PROFILE OF HCV GENOTYPES AND HIV-SUBTYPES AMONG HIV-COINFECTED PATIENTS IN SOUTHERN BRAZIL

Perfil dos genótipos do HCV e subtipos de HIV em pacientes coinfectados no Sul do Brasil

ABSTRACT

BACKGROUND:

Hepatitis B and C virus (HBV and HCV) are the two most common infections among human immunodeficiency virus (HIV)-infected patients.

OBJECTIVE:

To identify the frequency of HIV subtypes and HCV genotypes in HIV-coinfected patients.

METHODS:

A cross-sectional and retrospective study was carried out into two reference centers in Southern Brazil between January 1, 2002 and June 30, 2016. The Abbott Real Time HCV Genotype II system was used for routine diagnostics to determine the HCV genotype based on dual-target real-time PCR. Proviral HIV-1 RNA was extracted from serum samples and fragments of the pol gene were generated by PCR. The HIV-1 PT and RT gene sequences were submitted to Maximum Likelihood Phylogenetic analysis by collecting reference sequences from the HIV-1 group M subtype of the Los Alamos database.

RESULTS:

During the study period, 3340 patients with HIV were diagnosed at both referral centers, of which 4.97% (166/3340) had HBV and/or HCV coinfection. Seroprevalence of HIV-HBV, HIV-HCV and HIV-HBV-HCV was 37.4%, 58.4%, and 4.2%, respectively. HIV-HCV-coinfected patients had a lower median nadir CD4+ T-cell count when compared to HIV-HBV-coinfected patients (P=0.01). Among those coinfected with HCV, HCV-1 (HCV-1) and HCV-3 (HCV-3) genotypes were the most prevalent, being detected in 73.8% and 21.4%, respectively. Among the HCV-1 coinfected patients, 79.3% and 20.1% had subtypes 1a and 1b, respectively. HIV subtype B was the most prevalent in HIV-coinfected patients. There was no significant difference regarding nadir CD4+ T-cell count and HIV viral load when compared to coinfected with HCV-1 with HCV-3, as well as those co-infected with HCV-1a with HCV-1b.

CONCLUSION:

In the present study, a higher frequency of subtype B of HIV and HCV-1 were found in HIV-coinfected patients. Further larger-scale and long-term studies are needed to better understand the effect of HCV genotypes in HIV-infected patients.

HEADINGS:
HIV infections; Hepacivirus; Genotype; CD4 lymphocyte count

RESUMO

CONTEXTO:

Os vírus das hepatites B e C (VHB e VHC) são os causadores das duas infecções mais comuns entre os pacientes infectados pelo vírus da imunodeficiência humana (HIV).

OBJETIVO:

Identificar a frequência dos subtipos do HIV e genótipos de VHC em pacientes coinfectados com HIV.

MÉTODOS:

Estudo transversal e retrospectivo realizado em dois centros de referência do Sul do Brasil, entre 1º de janeiro de 2002 e 30 de junho de 2016. O sistema Abbott Real Time HCV Genótipo II foi utilizado para diagnósticos de rotina para determinar o genótipo do HCV com base na PCR em tempo real de duplo alvo. O RNA viral do HIV-1 foi extraído de amostras de soro e fragmentos do gene pol foram obtidos por PCR. As sequências do gene PT e RT do HIV-1 foram submetidas à análise filogenética por máxima verossimilhança através da coleta de sequências de referência do subtipo M do grupo HIV-1 da base de dados Los Alamos.

RESULTADOS:

Durante o período do estudo, 3340 pacientes foram diagnosticados com HIV em ambos os centros de referência, dos quais 4,97% (166/3340) possuíam coinfecção com HBV e/ou HCV. A soroprevalência de HIV-HBV, HIV-HCV e HIV-HBV-HCV foi de 37,4%, 58,4% e 4,2%, respectivamente. Pacientes HIV-VHC possuíam menor nadir de células T CD4+ quando comparados aos pacientes HIV-VHB (P=0,01). Entre os pacientes HIV-VHC, os genótipos VHC-1 e VHC-3 foram os mais prevalentes, sendo encontrados em 73,8% e 21,4%, respectivamente. Entre os coinfectados com VHC-1, 79,3% e 20,1% tinham subtipos 1a e 1b, respectivamente. O subtipo B do HIV foi o mais prevalente em pacientes coinfectados. Não houve diferença significativa em relação nadir de células T CD4+ e carga viral do HIV quando comparadas os coinfectados com o VHC-1 com o VHC-3, assim como, os coinfectados com HCV-1a quando comparados com o HCV-1b.

CONCLUSÃO:

No presente estudo, uma maior frequência do subtipo B do HIV e do VHC-1 foram encontrados em pacientes coinfectados com HIV. Outros estudos em larga escala e a longo prazo são necessários para entender melhor o efeito dos genótipos do HCV em pacientes infectados pelo HIV.

DESCRITORES:
Infecções por HIV; Hepacivirus; Genótipo; Contagem de linfócito CD4

INTRODUCTION

Globally, it has been estimated that 257, 71, and 36.7 million people are infected with chronic hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV), respectively^1,2. HIV, HBV, and HCV share similar forms of transmission, and thus, it has been estimated that 2.7 and 2.3 million people are living with HIV-HBV and HIV-HCV coinfections, respectively¹.

Infections by these viruses present a dynamic interaction, thereby amplifying each other and leading to increased morbidity in HIV coinfected patients³. Among HIV-HBV coinfected patients, liver-related complications were increased, and immune recovery is attenuated⁴. For HIV-HCV coinfections, several studies have shown effects of coinfection on the CD4+ T-cell count^5-8. However, despite evidence of the impact of HIV-HCV coinfection on CD4+ T-cell counts, it is unclear whether this effect was related to the different HCV genotypes.

