Background: The irregular use of antiretroviral therapy (ART) and late diagnosis still account for a large part of HIV-associated mortality in people living with HIV (PLHIV). Herein, we describe HIV-associated morbidity among hospitalised HIV/AIDS patients with advanced immunosuppression and assess the comorbidities, laboratory parameters, and immunological markers associated with mortality. Methods: The cross-sectional study was conducted at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) in Manaus, Brazil. In all, 83 participants aged between 12 and 70 years were enrolled by convenience within 72 h of their hospitalisation. Clinical and laboratory data were obtained from electronic medical records. We prospectively measured the cytokines Th1/Th2/Th17 and inflammatory cytokines IL-8, IL-1β, and IL-12 using cytometric bead array, and the soluble CD14 using in-house enzyme-linked immunosorbent assay. Results: The HIV/AIDS inpatients presented a scenario of respiratory syndromes as the most prevalent comorbidity. Almost all patients had CD4 T counts below 350 cells/mL and the mortality rate was 20.5%. Pulmonary tuberculosis, neurotoxoplasmosis and oropharyngeal–esophageal candidiasis were the most prevalent opportunistic infections. TB and weight loss were more prevalent in HIV/AIDS inpatients who died. The Mann Whitney analysis showed that those who died had higher platelet distribution width (PDW) on admission, which is suggestive for platelet activation. The Poisson multivariate analysis showed the prevalence of TB, digestive syndrome and increases in IL-8 and lactate dehydrogenase (LDH) associated to death. Conclusions: The advanced immunosuppression characterized by the opportunistic infections presented in these HIV/AIDS inpatients was the major factor of mortality. The role of platelet activation in worse outcomes of hospitalisation and the IL-8 associated with the context of advanced immunosuppression may be promising markers in the prediction of mortality in HIV/AIDS patients.
Keywords: (MeSH):HIV; Acquired immunodeficiency syndrome; AIDS-related opportunistic infections; Biomarkers; Morbidity; Mortality
Brazil has been successfully combating the HIV/AIDS epidemic by providing antiretroviral therapy (ART) in a unified, universal and free public health system that is widely available to the population [[
Since the beginning of the epidemic in 1982, more than 300,000 deaths caused by HIV/AIDS have been reported in Brazil. After the adoption of ART in 1996, there was a significant reversal in mortality. The deaths related to the HIV/AIDS epidemic have shown a downward trend in Brazil as a whole, however this trend is still subtle in the northern and northeastern regions [[
Located in northern Brazil, the State of Amazonas has peculiar characteristics, such as geographic isolation and, in the interior of the state, a lack of access to full health services. The capital, Manaus, is the largest city in the state and its health units are decentralized, located in the neighborhoods and semi-urban areas. These health units are essential for promoting the early diagnosis and the introduction of ART, and contribute to the decrease in morbidity and mortality caused by opportunistic diseases [[
Immune activation in HIV infection is a strong predictor of disease progression and is a result of dysregulated innate and adaptive immune responses to HIV with active participation of co-infections and microbial products [[
This cross-sectional study assessed the prevalence of comorbidities, laboratory parameters, and the immunological markers associated with the death in HIV/AIDS patients of either sex, who were admitted to the FMT-HVD between 2017 and 2018. Eighty-three participants aged between 12 and 70 were enrolled in this study by convenience within 72 h of admission. After signature of the informed consent form, the patient's blood was collected and the serum cytokines, chemokines, and soluble CD14 were measured.
During the first contact with the patients, socio-demographic data, such as name, age, gender, and use of ART, were collected. The following data were collected through the patient's electronic medical records:
- Clinic data: general health status, comorbidities, co-infections, treatment, clinical manifestations (weight loss, diarrhea, vomiting) and death.
- Laboratory data: blood count, the IgG serology test (cytomegalovirus—CMV, toxoplasmosis, Epstein Barr—EBV, herpes virus, hepatitis B virus—HBV, hepatitis C virus—HCV), immunological markers (viral load and T cell count CD4+ T and CD8+ T) and biochemical markers (bilirubin, creatinine, lactic dehydrogenase, GT range, albumin, alkaline phosphatase, GPT and OGT).
