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Molecular and epidemiological characterization of HIV-1 subtypes among Libyan patients

Molecular and epidemiological characterization of HIV-1 subtypes among Libyan patients Background: The epidemiological and clinical aspects of human immunodeficiency virus subtypes are of great inter ‑ est worldwide. These subtypes are rarely studied in North African countries. Libya is a large country with the longest coast on the Mediterranean Sea, facing the Southern European countries. Studies on the characterization of HIV‑ 1 subtypes are limited in Libya. This study aimed to determine the magnitude of the HIV problem among the Libyan population and to better understand the genetic diversity and the epidemiologic dynamics of HIV 1, as well as to cor‑ relate that with the risk factors involved. Methods: A total of 159 HIV‑ 1 strains were collected from 814 HIV positive patients from the four Libyan regions dur‑ ing a 16‑ year period (1995–2010). To determine the HIV‑ 1 subtypes, genetic analysis and molecular sequencing were carried out using provirus polygene. Epidemiologic and demographic information was obtained from each partici‑ pant and correlated with HIV‑ 1 subtypes using logistic regression. Results: The overall prevalence of HIV among Libyans ranged from 5 to 10 per 100,000 during the study period. It was higher among intravenous drug users (IVDUs) (53.9%), blood recipients (25.9%) and heterosexuals (17.6%) than by vertical transmission (2.6%). Prevalence was higher among males aged 20–40 years (M:F 1:6, P > 0.001). Among the 159 strains of HIV‑ 1 available for typing, 117 strains (73.6%) were subtype B, 29 (18.2%) were CRF02_AG, and 13 (8.2%) were subtype A. HIV‑ 1 subtype B was the most prevalent all over the country, and it was more prevalent in the North‑ ern region, particularly among IVDUs (P < 0.001). GRF02_AG was common in the Eastern region, particularly among blood recipients while subtype A emerged in the Western region, particularly among IVDUs. Conclusions: HIV‑ 1 infection is emerging in Libya with a shifting prevalence of subtypes associated with the chang‑ ing epidemiology of HIV‑ 1 among risk groups. A genetic analysis of HIV‑ 1 strains demonstrated low subtype hetero‑ geneity with the evolution of subtype B, and CRF_20 AG, as well as HIV‑ 1 subtype A. Our study highlights the impor‑ tance of expanded surveillance programs to control HIV infection and the necessity of introducing public health strategies to target the risk groups, particularly IVDUs. Keywords: HIV‑ 1 subtypes, Libya, Genetic diversity, Molecular epidemiology, AIDS, BG‑ S‑ Benghazi–Bulgarian Strains traced back to between the 1900s and early 1920s, but Background it was not until 1983 that the human immunodeficiency The emergence of the acquired immunodeficiency syn - virus (HIV) was identified as its cause. HIV belongs to drome (AIDS) among human populations has been the Retroviridae family, the Lentivirus genus. This genus comprises both types of HIV (HIV-1 and HIV-2), in addi- tion to many simian immunodeficiency viruses (SIV) *Correspondence: mohamedadaw@gmail.com Department of Medical Microbiology and Immunology, Faculty that naturally infect different primate species in Africa of Medicine, University of Tripoli, Tripoli, Libya [1, 2]. HIV types have distinct patterns of spread and Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Daw et al. BMC Res Notes (2017) 10:170 Page 2 of 9 progression to AIDS. HIV-2 infection is mainly restricted studies have been reported on the genetic variability to regions of Western and Central Africa and account for of HIV-1 in these countries. In Libya, epidemiological only 1.4% of HIV isolates [3]. HIV-1, which is responsi- data on the HIV-1 epidemic is lacking [24, 25]. The geo - ble for the AIDS pandemic, has been divided into four graphic location of the country, with the longest coast in groups (M, N, O and P), each of which is derived from a the Mediterranean basin facing the Southern European distinct introduction of simian immunodeficiency viruses countries, and its oil producer status make it an impor- that naturally infect chimpanzees (SIVcpz). HIV-1 group tant destination for Sub-Saharan immigrants [26, 27]. M (Major) alone is responsible for more than 95% of the Hence, studying the HIV-1 subtypes in Libya should be AIDS pandemic, and virtually all studies on HIV have a priority. The objectives of this study were to perform been conducted with representatives of this group [4, 5]. a comprehensive molecular epidemiological survey of The epidemiological and clinical features of HIV infec - HIV-1 diversity in Libya and to investigate the prevalence tion have changed considerably in recent years. This is of viral variants circulating among different risk groups clearly mirrored by changing in the modes of transmis- to better characterize the HIV-1 epidemic. sion and evident drop in related morbidity and mortality [6, 7]. The trajectory of the AIDS epidemic has been bro - Methods ken in most regions, and sustainable solutions are imple- Study population mented worldwide. However, despite all the progress, the A total of 814 persons diagnosed with HIV-1 infection AIDS epidemic is far from over, particularly in develop- in Libya were studied over a 16-year period from 1995 ing countries [8]. In those countries, which are rampant to 2010 (Table  1). Convenience specimens were col- with inequalities, political instability, and discriminatory lected from different vulnerable populations, including regulations, fragile communities affected by HIV and blood and blood product recipients, IVDUs, offspring of AIDS are found everywhere [9, 10]. Therefore, studies are infected mothers, and persons with promiscuous sexual needed to highlight the ever-changing epidemiology of contacts (Table  1). Venous blood samples were taken HIV among the infected populations in such countries. by qualified nurses and immediately tested for HIV by The global epidemiology of HIV-1 subtypes is very laboratory technicians who applied a three-serial rapid heterogeneous and varies greatly worldwide, even testing strategy using determine HIV-1/2 Kit (Inverness within regions of the same continent [11, 12]. During Medical Innovations) for initial testing, and subsequently 2004–2007, subtype C accounted for nearly half (48%) of all global infections [4, 13]. It is considered endemic in Table 1 Distribution by  age, gender, route of  infection sub-Saharan and Eastern Africa (where over two-thirds and  year of  diagnosis of  HIV-1 seropositive patients in Libya of