HCV is currently classified in seven genotypes (HCV-1 to HCV-7) and multiple subtypes according to their genetic sequence⁹. Globally, the occurrence of the HCV genotype is variable. HCV-1 is predominant in Australia, Europe, Latin America, and North America (53%-71% of all cases), whereas HCV-3 occurs predominantly in Asian countries (40% of all infections)¹⁰. In Brazil, there is a predominance of HCV-1 (64.9%), followed by HCV-3 (30.2%)¹¹. However, there is little information about the frequency of HCV genotypes among HIV-coinfected populations in Brazil.

Among HIV-coinfected patients, the presence of a hepatotropic virus can lead to an overload of the host’s immune system, which may lead to the emergence of specific HIV variants¹². However, there is little information on the genetic variability of HIV in HBV- and/or HCV-coinfected patients. Thus, we investigated the frequency of HIV subtypes and HCV genotypes/subtypes, and their relationship to the nadir CD4+ T-cell count among HIV-coinfected patients.

METHODS

Study area

This study was carried out into two reference centers for the diagnosis, treatment, and follow-up of patients with HIV and viral hepatitis: Specialized Center for Infectious and Parasitic Diseases (Centro Especializado de Doenças Infecto Parasitárias) in Cascavel city, and the Specialized Service of Sexually Transmitted Infections (Serviço Especializado de Infecções Sexualmente Transmissíveis) in Maringá city, both in Paraná State, Southern Brazil. These two centers serve 25 and 30 municipalities, respectively, with a total population of 1,189,062¹³, and are part of Unified Health System (Sistema Único de Saúde).

Study design

We developed a cross-sectional and retrospective study between April and December 2017. HIV-coinfected patients and who tested positive for HBV and/or HCV between January 1, 2002 and June 30, 2016 were included in the study. However, for HIV subtyping, only HIV-coinfected patients who had HIV viral loads between January 2014 and December 2016, regardless of whether they were treatment-naive or not were included. For calculations of the nadir CD4+ T-cell count and HIV viral load, only HIV-infected patients with a positive test for HBV or HCV at baseline were considered. Sociodemographic information of the enrolled participants were collected by trained nurses and physicians, using a structured and pre-tested questionnaire. The following variables were collected: date of birth, sex, ethnicity, use of antiretroviral drugs, level of educational attainment, sexual preference, fibrosis levels, time duration of HIV infection diagnosis and coinfection, use of injected drugs, and region. The classification of liver biopsies followed the METAVIR scale¹⁴.

Laboratory measurements

HIV infection status was based on positive test results from two peripheral blood samples, performed using an HIV Enzyme-linked Immunosorbent Assay (Abbott Diagnostics, Chicago, USA) and confirmed by western blotting (Bio-Rad, Marnes La Coquette, France). HBsAg, anti-HBs, anti-HBc, and anti-HCV were tested by commercially available enzyme immunoassay (Abbott Diagnostics). Positive results for anti-HCV were confirmed by amplification of HCV RNA using Reverse Transcription Polymerase Chain Reaction (RT-PCR) by COBAS Ampliprep/Cobas TaqMan48 real-time RT-PCR (Roche Diagnostics, Pleasanton, USA), as described elsewhere¹⁵. Anti-HIV, anti-HCV, HBV markers, and HCV-RNA data were entered into the patients’ medical records and subsequently collected.

The CD4+ T-cell count was performed by flow cytometry (BD Trucount^TM Tubes) using the FACSCalibur apparatus (Becton-Dickinson, New Jersey, USA). HIV viral load was determined by PCR using Abbott Real Time HIV-1 (Abbott Diagnostics), and the results were presented as base 10 logarithms. The minimum detection value for the HIV viral load was 50 copies/mL. Data of CD4 cell count and HIV viral load were stored and subsequently obtained for tabulation of data from the national network of the Sistema de Controle de Exames Laboratoriais (SISCEL; the Laboratory Test Control System), at the virology laboratory of the State University of Maringá. All information from SISCEL is stored using data encryption in its central database, which is located in the Department of Chronic Diseases and Sexually Transmitted Infections.

The Abbott Real Time HCV Genotype II system (Abbott Diagnostics) was used for routine diagnostics to determine the HCV genotype based on dual-target real-time PCR: a 5’ UTR target region was used to discriminate between HCV genotypes, and the NS5B gene was the target for 1a and 1b subtyping, using previously described methods¹⁶. The viral genotype was determined after phylogenetic analysis of the sequences obtained, along with established GenBank reference sequences¹⁷.

Proviral HIV-1 RNA was extracted from serum samples using the QIAamp Viral RNA mini Kit^® (Qiagen, Hilden, Germany) following the manufacturer’s instructions, and the cDNA was sequentially obtained using the Superscript III RT-PCR kit (Invitrogen, CA). Fragments of the pol gene were generated by PCR according to a previously reported^18,19. Strict laboratory precautions were taken to avoid cross contamination. The genes for protease (PR) and transcriptase (RT) were amplified, and the purified PCR products were sequenced with the ABI Prism^® BigDye™ Terminator version 3.1 cycle sequencing kit ready reaction (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions in the automatic sequencer AB 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA).

The HIV-1 PT and RT gene sequences were concatenated and submitted to Maximum Likelihood Phylogenetic analysis by collecting reference sequences from the HIV-1 group M subtype of the Los Alamos database (https://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html) and BioAfrica (http://www.bioafrica.net/rega-genotype/html/subtypinghiv.html). In addition, the HIV mutation profile associated with resistance to antiretroviral therapy (ART) was analyzed by subjecting the sequences obtained from PT and RT to the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/hivdb/by-sequences/).