Information regarding coinfections and comorbidities were obtained from the electronic medical database at the FMT-HVD, and the outcomes of interest were survival (hospital discharge) and death. Both were verified via either a death certificate or discharge authorization registered on the electronic medical record. Comorbidities or disorders were defined as signs and/or symptoms of respiratory, neurological, cardiovascular, and digestive origin, of infectious and non-infectious cause, with or without chronicity.
Respiratory disorders encompass a variety of pathogenic conditions that affect the respiratory tract, and include infectious and non-infectious signs and symptoms such as dyspnea (shortness of breath or difficulty in breathing), abnormal lung auscultation, long-term and/or productive cough, and pleural effusion. Neurological disorders include those of infectious and non-infectious etiology, signs and symptoms such as alteration in consciousness, sensory loss and/or movement disorders (poor coordination, tremors, asthenia), paralysis, and seizure. The cardiovascular disorders included hypertension, heart failure, peripheral edema, and anasarca. Gastrointestinal disorders include those of an infectious and non-infectious cause, signs and symptoms such as include odynophagia, dysphagia, esophagitis, gastritis, vomiting, and diarrhea.
Other comorbidities, such as chronic lymphocytic leukemia, Hodgkin's disease, aplastic anemia, and neurological disorders, such as multiple sclerosis and myasthenia gravis, were monitored.
On the same day as the patient's enrollment, after the interview and signing of the consent form, 5 mL of blood was collected by vacuum venipuncture. The samples were collected in dry tubes and centrifuged at 3500 rpm for 5 min at 25 °C to obtain the serum and aliquoted (1 mL) for analysis of the inflammatory markers. The whole blood that was collected in an anticoagulant tube was homogenized, aliquoted (1 mL), and then stored for future analysis. Samples were stored at − 80 °C until use.
The measurement of serum cytokines was performed using the flow cytometry technique CBA (cytometric bead array) with the human cocktail Th1/Th2/Th17 cytokine for IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL17 and inflammatory cytokines IL-8, IL-1β, and IL-12 (Biosciences, USA). Serum concentrations of soluble CD14 (sCD14) were determined by in-house enzyme-linked immunosorbent assay (ELISA). Initially, the plate was sensitized with the primary antibody (anti-human CD14 Antibody; Cat. Nº. MAB3833; Lot. AWI091610A; R&D Systems) diluted in carbonate–bicarbonate buffer (0.05 M; pH 9.6). After sensitization, blocking was performed with PBS/BSA blocking buffer (PBS pH 7.4; 3% bovine albumin). Subsequently, the sera from patients and controls were diluted into PBS/BSA dilution buffer (PBS pH 7.4; 0.5% bovine albumin) and incubated for 1 h at 37 °C. Three concentrations of recombinant human CD14 protein (Recombinant Human CD14; Cat. No. 383-CD; Lot. BCS1716091; R&D Systems) were added to each plate well in order to determine a standard-curve. After four lavage steps in PBS washing buffer (PBS pH 7.4; 0.05% Tween 20), the 1:1000 dilution secondary antibody (anti-human CD14 Biotinylated Antibody; Cat Nº BAF383; Lot. BAR0714031; R&D Systems) was added. After the lavage steps, 1:1000 dilution, 0.5 mg, peroxidase-labeled streptavidin (KPL) was added for 1 h at 37 °C. The reaction was developed with developing buffer (citrate-phosphate buffer + TMB + H
Data were tabulated in an Excel database created by the researchers and analyzed using the GraphPad Prism program, version 7. For data analysis, patients were defined as Death (those who died during hospitalisation) or Discharge (those who were discharged from hospital). Descriptive analysis was performed with mean and standard deviation (SD) for numerical variables when distribution was normal, or median and interquartile range for non-normal distributions. In the univariate analysis, Fisher's exact test was used to determine relative risk among categorical variables. Laboratory parameters, soluble CD14, cytokine and chemokines concentrations, and days of hospitalisation were compared between the Death and Discharge groups using the Mann Whitney test. The Poisson regression was performed to assess factors associated with death in HIV/AIDS inpatients among the most prevalent comorbidities, laboratory parameters and immunological markers. For this model, we used data from univariate analyses where the selected variables were those presenting p < 0.2. The statistical significance was defined as p < 0.05.
