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CLINICAL SCIENCE

Treatment outcome in dually HIV-1 and HIV-2 coinfected patients living in Spain

Requena, Silviaa; Caballero, Estrellab; Lozano, Ana Belénc; Ríos-Villegas, María Joséd; Benito, Rafaele; Rojo, Silviaf; Cabezas, Teresag; Macià, María Doloresh; Nieto, María del Carmeni; Soriano, Vicentej; de Mendoza, Carmena,k on behalf of the Spanish HIV-2 Study Group

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doi: 10.1097/QAD.0000000000002338
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Abstract

Introduction

Human immunodeficiency virus type 2 (HIV-2) is the result from multiple zoonotic transmissions to humans from sooty mangabeys infected with the simian immunodeficiency virus (SIVsm) in West Africa [1]. Compared with HIV-1 infection, the clinical course in HIV-2 carriers is characterized by a longer asymptomatic periods, lower plasma HIV-2 viremia, and lower mortality rate [2,3]. However, persons with HIV-2 infection can still progress to AIDS over time [3,4].

HIV-2 has circulated for decades in West Africa with relatively high endemicity in some regions, such as Guinea-Bissau and Ivory Coast [3]. This fact represented an auspicious scenario for the occurrence of dual HIV-1 and HIV-2 infections, once HIV-1 entered the region and begun spreading more efficiently than HIV-2 [5]. Indeed, the first dually infected individual was reported in Ivory Coast in 1988 [6]. Outside West Africa, countries with closer colonial links with Portugal, such as Brazil and India, have the largest HIV-2 populations [3,7]. More recently, important migration flows from HIV-2 endemic regions to Europe and North America account for reports of HIV-2 overseas [8,9]. The most recent estimate for HIV-2 globally is of one to two million infected people [7], with dual infections representing less than 5%.

In our study, we aimed to characterize the rate, main features and treatment outcome of HIV-1 and HIV-2 dually infected individuals living in Spain. To our knowledge, this work represents the largest series of dually coinfected patients treated in Europe.

Methods

The Spanish HIV-2 Study Group was founded in 1989. More than 40 clinics and/or laboratories distributed nationwide belong to the network and contribute recording periodical information from new HIV-2 diagnosed persons [10]. Main demographics, clinical and virological status of all HIV-2 infected individuals are recorded within a uniform database, from the moment of first diagnosis and regularly during their follow-up.

We retrospectively identified all HIV-2-infected individuals who were coinfected with HIV-1. The study included all cases identified from January 1989 up to December 2018. Currently HIV diagnosis is made following initial screening using a fourth generation antigen–antibody test. All initially reactive samples are subsequently tested using an HIV-1/HIV-2 differentiation serological assay, as recommended by the CDC HIV diagnosis algorithm [11]. In order to discriminate between serological cross-reactivity or dual infection, all samples with double reactivity are retested after serial serum dilution. Truly HIV-1 and HIV-2 dual infections keep antibody reactivity using the confirmatory test, whereas cross-reactivity is recognized because reactivity is lost for specific HIV-1 or HIV-2 antibodies on serial dilutions, as shown previously [12].

Plasma HIV-1 viremia was examined using the commercial viral load tests available at each of the participating clinics (Roche Taqman, Abbott Real Time or Siemens kPCR) whereas plasma HIV-2 RNA was measured using a noncommercial quantitative PCR (qRT-PCR) assay, as described elsewhere [13]. Briefly, the region amplified was the long terminal repeat (LTR) with specific primers and probe. Both HIV-2 groups A and B are reliably detected with this assay. Thermal cycling conditions consisted of an initial step of 2 min at 50 °C and an activation step at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min. For each run, a standard curve was generated using a stock of HIV-2 strain NIHZ, which was counted by electron microscopy and was used as the standard (Advanced Biotechnology Inc., Columbia, Maryland, USA). Prior to lysis, the stock solution contained 7.2 × 1010 virus particles/ml. The HIV-2 NIHZ stock solution was diluted to obtain 5 000 000; 500 000; 50 000; 5000; 500 and 50 copies/ml to generate the standard curve. The HIV-2 copy number in each clinical sample was estimated by interpolation from the regression curve. The results were expressed as HIV-2 RNA copies/ml. For the purpose of this study, data collected included year of diagnosis, sex, country of origin, risk group, baseline viremia, CD4+ cell counts, HIV subtype and antiretroviral therapy.

Statistical analysis

Main variables were recorded as number and percentages, or median and interquartile ranges (IQR) between 75 and 25%. Viral load was log-transformed using base 10. Plasma HIV-2 RNA and baseline CD4+ cell counts from HIV-2 monoinfected individuals recorded at the Spanish registry were used for comparisons with coinfected individuals. Mann–Whitney U test was performed for abnormally distributed variables. A Student t-test was used for variables displaying a normal distribution. All analyses were performed using the SPSS software version 20.0.