Statistical analysis

Pearson’s chi-square test or Fisher’s exact test were adopted for categorical variables, and the Mann-Whitney test was used in terms of quantitative variables. The level of significance was set at P<0.05. Statistical analysis was performed using Stata (version 12.0)²⁰. This study was reviewed and approved by the Research Ethical Committee of University Center of Assis Gurgacz Foundation (Report n. 1.397.212 of 28/01/2016). The norms for ethical research were followed by the researchers according to the requirements of the country, guaranteeing total confidentiality, and anonymity of the data.

RESULTS

During the study period, 3340 patients with HIV were diagnosed at both referral centers, of which 4.97% (166/3340) had HBV and/or HCV coinfection. Among them, 37.4% (62/166) were HIV-HBV, 58.4% (97/166) were HIV-HCV, and 4.2% (7/166) were HIV-HBV-HCV coinfected. Among all HIV-coinfected patients, 63.3% (105/166) were men (median age: 42 years, IQR 42-53 years) and 36.7% (61/166) were women (median age: 45 years, IQR 37-53 years).

Regarding HBV serological markers, 39.0% (30/77) and 37.5% (30/80) of HIV-HCV patients were positive for anti-HBc and anti-HBs, respectively; and 14.3%% (11/77) were positive for both anti-HBc and anti-HBs. The presence of HBeAg was detected in 50% (10/20) of HBV-coinfected patients. With regard to the risk factors for HCV coinfection acquisition, 31.6% (36/104) patients were illicit drug users, 21.2% (22/104) of which were people who inject drugs (PWID).

Of all patients, 91.6% (151/166) of HIV-coinfected patients had laboratory tests of CD4+ T-cell counts and HIV viral load determined, with a median nadir CD4+ T-cell count of 327 cells/mm³ (IQR 225-494), and log₁₀ HIV viral load of 4.5 (IQR 4-6) at the time of HIV diagnosis. In addition, there were no significant differences between HIV-HBV and HIV-HCV coinfection patients for median log HIV viral load, sex, ethnicity, education level, sexual preference, and number of sexual partners in last 12 months. However, compared to HIV-HBV patients, HIV-HCV patients had a lower median nadir CD4+ T-cell count, higher numbers of Illicit user drugs and PWID, and longer duration of HIV diagnosis and coinfection compared to the HBV-coinfected patients, all with statistical significance (Table 1).

TABLE 1
Characteristics of the study patients and association with HIV-HBV and HIV-HCV coinfection.

Variables	HIV-HBV	HIV-HCV	P-value
	n (%)	n (%)
n	62 (100.0)	97 (100.0)
Sex
Male	37 (59.7)	62 (63.9)	0.59
Female	25 (40.3)	35 (36.1)
Ethnicity
White	42 (67.7)	72 (77.4)	0.36
Black	4 (6.5)	3 (3.2)
Brown	16 (25.8)	18 (19.4)
Education
≤ 8 years	42 (68.8)	63 (67.7)	0.88
> 8 years	19 (31.2)	30 (32.3)
Sexual preference
Heterosexual	47 (78.3)	71 (85.5)	0.26
Homosexual/Bisexual	13 (21.7)	12 (14.5)
Time diagnostic HIV (years)
≤ 5	33 (53.2)	26 (26.8)	<0.001
> 5	29 (47.8)	71 (73.2)
Time diagnostic HBV or HCV (years)
≤ 5	38 (61.3)	32 (33.0)	<0.001
> 5	24 (38.7)	65 (67.0)
Number of sexual partners in last 12 months
≤ 1	30 (57.7)	38 (57.6)	0.92
2-5	3 (5.8)	5 (7.6)
> 5	19 (36.5)	23 (34.8)
Illicit drugs user
Yes	8 (12.9)	30 (30.9)	0.01
No	54 (87.1)	67 (69.1)
People who inject drugs
Yes	2 (3.2)	17 (17.5)	0.01
No	60 (96.8)	80 (82.5)
Reference center
10th Regional Health	37 (59.7)	49 (50.5)	0.26
15th Regional Health	25 (40.3)	48 (49.5)
Age (median; IQR)	45 (38-54)	47 (41-53)	0.17
Nadir CD4+ T-cell (median/mm3; IQR)	390 (287-559)	307 (197-443)	0.01
HIV viral load (log10 median; IQR)	5 (4-6)	5 (4-6)	0.96

• n: number of patients; IQR: interquartile range; HIV: human immunodeficiency virus; HBV: hepatitis B virus; HCV: hepatitis C virus.

Of all the HCV-coinfected patients (HIV-HCV and HIV-HBV-HCV), 40.4% (42/104) had liver biopsies. Of these, 26.2% (11/42) presented some type of hepatic alteration: 9.1% (1/11) A1F0, 18.2% (2/11) A1F1, 9.1% (1/11) A2F1, 18.2% (2/11) A2F2, 18.2% (2/11) A2F3, 9.1% (1/11) A2F4, 9.1% (1/11) A3F2, and 9.1% (1/11) A3F3.

Of all HCV-coinfected patients, 76.9% (80/104) were submitted to HCV genotyping. Of these, 81.3% (65/80) had minimal viral load that allowed genotyping of HCV. HCV-1, 2, 3, and 4 were detected in 73.8% (48/65), 1.6% (1/65), 21.4% (14/65), and 1.6% (1/65) of HIV-infected patients, respectively; 1.6% (1/65) of patients were infected by both HCV-1 and 2. In relation to HCV-1, 79.3% (23/29) and 20.1% (6/29) of patients had subtypes 1a and 1b, respectively.

