or by sequence analysis of the C2-V3 env region.

Results: SSEIA showed that 93% of the samples from horizontally infected children were serotype F, 1% were serotype B, and the remaining 6% were uninterpretable. In vertically infected children, 74% of strains were serotype F, 10% were serotype A, 3% were serotype B, and 3% were serotype E. Serotype F was also the dominant subtype in adults (68%), but serotypes A, B, C, D and E were also detected. SSEIA gave indeterminate results in 7% of cases. A strong correlation (90%) between serotyping and genotyping for subtype F was found. Analysis of the relative incidence of the different serotypes over a 7-year period (1990-1997) showed a stable distribution.

Conclusions: Subtype F largely dominates the epidemiology of HIV-1 infection in both children and adults in Romania, although other major subtypes are present. The predominance of subtype F in Romania may be a future potential source of HIV-1 variability in Europe.

Introduction

HIV-1 was first introduced into Romania in 1985, but only 13 AIDS cases had been reported by 1989 [¹]. In 1990, an HIV-1 outbreak was reported among children in hospitals and orphanages [²]. Seroepidemiological studies revealed (i) a uniform distribution of cases throughout the country [^3,4], (ii) a predominance of infection among children aged < 4 years, (iii) a close association with hepatitis B virus infection [^1,5], and (iv) the recent nature of the outbreak [⁴]. Because most HIV-1-infected children were born to uninfected mothers, HIV-1 transmission in this group was presumed to be related either to transfusion of unscreened blood or blood products, or to the re-use of unsterilized needles and syringes [^6,7]. However, the source(s) of the outbreak remained unclear because of the low prevalence of HIV-1 infection in adults, including blood donors [¹].

One of the most puzzling findings in the HIV-1 outbreak among Romanian children was described early in 1994, when the first report on Romanian HIV-1 strains was published [⁸]. A new HIV-1 subtype, subtype F, was defined. Several reports subsequently revealed that this subtype was present in former Zaïre [⁹], Cameroon [¹⁰], Brazil [¹¹], Argentina [¹²], Martinique [¹³], Cyprus [¹⁴], Russia [¹⁵], France [¹⁶], Belgium [¹⁷], and The Netherlands [¹⁸], but at a low prevalence. By contrast, this was the only subtype found in nosocomially infected children throughout Romania [^19,20]. Studies on HIV-1 strains circulating in Romanian adults also revealed the predominance of subtype F [²⁰], although it was found to cocirculate with subtypes B and G [²¹]. However, because genotyping methods have not been available in Romania, few strains have been characterized.

The objectives of this study were (i) to conduct a large-scale analysis of HIV-1 diversity in Romania in order to determine the origins of the outbreak in children, and (ii) to evaluate the prevalence and dynamics of HIV-1 subtypes in adults and children. HIV-1 diversity was investigated by determining the serotype in a peptide-based serological assay [²²], and by comparing the results of serological investigations with genotypic characterization.

Materials and methods

Samples

Serum or plasma samples from 1000 HIV-1-infected Romanians (579 adults and 421 children) collected between 1990 and 1997 were tested, and were confirmed by Western blot. The paediatric samples originated from the northeast (n = 198), northwest (n = 74), southwest (n = 79), south (n = 20), southeast (n = 8), and Bucharest (n = 42). The children had been infected either horizontally (n = 351) or vertically (n = 70). Horizontal infection was diagnosed when mothers tested HIV-1-seronegative or by documenting HIV-1 seroconversions in children.

The adult samples were from the northeast (n = 46), northwest (n = 33), southwest (n = 32), south (n = 89), southeast (n = 34), and Bucharest (n = 345). Samples were obtained from 39 of the 41 Romanian districts. No cases of HIV-1 infection have been reported to date in the remaining two districts. Most adults were infected by heterosexual contact, and some by homosexual contact (n = 4) or transfusion (n = 11). A total of 135 of the 179 Romanian blood donors who tested HIV-seropositive during 1990-1997 were included in the study. Information on age, sex and risk factors for HIV infection were collected when available.