Overall, respiratory disorders were the most prevalent (62.1%), followed by neurological (37.9%), gastrointestinal (21.0%) and cardiovascular (5.2%) disorders. Among the respiratory disorders of infectious cause, TB was the most prevalent (56.30%), followed by pneumocystosis (2.4%). The most prevalent signs and symptoms were abnormal lung auscultation (22.40%), followed by respiratory failure (5.30%), and severe cough (5.30%). Regarding neurological disorders, the most prevalent coinfections were neurotoxoplasmosis (44.4%), neurocriptococcosis (8.3%), and neurotuberculosis (2.8%). The most prevalent signs and symptoms were confusion, paralysis, and/or poor coordination (30.6%), tremors and asthenia (11.1%), and seizures (5.6%). The most prevalent gastrointestinal disorders of infectious cause were oral and esophageal candidiasis (55%). The most prevalent signs and symptoms were vomiting (39.7%), diarrhea (33.7%), and odynophagia (5.0%). A total of 25% of patients reported abdominal pain.
In all, 17 hospitalised patients died and 66 were discharged. The duration of hospitalisation did not differ between the two groups (p = 0.30), the median and 25% and 75% interquartiles of hospitalised days of those who died were 19 (14.5–26.5), while for those who were discharged the median was 25 (11.0–38.0). All the described disorders were assessed to identify factors associated with the mortality of the patients (Table 1). Tuberculosis was more prevalent among patients who died (p = 0.001). The IgG serological tests used for previous infections did not differ between HIV/AIDS patients who died or were discharged from hospital. Weight loss was associated with death (p = 0.013). The univariate analyses of HIV-RNA > 1000 copies and CD4 T < 350 cells/mL showed no association with death. The CD4 T counts were greatly reduced in both groups, the CD8 T cells presented normal values in the group of patients who died, while the Discharge group tended towards a slight increase, though this was not statistically significant. The ratio of CD4:CD8 cells below 0.20 due the reduction of CD4-T evidenced the advanced immune impairment of the patients (Table 2).
Table 1 Assessment of comorbidities in the outcomes of HIV–AIDS inpatients
Clinical data Death Discharge N = 17 N = 66 Age (mean, std.) 37.6 (9.9) 36.2 (9.4) 0.590 Gender (male) 11 (64.7) 54 (81.8) 0.184 ART 12 (70.6) 55 (83.3) 0.300 CD4 T < 350 cells/mL 14 (82.4) 58 (87.9) 0.688 HIV-RNA > 1000 copies 14 (82.4) 47 (71.2) 0.539 Weight loss 14 (82.4) 30 (45.5) 0.007** Very good condition 7 (41.2) 35 (53.0) 0.425 Lucid and space-oriented 8 (47.1) 40 (60.6) 0.410 Anemia 15 (88.2) 43 (65.2) 0.079 Respiratory disorder 14 (82.4) 42 (63.6) 0.244 Neurological disorder 8 (47.1) 29 (43.9) 0.999 Cardiovascular disorder 2 (11.8) 2 (3.0) 0.184 Gastrointestinal disorder 8 (47.1) 16 (24.2) 0.077 Active coinfections Tuberculosis 12 (70.6) 25 (37.9) 0.026* Parasites in stools 3 (17.6) 13 (19.7) 0.999 Toxoplasmosis IgM 1 (5.9) 7 (10.6) 0.999 Cytomegalovirus IgM 1 (5.9) 2 (3.0) 0.502 Epstein Barr virus IgM 17 (100) 66 (100) 0.999 Herpes virus IgM 0 (0) 3 (4.5) 0.999 Previous infections HCV IgG 1 (5.9) 2 (3.0) 0.502 Cytomegalovirus IgG 13 (76.5) 53 (80.3) 0.741 Toxoplasmosis IgG 13 (76.5) 52 (78.8) 0.999 Epstein Barr virus IgG 12 (70.6) 49 (74.2) 0.764 Herpes virus IgG 12 (70.6) 44 (66.7) 0.999