infected individuals reside) followed by the Indian Pacific and southern region of Brazil. HIV-1 subtype A1 a b Demographic characteristicsRegistered patients Ratio is more prevalent in Central Africa, Iran, Eastern Europe Studied Total and Central Asia, as it accounts for 12% of the total sub- types. Subtypes A2 and A3 are found primarily in Africa. Age (years) Subtype B is the most disseminated variant. It accounts <20 49 119 1:2 for 11% and found mainly in the European Union, USA, 20–40 87 623 1:7 Australia, North Africa and Japan [14, 15]. CRF02_AG >40 23 72 1:3 accounts for 8% and CRF01_AE for 5%, and they occur Gender mainly in Western Africa and Southeast Asia [2, 16]. Male 132 745 6:5 Other CRFs and URFs are responsible for 4% of global Female 27 69 1:3 infections, bringing the combined total of worldwide Transmission route CRFs to 16% and all recombinants (CRFs plus URFs) Blood or blood products 61 211 1:4 to 20% [17]. Other subtypes, such as subtype G and D, Sexual contact 17 143 1:9 account for 5 and 2%, respectively, while subtypes F, H, IVDU 72 439 1:7 J and K together cause fewer than 1% of HIV infections Vertical transmission 9 21 5:3 worldwide [18, 19]. Screening period The HIV epidemic in North African countries is grow - 1995–2000 48 311 1:7 ing faster nowadays due to political instability, which 2001–2005 52 217 5:4 resulted in massive population displacements and lack 2006–2010 59 286 1:5 of migration control [20, 21]. Most transmission within Total 159 814 1:5 these countries occurs among intravenous drug users Officially registered and coded cases of HIV in Libya (1995–2010) (IVDUs) and in some health-care settings [22, 23]. Few Ratio (Studied: Registered) Daw et al. BMC Res Notes (2017) 10:170 Page 3 of 9 Uni-Gold HIV-Kit (Trinity Biotech) and Bioline HIV- Declaration [32] and under the supervision of the Faculty 1/2-Kit (Standard Diagnostics) for confirmation of reac - of Medicine, University of Tripoli, Tripoli, Libya. Clinical tive results [29], The sera samples were collected from and epidemiological data were maintained only for ther- each of the four regions according to the Libyan Min- apeutic and research purposes. All participants signed istry of Housing & Planning, as recently described by an informed consent form witnessed by the local health Daw et  al. [28]. Of the 814 infected samples, only 159 officer before collection of data and blood samples. The samples (19.5%) were found suitable for genotyping and questionnaire used to collect demographic and epide- subtype specification. Of these, 35 (22%) were from the miological data was anonymous and linked to the blood Eastern Region, 42 (26.4%) from the Western Region, 59 sample tube only by a code. This study was conducted in (37.1%) from the Northern Region, and 23 (14.5%) from collaboration with both the Libyan Study Group of Hepa- the Southern Region. Demographics and relevant clini- titis & HIV and the Libyan Society of Hospital Infection cal data were taken from the questionnaire database and (LSHI). Both organizations have a strong human rights cross-referenced with the patients’ medical files, as previ - focus as well as extensive involvement in hepatitis and ously described [28]. AIDS prevention activities in the region and consequently have managed to establish strong collaborative links with Genetic analysis government, public institutions, and civil society. The HIV-1 genotyping and genetic analysis was car - ried out using partial PCR amplification was performed Results on a convenience sample of 159 collected from different A total of 814 HIV-seropositive cases from all the four patients all over Libyan regions. A multiple alignment of regions of the country were followed. Of these, 745 the newly derived protease/RT sequences and full-length (91.5%) were males (M:F ration 10.8:1). The largest num - genome sequences with selected reference sequences was ber of infections were attributed to IVDUs (439, 53.9%) constructed and the origin of the subtypes were traced followed by those who had received blood or blood and analysed as previously described [30]. Phylogenetic products (211, 25.9%), sexual contact (143, 17.6%) and trees were generated, and the consistency of branching mother-to-child transmission (21, 2.6%). The preva - order was evaluated using Phylogeny Inference Package lence was significantly higher among males (M:F ratio V3.5c. Recombinant analysis used Simplot, version 3.4,20 1:6, P < 0.001) and among those aged 20–40 years. It was and alignment examination determined precise break- lower among those <20 years old and those >40 years old points. After breakpoint identification, each segment (P  >  0.01). The prevalence of HIV-1 among the studied was extracted and analyzed phylogenetically to confirm population changed over the study period by a ratio (R) the assignment of the subtype. Serologic test results were of 1:1.5 (1995‒2000 vs. 2005‒2010). linked to the questionnaire only by a unique numeric The trends in the prevalence of HIV infections among code to preserve confidentiality and anonymity of the major risk groups during 1995‒2010 are shown in study participants [31]. Fig.  1. During the early part of the study, HIV-1 preva- lence was high among persons receiving blood or blood Statistical analysis The data were coded and entered in an Excel sheet. The database was cleaned and verified. The data were ana - lyzed by using Microsoft Excel 2007, and statistical tests were done using Minitab version 15 and SPSS version 16. A Chi square test was used to determine the difference in risk factors between seropositive and sero-negative viral status. Odds ratios (OR) and 95% confidence intervals (CI) in univariate analysis were calculated using seron- egative persons as reference. Multiple logistic regression was used for multivariate analysis to determine the inde- pendent risk factors for exclusively all HIV-1 subtypes involved. Ethical considerations The study was approved by the Libyan National Ethical Committee (Approval No. LY NS, AIDS-HIV-310153). Fig. 1 Prevalence of HIV‑1 subtypes among risk groups in Libya 1995–2010 It was conducted in accordance with the Helsinki Daw et al. BMC Res Notes (2017) 10:170 Page 4 of 9 products and via vertical transmission (P < 0.001). These South, 2 (10.5%) from the North, and only 1 (5.3%) from prevalence rates declined considerably by the end of the the West. Subtype B was predominant in the North (11; study period, possibly due to strict adherence to the uni- 47.8%) and West (7; 30.4%), and only a few strains were versal obligatory blood screening program