Introduction

Human Immunodeficiency Virus (HIV) has exerted a profound historical impact on human health and socioeconomic systems. As of 2019, HIV/AIDS is classified as a pandemic, affecting an estimated 38 million people worldwide, with a disproportionate burden exceed 25.7 million individuals residing in sub-Saharan Africa including Sudan1,2. The most significant hallmark of HIV infection is the gradual and cumulative deterioration of the immune system, which ultimately leads to the development of acquired immunodeficiency syndrome (AIDS)3. The intromission of antiretroviral therapy (ART) into the HIV treatment program has resulted in a significant improvement in the overall course of the disease and a gradual improvement in survival rates4. Throughout history, a characteristic and surprising immunopathogenic feature of HIV infection directly impacts the immune system by significantly reducing the number of CD4 + T lymphocytes, which are the master of all aspects that regulate immune defences against all pathogenic microorganisms5. Few studies have focused on and emphasized a proper understanding of the mechanisms underlying the potential effects of HIV infection on induced autoimmune diseases (AD)5. AD is a group of chronic inflammatory diseases characterized by irregular polyreactivity of B and T cells to host tissue antigens6. The characteristic feature of autoimmunity and the best-known immunological manifestation is the production of autoantibodies against autoantigens, which provide distinct biomarkers for autoimmune disease detection, diagnosis, classification, monitoring, and progression7. In general, the autoimmune phenomenon is a multifactorial disease caused by the interaction of genetic predisposition factors and environmental factors such as viral infections8. Many proposals illustrate the proximate mechanisms of how HIV infection can trigger and irritate autoimmunity6. Increasing knowledge in the same context of HIV infection can stimulate and deregulate the master of immune cells, mostly B and T lymphocytes; however, HIV infection triggers polyclonal cloning and polyreactive B cells, leading to the production of autoantibodies. Furthermore, there is molecular mimicry between HIV proteins and self-antigens, which may also play an important role in the potential of HIV to cause Systemic autoimmune rheumatic diseases (SARDs)9. The conceptualization of the distinct ability of HIV to cause AD has a strong resonance in the field of infections and autoimmune diseases10. Therefore, the dark mystery of HIV and autoimmune diseases, which is interesting from a clinical and pathophysiological perspective, needs to be solved and demystified from a clinical and diagnostic perspective. One of the unusual features of autoimmunity is the presence of high levels of circulating autoantibodies10. Current evidence shows that the incidence of rheumatologic diseases in HIV patients reaches 60%11.

A complete picture of HIV and autoimmune diseases is lacking, although numerous case reports and small case series are increasingly recognized as important comorbidities of HIV infection11,12,13. Interestingly, autoimmunity may also occur after the introduction of combined antiretroviral therapy (cART) due to a gradual recovery of CD4 + T cell activity and a significant decrease in viral load, which can lead to the initiation of immune reconstitution inflammatory syndrome (IRIS), a phenomenon characterized by a substantial increase in inflammatory levels and biomarkers, paradoxically leading to a worsening of the clinical picture14,15. Previous studies have shown a high prevalence of antinuclear antibodies (ANA) in HIV-infected patients before the start of the HART era, and few studies have focused on the prevalence of ANA after the cART era due to IRIS16.

To date, no published studies in our country have reported on the prevalence of ANA patterns among people living with HIV/AIDS (PLWHA), resulting in a significant knowledge gap that impedes and prevents a comprehensive interpretation of clinical outcomes. Therefore, this study was considered the first study to evaluate and determine the prevalence of ANA in PLWHA, as a result, we believe that our study might be addresses this critical gap by systematically evaluating the dynamic distribution of distinct ANA patterns and their correlation with estimated endpoint titers of ANA. By exploring such associations, we intend to better understand the role of HIV-driven chronic inflammation in shaping autoimmune serological profiles, providing new insights into disease mechanisms and their consequences for this susceptible population. This study not only advances our understanding of HIV-related immune dysregulation, but it also emphasizes the clinical utility of ANA patterns as potential biomarkers of chronic inflammatory processes particularly during ART due to development of IRIS. We systematically evaluating the distribution of different ANA patterns and their correlation of estimated endpoint titers by using indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) which was considered most frequently method and gold standard for screening for the presence of a vast array of autoantibodies and was by American College of Rheumatology (ACR), in which the titer of the HEp-2-IFA indicates to the relative concentration of particular autoantibody and tends to be higher in patients with systemic autoimmune diseases (SAD) than healthy peoples17. Furthermore, the HEp-2-IFA represents the target antigens’ unique topographic distribution across the several stages of the cell cycle. The HEp-2 IFA patterns have additional clinical utility since they reflect autoantibody specificities with clinical relevance17.