Genotyping

Whole blood-EDTA samples were obtained from 86 HIV-1-infected individuals (66 children and 20 adults). Peripheral blood mononuclear cells were separated and cocultured as previous described [²³]. Cocultures yielded 84 HIV-1 isolates (from 64 out of 66 children and all 20 adults). Proviral DNA was genotyped on the basis of the C2-V3 env region by sequencing with or without heteroduplex mobility assay; genotyping results have been published elsewhere [^19,20]. Corresponding plasma samples from these patients were subjected to serotyping; genotyping and serotyping results were compared in order to assess the reliability of the serotyping assay.

Serological subtype differentiation

Serotyping was performed using an HIV-1 subtype-specific enzyme immunoassay (SSEIA), as described by Barin et al. [²²] and Plantier et al. [²⁴]. SSEIA avoids the major cross-reactivity of peptide binding that is usually observed in an indirect enzyme-linked immunosorbent assay (ELISA) V3 format by blockade with excess peptide in the liquid phase.

Polyvinyl microtitre plate (Falcon) wells were coated with a mixture of peptides corresponding to HIV-1 subtype A-F V3-loop consensus sequences [^25,26]: subtype A, NNTRKSVHIGPGQAFYATGDIIG DIRQAHC; subtype B, NNTRKSIHIGPGRAFY TTGEIIGDIRQAHC; subtype C, NNTRKSIRI GPGQTFYATGDIIGDIRQAHC; subtype D, NNTRQRTHIGPGQALYTT.RIIGDIRQAHC; subtype E, NNTRTSITIGPGQVFYRTGDIIGDIR QAHC; subtype F, NNTRKSIHLGPGQAFYATG DIIGDIRKAHC. Peptides were synthesized by the Virus Laboratory of the F. Rabelais University (Tours, France) and SSEIA was performed as previously described [²²]. Only post-seroconversion samples were subjected to serotyping assays, because previous studies have shown that SSEIA lacks sensitivity in the early phase of seroconversion [¹⁶].

Results

SSEIA subtype determination

A total of 932 cases (93%) were unambiguous in SSEIA, and it was not possible to interpret 68 (7%) cases (Table 1). Of the 351 samples from horizontally infected children, 327 (93%) were serotype F, two were serotype B (1%), and the remaining 22 (6%) were not interpretable. Most of the samples that could not be serotyped (n = 18) originated from patients with AIDS and were characterized by the absence of anti-V3 reactivity. In the remaining four cases, strong cross-reactivity hampered subtype determination by SSEIA. Fifty-two samples (74%) from 70 vertically infected children were serotype F, seven (10%) were serotype A, two (3%) were serotype B, and two (3%) were serotype E. Seven samples from vertically infected children gave indeterminate results and originated from AIDS patients.

Table 1

A total of 540 (93%) out of 580 samples from adults gave unambiguous results: 398 (68%) were serotype F, 78 (14%) were serotype A, 53 (9%) were serotype B, five (1%) were serotype C, one (0.17%) was serotype D, five (1%) were serotype E, and the remaining 45 (7%) samples were ambiguous (Table 1). Among the ambiguous samples, 23 (10 from seroconverters and 13 from AIDS patients) had no anti-V3 reactivity. In the remaining 22 samples, a high level of cross-reactivity hampered subtype determination by SSEIA.

Comparison between SSEIA results and genotype determination

Eighty-four samples were genotyped. The comparison between serotyping and genotyping results was limited by the absence of peptides representing HIV-1 subtypes G-J in the SSEIA test. In patients genotyped as subtype F (n = 78), a strong correlation between SSEIA and genotyping was found (90% concordance). Two nosocomially infected children were repeatedly serotype D but genotype F. Sequence analysis confirmed that these strains belonged to subtype F but had a GPGR motif on the crown of the V3 loop [¹⁹] characteristic of subtype B strains [²⁵]. Samples from six children with AIDS who were genotyped as subtype F could not be serotyped by SSEIA. Two serotype B samples from adults were genotyped as subtype B, and three serotype A samples were genotype A. A sample from an adult that was genotyped as subtype G gave indeterminate results by SSEIA.