introduced isolated from the East (3; 13%) and South (2; 8.7%). Only in Libya in the early 1980s. On the other hand, the fre- two strains of subtype A were emerging in the Western quency of infected IVDUs increased significantly during region. Between 2001 and 2010, the contribution of sub- the study period, from 2.5% during 1995‒2000 to 26.4% type B increased up to three folds in most of the regions. in 2005‒2010 (1:10.5). There was a slight increase in the This was paralleled by a reduction of subtype CRF02_ frequency of those who contracted HIV via sexual behav- AG, particularly in the Eastern region, where it was the ior from 2.5 to 5.7% (R 1:2.3). The decline among those main subtype during 1995‒2000. However, a few strains receiving blood products was considerable, from 21.4 to of subtype A were emerging in the West and South. The 0.5% (R: 4.3:1). location of the various genotypes was plotted on a map Neighbor-joining trees of the HIV sequences of the of Libya to examine the trends in their geographic dis- 159 available samples and reference sequences of HIV-1 tribution as illustrated in Fig.  4. The arrows on the map group M (subtypes A, B, C, D, G, and CRF02_ A/G) indicate the plausible route through which these strains revealed three distinct subtypes, including subtype A, may have entered Libya, including the Benghazi‒Bulgar- subtype B and CRF02_AG. Figure  2 shows the phyloge- ian strains (BG-S). Most infections and the greatest het- netic tree built using the sequence analysis of 159 strains erogeneity was observed all over the country, but they collected from all regions of Libya with reference samples particularly more heterogeneous in the Northern region, are those named preceded by the HIV-1 subtype. where the capital Tripoli is located, followed by the three Subtype B was the most frequent subtype, account- other regions. The most common B subtypes showed the ing for 73.5% of the samples, followed by CRF02_AG widest distribution across the country. Most CRF02_AG (18.3%). Subtype A accounted for 8.2%. According cases were located in and around Benghazi in the Eastern to patient retrieval data, all persons involved in this region of Libya. study were presumed to have been infected in Libya. The prevalence of the reported subtypes varied greatly Discussion over the study period as shown in Fig.  3. There were 16 Despite the overwhelming progress in understanding the CRF02_AG strains (10.1%) in 2000 but only five (3.1%) in global epidemiology of HIV, few accurate data are availa- 2010, whereas subtype B represented 16.4% in 2000 and ble on the prevalence of HIV-1 infection in North African reached 32.7% in 2010. However, the prevalence of a few countries. Studies in this region have been hampered by strains of subtype A did not change significantly during fears of stigma and discrimination against people living the study period. with HIV/AIDS [33]. In Libya, a few studies were carried The association of HIV-1 subtypes with demographic out on the prevalence of co-infection with HIV and hepa- and risk factors were analyzed. These included age, gen - titis B or hepatitis C virus, but no study has focused on der, mode of transmission, and time of diagnosis as the magnitude of the HIV problem in the Libyan popula- shown in Table  2. HIV-1 CRF02_AG was most frequent tion [29]. Consequently, we performed a comprehensive among those who received blood or blood products, and cohort study over a 16-year period of individuals newly among those  <20  years old, followed by those who were diagnosed with HIV-1 from all geographical regions infected by vertical transmission. Subtype B was more of Libya. The overall prevalence of HIV-1 in our study frequent among IVDUs, followed by those with promis- ranged from 5 to 10 per 100,000 according to the cases cuous sexual contact predominantly among the sexually registered in Libya. Furthermore, the prevalence varied active group (20‒40  years). A few strains of subtype A from one risk group to another. It was highest among were identified, mainly among IVDUs aged 20‒40  years. IVDUs, followed by blood recipients, and to a lower The proportions of younger persons infected with extent people with promiscuous sexual behavior. In our CRF02_AG declined significantly over the study period, study, the number of people infected via IVDU increased whereas subtype B increased (P  <  0.001), particularly steadily from the late 2000 and doubled by the end of among IVDUs and those practicing sexual behav- 2010. Most cases newly infected with HIV were among ior. There was a significant spike in the relative preva - IVDUs, only 10% were attributable to heterosexual expo- lence of subtype B during 2006–2010, while CRF02_AG sure, and rarely to other risk factors. This is in agreement declined (OR 4.1; P < 0.001). Table 3 shows the distribu- with other studies conducted in the Middle East and tion of HIV-1 subtypes by region of residence. During North Africa (MENA) region. There is documented evi - 1995‒2000, 11 (57.9%) strains of CRF02_AG were iso- dence of concentrated epidemics of HIV among IVDUs lated from the Eastern region, 5 (26.3%) strains from the in Egypt, Morocco and Algeria, and suggestive evidence Daw et al. BMC Res Notes (2017) 10:170 Page 5 of 9 Fig. 2 Phylogenetic tree built using the sequence analysis of 159 strains collected from all regions of Libya. Subtype B (orange), subtype A (red), and CRF02_AG (yellow). Reference samples are those named, preceded by the subtype Daw et al. BMC Res Notes (2017) 10:170 Page 6 of 9 of epidemics among heterosexuals in Sudan and Egypt [34, 35]. However, most infections in these countries seem to be concentrated in high risk groups and there is no evidence of a sustainable general population epi- demic. Therefore, large-scale cross-sectional studies are needed to verify that assumption, with further validity assessments for extrapolations in these countries [36, 37]. Molecular analysis of HIV-1 subtypes among Libyan patients showed three distinct subtypes. Subtype B was the most prevalent genotype, representing over 70% of HIV-1 infections in Libya, followed by CRF02_AG (18.3%) and subtype A (8.2%). This is in an agreement with other studies from neighboring Northern African countries. In Egypt, subtype B was the commonest strain, Fig. 3 Distribution of HIV 1 subtypes among Libyan patients accounting for 91.7% of cases, followed by O (4.2%) and 1995–2010 CRF01_AE (4.2%) [38]. In Tunisia, subtype B accounted Table 2 Association between infection with different HIV-1 subtypes and certain influencing variables among the Libyan population