Results

Up to December 2018, 373 persons with HIV-2 infection had been reported at the Spanish HIV-2 registry. A total of 34 (9.1%) were coinfected with HIV-1. RNA from both viruses could be detected in plasma at least once in only 10 (29.4%) of these individuals during their follow-up. For the subset of nine patients diagnosed before year 2000, there were no available specimens for testing HIV-2 viremia with sensitive and reliable assays. The diagnosis of dual infection in the rest of coinfected persons was made using serological dilutions, as described elsewhere [12]. Confirmation using proviral DNA testing was further obtained in five cases, reassuring about the value of the serological procedure.

As shown in Table 1, men represented 67.6% of dually infected individuals. Their median age was 39 years, significantly younger than in HIV-2 monoinfected persons (43 years; P = 0.035). Most coinfected patients came from West African countries (21, 61.7%), although 6 (17.6%) were native Spaniards. The remaining seven persons came from other African countries (six) and Brazil (one). At the time of diagnosis, nine (26.5%) presented with AIDS-related events.

T1-6
Table 1:
Main epidemiological features of HIV-1 and HIV-2 dually infected individuals.

Genotyping for HIV-1 and HIV-2 could be achieved successfully in 12 (4 CRF02_AG; 3 B; 2 G; 1D; 1F and 1J) and 11 (10 A and 1 B) patients, respectively. In five patients with subtyping information for both viruses, variants were as follows: G and A (two), F and A (one), CRF02_AG and B (one), and B and A (one), respectively. Two native Spaniard women were infected with HIV-1 G and CRFAG_02 variants, respectively. HIV-1 subtype B was found in three men from Brazil, Gambia, and Guinea Bissau, respectively. Interestingly, one Spanish woman infected with HIV-2 subtype B was superinfected with HIV-1 12 years later. She was a commercial sex worker.

Coinfection with hepatitis B virus (HBV), as shown by persistent positive serum HBsAg, was found in three dual HIV-1/HIV-2-coinfected patients. They came from Guinea-Bissau (two) and Senegal (one). The first case was a 39-year old woman from Guinea-Bissau who had been diagnosed with HIV-1 in 1999. Ten years later, she was found to be positive for HIV-2 and HBsAg. At that time, she had CD4+ cell counts of 62 cells/μl. She started treatment with tenofovir/emtricitabine and lopinavir/ritonavir. Despite it, she developed pneumonia with Pneumocystis jirovecii and died shortly thereafter. The second patient was a 45-year old man from Guinea-Bissau who had been diagnosed in 2009 with HIV-1, HIV-2, and HBsAg. He started tenofovir/emtricitabine and lopinavir/ritonavir. Within 3 months, he achieved undetectable viremia and kept it for at least 10 months. The last patient was a 50-year old man from Senegal who had been diagnosed with HIV-1 along with HIV-2 in 2010. One year later, he was found to be positive for HBsAg. He started treatment with tenofovir/emtricitabine combined with darunavir/ritonavir and raltegravir. He achieved undetectable viremia for all viruses within 3 months, but he was lost to follow-up since year 2015. Chronic hepatitis C virus (HCV) infection was demonstrated in only one individual coming from Guinea-Bissau. No information regarding prescription of HCV therapy has been documented to date.

Before initiating antiretroviral therapy, the median CD4+ cell count in dually infected patients was of 204 (97–427) cells/μl, significantly lower than for HIV-2 monoinfected individuals [445 (223–668) cells/μl; P < 0.001). At baseline, plasma viremia was undetectable in 5% for HIV-1 and in 45% for HIV-2. Mean plasma HIV-RNA in virologically nonsuppressed individuals was of 4.9 and 3.7 log copies/ml, respectively.

Information on antiretroviral therapy prescribed could be collected for 20 individuals. Regimens based on ritonavir-boosted protease inhibitors were prescribed in 13 individuals (7 lopinavir, 3 darunavir, 1 atazanavir, 1 fosamprenavir, and 1 indinavir). Three patients were treated with integrase inhibitors (dolutegravir), another three with integrase inhibitors and boosted protease inhibitors (raltegravir and darunavir) and one with Trizivir. Overall, the median follow-up on antiretroviral therapy was of 32 (13–48) months. At last visit, the median CD4+ cell count was of 418 (190–739) cells/μl. From baseline, individuals with CD4+ cell counts less than 200 cells/μl fell to 22% on antiretroviral therapy, whereas the proportion with CD4+ cell counts greater than 500 cells/μl reached up above half of patients. Overall, undetectable plasma HIV-RNA was attained by 85 and 80% for HIV-1 and HIV-2, respectively. Viral suppression for both viruses was seen in 70% (Fig. 1).