Distribution of HIV-1 serotypes in Romania

Risk-group distribution

Epidemiological data supported the results obtained by SSEIA. Four HIV-1-infected homosexual men and four female heterosexual contacts of Western European HIV-1-infected patients were infected by serotype B. One patient who worked for several years in Israel and reported sexual contact with Ethiopian prostitutes harboured a serotype C strain. A merchant sailor, who was probably infected by an African prostitute, was serotype A. Four haemophiliacs and seven other patients infected before 1990 by transfusion with unscreened blood were infected by serotype F. Subtype A prevailed in African patients living in Romania (n = 5), whereas patients originating from the Middle East were infected by serotypes A (n = 1), B (n = 2), or F (n = 2). Two African patients were infected by serotype F, but data concerning the time of infection (before or after entering Romania) were not available. Six prostitutes were included in this study and were serotyped as subtype F (n = 4) and subtype B (n = 2); those who harboured subtype B were infected in western Romania. In 28 heterosexual couples, transmission included serotypes F (n = 20), A (n = 4), and B (n = 3), and one sample that could not be typed.

All horizontally infected children were infected by serotype F. Conversely, serotyping of samples from vertically infected children showed the cocirculation of several serotypes in this group, including subtypes F (n = 52), A (n = 7), B (n = 2), and E (n = 2), in addition to seven cases in which SSEIA gave indeterminate results.

Geographical distribution

HIV-1 serotype F dominated the adult HIV-1 epidemic from all regions of Romania (Fig. 1). The geographic distribution was variable, with a prevalence ranging from 50% in the western region to 84% in northeast Romania. Subtype B was frequent (31%) in the western regions. Almost all major viral subtypes were detected in southeast Romania and in Bucharest (Fig. 1).

Fig. 1

All nosocomially infected children were infected by subtype F, regardless of their geographic origin (Fig. 1). Subtype F also dominated the cases of vertical transmission in all geographical regions, although other major HIV-1 subtypes were also present (Fig. 1). HIV-1 diversity in vertically infected children generally reflected diversity in adult patients. In northeast Romania there were no cases of vertical transmission involving subtypes other than F, probably because of the high prevalence of this subtype in the adult population in this region (84%).

Incidence of HIV-1 subtypes in 1990-1997

The samples were also analysed according to the date of collection in order to investigate the dynamics of HIV-1 diversity in Romania (Table 2). Although variations in relative prevalence were observed in the first 2 years of the study, probably because of the small number of samples, a stable prevalence of the different subtypes was recorded in the last 7 years. Thus, subtype F represents 67-74% of the HIV-1 strains circulating in Romanian patients. The lower prevalences determined in 1996 and 1997 were probably due to a higher proportion of samples originating from recently HIV-1-infected subjects (post-seroconvertors) that could not be subtyped (Table 2). The prevailing subtype F was also associated with subtypes A (7-23%) and B (5-11%). The incidence of subtypes C, D, and E seemed to be very low and their occurrence in the Romanian population was a result of strain trafficking.

Table 2

Discussion

We used a recently developed blocking assay [^22,24] to characterize HIV-1 diversity in Romania. Peptide immunoassays based on HIV-1 V3 sequences are thought to be an effective subtyping approach for investigating geographic distribution [^27,28]. However, cross-reactivity is frequent in indirect ELISA formats [²⁹], and previous serotyping attempts based on indirect ELISA have failed to discriminate subtype F from other HIV-1 group M subtypes. In the first serotyping study, the serological profile in Romania was similar to that obtained in the United Kingdom and the United States (i.e., B subtype [³⁰]), whereas in the second study it was concluded that subtype A dominated the epidemiology of HIV-1 infection in Romania [³¹].

The results we obtained by using the serotyping assay developed by Barin et al. [²²] were compared with genotyping results in 84 patients. A strong correlation between genotyping and serotyping methods was obtained. Sixty-eight samples were not serotyped for the following reasons: (i) serotype B reactivities for subtype F strains (n = 2) with GPGR motifs in the V3 crown sequence [¹⁹]; (ii) lack of V3 reactivity in recent adult seroconverters (n = 10) or AIDS patients (n = 38); and (iii) multiple reactivity hampering identification in the remaining cases (n = 18). Because the format of the serotyping assay used in this study did not allow the detection of serotypes G-J, it was possible that some of the untyped samples yielding ambiguous results belonged to these subtypes. Indeed, one sample was genotyped as subtype G.