CRF02_AG Subtype B Subtype A OR 95% CI OR 95% CI OR 95% CI Lower Upper Lower Upper Lower Upper Age group (years) <20 2.3 1.34 2.62 0.60 0.35 0.95 0.0 0.0 0.0 21–40 0.20 0.09 0.24 2.89 1.95 3.95 0.70 0.43 1.02 >40 0.0 0.0 0.0 2.91 2.21 3.9 0.59 0.39 0.91 Sex M vs. F 0.75 0.63 0.89 1.71 1.68 1.98 1.62 1.46 1.79 Risk factors Blood transfusion 1.22 1.12 1.87 0.3 0.12 0.34 0 0 0 IVDU 0.0 0.0 0.0 6.31 2.15 17.15 5.65 1.75 17.06 Sexual contact 0.0 0.0 0.0 1.21 1.05 1.43 0 0 0 Vertical transmission 0.9 0.8 1.90 0 0 0 0 0 0 Screening period 1995–2000 1.06 0.8 2.4 1.9 0.6 3.4 0.0 0.0 0.0 2001–2005 0.02 0.01 1.02 2.7 1.2 5.6 0.01 0.01 0.01 2006–2010 0.01 0.0 0.42 4.1 2.2 6.8 0.01 0.01 0.04 OR odds ratio, CI confidence interval, M males, F females, IVDU intravenous drug use Table 3 Distribution of HIV-1 subtypes by regions of residence among Libyans infected with HIV 1995‒2010 Region 1995–2000 2001–2005 2006–2010 HIV 1 subtypes CRF02_AG A B CRF02_AG A B CRF02_AG A B East 11 0 3 3 0 5 2 0 11 West 1 2 7 1 2 10 0 3 16 North 2 0 11 1 2 17 0 1 25 South 5 0 2 2 0 3 1 3 7 Total 19 2 23 7 4 35 3 7 59 Daw et al. BMC Res Notes (2017) 10:170 Page 7 of 9 treated at Benghazi Pediatric Hospital in Benghazi [25]. There is an evident speculation that this was caused by an unannounced testing of an attenuated vaccine strain by foreign medical staff, similar to vaccine trials early last century, in which children from developing countries were used [47–49]. In any case, HIV 1 subtypes distribu- tion in Libya shows limited diversity, and further epide- miological studies are needed to clarify the overlapping chains of transmission across North African countries. The prevalence of HIV 1 subtypes among risk groups varies greatly from one part of the world to another. In Europe and North America, subtype B was found to be endemic among IVDUs, but in Cambodia and Vietnam subtype E was the most common [50, 51]. In our study, the initial epidemic among IVDUs was largely due to sub- type B, whereas subtype CRF_02 AG was more prevalent among blood product recipients and a those infected by vertical transmission. Subtype A emerged among all risk groups, particularly those prone to promiscuous sexual Fig. 4 Map of Libya shows the distribution of HIV‑1 subtypes among contacts. This is in agreement with other studies from the Libyan regions. The arrows indicate the plausible route of HIV‑1 Africa, USA and EU on subtype B, though CRF_20AG subtypes entry into Libya, including BG‑S (Benghazi‒Bulgarian Strain) and subtype A were common among Sub Saharan Afri- can countries [49, 52]. On the population level, none of our patients had mul- for 95.8% and CRF02_AG for 4.2% [39]. Similarly, in tiple infections with HIV-1 subtypes. This observation Morocco HIV 1-subtype B accounted 93.5%, subtype A of low inter-patient diversity has also been reported in for 1%, and subtype F for 0.5% [40]. In Algeria, subtype epidemics of HIV-1 subtypes in the former Soviet Union B accounted for 56.3%, followed by CRF02_AG (18.0%), and Ukraine [53]. Furthermore, differences in patho - along with other contributing strains (CRF06_cpx 17.2, genicity among HIV-1 genetic subtypes were not noted G 2.7, 206 2.7, D 1.3, and others 1.9% [41]. CRF02_AG in the current study. However, Zhang et al. [54] reported and subtype A strains were also common in Sub-Saharan higher initial viral loads, with similar CD4 cell counts and West and Central Africa. However, the CRF02_AG in patients with subtype CRF_01AE and patients with strains found in our study were monophyletic, with low subtype B. Thus, further studies are needed to evaluate genetic diversity, and so are likely descended from a com- antiretroviral therapy of HIV-infected patients in Libya. mon ancestor [42, 43]. Similar results were reported from Despite the dominance of subtype B in the Libyan European countries in the Mediterranean basin, particu- population, the prevalence of subtypes is both periodi- larly Italy, Spain and Greece, with local expansion in cer- cally and geographically variable. CRF_02 AG, which tain populations, particularly IVDUs and heterosexuals was associated mainly with blood products, was more [44, 45]. prevalent at the start of the study (1995) but was rarely The reasons for the expansion and spread of HIV-1 reported by the end of the study (2010). This subtype subtypes in Libyan regions are not clear, and it is dif- was dominant mainly in the Eastern region of the coun- ficult to trace the possible evolutionary history of the try, where it accounted for over 45% of HIV-1 infection Libyan subtypes. Among the speculative factors are followed by the Southern region (17%), but it was rarely immigration, scandals and population integration within reported in the other regions (<8%). This was not the case sub-Saharan and North African countries [36, 46]. The for subtype B, the prevalence of which rose significantly substantial and continuous increase in subtype B strain throughout the study period, particularly among IVDUs among Libyans observed in this study could be attrib- and all over the Libyan regions, but particularly in West uted to the large number of legal and illegal workers who and South regions. Subtype A first appeared in 2005 and come to Libya from Egypt and neighboring Maghreb its prevalence increased, particularly in the Western and countries, where this strain is dominant [28, 29]. In the Northern regions. Eastern region of Libya in 1998, a unique circulating It has been suggested that mobile populations play a form was suddenly identified among 400 children with no special role in HIV-1 epidemics, particularly in devel- known previous exposure or risk factors who had been oping countries. HIV-1 prevalence in communities Daw et al. BMC Res Notes (2017) 10:170 Page 8 of 9 Consent to publish located on commercial transport routes in Africa was We confirm that the data are freely available and can be published according found to be higher than that in communities elsewhere to BMC regulations without needing consent. in the same region [55]. Libya is considered a major Ethics approval and consent to participate transit area of migration from Sub-Saharan Africa Ethical clearance was obtained from the Libyan National Ethical Committee towards Western Europe for people seeking better liv- and written informed consent was taken from all participants. ing conditions. 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Molecular and epidemiological characterization of HIV-1 subtypes among Libyan patients

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Springer Journals