*, **: p-value; statistically significant difference
Table 2 Univariate analyses with laboratory parameters in the outcomes in HIV/AIDS patients
Laboratory tests Death Discharge N = 17 N = 66 Hematological data (NVA) HIV-RNA copies/mLb 126,578 (3247–273,012) 25,258.5 (391.5–145,976.8) 0.590 Lymphocytes (percent)a (25.0–40.0%) 20.5 (10.3) 27.4 (13.5) 0.055 CD4-T cells/µLb (600 to 1500/mm3) 44 (7–232) 79 (30–198.2) 0.140 CD8-T cells/µLb (200 to 800/mm3) 402 (198–776) 544.5 (359.2–1143.8) 0.312 CD4/CD8 ratiob (1.0 and 4.0) 0.1 (0–0.4) 0.1 (0.1–0.3) 0.123 Hemoglobin (13.0–18.0 g/dL) 9.3 (2.7) 10.8 (2.4) 0.165 Leukocytesb (4500–11,000/mL) 4740 (3510–8600) 4215 (3275–5,757.5) 0.527 Neutrophilsb (1800–7700/mL) 2660 (1679–3234) 3118 (2109–4907.5) 0.113 Monocytesb (80–1100/mL) 249 (161–379) 329 (187.2–488.2) 0.370 Eosinophilsb (40–550/mL) 2 (2–4) 4 (2.6–7) 0.030* Plateletsa (150,000–400,000 × 103/mL) 299,352.9 (207,630.4) 273,947.0 (132,454.6) 0.536 MPVb (8.8 fL to 12.5 fL) 8.2 (7.8–8.8) 8.1 (7.4–8.7) 0.446 PDWb (9.3 fL to 16.0 fL) 15.8 (13–18.7) 13.5 (11.2–16.5) 0.037* Biochemistry parameters (NVA) Bilirubinb (< 1.0 mg/dL) 0.4 (0.2–0.9) 0.4 (0.3–0.7) 0.999 Creatinineb (0.6–1.35 mg/dL) 1 (0.7–1.3) 0.8 (0.6–0.9) 0.069 LDHb (120 and 246 U/uL) 435 (332–534) 367.5 (300.2–474.2) 0.159 Gamma-glutamyltransferaseb,c 88 (42–203) 103.5 (57.8–290) 0.876 Albuminb (3.5–5.0 g/dL) 3.4 (2.8–4) 3.8 (3.3–4.5) 0.129 Alkaline phosphataseb (65.0–330.0 U/uL) 282 (200–459) 281 (201–386) 0.835 Aspartate aminotransferaseb (2–38.0 U/uL) 44 (26–82) 34 (25–58) 0.280 Alanine aminotransferaseb (2–44.0 U/uL) 31 (22–41) 43 (31–77) 0.038*
NVA Normal values in adults, PDW Platelet distribution width, MPV Mean platelet volume, LDH lactate dehydrogenase
The low hemoglobin level was present in both groups; however, it was more prevalent in those who died. The total leukocytes, neutrophils, and monocytes did not differ between groups and remained in normal ranges in most patients. The percentage of lymphocytes in those who died showed a tendency to be lower. The eosinophil counts were reduced in both groups, though they were even lower (p = 0.03) in those who died. The platelet distribution width (PDW) was higher among those who died.
Overall, the patients preserved their liver function, with no significant alterations in the hepatic markers. The alkaline phosphatase levels and the transaminase levels were slightly elevated in both groups. The renal function was able to maintain the creatinine levels in normal ranges (Table 2).
The soluble CD14, the chemokines and Th1/Th2/Th17 cytokine levels at the time of hospitalisation revealed no difference between the two groups, while IL-8 tended to be associated with death (p = 0.06) (Fig. 1). It is worth mentioning the stratification of immunological markers in descending order of plasma levels.