Material and methods

Study design and patient selection

This cross-sectional study recruited patients with confirmed HIV infection who were subsequently admitted to two major voluntary HIV counseling and testing clinics (VTC) at the Military Hospital and the Bahri Hospital in Khartoum, Sudan. Data have been extracted from hospital registries, ensuring the inclusion of relevant clinical and demographic variables. Demographic variables included age at HIV diagnosis and gender. Clinical variables included treatment modalities, Clinical manifestation, Comorbidities and WHO clinical stages. all medical report forms required for the patients, were completed by qualified physicians. We included patients with confirmed positivity of HIV infection using all standard tests selected by the National Public Health Laboratory (NPHL). Currently, we used in Sudan: SD bioline for the first line test, UniGold for the second test and Colloidal gold for the third test. Any patient with a known autoimmune disease was excluded from the study.

Laboratory works

Sample collection

Blood samples were taken during a regular follow-up visit to a well-known HIV clinical centre. The samples used in this investigation were obtained during routine clinical care and would otherwise have been discarded. Following rigorous aseptic protocols, competent venipuncture operators collected 3 to 5 mL of blood samples on-site and transported them to vacuum containers for preservation. The samples were centrifuged at 3000 RPM for 5 min at room temperature. The materials were then stored at − 20 °C for additional analysis.

Antinuclear autoantibody (ANA) detection

This assay is designed exclusively for the in vitro determination of human antibodies in serum or plasma. For the detection of antinuclear antibodies (ANA). The serum of all participants was analyzed for ANA analysis using commercial slide biochips with ethanol-fixed cultured HEp-2-12 slides from AESKU Diagnostics.

The protocol was carried out on a fully automated HELMED® indirect immunofluorescence system (IIF) 'AESKULISA®, AESKU Diagnostics, Wendelsheim, Germany”. Briefly, Cultured HEp-2-12 slides were incubated with 30 µl of diluted patient serum for 30 min at room temperature, and then the slide was rinsed with a stream of PBS-Tween for 5 min after washing the HEp-2-12. Slides were treated with fluorescein-labelled anti-human immunoglobulin for 30 min (second incubation) and then washed with PBS-Tween for 5 min, then the mounting medium was added and covered with a coverslip. The microscopic pattern of ANA was determined compared to positive controls for the homogeneous nuclear pattern according to the AESKU pattern library17,29.

Statistical analyses

ANA positivity is defined using the laboratory cutoff titer or endpoint titer (for ANA) at dilution 1:80, in which any specimens with positive reactivity at this dilution were evaluated for ANA pattern29.

All the statistical analyses were performed using SPSS for Windows version 20.0 and R software version 4.4.2. The Gaussian distribution of the data was tested using the Shapiro-Wilks test. Where appropriate, mean [± SD)] or median [interquartile range (IQR)] and related nonparametric statistics (Mann Whitney U and Kruskal Wallis) were used. For categorical variables, Chi-square tests (χ2) were used. Packages ‘ggplot2’ were used to visualize the statistical analysis results. Statistical significance was established at a p-value less than 0.05.

