Major Histocompatibility Complex Class I and Dengue Hemorrhagic Fever: A Meta-Analysis of Human Leukocyte Antigens A*24 and B*44

Introduction: Dengue fever (DF) is a disease caused by dengue virus (DENV) from the family Flaviviridae. The role of human leukocyte antigens (HLAs) in dengue fever (DF) and its more severe manifestation, dengue hemorrhagic fever (DHF), has been a topic of great research interest. In addition to HLA profile, race, age, DENV serotype, infection while having certain chronic diseases, and secondary infection are risk factors for DHF susceptibility. Antibody-dependent enhancement (ADE) of dengue virus infection is a mechanism for DHF infection. Individual studies have examined the effects of HLA-A*24 and HLA-B*44 presence on DHF, but none have analyzed these in a meta-analysis. The objective of this study was to determine the effects of HLA-A*24 and HLA-B*44 presence on DHF and DF susceptibility. Materials and methods: A meta-analysis on DHF and DF susceptibility in patients with HLA-A*24 and HLA-B*44 was conducted. Google Scholar was used to find studies that contained patients with HLA-A*24 or HLA-B*44 that were diagnosed with DHF or DF. Studies containing patients diagnosed using the 1997 WHO guidelines and possessing HLA-A*24 or HLA-B*44 that were diagnosed with DHF or DF, including primary or secondary infection, and studies measuring odds ratios (ORs) were included. Patients diagnosed using the 2009 WHO guidelines and studies in a foreign language, using animals, or lacking odds ratios were excluded. The National Institutes of Health (NIH) quality assessment of the case-control study tool was used, and a Doi plot was generated using MetaXL to assess for risk of bias. Review Manager version 5.4 was used to generate odds ratios and forest plots with subgroup analysis from allele and phenotype frequency data. Ten studies from 2001 to 2015 met the inclusion criteria. The studies included 2837 DHF/DF patients and 4880 healthy control (HC) patients. Results: HLA-A*24 was associated with a 1.39 times susceptibility to DHF while those possessing HLA-B*44 were 0.62 times susceptible to DHF (OR=1.39 and 95% CI=1.17-1.66; OR=0.62 and 95% CI=0.39-0.99). Neither HLA-A*24 nor HLA-B*44 presence was associated with DF susceptibility (OR=1.04 and 95% CI=0.82-1.33; OR=0.88 and 95% CI=0.68-1.14). Conclusion: These results indicate that two different major histocompatibility complex (MHC) class I alleles, HLA-A*24 and HLA-B*44, have opposing effects on DHF susceptibility but none on DF susceptibility. The study's specificity is limited in that it examines HLA allele groups and not specific HLA proteins. The results of this study can be used clinically to identify patients that may be at a higher risk of developing DHF based on their HLA profile.