Patients coinfected with HIV-HCV-1b had lower nadir CD4+ T-cell counts (median, 111 cells/mm³; IQR: 107-424) when compared to those with HIV-HCV-1a (median, 309 cells/mm³; IQR, 197-528), but without significant difference (P=0.25). Patients coinfected with HIV-HCV-1a had a higher median log HIV viral load (median, 5.5; IQR, 4-6) compared to those with HIV-HCV-1b (median, 4.5; IQR, 3.5-5.5), but also with no statistical difference (P=0.87). There was also no significant difference in HCV-RNA levels between the genotypes of HCV-coinfected patients for HIV-HCV-1a (median, 6; IQR, 5-6) and HIV-HCV-1b (median, 6; IQR, 5-6), but also with no statistical difference (P=0.35). HIV-HCV-1 patients had a higher median nadir CD4+ T-cell count and lower levels of HIV viral load when compared to those with HIV-HCV-3, but both had no significant difference (P=0.47). Regarding the other characteristics, there were also no significant differences between HCV-1 and 3 (Table 2).

TABLE 2
Characteristics of HCV-coinfected patients and association with HCV-1 and 3.

Variables	Genotype 1	Genotype 3	P-value
	n (%)	n (%)
n	48 (100.0)	14 (100.0)
Sex
Male	28 (58.3)	10 (71.4)	0.38
Female	20 (41.7)	4 (28.6)
Ethnicity
White	38 (82.6)	10 (71.4)	0.66
Black	2 (4.3)	1 (7.2)
Brown	6 (13.1)	3 (21.4)
Education
≤ 8 years	29 (63.1)	8 (57.1)	0.69
> 8 years	17 (36.9)	6 (42.9)
Sexual preference
Heterosexual	33 (82.5)	9 (75.0)	0.56
Homosexual/Bisexual	7 (17.5)	3 (25.0)
Time diagnostic HIV (years)
≤ 5	9 (18.8)	4 (28.6)	0.43
> 5	39 (81.2)	10 (71.4)
Time diagnosis HCV (years)
≤ 5	11 (22.9)	5 (35.7)	0.34
> 5	37 (77.1)	9 (64.3)
Number of sexual partners in last 12 months
≤ 1	19 (55.9)	2 (20.0)	0.13
2-5	3 (8.8)	2 (20.0)
> 5	12 (35.3)	6 (60.0)
Illicit drugs user
Yes	15 (31.3)	4 (28.6)	0.85
No	33 (68.7)	10 (71.4)
People who inject drugs
Yes	10 (20.4)	1 (7.1)	0.24
No	38 (79.6)	13 (92.9)
Reference center
10th Regional Health	14 (29.2)	5 (35.7)	0.64
15th Regional Health	34 (70.8)	9 (64.3)
Age (median; IQR)	50 (42.5-54.5)	49 (45-54)	0.88
Nadir CD4+ T-cell (median/mm3; IQR)	304 (190-424)	234 (129-317)	0.29
HIV viral load (log median; IQR)	5.5 (4.2-6)	6 (5.5-6)	0.73
HCV viral load (log median; IQR)	6.2 (5.9-6.5)	6.2 (5.7-6.3)	0.70

• n: number of patients; IQR: interquartile range; HIV: human immunodeficiency virus; HCV: hepatitis C virus. Pearson’s chi-square test for comparison between groups.

Among the all HIV-coinfected patients, 23.5% (39/166) were subjected to HIV subtyping. Of these, 43.6% (17/39) HIV-coinfected patients had sufficient HIV viral load for subtyping. Subtype B was found in 47.1% (8/17) of the HIV-coinfected patients, subtype C in 11.8% (2/17) of the HIV-coinfected patients, and subtype F in 17.6% (3/17) of the of the HIV-coinfected patients. The other strains (23.5%) presented recombinant structure and were classified as BC (11.7%), BDF1 (5.9%) and circulating recombinant forms (CRFs) 29-BF (5,9%). When patients were grouped by type of coinfection, those that were HBV-coinfected had the following HIV subtypes: 66.7% (4/6) were of the HIV subtype B, 16.7% (1/6) of the subtype F, and 16.7% (1/6) unique recombinant forms (URF) type 31-BC. However, the group of HCV-coinfected patients showed the following profile: 40% (4/10) were of subtype B, 10% (1/10) was of subtype C, 20% (2/10) were of subtype F1, 20% (2/10) were URFs (31-BC and BDF1), and 10% (1/10) was CRF (29-BF). One patient with triple infection (HIV-HBV-HCV) had subtype C.

There was no significant difference (P=0.82) in the nadir CD4+ T-cell count between subtype B (median, 424 cell/mm³; IQR, 242-608) and non-B groups (median, 325 cell/mm³; IQR, 242-826) of HIV. Correspondingly, there was also no significant difference (P=0.85) in the log₁₀ HIV viral load levels between subtype B (median, 4; IQR, 3-6) and non-B (median, 3.5; IQR, 3-4). In the ART resistance analysis, 29.4% (5/17) HIV-coinfected patients showed resistance-related mutations, with 40% (2/5) nucleoside reverse transcriptase inhibitors (NRTIs) resistance; 20% (1/5) presented mutations related to non-NRTI resistance; 10% (1/5) presented mutations related to resistance to NRTI and non-NRTI; and 10% (1/5) for NRTIs, non-NRTI, and protease inhibitors were found.

DISCUSSION

The genetic diversity of HCV has been linked to treatment responses. Direct-acting antivirals (DAAs) have been successfully used in the treatment of HCV, all of which have been strictly correlated with the genotype¹⁰. In 2015, the Brazilian Ministry of Health incorporated the first DAAs for the treatment of hepatitis C. The current guidelines in Brazil recommend that HIV-HCV-coinfected patients must be treated using the approach that non-HIV-infected individuals follow because the efficacy of the currently licensed DAA regimens does not appear to differ between HCV-monoinfected and coinfected individuals²¹. The treatment for hepatitis C and coinfections currently indicated in Brazil has two new therapeutic options that joins the scheme comprising the association of sofosbuvir/daclatasvir, pan-genotypic already offered since 2015. In addition to these options, the Brazilian Ministry of Health maintained the indications for ledipasvir/sofosbuvir (genotype 1), elbasvir/pibrentasvir (genotypes 1 and 4), ribavirin and alfapeginterferon (for some pediatric situations)²¹. Treatment schedules are defined based on genotype and sub genotype of HCV, and knowledge of HCV genotypes helps to predict therapeutic responses and determines the durations of drug treatments²².