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2017 The Author(s)
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1756-0500
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10.1186/s13104-017-2491-2
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Abstract

Background: The epidemiological and clinical aspects of human immunodeficiency virus subtypes are of great inter ‑ est worldwide. These subtypes are rarely studied in North African countries. Libya is a large country with the longest coast on the Mediterranean Sea, facing the Southern European countries. Studies on the characterization of HIV‑ 1 subtypes are limited in Libya. This study aimed to determine the magnitude of the HIV problem among the Libyan population and to better understand the genetic diversity and the epidemiologic dynamics of HIV 1, as well as to cor‑ relate that with the risk factors involved. Methods: A total of 159 HIV‑ 1 strains were collected from 814 HIV positive patients from the four Libyan regions dur‑ ing a 16‑ year period (1995–2010). To determine the HIV‑ 1 subtypes, genetic analysis and molecular sequencing were carried out using provirus polygene. Epidemiologic and demographic information was obtained from each partici‑ pant and correlated with HIV‑ 1 subtypes using logistic regression. Results: The overall prevalence of HIV among Libyans ranged from 5 to 10 per 100,000 during the study period. It was higher among intravenous drug users (IVDUs) (53.9%), blood recipients (25.9%) and heterosexuals (17.6%) than by vertical transmission (2.6%). Prevalence was higher among males aged 20–40 years (M:F 1:6, P > 0.001). Among the 159 strains of HIV‑ 1 available for typing, 117 strains (73.6%) were subtype B, 29 (18.2%) were CRF02_AG, and 13 (8.2%) were subtype A. HIV‑ 1 subtype B was the most prevalent all over the country, and it was more prevalent in the North‑ ern region, particularly among IVDUs (P < 0.001). GRF02_AG was common in the Eastern region, particularly among blood recipients while subtype A emerged in the Western region, particularly among IVDUs. Conclusions: HIV‑ 1 infection is emerging in Libya with a shifting prevalence of subtypes associated with the chang‑ ing epidemiology of HIV‑ 1 among risk groups. A genetic analysis of HIV‑ 1 strains demonstrated low subtype hetero‑ geneity with the evolution of subtype B, and CRF_20 AG, as well as HIV‑ 1 subtype A. Our study highlights the impor‑ tance of expanded surveillance programs to control HIV infection and the necessity of introducing public health strategies to target the risk groups, particularly IVDUs. Keywords: HIV‑ 1 subtypes, Libya, Genetic diversity, Molecular epidemiology, AIDS, BG‑ S‑ Benghazi–Bulgarian Strains traced back to between the 1900s and early 1920s, but Background it was not until 1983 that the human immunodeficiency The emergence of the acquired immunodeficiency syn - virus (HIV) was identified as its cause. HIV belongs to drome (AIDS) among human populations has been the Retroviridae family, the Lentivirus genus. This genus comprises both types of HIV (HIV-1 and HIV-2), in addi- tion to many simian immunodeficiency viruses (SIV) *Correspondence: mohamedadaw@gmail.com Department of Medical Microbiology and Immunology, Faculty that naturally infect different primate species in Africa of Medicine, University of Tripoli, Tripoli, Libya [1, 2]. HIV types have distinct patterns of spread and Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Daw et al. BMC Res Notes (2017) 10:170 Page 2 of 9 progression to AIDS. HIV-2 infection is mainly restricted studies have been reported on the genetic variability to regions of Western and Central Africa and account for of HIV-1 in these countries. In Libya, epidemiological only 1.4% of HIV isolates [3]. HIV-1, which is responsi- data on the HIV-1 epidemic is lacking [24, 25]. The geo - ble for the AIDS pandemic, has been divided into four graphic location of the country, with the longest coast in groups (M, N, O and P), each of which is derived from a the Mediterranean basin facing the Southern European distinct introduction of simian immunodeficiency viruses countries, and its oil producer status make it an impor- that naturally infect chimpanzees (SIVcpz). HIV-1 group tant destination for Sub-Saharan immigrants [26, 27]. M (Major) alone is responsible for more than 95% of the Hence, studying the HIV-1 subtypes in Libya should be AIDS pandemic, and virtually all studies on HIV have a priority. The objectives of this study were to perform been conducted with representatives of this group [4, 5]. a comprehensive molecular epidemiological survey of The epidemiological and clinical features of HIV infec - HIV-1 diversity in Libya and to investigate the prevalence tion have changed considerably in recent years. This is of viral variants circulating among different risk groups clearly mirrored by changing in the modes of transmis- to better characterize the HIV-1 epidemic. sion and evident drop in related morbidity and mortality [6, 7]. The trajectory of the AIDS epidemic has been bro - Methods ken in most regions, and sustainable solutions are imple- Study population mented worldwide. However, despite all the progress, the A total of 814 persons diagnosed with HIV-1 infection AIDS epidemic is far from over, particularly in develop- in Libya were studied over a 16-year period from 1995 ing countries [8]. In those countries, which are rampant to 2010 (Table  1). Convenience specimens were col- with inequalities, political instability, and discriminatory lected from different vulnerable populations, including regulations, fragile communities affected by HIV and blood and blood product recipients, IVDUs, offspring of AIDS are found everywhere [9, 10]. Therefore, studies are infected mothers, and persons with promiscuous sexual needed to highlight the ever-changing epidemiology of contacts (Table  1). Venous blood samples were taken HIV among the infected populations in such countries. by qualified nurses and immediately tested for HIV by The global epidemiology of HIV-1 subtypes is very laboratory technicians who applied a three-serial rapid heterogeneous and varies greatly worldwide, even testing strategy using determine HIV-1/2 Kit (Inverness within regions of the same continent [11, 12]. During Medical Innovations) for initial testing, and subsequently 2004–2007, subtype C accounted for nearly half (48%) of all global infections [4, 13]. It is considered endemic in Table 1 Distribution by  age, gender, route of  infection sub-Saharan and Eastern Africa (where over two-thirds and  year of  diagnosis of  HIV-1 seropositive