F1-6
Fig. 1:
Treatment outcomes in 20 HIV-1 plus HIV-2 dually infected patients.Black bar: HIV-1; light grey bar: HIV-2; dark grey bar: dual HIV-1 and HIV-2. cART, combination antiretroviral therapy.

Genotyping was planned for all patients who experienced virological failure. For detectable HIV-2 infection, two patients died soon after virological failure. Of the remaining two, only one depicted M184V in HIV-2 after failing under tenofovir/emtricitabine with darunavir/ritonavir. The last patient, a female sex worker, never reached undetectable viremia despite being treated with tenofovir/emtricitabine and lopinavir/ritonavir. She did not harbour drug-resistant mutant viruses but she acknowledged poor drug adherence.

Discussion

Given that HIV infection does not elicit protective immunity, superinfection with HIV-1 in persons carrying HIV-2, or viceversa, may occur in places where both viruses co-circulate [6,14–16]. In Spain, the first confirmed case of dual HIV-1 and HIV-2 coinfection was reported in 1993 [9]. To date a total of 34 cases (9.1%) of dual infection out of 373 total number of HIV-2 cases have been accumulated.

Clinical suspicion of dual HIV-1 and HIV-2 coinfection should especially be considered for seropositive West African natives, people who has traveled to (or lived in) HIV-2 endemic regions acknowledging risk behaviors, and anyone with sexual partners from West Africa. Confirmation is required for presumably dual infections, as unique considerations for antiretroviral therapy exist for this population [17–19]. Demonstration of RNA and/or proviral DNA for both HIV-1 and HIV-2 constitute the most definitive evidence of dual infection, although discriminatory serological tests may be helpful as well [11,20].

Drugs exhibiting activity against HIV-2 should be ensured in any antiretroviral combination chosen to treat dually infected patients. All integrase strand transfer inhibitors, most nucleos(t)ide reverse transcriptase inhibitors, and some protease inhibitors, such as darunavir, lopinavir, or saquinavir depict activity against HIV-2 [21]. However, frequent selection of HIV-2-resistant variants along with slower immune restoration are relatively common whenever treating HIV-2 patients [22].

In agreement with prior studies conducted in West Africa [16–18,23], dually infected patients in Spain were younger and had lower CD4+ cell counts than HIV-2 monoinfected persons. Thus, antiretroviral therapy should not be deferred in this population and attention to include drugs active against both viruses should be ensured in order to minimize the risk of viral escape [17,18]. Furthermore, viral load monitoring should be mandatory for both HIV-1 and HIV-2 patients undergoing antiretroviral therapy.

In our series, up to 80% of dually infected patients treated achieved viral suppression for HIV-2, a rate similar to the 85% seen for HIV-1. Three of the HIV-2 virologic failures occurred in patients treated with optimal regimens, including two nucleoside analogues and one protease inhibitor (one) and one unique individual with CD4+ cell counts below 100 cells/μl, despite being treated with nucleoside analogues, protease inhibitors and raltegravir. The remaining HIV-2 failure corresponded to one person back to 2007 treated with two nucleoside analogues with fosamprenavir who failed to suppress both viruses.

Overall, the proportion of patients who successfully controlled HIV-1 and HIV-2 in our series was greater than in prior reports, in which inadvertently some individuals received nonnucleoside reverse transcriptase inhibitors [14–16]. Likewise, the CD4+ cell count gain of our dual coinfected patients was satisfactory (median increase of 212 cells/μl). It should be noted that most patients in our series received HIV-2 active protease inhibitors and/or integrase inhibitors, whose antiviral activity has been well documented [21,22].

In our knowledge, this is the first report showing the efficacy of integrase inhibitors in a small series of HIV-1/HIV-2 dually coinfected patients. A report from 2009 described one coinfected individual treated with raltegravir with nucleoside analogues that experienced a favourable treatment response [19].

The recognition of the CRF02_AG subtype as the most frequent HIV-1 variant in our dually infected patients might suggest that most coinfections could have occurred in West Africa rather than within Spain as HIV-1 superinfections of originally HIV-2 carriers [10,24]. However, local transmissions might have occurred as well, given that circulation of HIV-1 non-B subtypes is not rare among Africans living in Spain [25]. At least for one coinfected individual, a Spanish native female sex worker, we had evidence of HIV-1 superinfection whereas she continued having sexual risky behaviors. This case underlines the need for repeated testing and excluding HIV-1 periodically in the subset of HIV-2 carriers that continue having sexual risky behaviors.