Results

Demographic and clinical characteristics of HIV participants

The demographic and clinical characteristics of HIV patients enrolled in this study are summarized in Table 1. A total of 116 confirmed HIV patients participated in the study. All patients (100%) received an antiretroviral regimen (Dolutegravir (DGT), Lamivudine (3TC), Tenofovir disoproxil fumarate (TDF)) as well as Isoniazid and Rifapentine as tuberculosis (TB) prophylaxis. Furthermore, the common comorbidity among people living with HIV (PLWHA) was depression (69.8%), which was most common among female patients since all female patients had developed depression ranging from mild depression to severe depression. The second most common comorbidity associated with PLWHA was TB (30%), which was most common among male patients since all TB/HIV co-infections were male patients. The third common comorbidity was cardiovascular disease (CVD) (21.6%). Moreover, the most clinical manifestation in PLWHA was weight loss (100%), followed by seborrheic dermatitis (40.5%), recurrent upper respiratory tract infections (38.8%) and angular cheilitis (25.9%). The study included 65/116 (56%) males and 51/116 (44%) females. The mean ± SD age of the evaluated patients was 36.55 ± 12.8 years, and the Median [IQR] was 35 [28.3–43]. And the mean ± SD since the diagnosis of HIV infection was 1.28 ± 0.449 years. The majority of patients (53.4%) were in the age range of 26 to 41 years (Table 1).

Table 1 Demographic and clinical background characteristics of PLWHA in Sudan, 2020-2021.
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ANA positivity among study participants

Among the study participants analyzed in this study, ANA positivity was observed in 85 (73.3%) HIV patients. Interestingly, the results showed a slightly higher proportion of ANA positivity among the male group, with 49/65 (75.4%), compared to the female group, with 36/45 (70.6%). However, this difference was statistically insignificant (p = 0.7) (Table 2). Furthermore, the results indicated that the positivity of ANA across age groups was higher among older patients aged 26–41 and ≥ 57 years, both with an estimate of 80%, followed by those aged ≤ 25 years 78.9% and those aged 42–56 years 23.5% 67.7%. However, there were no significant differences between the positivity for ANA and the age group (p = 0.56) (Table 2).

Table 2 Demographic correlates of ANA positivity among PLWHA in Sudan, 2020–2021.
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ANA patterns among study participants

Anti-cellular antibodies-4 (AC-4) was most prevailed ANA pattern 48/85 (56.5%) among HIV patients, followed by AC-20 pattern 21/85 (24.7%), AC-15 (3/85, 3.5%) and AC-21 (2/85, 2.4%). Other patterns included AC-11 (4/85, 4.7%), AC-8 (4/85, 4.7%), AC-25 (2/85, 2.4%), and AC-7 (1/85, 1.2%) (Fig. 1).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Mapping the spectrum of ANA staining patterns among PLWHA in Sudan, 2020–2021. AC: anti-cellular antibodies this nomenclature systems according to the International Consensus on ANA Patterns (ICAP).

Our study showed the frequency of ANA patterns across sex groups, and interestingly, the majority of ANA patterns was detected among male compared to female patients. On the other hand, AC-4 pattern was a commonly identified pattern in male groups (58.3%) compared to female groups (41.7%); in contrast, AC-20 was commonly predominant ANA pattern among female groups (61.9%) compared to male groups (38.1%) (Table 3). Furthermore, our study also showed the frequency of ANA patterns across age groups, which revealed that the highest frequency of ANA patterns was shown at ages 26–41 years, followed by the ages of 42–56 years, aged ≤ 25 years and  ≥ 57 years, and the predominant patterns in all age group were AC-4 and AC-20 (Table 3). No significant differences were found in the prevalence of ANA according to factors such as age and sex. Furthermore, our study observed that about half (45/85, 52.9%) of ANA patterns positive at cutoff titer or low estimated endpoint titer (1:80), and about (40/85, 47.1%) of ANA pattern were showed high dilution estimated endpoint titer of autoantibody at 1:160 and over (Table 3 and Fig. 2). Interestingly, the high dilution endpoint titer was more predominant in the AC-20 pattern flowed by AC-16 and AC-21. Whereas the low-end point titer was predominantly in the AC-4 Pattern (Table 3), and there were significant differences were found in the distribution of different ANA patterns and estimated endpoint titer (p= 0.007). Moreover, the median [IQR] of log10 estimated end pint titer was higher in AC-16 pattern median [IQR] 3 [2.45–3.050], followed by AC-21 median [IQR] 2.65 [2.57–2.72], AC-25 median [IQR] 2.50 [2.50.], AC-20 median [IQR] 2.50 [2.20–2.72] and AC-8 median [IQR] 2.50 [2.20–2.65], while AC-7, AC -11 and AC-4 had lower median [IQR] 1.9 [1.900] respectively (Fig. 3A).