Introduction
Dengue fever (DF) is a disease caused by dengue virus (DENV) from four different possible serotypes: DENV-1, DENV-2, DENV-3, or DENV-4 [1]. Dengue virus is a member of the family Flaviviridae, which are positivestrand RNA viruses [1]. Other members of this family include West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, zika virus, hepatitis C virus, and yellow fever virus [1]. Dengue fever affects 100-400 million individuals per year globally, with 70% of cases from Asia [1]. However, it affects countries from the Americas, Western Pacific, Africa, and Eastern Mediterranean [1]. Aedes aegypti and Aedes albopictus are the vectors for dengue virus, which transmit it to humans upon biting the subject [1].
According to the 1997 WHO classification guidelines for dengue clinical diagnosis, symptomatic infections are subdivided into undifferentiated fever, dengue fever (DF) syndrome, or dengue hemorrhagic fever (DHF) [2]. Eighty percent of dengue virus infections are asymptomatic [1]. DF is the mild presentation of the disease and can be characterized as without hemorrhage or with hemorrhage [2]. At least two of the following symptoms are required for diagnosis: leukopenia, hemorrhage, rash, arthralgia, headache, retro-orbital pain, or myalgias [2]. However, some individuals develop the more clinically severe disease, DHF [2]. DHF diagnosis requires the following symptoms: plasma leakage, high fever, hemorrhagic phenomena, thrombocytopenia with hemoconcentration, or circulatory failure [2]. DHF can be further subdivided into without shock or dengue shock syndrome (DSS) [2]. DSS is a more severe form of DHF resulting in circulatory failure, hypovolemic shock, lethargy, or acute abdominal pain [2]. The diagnosis of DSS requires all signs of DHF plus shock [2]. In 2009, the WHO updated the dengue fever classification system [3]. Dengue fever is classified as non-severe or severe [3]. Severe dengue is determined by plasma leakage, severe bleeding, and severe organ impairment [3]. Non-severe dengue is further classified with or without warning signs [3]. These warning signs include abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, hepatomegaly, or increased hematocrit with a decrease in platelet count [3]. These guidelines were updated to more practically guide clinicians in the diagnosis of a patient with severe dengue without the strict parameters of the 1997 protocol [3]. However, researchers continue to use both classifications.
In addition to HLA profile, DHF susceptibility is increased with secondary infection [15] and DENV-2 serotype [15], in young infants and the elderly [16], in pregnant patients [17], and in patients with bronchial asthma, diabetes mellitus, and sickle cell anemia [18]. DHF susceptibility is also affected by race; African descent was shown to decrease the risk of DHF infection [19].
Antibody-dependent enhancement (ADE) is the mechanism by which dengue virus infection can lead to the more severe disease state, DHF [20]. This is due to the dengue virus attaching to the dengue antibody, which is incapable of neutralizing the virus [20]. As a result, the active virus is internalized into the cell via the Fc gamma receptors (FcgammaRs) [20]. This leads to an increased dengue viremia and worse clinical manifestations such as DHF [20].
In this study, we conducted a meta-analysis of the MHC class I allele groups, HLA-A*24 and HLA-B*44, in its role in DHF susceptibility. MHC class I plays a role in viral immunity [21]. HLA-A*24 and HLA-B*44 are frequently mentioned in the literature and warrant further research in a meta-analysis to determine their role in dengue immunity. For example, in a Vietnamese population, Nguyen et al. found HLA-A*24 presence to lead to DHF susceptibility [7]. For HLA-B*44, Vejbaesya et al. found it to be protective against DHF [13].

Materials And Methods
A meta-analysis of DF and DHF patients with HLA-A*24 and HLA-B*44 was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The following inclusion criteria were used: patients possessing HLA-A*24 or HLA-B*44 that were diagnosed with DHF or DF, including primary or secondary infection; studies using odds ratio (OR) or sufficient data available to calculate an odds ratio; and studies using the 1997 WHO guidelines [2]. The following exclusion criteria were implemented: studies including patients diagnosed with dengue shock syndrome (DSS), patients diagnosed according to the 2009 WHO guidelines [3] of severe dengue and non-severe dengue, studies in a foreign language, studies using animals as subjects, and studies not using odds ratio or studies with insufficient data available to calculate odds ratios. The 1997 guidelines [2] were used to encompass all studies within the full publication date range. The 2009 guidelines [3] were excluded to avoid confounding the two guidelines. Google Scholar database was used in the literature review search. The following search terms were used: "HLA-A*24 Dengue Hemorrhagic fever" and "HLA-B*44 Dengue Hemorrhagic fever" ( Figure  1). Limitations on study publication date from 2000 to 2022 will be applied. The protocol was published prior to the research on May 20, 2022 [22]. Version 2 was published on May 29, 2022 [22]. The protocol correction was implemented to note the collection and analysis of phenotype frequencies in addition to allele frequencies in this research, as well as clarification on the use of sensitivity testing [16]. Two independent reviewers selected studies in this research according to the following criteria. If a decision could not be reached by the two reviewers, the principal investigator (PI) made the final decision. Data was extracted from figures in the articles. This data included ORs with 95% CIs if directly provided. If this was not directly provided, allele frequencies or phenotype frequencies were collected to generate ORs and 95% CIs. This data was used to determine the odds of DF and DHF given the presence of HLA-A*24 or HLA-B*44.
A combined odds ratio and 95% CI with subgroup analysis of DF versus HC and DHF versus HC for each HLA examined in this study was generated using the inverse variance (IV) random effect model. Review Manager version 5.4 was used in the generation of these analyses. Figure 3 shows that in the DF versus HC with HLA-A*24 analysis, HLA-A*24 is not associated with susceptibility or protection to DF (OR=1.04 and 95% CI=0.82-1.33). The DHF versus HC with HLA-A*24 analysis shows that HLA-A*24 is associated with a 1.39 times susceptibility to DHF (OR=1.39 and 95% CI=1.17-1.66) (Figure 3). The DF/DHF versus HC with HLA-A*24 analyses found a 1.26 times susceptibility to DF/DHF (OR=1.26 and 95% CI=1.09-1.45) (Figure 3). Figure  4 shows that in the DF versus HC with HLA-B*44 analysis, HLA-B*44 is not associated with susceptibility or protection to DF (OR=0.88 and 95% CI=0.68-1.14). The DHF versus HC with HLA-B*44 analysis shows that HLA-B*44 is protective to DHF with a 0.62 times susceptibility to DHF (OR=0.62 and 95% CI=0.39-0.99) (Figure 4). The DF/DHF versus HC with HLA-B*44 analysis shows a protective effect with a 0.77 susceptibility to DF/DHF (OR=0.77 and 95% CI=0.60-0.98) (Figure 4).