In conclusion, we report a 9% dual HIV-1/HIV-2 coinfection among HIV-2 carriers in Spain. The continuous immigration flow from West African countries to Spain seems to largely account for this relatively high rate of coinfection. A proper characterization of all HIV-seroreactive patients is crucial to ensure an optimal clinical and therapeutic care of this unique population. Regimens containing integrase inhibitors or active protease inhibitors, may effectively suppress both HIV-1 and HIV-2 replications.

Acknowledgements

We would like to thank the participation of all members of the Spanish HIV-2 Network. This work was supported in part by grants from Fundación Investigación y Educación en SIDA (F-IES); FIPSE (36742/08); Fondo de Investigación Sanitaria-Fondos FEDER (FIS EC10/277, CES12/003, FI14/00264, CD14/00243), and the European Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN, project FP7–223131).

Spanish HIV-2 Network: C. Rodríguez, M. Vera and J. del Romero (Centro Sanitario Sandoval, Madrid); M.D. Ocete (Hospital General Universitario, Valencia); E. Caballero (Hospital Vall d’ Hebrón, Barcelona); A. Aguilera (Hospital Conxo-CHUS, Santiago); M.J. Amengual and M. Cervantes (Corporación Sanitaria Parc Taulí, Barcelona); R. Benito and S. Algarate (Hospital Clínico Universitario Lozano Blesa, Zaragoza); R. Ortiz de Lejarazu and S. Rojo (Hospital Clínico Universitario, Valladolid); J.M. Eirós and C. Ramos (Hospital Rio Hortega, Valladolid); J. García-Costa (Hospital Cristal-Piñor, Orense); E. Calderón (Hospital Virgen del Rocío, Sevilla; CIBER de Epidemiología y Salud Pública); M.J. Ríos-Villegas (Hospital Virgen Macarena, Sevilla). M. Trigo, J. Diz and M. García-Campello (Complejo Hospitalario, Pontevedra); M. Rodríguez-Iglesias (Hospital Universitario Puerta del Mar, Cádiz); A. Hernández-Betancor (Hospital Insular Universitario, Las Palmas de Gran Canaria); J.M. Ramos and A. Gimeno (Hospital Universitario, Alicante); V. Sánchez (Hospital General, Elche); C. Gómez-Hernando (Complejo Hospitalario Virgen de la Salud, Toledo); M.J. Echeverria, G. Cilla and E. Pérez-Trallero (Hospital Donostia, San Sebastián); L. Fernández-Pereira (Hospital San Pedro de Alcántara, Cáceres); J. Niubó (Ciudad Sanitaria de Bellvitge, Barcelona); N. Margall (Hospital Santa Creu i Sant Pau, Barcelona); M. Hernández, A.M. López-Lirola and J.L. Gómez-Sirvent (Hospital Universitario La Laguna, Tenerife); L. Force and M. Sauca (Hospital General, Mataró); S. Pérez and L. Morano (Hospital do Meixoeiro, Vigo); C. Raya (Hospital del Bierzo, Ponferrada); A. González Praetorius (Hospital Universitario, Guadalajara); C. Cifuentes, M. Peñaranda and María Dolores Macià (Hospital Son Espases, Palma Mallorca); M.C. Nieto (Hospital de Basurto, Bilbao); J.M. Montejo (Hospital de Cruces, Bilbao); L. Roc and A. Martinez-Sapiña (Hospital Miguel Servet, Zaragoza); I. Viciana (Hospital Virgen de la Victoria, Málaga); A.B. Lozano, I. Pérez-Camacho, E. Fernández-Fuertes and J.M. Fernández (Hospital de Poniente, Almería); T. Cabezas (Complejo Hospitalario Torrecárdenas, Almería); I. García Bermejo and G. Gaspar (Hospital Universitario de Getafe, Madrid); M. Górgolas, C. Vegas, J. Blas and R. Téllez (Fundación Jiménez Díaz, Madrid); L. Pérez, M. Valeiro and T. Aldamiz (Hospital Gregorio Marañón, Madrid); F. García (Hospital Clínico Universitario, Granada); A. Suárez and I. Rodríguez-Avial (Hospital Clínico San Carlos, Madrid); P. Barreiro (Hospital Universitario La Paz, Madrid); V. Soriano, F. Gómez-Gallego and O. Corral (UNIR Health Sciences School, Madrid); S. Requena, L. Benítez-Gutiérrez, V. Cuervas-Mons and C. de Mendoza (IIS Hospital Universitario Puerta de Hierro, Majadahonda).

Conflicts of interest

There are no conflicts of interest.

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* Members are listed in the Acknowledgements section.

Keywords:

antiretrovirals; HIV-1; HIV-2

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