Table 3 Distribution and frequency of ANA staining pattern according to ICAP classification by gender, age and estimated endpoint titer.
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Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Heat-map analysis of estimated endpoint gradients titer across heterogeneous ANA patterns.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Box plots of Log10 ANA estimated endpoint titer among A: heterogeneous ANA patterns, B: Sex, C: age groups of newly diagnosed PLWHA in Sudan.

Interestingly, the higher clinically significant estimated endpoint titer of ANA (1: 160, 1:320, 1:640, and 1:1280) was more prevalent among female patients than male patients (Table 4 and Fig. 3B). Regarding age groups, the predominant clinically significant estimated endpoint titer of ANA was more prevalent in age 26–41 years compared to other age groups (Table 4 and Fig. 3C).

Table 4 Distribution and frequency of Estimated endpoint titer by sex, and age.
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Regarding sex groups, the median [IQR] of log10 estimated endpoint titer was relatively higher in female patients median [IQR] 2.2 [1.9 –2.5] compared to male patients 1.9 [1.9–2.5], but no statistically significant different median between male and female (p = 0.21) (Fig. 3B). while in age groups, no statistically significant difference in median [IQR] of log10 estimated endpoint titer between age groups (p = 0.83), which was approximately the same in all age groups 1.9 [1.9–2.50] (Fig. 3C).

Discussion

The idea of how viral infections can trigger and alter the clinical manifestations of various broad spectrums of AD is well-known and has received considerable attention and interest in the scientific community18. However, it is difficult to definitively prove the link between autoimmune phenomena and viral infections19. Meanwhile, more research is needed to explore the mechanisms by which HIV infection alters the counterbalance of the immune response and causes autoimmunity disorder18,20. Several scenarios of immunological aberrations lead to altered immunological tolerance during HIV infection, including pyroptosis-induced death of CD4 + T cells, autoreactivity of CD8 + T cells, failure of regulatory T cell, polyclonal B cell activation, and molecular mimicry, all contribute to the production of autoantibodies and ultimately the formation of immune complexes21,22. HIV-related systemic and persistent background inflammation may independently lead to poor control of inflammatory activation, contributing to systemic homeostatic disturbances16,23. Today, autoimmunity is generally diagnosed at the same time as primary HIV infection, when CD4 T cells still have an excellent immune response24. On the other hand, autoimmunity may also occur after the initiation of HAART due to immune reconstitution inflammatory syndrome (IRIS)25, especially in the first six months after the initiation of ART or in stage IV of chronic HIV infection26. While reconstitution of the immune system with ART plays an important role in the decrease of mortality in PLWH, uncontrolled inflammation through the development of IRIS may swiftly lead to clinical deterioration and triggering of autoimmunity16. A previous study shows that the prevalence of rheumatic manifestations in HIV-infected patients before the introduction of HAART ranged from 3 to 71% and was associated with late stages of immunosuppression in HIV infection11. The prevalence of rheumatic manifestations associated with HIV infection has declined significantly in the era of HAART27. Still, of great interest and importance, a new group of rheumatic diseases has emerged, covering the spectrum of systemic autoimmune and auto-inflammatory diseases, and a new clinical potential presents a challenge due to the IRIS phenomenon26.

In Sudan, there is limited data on the incidence and prevalence of ANA in the general population, and few Sudanese reports investigate the occurrence of these autoantibodies. One previous case–control study was conducted on 115 SLE patients and 106 matched controls, and the results showed that ANA was positive in 58% of Sudanese patients and 9% of Sudanese controls36. Another study conducted by the same researcher included 93 Sudanese SLE patients to observe the prevalence of antinuclear-associated antibodies among these patients. The study revealed that anti-SSA/Ro60 was the most predominant autoantibody (36.6%), followed by anti-dsDNA (33.3%), SSA/Ro52 (32.2%), SSB/la (16.1%), anti-U1RNP (21.5%), anti-Sm, (12.9%), anti-histone (10.7%), ribosomal P antigens (4.3%), and PCNA (1.1%)37

This study examined the appearance of laboratory evidence of autoantibodies in PLWHA by determining the distribution of antinuclear antibodies (ANA). Amongst the patients analyzed in this study, the proportion of patients with positive ANA was 73.2%, which was higher than reported in Thailand (25%11, France (33%27, and the Amazon region (32%38). Furthermore, a recent study conducted by Hasan et al. reported the detection of ANA in a case of HIV-associated systemic sclerosis (SS)28.