The geographic distribution of HCV genotypes is complex; the epidemic subtypes - especially, 1a, 1b, 2a, and 3a - are widely distributed around the world and account for a significant share of HCV cases²³. In this study, HCV-1 presented the highest frequency (73.8%), followed by HCV-3 (21.4%) among HIV-coinfected patients. The frequency of HCV genotypes reported here was similar to that found by other authors among HIV-coinfected and HCV-monoinfected patients in Brazil^24-26 as well as in Greece²⁷, Northern and Central Asia, and in Central and Western Europe²³. We did not detect HCV-5 or 6, which was in agreement with other studies^28-30. More studies are needed to add data on the genetic diversity of HCV among HIV-infected patients and may provide important evidence for the understanding of the origin and spread of HCV infections in Southern Brazil.

The high frequency of HCV-1 has been associated with the transmission of HCV by PWID, which is responsible for increasing the risk of acquiring HCV^31,32. In our study, PWID were the most infected with HCV-1 among the HIV-coinfected patients. These results suggest that HCV-1 was introduced into PWID and spread among HIV-infected patients. Considering that the administrative route of the drug was the only difference between PWID and non-PWID, these data emphasize the importance of not sharing needles to prevent the spread of HCV-1 among HIV-infected patients. However, the origins of HCV-1 have not yet been specified and further investigations are required.

HIV is characterized by high genetic diversity and extensive heterogeneity. This characteristic is due to multiple factors, including multiple human populations, high rates of viral evolution/recombination, selective pressure of the host immune system and/or antiretroviral therapy^12,33. Although subtype B has been showing a declining trend in sexual transmission, it still accounts for a large proportion. In the United States and Western Europe, HIV-1 subtype B is the most common variant. In Brazil, HIV subtypes C, F1, and recombinants BC and BF are generally observed at low frequencies as previously described³⁴. However, there have been a few studies showing the genetic variability of HIV-1 among patients with HIV-hepatitis coinfections. A study in HIV-coinfected patients in Brazil reported that HIV-1 subtype C and B were present in 47.4% and 31.6% of HIV-coinfected patients, respectively³⁵; another Brazilian study reported that HIV-1 subtype B was the most prevalent¹². Our study corroborates the results of these studies. In addition, the results of the present study were in accordance with previous investigations, which report that B recombinant and CRFs of HIV-1 circulate among HIV-coinfected patients^12,35. The detailed dynamics of HIV subtypes viral propagation in the study region is not well understood, although two phylogeographic studies have demonstrated possible routes for the origin and spread of subtype C in the Southern region of the country^36,37.

Studies on HIV-HBV coinfection have focused on the screening of HBsAg as a marker of HBV infections. However, information on other markers remains unclear³⁸. In Brazil, studies among HIV-infected individuals have found positive rates for anti-HBc ranging from 38.6% to 55.1%^39-42. These differences likely reflect the percentage of risk factors in the groups studied and the pattern of endemicity of HBV in different regions. Our study reported that 39.0% of HIV-HCV-coinfected people had anti-HBc reactivity. Anti-HBc alone in the HIV population can be interpreted as a marker of occult hepatitis B⁴³, a phenomenon that may be caused by either a resolved HBV infection⁴⁴, or loss of anti-HBs over time⁴⁵; other studies have reinforced these findings^43,46. In addition, in our study, we found a high frequency of patients with HBeAg (50%) among HIV-HBV-coinfected patients. The AIDS-related immunosuppression increases the frequency of reactivation of HBV, with the recurrence of HBeAg and reversion to the immunoactive phase among HIV-HBV-coinfected patients⁴⁷.

Assessing the severity and predicting the course of HIV infections with both CD4 + T cell counts and HIV viral load levels are essential for estimating the severity of HIV-related immunodeficiency. The present study showed a lack of statistical association between HCV-1 and HCV-3 for nadir CD4+ T-cell count and HIV viral loads. Few studies have systematically investigated the CD4+ T-cell count among HCV genotypes in HIV-infected patients. In a study carried out in Brazil, no significant differences were observed in the CD4+ T-cell count for HCV genotypes 1 and 3⁴⁸; our data corroborate this finding. Our study was also in line with data already published in the EuroSIDA study⁴⁹. The authors of this study report that they found no significant differences in the CD4+ T-cell count and HIV viral load in relation to the HCV genotype.

HCV infection could impact the course of HIV infections via chronic immune activation and cytokine production in coinfected individuals^50,51, which may result in diminished CD4+ T-cell counts⁵². In our study, patients with HIV-HCV had a significantly lower median nadir CD4+ cell count when compared with those with HIV-HBV; this result is supported by the biology of the coinfection⁵². Thus, HCV in HIV patients can cause damage to their immune system, which could subsequently increase viral replication of HIV and HCV, further contributing to an impaired immune system and consequently lower CD4+ T-cell count^53,54. However, caution should be taken in the interpretation of CD4+ T-cell counts since it may not truly reflect the immunological status of HIV-infected patients. Factors such as medications, advanced liver disease, splenomegaly, and viral infections such as Epstein Barr Virus, cytomegalovirus, and HTLV-1, as well as bacterial infections such as tuberculosis may cause the absolute CD4+ T-cell count to decrease⁵⁵. In addition, the lymphoid tissue fibrosis and liver fibrosis contributes to CD4+ T-cell depletion^56-58, and a low CD4+ T-cell count was associated with advanced and major liver disease histological index⁵⁹. In this sense, our reports showed that patients HCV coinfected and with Fibrosis F3 had a higher median nadir T CD4 + when compared to individuals HCV coinfected with Fibrosis F4. However, due to the low amount individuals, we cannot position ourselves on this finding.