patients in Libya of infected individuals reside) followed by the Indian Pacific and southern region of Brazil. HIV-1 subtype A1 a b Demographic characteristicsRegistered patients Ratio is more prevalent in Central Africa, Iran, Eastern Europe Studied Total and Central Asia, as it accounts for 12% of the total sub- types. Subtypes A2 and A3 are found primarily in Africa. Age (years) Subtype B is the most disseminated variant. It accounts <20 49 119 1:2 for 11% and found mainly in the European Union, USA, 20–40 87 623 1:7 Australia, North Africa and Japan [14, 15]. CRF02_AG >40 23 72 1:3 accounts for 8% and CRF01_AE for 5%, and they occur Gender mainly in Western Africa and Southeast Asia [2, 16]. Male 132 745 6:5 Other CRFs and URFs are responsible for 4% of global Female 27 69 1:3 infections, bringing the combined total of worldwide Transmission route CRFs to 16% and all recombinants (CRFs plus URFs) Blood or blood products 61 211 1:4 to 20% [17]. Other subtypes, such as subtype G and D, Sexual contact 17 143 1:9 account for 5 and 2%, respectively, while subtypes F, H, IVDU 72 439 1:7 J and K together cause fewer than 1% of HIV infections Vertical transmission 9 21 5:3 worldwide [18, 19]. Screening period The HIV epidemic in North African countries is grow - 1995–2000 48 311 1:7 ing faster nowadays due to political instability, which 2001–2005 52 217 5:4 resulted in massive population displacements and lack 2006–2010 59 286 1:5 of migration control [20, 21]. Most transmission within Total 159 814 1:5 these countries occurs among intravenous drug users Officially registered and coded cases of HIV in Libya (1995–2010) (IVDUs) and in some health-care settings [22, 23]. Few Ratio (Studied: Registered) Daw et al. BMC Res Notes (2017) 10:170 Page 3 of 9 Uni-Gold HIV-Kit (Trinity Biotech) and Bioline HIV- Declaration [32] and under the supervision of the Faculty 1/2-Kit (Standard Diagnostics) for confirmation of reac - of Medicine, University of Tripoli, Tripoli, Libya. Clinical tive results [29], The sera samples were collected from and epidemiological data were maintained only for ther- each of the four regions according to the Libyan Min- apeutic and research purposes. All participants signed istry of Housing & Planning, as recently described by an informed consent form witnessed by the local health Daw et  al. [28]. Of the 814 infected samples, only 159 officer before collection of data and blood samples. The samples (19.5%) were found suitable for genotyping and questionnaire used to collect demographic and epide- subtype specification. Of these, 35 (22%) were from the miological data was anonymous and linked to the blood Eastern Region, 42 (26.4%) from the Western Region, 59 sample tube only by a code. This study was conducted in (37.1%) from the Northern Region, and 23 (14.5%) from collaboration with both the Libyan Study Group of Hepa- the Southern Region. Demographics and relevant clini- titis & HIV and the Libyan Society of Hospital Infection cal data were taken from the questionnaire database and (LSHI). Both organizations have a strong human rights cross-referenced with the patients’ medical files, as previ - focus as well as extensive involvement in hepatitis and ously described [28]. AIDS prevention activities in the region and consequently have managed to establish strong collaborative links with Genetic analysis government, public institutions, and civil society. The HIV-1 genotyping and genetic analysis was car - ried out using partial PCR amplification was performed Results on a convenience sample of 159 collected from different A total of 814 HIV-seropositive cases from all the four patients all over Libyan regions. A multiple alignment of regions of the country were followed. Of these, 745 the newly derived protease/RT sequences and full-length (91.5%) were males (M:F ration 10.8:1). The largest num - genome sequences with selected reference sequences was ber of infections were attributed to IVDUs (439, 53.9%) constructed and the origin of the subtypes were traced followed by those who had received blood or blood and analysed as previously described [30]. Phylogenetic products (211, 25.9%), sexual contact (143, 17.6%) and trees were generated, and the consistency of branching mother-to-child transmission (21, 2.6%). The preva - order was evaluated using Phylogeny Inference Package lence was significantly higher among males (M:F ratio V3.5c. Recombinant analysis used Simplot, version 3.4,20 1:6, P < 0.001) and among those aged 20–40 years. It was and alignment examination determined precise break- lower among those <20 years old and those >40 years old points. After breakpoint identification, each segment (P  >  0.01). The prevalence of HIV-1 among the studied was extracted and analyzed phylogenetically to confirm population changed over the study period by a ratio (R) the assignment of the subtype. Serologic test results were of 1:1.5 (1995‒2000 vs. 2005‒2010). linked to the questionnaire only by a unique numeric The trends in the prevalence of HIV infections among code to preserve confidentiality and anonymity of the major risk groups during 1995‒2010 are shown in study participants [31]. Fig.  1. During the early part of the study, HIV-1 preva- lence was high among persons receiving blood or blood Statistical analysis The data were coded and entered in an Excel sheet. The database was cleaned and verified. The data were ana - lyzed by using Microsoft Excel 2007, and statistical tests were done using Minitab version 15 and SPSS version 16. A Chi square test was used to determine the difference in risk factors between seropositive and sero-negative viral status. Odds ratios (OR) and 95% confidence intervals (CI) in univariate analysis were calculated using seron- egative persons as reference. Multiple logistic regression was used for multivariate analysis to determine the inde- pendent risk factors for exclusively all HIV-1 subtypes involved. Ethical considerations The study was approved by the Libyan National Ethical Committee (Approval No. LY NS, AIDS-HIV-310153). Fig. 1 Prevalence of HIV‑1 subtypes among risk groups in Libya 1995–2010 It was conducted in accordance with the Helsinki Daw et al. BMC Res Notes (2017) 10:170 Page 4 of 9 products and via vertical transmission (P < 0.001). These South, 2 (10.5%) from the North, and only 1 (5.3%) from prevalence rates declined considerably by the end of the the West. Subtype B was predominant in the North (11; study period, possibly due to strict adherence to the uni- 47.8%) and West (7; 30.4%), and only a few strains were versal obligatory blood