Our findings also revealed that male patients were more likely to have ANA positivity than female patients; this conclusion contradicts prior research in the general population, which has consistently shown a greater ANA positive rate in women. One possible explanation for this discrepancy is that HIV infection has immunomodulatory effects that affect usual gender-based immune responses. HIV is known to produce considerable immunological dysregulation in men, which may result in increased autoantibody production. Furthermore, antiretroviral medication (ART) may alter autoimmune responses differently in males and females, which could explain this surprising finding26,27. This data defies conventional thinking and emphasizes the need for more study into how HIV, sex hormones, and chronic immunological activation interact in generating autoantibody responses, and this may be due to comorbidities, particularly TB since all HIV/TB co-infections were male patients. Furthermore, a Previous study in non-HIV individuals showed that testosterone is frequently considered immunosuppressive, resulting in reduced autoimmune responses in males. However, in HIV patients, prolonged inflammation and persistent immunological activation may reverse testosterone’s suppressive effects, boosting ANA generation. On the other hand, previous studies also suggest that men and women react differently to ART, especially men who exhibit more immunological activation, which may contribute to increased ANA production31,32. Furthermore, we observed a higher ANA positivity in older patients. This finding is consistent with the existing literature reporting a higher prevalence of positivity for ANA in older people26. Therefore, this study needs future studies in older adults to explore the possibility of effect modification of age to induce ANA production32.

On the other hand, the findings presented in this study reveal that the most common ANA pattern observed in PLWHA was nuclear fine speckled (AC-4) (56.5%), which was the most common ANA pattern in male patients aged 26–40 years, followed by the cytoplasmic pattern (AC-20,21 and 16) (30.6%), which is categorized as cytoplasmic fine granules (AC-20) (24.7%%), cytoplasmic fibrillary/ filamentous (AC-16) (3.5%), and cytoplasmic Reticular/AMA (AC-21) (2.4%), which was most common ANA pattern in female patients and aged 26–40 years.

Unprecedentedly, our study reported two younger male patients with anti-mitochondrial antibody type 2 (AC-21) with a high estimated endpoint titer, which might be considered a pivotal biomarker associated with primary biliary cholangitis (PBC). Interestingly, PBC typically affects middle-aged women and has never been reported in previously healthy young children34,35. These uncommon instances should be further researched to understand their possible implications in the pathophysiology of HIV in PLWHA.

Although the frequency of individuals with positive ANA was considerable, the 1:80 (low-endpoint titer) may not firmly imply a link between ANA positivity and autoimmunity. Given the cross-sectional character of this investigation.it is recommended that ANA levels be tracked longitudinally in this patient cohort to provide more evidence. Nevertheless, our study found about 47.1% (40 /85) individuals positive for ANA who were positive with an ANA titer of  ≥ 1:160 (higher estimated endpoint titer), which may indicate strong support for the association of ANA with HIV and autoimmunity, therefore, it is necessary to be interested in following these subjects for any sign of autoimmune disease based on the cutoff titer 1:160 which consider the most common clinical cut off for diagnosis of autoimmunity33,39. Interestingly, the median [IQR] of the estimated endpoint titer of ANA was higher among female patients than male patients, which aligns with previous literature studies26,32. Regarding age groups, the median [IQR] and predominant clinically significant ANA titers were higher in the middle-aged patients (26–41 years) and younger ages ≤ 25 years compared to older patients; understanding sex variations in HIV and age-related ANA positivity will enhance our understanding of the pathophysiologic processes that lead to clinically relevant ANA positivity in middle-aged and younger patients in PLWHA32. Our results do not support the idea that older patients had a higher ANA titer32. Given the potential for confounding, such as chronic systemic inflammation induced by HIV, particularly post-HAART era due to IRIS, our findings highlight the need to understand the role of age factors, especially in the middle-aged and younger patients in PLWHA and the development of IRIS.

