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Correlation between HIV-1 genotype and clinical progression in HIV/AIDS patients in Surabaya, Indonesia

Several factors such as host and viral factors can affect the progression of HIV/AIDS. This study aims to identify the correlation viral factors, especially the HIV-1 subtype with HIV/AIDS progression. Inpatient HIV/AIDS during the period March to September 2017 and willing to participate are included in the study. Historical data of disease and treatment was taken by medical record. Blood samples were amplified, sequenced and undergone phylogenetic analysis. Linear regression analysis was used to estimate beta coefficient (β) and 95%CI of HIV/AIDS progression (measured by the CD4 change rate, ΔCD4 cell count/time span in months).This study has 17 samples. The HIV-1 subtype was dominated by CRF01_AE (81.8%) followed by subtype B (18.2%). There was significant correlation between subtype HIV-1 (p = 0.04) and body mass index (p = 0.038) with HIV/AIDS clinical stage. Many factors were assumed to be correlated with increased rate of CD4, but we only subtype HIV-1 had a significant correlation (p = 0.024) with it. From multivariate analysis, we also found that subtype HIV-1 had a significant correlation (β = 0.788, 95%CI: 17.5-38.6, p = 0.004).


Introduction
[11]Information on the factors that affect the progression of the disease will be a valuable guide to manage the disease.
HIV can be classified by type, i.e. HIV-1 and HIV-2. HIV-1 itself can be subdivided by groups (M, N, and O) and subtypes (A, B, C, D, F, G, H, J, and K). Many studies have reported a correlation between HIV-1 and HIV/AIDS progression, but the further relevance of subtypes still results in varying outcomes. Moreover, there is limited data on the relationship of HIV-1 subtypes to disease progression in Indonesia. This study aims to determine the viral subtype and its correlation with disease progression. The HIV-1 pol gene encoding protease (PR gene) and Reverse Transcriptase (RT gene) were then amplified from DNA extracted from PBMCs by nested polymerase chain reaction(PCR) using Ex Taq (Takara Bio, Shiga, Japan) and primer sets as follows. To amplify the viral PR gene fragment, theprimers DRPRO5 [5'-AGACAGGYTAATTTTTTAGGGA-3'; corresponding to nucleotides (nt) 2074 to 2095 of anHIV-1 reference strain, HXB2 (GenBankaccession no.K03455)] and DRPRO2L (5'-TATGGATTTTCAGGCCCAATTTTTGA-3'; nt 2716 to 2691) were for the first PCR, and the primers DRPRO1M (5'-AGAGCCAACAGCCCCACCAG-3'; nt 2148 to 2167) and DRPRO6 (5'-ACTTTTGGGCCATCCATTCC-3'; nt 2611 to 2592) were for the nested PCR. In addition, to amplify the viral RT gene, RT1L(5'-ATGATAGGGGGAATTGGAGGTTT-3'; nt 2388 to2410) and GPR2M (5'-GGACTACAGTCYACTTGTCCATG-3'; nt 4402 to 4380) were for the first PCR and RT7L(5'-GACCTACACCTGTCAACATAATTGG-3'; nt 2485 to2509) and GPR3L (5'-TTAAAATCACTARCCATTGYTCTCC-3'; nt 4309 to 4285) were for the nested PCR.PCR products amplified at the end-point dilution of DNA templates were subjected to sequencing analysis to examine the genomic fragment of the major viral population in a sample.

Material and Methods
Sequencing analysis of the amplified HIV-1 PR and RT genes was carried out using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM3500xL genetic analyzer (Applied Biosystems, Foster City, CA), and data were assembled using Genetyx version 10 software (Genetyx, Tokyo, Japan). The full length of the PR gene (297 bp; nt 2253 to 2549) and the N-terminus of the RT gene (762 bp; nt 2550 to 3311) were sequenced and subjected to subsequent analyses. HIV-1 subtyping was carried out using the Recombinant Identification Program (RIP) available on the HIV sequence database website (www.hiv.lanl.gov/). Also, neighbor-joining (NJ) trees with the Kimura two-parameter model were constructed using theMEGA6.2 software.[12,13] Bootstrap values (1,000 replicates) for relevant nodes were reported on a representative tree. If one of the PR and RT genes failed to be sequenced, the subtype was assigned based on the other gene. Furthermore, if there was a discrepancy in the subtype between PR and RT, it was defined as a recombinant of more than two HIV-1 subtypes and circulating recombinant forms (CRFs).
Linear regression analysis was used to estimate beta coefficient (β) and 95% confidence intervals (CIs) of HIV/AIDS progression (measured by the rate of change of CD4, ∆CD4 cell count/span of time in months).  (Figure 1).

Factors related to HIV / AIDS progression
From baseline characteristics and clinical stage, after analysis there was significant correlation between subtype HIV-1 (p = 0.04) and body mass index (p = 0.038) ( Table 2). From the analysis in relation to the increasing rate of CD4, it was found only subtype HIV-1 has a significant correlation (p = 0.024). From multivariate analysis, it was found that the subtype HIV-1 still had a significant correlation (β = 0.788, 95% CI: 17.5-38.6, p = 0.004) ( Table 3).  [14] This difference shows that there are more influential factors on HIV / AIDS-related deaths in our study. Correlation between HIV-1 subtype and disease progression remains controversial. Some studies report the correlation between HIV-1 subtype and HIV / AIDS progression by relating the shortening of time needed by HIV to become AIDS, low rates of CD4 change, high viral load, and HIV / AIDS-related deaths. A meta-analysis shows that people infected with subtype C have HIV / AIDS progression faster than other subtypes, followed by subtypes D, AE, G and A.
[15] The study by Chu et al. reported that the low rate of CD4 change was related to the CRF01_AE subtype.
[1] The previous studies in Indonesia had reported that this CRF01_AE subtype also has a higher prevalence than other subtypes [16], and in this study, CRF01_AE is also associated with the rate of change CD4 cells in patients who had received ART (p = 0.004). Ng et al. reported that the viral load was significantly higher in those who got infected with CRF01_AE than with non-CRF01_AE (P<0.015). [17] Viral load testing itself has not been being a routine check-up, so there are limitations in the availability of data. Subtype linkages with HIV / AIDS-related deaths are still contradictory. Costello et al. reported that subtype CRF01_AE had estimated median time to death 7.8 (7.0-9.1) years. [18] While Alaeus et al. reported no difference in survival rates in all subtypes of HIV-1. [19] In this study, the HIV-1 subtype was not associated with death (data not shown).
In conclusion, HIV-1 subtype especially CRF01_AE is related to HIV / AIDS progression. But further research is needed with obtaining more sample quantities and including more factors involved in the correlation with disease progression and maybe we can use viral load as abetter marker to reflect disease progression.