screening program introduced isolated from the East (3; 13%) and South (2; 8.7%). Only in Libya in the early 1980s. On the other hand, the fre- two strains of subtype A were emerging in the Western quency of infected IVDUs increased significantly during region. Between 2001 and 2010, the contribution of sub- the study period, from 2.5% during 1995‒2000 to 26.4% type B increased up to three folds in most of the regions. in 2005‒2010 (1:10.5). There was a slight increase in the This was paralleled by a reduction of subtype CRF02_ frequency of those who contracted HIV via sexual behav- AG, particularly in the Eastern region, where it was the ior from 2.5 to 5.7% (R 1:2.3). The decline among those main subtype during 1995‒2000. However, a few strains receiving blood products was considerable, from 21.4 to of subtype A were emerging in the West and South. The 0.5% (R: 4.3:1). location of the various genotypes was plotted on a map Neighbor-joining trees of the HIV sequences of the of Libya to examine the trends in their geographic dis- 159 available samples and reference sequences of HIV-1 tribution as illustrated in Fig.  4. The arrows on the map group M (subtypes A, B, C, D, G, and CRF02_ A/G) indicate the plausible route through which these strains revealed three distinct subtypes, including subtype A, may have entered Libya, including the Benghazi‒Bulgar- subtype B and CRF02_AG. Figure  2 shows the phyloge- ian strains (BG-S). Most infections and the greatest het- netic tree built using the sequence analysis of 159 strains erogeneity was observed all over the country, but they collected from all regions of Libya with reference samples particularly more heterogeneous in the Northern region, are those named preceded by the HIV-1 subtype. where the capital Tripoli is located, followed by the three Subtype B was the most frequent subtype, account- other regions. The most common B subtypes showed the ing for 73.5% of the samples, followed by CRF02_AG widest distribution across the country. Most CRF02_AG (18.3%). Subtype A accounted for 8.2%. According cases were located in and around Benghazi in the Eastern to patient retrieval data, all persons involved in this region of Libya. study were presumed to have been infected in Libya. The prevalence of the reported subtypes varied greatly Discussion over the study period as shown in Fig.  3. There were 16 Despite the overwhelming progress in understanding the CRF02_AG strains (10.1%) in 2000 but only five (3.1%) in global epidemiology of HIV, few accurate data are availa- 2010, whereas subtype B represented 16.4% in 2000 and ble on the prevalence of HIV-1 infection in North African reached 32.7% in 2010. However, the prevalence of a few countries. Studies in this region have been hampered by strains of subtype A did not change significantly during fears of stigma and discrimination against people living the study period. with HIV/AIDS [33]. In Libya, a few studies were carried The association of HIV-1 subtypes with demographic out on the prevalence of co-infection with HIV and hepa- and risk factors were analyzed. These included age, gen - titis B or hepatitis C virus, but no study has focused on der, mode of transmission, and time of diagnosis as the magnitude of the HIV problem in the Libyan popula- shown in Table  2. HIV-1 CRF02_AG was most frequent tion [29]. Consequently, we performed a comprehensive among those who received blood or blood products, and cohort study over a 16-year period of individuals newly among those  <20  years old, followed by those who were diagnosed with HIV-1 from all geographical regions infected by vertical transmission. Subtype B was more of Libya. The overall prevalence of HIV-1 in our study frequent among IVDUs, followed by those with promis- ranged from 5 to 10 per 100,000 according to the cases cuous sexual contact predominantly among the sexually registered in Libya. Furthermore, the prevalence varied active group (20‒40  years). A few strains of subtype A from one risk group to another. It was highest among were identified, mainly among IVDUs aged 20‒40  years. IVDUs, followed by blood recipients, and to a lower The proportions of younger persons infected with extent people with promiscuous sexual behavior. In our CRF02_AG declined significantly over the study period, study, the number of people infected via IVDU increased whereas subtype B increased (P  <  0.001), particularly steadily from the late 2000 and doubled by the end of among IVDUs and those practicing sexual behav- 2010. Most cases newly infected with HIV were among ior. There was a significant spike in the relative preva - IVDUs, only 10% were attributable to heterosexual expo- lence of subtype B during 2006–2010, while CRF02_AG sure, and rarely to other risk factors. This is in agreement declined (OR 4.1; P < 0.001). Table 3 shows the distribu- with other studies conducted in the Middle East and tion of HIV-1 subtypes by region of residence. During North Africa (MENA) region. There is documented evi - 1995‒2000, 11 (57.9%) strains of CRF02_AG were iso- dence of concentrated epidemics of HIV among IVDUs lated from the Eastern region, 5 (26.3%) strains from the in Egypt, Morocco and Algeria, and suggestive evidence Daw et al. BMC Res Notes (2017) 10:170 Page 5 of 9 Fig. 2 Phylogenetic tree built using the sequence analysis of 159 strains collected from all regions of Libya. Subtype B (orange), subtype A (red), and CRF02_AG (yellow). Reference samples are those named, preceded by the subtype Daw et al. BMC Res Notes (2017) 10:170 Page 6 of 9 of epidemics among heterosexuals in Sudan and Egypt [34, 35]. However, most infections in these countries seem to be concentrated in high risk groups and there is no evidence of a sustainable general population epi- demic. Therefore, large-scale cross-sectional studies are needed to verify that assumption, with further validity assessments for extrapolations in these countries [36, 37]. Molecular analysis of HIV-1 subtypes among Libyan patients showed three distinct subtypes. Subtype B was the most prevalent genotype, representing over 70% of HIV-1 infections in Libya, followed by CRF02_AG (18.3%) and subtype A (8.2%). This is in an agreement with other studies from neighboring Northern African countries. In Egypt, subtype B was the commonest strain, Fig. 3 Distribution of HIV 1 subtypes among Libyan patients accounting for 91.7% of cases, followed by O (4.2%) and 1995–2010 CRF01_AE (4.2%) [38]. In Tunisia, subtype B accounted Table 2 Association between infection with different HIV-1 subtypes and certain influencing variables among the