Graph: Fig. 1 Serological markers as predictors of mortality. The soluble CD14, the chemokine IL-8 and the cytokines Th1/Th2/Th17 were compared among the patients who died and those who survived. A sCD14; B IL-8; C IL-6; D IFNγ; E IL-12; F IL-2; G IL-4; H IL-17A; I IL-10; J TNFα and L IL-1β. Concentrations were compared between Death and Survival groups using the Mann Whitney test
When isolated, the prevalence of TB showed a relative risk (RR) of 4.62, though, when associated with digestive syndrome, it presented RR = 3.07. In relation to laboratory parameters and immunological markers, the risk of death increases along with the increment in IL-8 and LDH levels, e.g., IL-8 (RR = 1.0001) and LDH (RR = 1.0007). The mean IL-8 in univariate analysis showed no differences between groups; however, most patients who died had higher levels of IL-8 than those who were discharged (Fig. 1B). The RR of the lymphocyte percentage was 0.9503, indicating that the risk of death was higher as the percentage decreased (Table 3).
Table 3 Relative risk of comorbidities, laboratory parameters and immunological markers in the mortality of HIV/AIDS patients
Characteristic Death RR 95% CI p-value Yes No N = 17 N = 66 Digestive syndromea 8/17 (47%) 16/66 (24%) 3.05 (1.55–6.06) 0.001 Tuberculosisa 12/17 (71%) 25/66 (38%) 4.62 (1.87–11.36) 0.001 IL.8 pg/mLb 90.4 (20–189.0) 26.1 (14.0–90.1) 1.01 (1.00–1.01) 0.007 Lymphocytesb in percentage 20.5 (10.3) 27.4 (13.5) 0.95 (0.91–0.98) 0.003 LDH U/uL** (120 and 246 U/uL) 435 (332–534) 367.5 (300.2–474.2) 1.01 (1.00–1.01) 0.001
Relative risk; CI: confidence interval; and the p-value was calculated using the Poisson regression; LDH: lactate dehydrogenase
After the advent of ART, HIV infection has changed from an illness that causes rapid deterioration to a complex chronic disease. Brazil has responded to the HIV pandemic in innovative ways, and was the world's first developing country to offer ART free of charge to all PLHIV and the third developing country to provide ART regardless of CD4+ T cell counts [[
This cross-sectional study has found that the HIV/AIDS epidemiological scenario has not changed in the few last years; respiratory disorders are still the most prevalent complication, while TB, neurotoxoplasmosis, and oropharyngeal–esophageal candidiasis are the most prevalent opportunistic infections. During the last decade, TB has been highly prevalent among HIV/AIDS patients who died [[
Another challenge is the patients' low adherence to ART. Despite the free access to the antiretroviral drugs, due to several reasons including social factors, side effects or skepticism, since many have experienced previous treatment failure, some patients abandon ART [[
As almost all HIV-infected patients from both groups showed the CD4 counts lower than 200 cells/uL. However, the risk of death associated with decreased CD4 was especially a result of TB prevalence associated with oropharyngeal–esophageal candidiasis. The univariate analysis showed higher PDW at the time of admission of the patients who died. PDW is a platelet activation marker, since, under activation, platelets increase in number and size of pseudopodia, affecting their distribution width [[
HIV/AIDS is characterized by a state of chronic immune activation and inflammation. Elevated levels of IL8 have been reported in plasma, serum and cerebrospinal fluid in HIV/AIDS patients with CD4+ T cell counts < 350 cells/mm
IL-8 is a prominent neutrophil chemoattractant produced by macrophages and plays a dual role in the pathogenesis of TB and HIV-1 coinfection. On the one hand, IL-8 performs a key role in killing M. tuberculosis by phagocytosis in in vitro studies [[
The sample size was one limitation of this study, and restricted the detection of other associations. Several factors contributed to the limitation of the sample, one of which is that the study was carried out in a medium-sized hospital. In addition to the structural limitations, some patients were hospitalised for up to 2 months, and others were re-hospitalised. Nevertheless, our limited sample size (by convenience) is still representative of the HIV/AIDS population in the region. Before ART, Kaposi's sarcoma and pneumocystosis were the most prevalent opportunistic diseases; after ART, TB, bacterial pneumonia and histoplasmosis became the most frequent causes of death. This study sampling was able to represent the same epidemiological scenario regarding HIV/AIDS patients that has been prevalent in northern Brazil in recent years.