Our study presents some limitations. First, convenience sampling for HIV-1 subtyping among HIV-coinfected patients was the main limitation of our study. A more systematic sampling, spanning all coinfected patients, would certainly provide a more accurate picture for the diversity of HIV-1 in our region. Secondly, because of a few patients with HCV-3, the power of the study may be insufficient to compare HCV-1 and 3 in HIV-infected patients who may be masked by potential confounding factors and to allow for more precise conclusions. Thirdly, information on epidemiology was collected from questionnaires administered during individual interviews, all of which may not have been completed with the same level of detail, potentially generating biases.

CONCLUSION

HCV-1 was the most prevalent among HIV-HCV-coinfected patients, followed by HCV-3, as well as subtype B of HIV-1 among HIV-coinfected patients. Significantly lower values for nadir CD4+ T-cell counts in the HIV-HCV group were found when compared to those with HIV-HBV. There was no statistically significant association between nadir CD4+ T-cell counts and HIV viral load among HCV-1 and HCV-3 among HIV-infected patients. Prospective studies should be conducted to better understand the effect of HCV genotypes in HIV-infected patients.

ACKNOWLEDGEMENTS

The authors extend their thanks to the Secretariat of Health of the City of Cascavel and Maringá.

REFERENCES

• ¹
  World Health Organization. Global Hepatitis Report, 2017. [Internet]. [Accessed 2018 June 10]. Available from: Available from: http://apps.who.int/iris/bitstream/10665/255016/1/9789241565455-eng.pdf?ua=1
  » http://apps.who.int/iris/bitstream/10665/255016/1/9789241565455-eng.pdf?ua=1
• ²
  Joint United Nations Programme on HIV/AIDS. AIDS by the numbers, 2016. [Internet]. [Accessed 2018 June 10]. Available from: http://www.unaids.org/sites/default/files/media_asset/AIDS-by-the-numbers-2016_en.pdf
  » http://www.unaids.org/sites/default/files/media_asset/AIDS-by-the-numbers-2016_en.pdf
• ³
  Oliveira SB, Merchán-Hamann E, Amorim LDAF. HIV/AIDS coinfection with the hepatitis B and C viruses in Brazil. Cad Saude Publica. 2014;30:433-8.
• ⁴
  Thio CL, Smeaton L, Saulynas M, Hwang H, Saravan S, Kulkarni S, et al: Characterization of HIV-HBV coinfection in a multinational HIV-infected cohort. AIDS. 2013;27:191-201.
• ⁵
  Chandra N, Joshi N, YSN Raju, Kumar A, Teja VD. Hepatitis B and/or C co-infection in HIV infected patients: a study in a tertiary care centre from South India. Indian J Med Res. 2013;138:950-954.
• ⁶
  Muriuki BM, Gicheru MM, Wachira D, Nyamache AK, Khamadi SA. Prevalence of hepatitis B and C viral co-infections among HIV-1 infected individuals in Nairobi, Kenya. BMC Res Notes. 2013;6:363.
• ⁷
  Mallet V, Vallet-Pichard A, Pol S. The impact of human immunodeficiency virus on viral hepatitis. Liver Int. 2011 (Suppl 1):S135-S139.
• ⁸
  Otegbayo J, Taiwo BO, Akingbola TS, Odaibo GN, Adedapo KS, Penugonda S, et al. Prevalence of hepatitis B and C seropositivity in a Nigerian cohort of HIV-infected patients. Ann Hepatol. 2008;7:152-6.
• ⁹
  Scheel TKH, Prentoe J, Carlsen THR, Mikkelsen LS, Gottwein JM, Bukh J. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems. PLoS Pathog. 2012;8:e1002696.
• ¹⁰
  Petruzziello A, Marigliano S, Loquercio G, Cozzolino A, Cacciapuoti C. Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol. 2014;61:S45-57.
• ¹¹
  Campiotto S, Pinho JRR, Carrilho FJ, Da Silva LC, Souto FJD, Spinelli V, et al. Geographic distribution of hepatitis C virus genotypes in Brazil. Braz J Med Biol Res. 2005;38:41-9.
• ¹²
  Cantão NM, Fogaça De Almeida L, Rodrigo Wolf I, Oliveira Almeida R, Alves De Almeida Cruz A, Nunes C, et al. HIV Reverse Transcriptase and Protease Genes Variability Can Be a Biomarker Associated with HIV and Hepatitis B or C Coinfection. Sci Rep. 2018;8:8280.
• ¹³
  Regionais de Saúde - Centro de Apoio Operacional das Promotorias de Proteção à Saúde Pública. [Internet]. [Accessed 2018 May 23]. Available from: http://www.saude.mppr.mp.br/modules/conteudo/conteudo.php?conteudo=522
  » http://www.saude.mppr.mp.br/modules/conteudo/conteudo.php?conteudo=522
• ¹⁴
  Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996;24:289-93.
• ¹⁵
  Gupta E, Bajpai M, Choudhary A. Hepatitis C virus: Screening, diagnosis, and interpretation of laboratory assays. Asian J Transfus Sci. 2014;8:19-25.
• ¹⁶
  van de Laar TJW, Langendam MW, Bruisten SM, Welp E a E, Verhaest I, van Ameijden EJC, et al. Changes in risk behavior and dynamics of hepatitis C virus infections among young drug users in Amsterdam, the Netherlands. J Med Virol. 2005;77:509-18.
• ¹⁷
  Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, Feinstone S, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology. 2005;42:962-73.
• ¹⁸
  Bello G, Passaes CP, Guimarães ML, Lorete RS, Matos Almeida SE, Medeiros RM, et al. Origin and evolutionary history of HIV-1 subtype C in Brazil. AIDS. 2008;22:1993-2000.
• ¹⁹
  Telele NF, Kalu AW, Gebre-Selassie S, Fekade D, Marrone G, Grossmann S, et al. A viral genome wide association study and genotypic resistance testing in patients failing first line antiretroviral therapy in the first large countrywide Ethiopian HIV cohort. BMC Infect Dis. 2019;19:569.
• ²⁰
  StataCorp L. Stata version 12.0. College station. TX StataCorp LP. 2011.
• ²¹
  Brasil. Ministério da Saúde. Protocolo Clínico e Diretrizes Terapêuticas para Hepatite C e Coinfecções. [Internet]. [Accessed July 29, 2019]. Available from: http://www.aids.gov.br/pt-br/pub/2017/protocolo-clinico-e-diretrizes-terapeuticas-para-hepatite-c-e-coinfeccoes
  » http://www.aids.gov.br/pt-br/pub/2017/protocolo-clinico-e-diretrizes-terapeuticas-para-hepatite-c-e-coinfeccoes
• ²²
  Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Gonçales FL, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med. 2002;347:975-82.
• ²³
  Petruzziello A, Marigliano S, Loquercio G, Cozzolino A, Cacciapuoti C. Global epidemiology of hepatitis C virus infection: An up-date of the distribution and circulation of hepatitis C virus genotypes. World J Gastroenterol. 2016;22:7824-40.
• ²⁴
  Mendes-Correa MC, Cavalheiro NP, Mello C, Barone AA, Gianini RJ. Genotypic distribution of hepatitis C among hepatitis C and HIV co-infected patients in Brazil. Int J STD AIDS. 2008;19:595-9.
• ²⁵
  Silva CM, Costi C, Krug LP, Ramos AB, Grandi T, Gandolfi VL, et al. High proportion of hepatitis C virus genotypes 1 and 3 in a large cohort of patients from Southern Brazil. Mem Inst Oswaldo Cruz. 2007;102:867-70.
• ²⁶
  Freitas SZ, Teles SA, Lorenzo PC, Puga MAM, Tanaka TSO, Thomaz DY, et al. HIV and HCV coinfection: prevalence, associated factors and genotype characterization in the Midwest Region of Brazil. Rev Inst Med Trop Sao Paulo. 2014;56:517-24.
• ²⁷
  Triantos C, Konstantakis C, Tselekouni P, Kalafateli M, Aggeletopoulou I, Manolakopoulos S. Epidemiology of hepatitis C in Greece. World J Gastroenterol . 2016;22:8094-102.
• ²⁸
  Valle Tovo C, Alves de Mattos A, Ribeiro de Souza A, Ferrari de Oliveira Rigo J, Lerias de Almeida PR, Galperim B, et al. Impact of human immunodeficiency virus infection in patients infected with the hepatitis C virus. Liver Int . 2007;27:40-6.
• ²⁹
  Krug LP, Lunge VR, Ikuta N, Fonseca AS, Cheinquer H, Ozaki LS, et al. Hepatitis C virus genotypes in Southern Brazil. Braz J Med Biol Res . 1996;29:1629-32.
• ³⁰
  Galperim B, Cheinquer H, Stein A, Fonseca A, Lunge V, Ikuta N. Prevalence of hepatitis C virus in alcoholic patients: Role of parenteral risk factors. Arq Gastroenterol. 2006;43:81-4.
• ³¹
  Alter MJ. Epidemiology of viral hepatitis and HIV co-infection. J Hepatol. 2006;44 (Suppl 1):S6-9.
• ³²
  Wolff FH, Fuchs SC, Barcellos NT, Falavigna M, Cohen M, Brandão AB, et al. Risk factors for hepatitis C virus infection in individuals infected with the HIV. Dig Liver Dis. 2008;40:460-7.
• ³³
  Hemelaar J. Implications of HIV diversity for the HIV-1 pandemic. J Infect. 2013;66:391-400.
• ³⁴
  Librelotto CS, Graf T, Simon D, de Almeida SEM, Lunge VR. HIV-1 epidemiology and circulating subtypes in the countryside of South Brazil. Rev Soc Bras Med Trop. 2015;48:249-57.
• ³⁵
  Avanzi VM, Vicente BA, Beloto NCP, Gomes-Da-Silva MM, Ribeiro CEL, Tuon FF, et al. Profile of HIV subtypes in HIV/HBV- and HIV/HCVcoinfected patients in Southern Brazil. Rev Soc Bras Med Trop . 2017;50:470-77.
• ³⁶
  Bello G, Zanotto PM de A, Iamarino A, Gräf T, Pinto AR, Couto-Fernandez JC, et al. Phylogeographic analysis of HIV-1 subtype C dissemination in Southern Brazil. PLoS One. 2012;7:e35649.
• ³⁷
  Delatorre E, Couto-Fernandez JC, Guimarães ML, Vaz Cardoso LP, de Alcantara KC, Martins de Araújo Stefani M, et al. Tracing the Origin and Northward Dissemination Dynamics of HIV-1 Subtype C in Brazil. PLoS One . 2013;8:e74072.
• ³⁸
  Chen X, He J-M, Ding L-S, Zhang G-Q, Zou X-B, Zheng J. Prevalence of hepatitis B virus and hepatitis C virus in patients with human immunodeficiency virus infection in Central China. Arch Virol. 2013;158:1889-94.
• ³⁹
  Mendes-Corrêa MCJ, Barone AA, Cavalheiro NP, Tengan FM, Guastini C. Prevalence of hepatitis B and C in the sera of patients with HIV infection in Sao Paulo, Brazil. Rev do Inst Med Trop São Paulo. 2000;42:81-85.
• ⁴⁰