Libyan population CRF02_AG Subtype B Subtype A OR 95% CI OR 95% CI OR 95% CI Lower Upper Lower Upper Lower Upper Age group (years) <20 2.3 1.34 2.62 0.60 0.35 0.95 0.0 0.0 0.0 21–40 0.20 0.09 0.24 2.89 1.95 3.95 0.70 0.43 1.02 >40 0.0 0.0 0.0 2.91 2.21 3.9 0.59 0.39 0.91 Sex M vs. F 0.75 0.63 0.89 1.71 1.68 1.98 1.62 1.46 1.79 Risk factors Blood transfusion 1.22 1.12 1.87 0.3 0.12 0.34 0 0 0 IVDU 0.0 0.0 0.0 6.31 2.15 17.15 5.65 1.75 17.06 Sexual contact 0.0 0.0 0.0 1.21 1.05 1.43 0 0 0 Vertical transmission 0.9 0.8 1.90 0 0 0 0 0 0 Screening period 1995–2000 1.06 0.8 2.4 1.9 0.6 3.4 0.0 0.0 0.0 2001–2005 0.02 0.01 1.02 2.7 1.2 5.6 0.01 0.01 0.01 2006–2010 0.01 0.0 0.42 4.1 2.2 6.8 0.01 0.01 0.04 OR odds ratio, CI confidence interval, M males, F females, IVDU intravenous drug use Table 3 Distribution of HIV-1 subtypes by regions of residence among Libyans infected with HIV 1995‒2010 Region 1995–2000 2001–2005 2006–2010 HIV 1 subtypes CRF02_AG A B CRF02_AG A B CRF02_AG A B East 11 0 3 3 0 5 2 0 11 West 1 2 7 1 2 10 0 3 16 North 2 0 11 1 2 17 0 1 25 South 5 0 2 2 0 3 1 3 7 Total 19 2 23 7 4 35 3 7 59 Daw et al. BMC Res Notes (2017) 10:170 Page 7 of 9 treated at Benghazi Pediatric Hospital in Benghazi [25]. There is an evident speculation that this was caused by an unannounced testing of an attenuated vaccine strain by foreign medical staff, similar to vaccine trials early last century, in which children from developing countries were used [47–49]. In any case, HIV 1 subtypes distribu- tion in Libya shows limited diversity, and further epide- miological studies are needed to clarify the overlapping chains of transmission across North African countries. The prevalence of HIV 1 subtypes among risk groups varies greatly from one part of the world to another. In Europe and North America, subtype B was found to be endemic among IVDUs, but in Cambodia and Vietnam subtype E was the most common [50, 51]. In our study, the initial epidemic among IVDUs was largely due to sub- type B, whereas subtype CRF_02 AG was more prevalent among blood product recipients and a those infected by vertical transmission. Subtype A emerged among all risk groups, particularly those prone to promiscuous sexual Fig. 4 Map of Libya shows the distribution of HIV‑1 subtypes among contacts. This is in agreement with other studies from the Libyan regions. The arrows indicate the plausible route of HIV‑1 Africa, USA and EU on subtype B, though CRF_20AG subtypes entry into Libya, including BG‑S (Benghazi‒Bulgarian Strain) and subtype A were common among Sub Saharan Afri- can countries [49, 52]. On the population level, none of our patients had mul- for 95.8% and CRF02_AG for 4.2% [39]. Similarly, in tiple infections with HIV-1 subtypes. This observation Morocco HIV 1-subtype B accounted 93.5%, subtype A of low inter-patient diversity has also been reported in for 1%, and subtype F for 0.5% [40]. In Algeria, subtype epidemics of HIV-1 subtypes in the former Soviet Union B accounted for 56.3%, followed by CRF02_AG (18.0%), and Ukraine [53]. Furthermore, differences in patho - along with other contributing strains (CRF06_cpx 17.2, genicity among HIV-1 genetic subtypes were not noted G 2.7, 206 2.7, D 1.3, and others 1.9% [41]. CRF02_AG in the current study. However, Zhang et al. [54] reported and subtype A strains were also common in Sub-Saharan higher initial viral loads, with similar CD4 cell counts and West and Central Africa. However, the CRF02_AG in patients with subtype CRF_01AE and patients with strains found in our study were monophyletic, with low subtype B. Thus, further studies are needed to evaluate genetic diversity, and so are likely descended from a com- antiretroviral therapy of HIV-infected patients in Libya. mon ancestor [42, 43]. Similar results were reported from Despite the dominance of subtype B in the Libyan European countries in the Mediterranean basin, particu- population, the prevalence of subtypes is both periodi- larly Italy, Spain and Greece, with local expansion in cer- cally and geographically variable. CRF_02 AG, which tain populations, particularly IVDUs and heterosexuals was associated mainly with blood products, was more [44, 45]. prevalent at the start of the study (1995) but was rarely The reasons for the expansion and spread of HIV-1 reported by the end of the study (2010). This subtype subtypes in Libyan regions are not clear, and it is dif- was dominant mainly in the Eastern region of the coun- ficult to trace the possible evolutionary history of the try, where it accounted for over 45% of HIV-1 infection Libyan subtypes. Among the speculative factors are followed by the Southern region (17%), but it was rarely immigration, scandals and population integration within reported in the other regions (<8%). This was not the case sub-Saharan and North African countries [36, 46]. The for subtype B, the prevalence of which rose significantly substantial and continuous increase in subtype B strain throughout the study period, particularly among IVDUs among Libyans observed in this study could be attrib- and all over the Libyan regions, but particularly in West uted to the large number of legal and illegal workers who and South regions. Subtype A first appeared in 2005 and come to Libya from Egypt and neighboring Maghreb its prevalence increased, particularly in the Western and countries, where this strain is dominant [28, 29]. In the Northern regions. Eastern region of Libya in 1998, a unique circulating It has been suggested that mobile populations play a form was suddenly identified among 400 children with no special role in HIV-1 epidemics, particularly in devel- known previous exposure or risk factors who had been oping countries. HIV-1 prevalence in communities Daw et al. BMC Res Notes (2017) 10:170 Page 8 of 9 Consent to publish located on commercial transport routes in Africa was We confirm that the data are freely available and can be published according found to be higher than that in communities elsewhere to BMC regulations without needing consent. in the same region [55]. Libya is considered a major Ethics approval and consent to participate transit area of migration from Sub-Saharan Africa Ethical clearance was obtained from the Libyan National Ethical Committee towards Western Europe for people seeking better liv- and written informed consent was taken from all participants. ing conditions. 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Published: Dec 1, 2017

Keywords: biomedicine, general; medicine/public health, general; life sciences, general

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