Another limitation was related to the study design, which did not include patients' follow up, only one single blood sample collection. The information regarding the deaths of patients was obtained solely via electronic medical records.
This cross-sectional study found that the HIV/AIDS epidemiological scenario remains unchanged in northern Brazil, e.g., greater risk of death among the TB-HIV coinfected patients and among those with advanced immunosuppression, characterized by the presence of oropharyngeal–esophageal candidiasis. The univariate analysis showed higher PDW on hospital admission, indicating platelet activation, though further studies are needed to assess their relationship with the severity of the pulmonary infection in HIV/AIDS patients. Nonetheless, the use of IL-8 as a serological predictor of death in the HIV/AIDS patients is promising.
We gratefully acknowledge the partnership with the INProg-HIV group.
WMG, GM, DSS, DFS, PPO, FRP, GFM, BJB, VLOS, and RLG were responsible for the data collection from medical records. WMG, YOC, and AGC performed immunoassays. TVRA, AGC, and AASB performed the statistical analyses. MCS, MVGL, TVRAA, MFS, LCLF and PAN participated in study design. TVRA, MFS, LCLF and PAN wrote the first draft of the manuscript. CHMF, TVRA, and PAN elaborated the final version of manuscript. All authors read and approved the final manuscript.
TVRA and BJB received a post-doctoral fellowship from "Programa Nacional de Cooperação Acadêmica na Amazônia" of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (PROCAD-AM 88887.321243/2019-00). WMG and DFS each received a master's grant 119 from Programa Pos-Graduação em Immunologia Basica e Aplicada" at the Universidade Federal 120 do Amazonas. MVGL and PAN are CNPq fellows, levels I and II, respectively. This work was funded by a grant from Fundação Oswaldo Cruz—Programa INOVA_Geração do Conhecimento number VPPCB-007-FIO-18, call 28/2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
All protocols and consent forms were approved by the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado Ethics Review Board (CAAE: 57330116.6.0000.0005). All consent forms were signed by the research participants and filed for future verification.
Not applicable.
The authors declare that they have no competing interests.
• AIDS
- Acquired immunodeficiency syndrome
• ART
- Antiretroviral therapy
• CBA
- Cytometric bead array
• CI
- Confidence interval
• CMV
- Cytomegalovirus
• EBV
- Epstein Barr virus
• FMT-HVD
- Fundação de Medicina Tropical Dr. Heitor Vieira Dourado
• HBV
- Hepatitis B virus
• HCV
- Hepatitis C virus
• HIV
- Human immunodeficiency virus
• IFN
- Interferon
• IL
- Interleukin
• MPV
- Mean platelet volume
• NVA
- Normal values in adults
• PDW
- Platelet distribution width
• RR
- Relative risk
• TB
- Tuberculosis
• TNF
- Tumor necrosis factor
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By Wellington Mota Gama; Carlos Henrique Michiles Frank; Taynná Vernalha Rocha Almeida; Daniel Silva dos Santos; Yury Oliveira Chaves; Danielle Furtado da Silva; Patrícia Puccinelli Orlandi; Flávio Ribeiro Pereira; Gleicienne Feliz Magalhães; Bárbara Jóse Baptista; Viviane Lago de Oliveira Silva; Antônio Alcirley da Silva Balieiro; Monique Freire Santana; Roberta Lins Gonçalves; Allyson Guimarães da Costa; Marcelo Cordeiro dos Santos; Luís Carlos de Lima Ferreira; Marcus Vinicius Guimaraes Lacerda and Paulo Afonso Nogueira
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