Despite the fact that results were indeterminate in 7% of cases, we would consider the serotyping assay to be the method of choice for HIV-1 subtyping in countries in which a particular HIV-1 subtype prevails. The method is simple and does not require sophisticated technical facilities or specially trained personnel. It is also cost-effective and can be used to screen a large number of samples rapidly. The technique is therefore suitable for use in developing countries. Knowledge of HIV-1 diversity in different regions [^16,17,32-35] and its consequences on virus biology, pathogenesis and the transmission rate of different subtypes [^36-38], diagnostics [^39,40], epidemiology [⁴¹], vaccination strategies [⁴²] and patient follow-up [^43,44] suggests that a method such as SSEIA could be used on a large scale.

Viral trafficking linked to human behaviour has been proposed as the main factor behind the geographic distribution of different HIV subtypes [^16,45]. A founder effect was described for HIV-1 subtype F in Romania, resulting in the unique epidemic among nosocomially infected children [⁴⁶]. This study confirmed the frequency of subtype F, not only in children but also in adults. In addition, our results underline the importance of strain trafficking and document the cocirculation of subtype F with subtypes A, B, C, D and E. The presence of internationally prevalent subtypes in Romania is logical, because the main route of transmission among Romanian adults is heterosexual, with some HIV-1-seropositive adults being infected through contacts with foreign visitors or with Romanians who have travelled abroad. The regions most affected by the AIDS epidemic are those around the major portals into Romania, the capital and the most important port (Constanta); extensive viral diversity is also observed in these regions. Subtype B is more frequent in the northwest regions, which have the closest contact with Western Europe.

HIV-1-seropositive blood donors revealed a similar profile of viral diversity and a higher prevalence than in neighbouring Eastern European countries. The high prevalence of non-B subtypes among blood donors (86%) might pose problems for the safety of blood donations, because many screening tests lack sensitivity during seroconversion to these particular subtypes [⁴⁰].

Studies on the dynamics of the spread of various HIV-1 subtypes in western countries have yielded contradictory data on the evolution of viral diversity [^17,47,48]. Our observations over a period of 7 years showed that there was a stable relative prevalence of different HIV-1 subtypes amongst adults, probably because transmission in Romania occurred primarily through heterosexual contact, international travel was not widespread, and no further nosocomial epidemic was documented.

HIV-1 subtype F was also the main viral variant involved in vertical transmission, although subtypes A, B and E were also detected. Conversely, only subtype F was detected in this study in nosocomially infected children from all over the country. We have previously documented a high degree of sequence homology among strains obtained from horizontally infected children in different regions of Romania, pointing to a single introduction of subtype F in this risk group [^20,49], probably related to the use of infected blood products [²⁰]. The subsequent explosive spread in orphanages resulted from improper medical practices [^6,50]. Epidemiological, serological and molecular data [^8,19,20], which reveal a uniform distribution of HIV-1 subtype F in nosocomially infected children, blood recipients and haemophiliacs throughout the country, reinforce the hypothesis that this virus subtype was introduced to Romania through an HIV-infected blood product.

However, the precise source of subtype F in Romania remains to be established. Numerous studies have shown worldwide circulation of this subtype [^9-15,19,20]. Phylogenetic analyses of the env genes of subtype F strains with different geographical origins [^15,46] suggest that separate epidemiological events have contributed to the worldwide emergence of this subtype [²⁰]. Unlike other HIV-1 subtypes, the origin of which has been traced to Africa [^51,52], the ancestry of subtype F strains has not yet been determined. However, whatever the origin of subtype F in Romania, the widespread circulation of a divergent subtype in a European country should be considered as a potential source of further strain variability.

Acknowledgements

The authors are grateful to the staff of Laboratories of the Romanian Network of Blood Transfusion Centre laboratories, the staff of the Clinics for Infectious Diseases in Romania and Professor Lütz Gürtler (Max von Pettenkofer Institute, Munich, Germany) for providing samples.