  de Almeida Pereira RAR, Mussi ADH, de Azevedo e Silva VC, Souto FJD. Hepatitis B Virus infection in HIV-positive population in Brazil: results of a survey in the state of Mato Grosso and a comparative analysis with other regions of Brazil. BMC Infect Dis . 2006;6:34.
• ⁴¹
  Marchesini AM, Pra-Baldi ZP, Mesquita F, Bueno R, Buchalla CM. Hepatitis B and C among injecting drug users living with HIV in Sao Paulo, Brazil. Rev Saude Publica. 2007;41 (Suppl 2):57-63.
• ⁴²
  Freitas SZ, Soares CC, Tanaka TSO, Lindenberg ASC, Teles SA, Torres MS, et al. Prevalence, risk factors and genotypes of hepatitis B infection among HIV-infected patients in the State of MS, Central Brazil. Braz J Infect Dis. 2014;18:473-80.
• ⁴³
  Gandhi RT, Wurcel A, Lee H, McGovern B, Boczanowski M, Gerwin R, et al. Isolated antibody to hepatitis B core antigen in human immunodeficiency virus type-1-infected individuals. Clin Infect Dis. 2003;36:1602-5.
• ⁴⁴
  Thio CL. Hepatitis B in the human immunodeficiency virus-infected patient: Epidemiology, natural history, and treatment. Semin Liver Dis. 2003;23:125-36.
• ⁴⁵
  Collier MG, Drobeniuc J, Cuevas-Mota J, Garfein RS, Kamili S, Teshale EH. Hepatitis A and B among young persons who inject drugs-Vaccination, past, and present infection. Vaccine. 2015;33:2808-12.
• ⁴⁶
  French AL, Operskalski E, Peters M, Strickler HD, Tien PC, Sharp GB, et al. Isolated hepatitis B core antibody is associated with HIV and ongoing but not resolved hepatitis C virus infection in a cohort of US women. J Infect Dis. 2007;195:1437-42.
• ⁴⁷
  Hoffmann CJ, Thio CL. Clinical implications of HIV and hepatitis B co-infection in Asia and Africa. Lancet Infect Dis. 2007;7:402-9.
• ⁴⁸
  Wolff FH, Fuchs SC, Barcellos NNT, de Alencastro PR, Ikeda MLR, Brandão ABM, et al. Co-infection by Hepatitis C virus in HIV-infected patients in Southern Brazil: Genotype distribution and clinical correlates. PLoS One . 2010;5.e10494.
• ⁴⁹
  Soriano V, Mocroft A, Rockstroh J, Ledergerber B, Knysz B, Chaplinskas S, et al. Spontaneous viral clearance, viral load, and genotype distribution of hepatitis C virus (HCV) in HIV-infected patients with anti-HCV antibodies in Europe. J Infect Dis . 2008;198:1337-44.
• ⁵⁰
  Gonzalez VD, Falconer K, Blom KG, Reichard O, Mørn B, Laursen AL, et al. High levels of chronic immune activation in the T-cell compartments of patients coinfected with hepatitis C virus and human immunodeficiency virus type 1 and on highly active antiretroviral therapy are reverted by alpha interferon and ribavirin treatment. J Virol. 2009;83:11407-11.
• ⁵¹
  Graham CS, Curry M, He Q, Afdhal N, Nunes D, Fleming C, et al. Comparison of HCV‐specific intrahepatic CD4+ T cells in HIV/HCV versus HCV. Hepatology. 2004;40:125-32.
• ⁵²
  Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, Lampiris H, et al. T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis . 2003;187:1534-43.
• ⁵³
  Adewole OO, Anteyi E, Ajuwon Z, Wada I, Elegba F, Ahmed P, et al. Hepatitis B and C virus co-infection in Nigerian patients with HIV infection. J Infect Dev Ctries. 2009;3:369-75.
• ⁵⁴
  Netski DM, Mosbruger T, Astemborski J, Mehta SH, Thomas DL, Cox AL. CD4+ T cell-dependent reduction in hepatitis C virus-specific humoral immune responses after HIV infection. J Infect Dis . 2007;195:857-63.
• ⁵⁵
  Schechter M, Harrison LH, Halsey NA, Trade G, Santino M, Moulton LH, et al. Coinfection with human T-cell lymphotropic virus type I and HIV in Brazil. Impact on markers of HIV disease progression. JAMA. 1994;271:353-7.
• ⁵⁶
  Sanchez JL, Hunt PW, Reilly CS, Hatano H, Beilman GJ, Khoruts A, et al. Lymphoid fibrosis occurs in long-term nonprogressors and persists with antiretroviral therapy but may be reversible with curative interventions. J Infect Dis . 2014;211:1068-75.
• ⁵⁷
  Rockstroh JK, Spengler U, Sudhop T, Ewig S, Theisen A, Hammerstein U, et al. Immunosuppression may lead to progression of hepatitis C virus-associated liver disease in hemophiliacs coinfected with HIV. Am J Gastroenterol. 1996;91:2563-8.
• ⁵⁸
  Puoti M, Bonacini M, Spinetti A, Putzolu V, Govindarajan S, Zaltron S, et al. Liver fibrosis progression is related to CD4 cell depletion in patients coinfected with hepatitis C virus and human immunodeficiency virus. J Infect Dis . 2001;183:134-7.
• ⁵⁹
  Mohsen AH, Easterbrook PJ, Taylor C, Portmann B, Kulasegaram R, Murad S, et al. Impact of human immunodeficiency virus (HIV) infection on the progression of liver fibrosis in hepatitis C virus infected patients. Gut. 2003;52:1035-40.

• Disclosure of funding: no funding received

Publication Dates

• Publication in this collection
  28 Nov 2019
• Date of issue
  Oct-Dec 2019

History

• Received
  22 Apr 2019
• Accepted
  07 Sept 2019