FIGURE 3: HLA-A*24 forest plots with subgroup analysis of DF versus healthy control (HC) and DHF versus HC
HLA: human leukocyte antigen; DF: dengue fever; DHF: dengue hemorrhagic fever Doi plots were generated for individual subgroups, as well as overall combined groups for both HLA-A*24 and HLA-B*44 (Figure 2). Although the individual HLA-A*24 Doi plots showed minor (hemorrhagic fever versus healthy controls) and major (dengue versus healthy controls) asymmetry, combined subgroup analysis indicated a Luis Furuya-Kanamori (LFK) index of -0.38 (Figure 2). HLA-B*44 Doi plots showed minor asymmetry (hemorrhagic fever versus healthy controls) and no asymmetry (dengue versus healthy controls); there was a combined subgroup analysis Doi plot indicating no asymmetry with an LFK index of -0.64 (Figure 2).

Discussion
The results of this meta-analysis indicate differing effects of HLA-A*24 and HLA-B*44 presence on DHF susceptibility but not DF. HLA-A*24 presence led to a 1.39 times susceptibility to DHF as compared to HC (OR=1.39 and 95% CI=1.17-1.66). HLA-B*44 had a protective effect from DHF as indicated by the 0.62 times susceptibility to DHF as compared to HC (OR=0.62 and 95% CI=0.39-0.99). Both HLA-A*24 and HLA-B*44 presence did not have a protective or increased susceptibility effect on DF infection compared to HC (OR=1.04 and 95% CI=0.82-1.33; OR=0.88 and 95% CI=0.68-1.14).
The studies used in this meta-analysis were subject to the following limitations. The studies used only contained specificity up to the HLA allele group rather than the specific HLA protein. These factors limit the study in its specificity of HLA-A*24 and HLA-B*44 on DHF. An additional limitation of this study includes potential disease heterogeneity. Future studies should examine HLA associations with DHF in additional different populations. This could investigate whether certain populations are associated with certain HLA profiles for DHF susceptibility compared to others.

Conclusions
This study showed that two different MHC class I allele groups, HLA-A*24 and HLA-B*44, had opposing effects on DHF susceptibility but no effect on DF. HLA-A*24 presence leads to an increase in DHF susceptibility, whereas HLA-B*44 had a protective effect. More data should be collected on isolating the specific HLA alleles involved among the HLA-A*24 and HLA-B*44 allele groups. Then, once this data is available, further research should be conducted to determine which specific HLA allele is involved in DHF susceptibility. This research can be clinically significant in identifying a patient's risk based on their HLA profile to DHF.

Additional Information Disclosures
Human subjects: All authors have confirmed that this study did not involve human participants or tissue.
Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.