According to the International Consensus on ANA Patterns (ICAP) [www.anapatterns.org] in HEp-2 cells, ANA with nuclear fine might be linked to systemic autoimmune rheumatic diseases (SARD), in particular systemic lupus erythematosus (SLE), Sjögren syndrome (SjS), systemic sclerosis (SSc) and overlap syndromes (OSs)17, while nucleolar pattern associated SSc and systemic sclerosis -polymyositis overlapping syndrome, while smooth nuclear envelope might be associated with autoimmune liver diseases, Anti-Phospholipid Syndrome (APS)29, while cytoplasmic fine granules and cytoplasmic fibrillar /cytoskeletal pattern typically associated with Myositis, in contrast, cytoplasmic filamentous linear pattern commonly found in type 1 autoimmune hepatitis and cytoplasmic reticular /AMA typically associated with PBC, also reported in a tiny fraction of patients with autoimmune chronic active hepatitis, PBC-SSc and PBC-SjS overlap syndrome (Table 5)30.

Table 5 Classification of determined ANA pattern in the study according to International Consensus on ANA Patterns nomenclature and antigen association.
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Conclusion

Our study demonstrated that the proportion of ANA-positive individuals in the HIV-infected group was higher than previously reported. A significant ANA prevalence persisted during the HAART era, indicating a strong immunological response which might be associated with immune reconstitution inflammatory syndrome (IRIS). Our findings also revealed several noteworthy observations:

Sex-based differences: Our study showed that male patients had a higher percentage of ANA positive. This unexpected finding shows that HIV-related changes in immune modulation merit further investigation.

Age-related differences: Median ANA titers were greater in younger (≤ 25 years) and middle-aged (26–41 years) patients than in older people. These data indicate that HIV infection and immunological reconstitution may interact with age-related immune dynamics, impacting autoantibody production.

Clinical implications: By identifying distinct ANA patterns and their correlation with titers, our study adds to the growing body of evidence that ANA profiles in PLWHA may reflect underlying chronic inflammation, immune dysregulation, or even immune reconstitution inflammatory syndrome (IRIS), particularly in the context of antiretroviral therapy. Therefore, our findings should serve as a framework for physicians to interpret and apply what comes out of ANA testing in HIV-infected patients undergoing HAART prescription drugs. Furthermore, we strongly support routine follow-up of HIV patients who test positive for ANA to monitor the possible development of autoimmune disorders, particularly in those with higher estimated endpoint titer. Finally, our study is a preliminary investigation, therefore, larger longitudinal studies including pre and post treatment data are required to validate and expand on our findings.

To summarize, whereas ANA testing is nonspecific for classified autoimmune disorders, our work provides novel, population-specific data that fills a significant gap in the regional literature. The observed differences in ANA patterns, titers, gender, and age relationships may provide important insights into HIV-associated autoimmunity and inform future clinical evaluations.

Limitations of the study

Finally, the relatively small sample size of HIV patients limited the statistical analysis. The sample size is small due to difficulties in obtaining patients with HIV in my country due to the small number of HIV centres. Furthermore, we consider the lack of pre-HAART ANA data as a limitation. However, in our setting, including untreated HIV-positive people was not practical for a variety of reasons. To begin, the majority of HIV patients in Sudan are diagnosed at a late stage of infection due to poor healthcare resources and infrastructure, making early identification difficult. Second, societal variables, such as the stigma associated with HIV, frequently cause individuals to postpone testing and, once diagnosed, begin HAART quickly. These issues indicate the need for more comparative studies in future research to determine the prevalence of ANA both pre-HAART and post-HAART and their clinical significant. Furthermore, we acknowledge the lack of extractable nuclear antigen (ENA) profiling as a limitation. Unfortunately, ENA testing was not feasible due to resource limitations.