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Abstract INTRODUCTION:
Syphilis, a sexually transmitted infection caused by bacteria Treponema Pallidum, has resurged as a major public health concern in U.S. There is an alarming increase in the prevalence of syphilis in HIV-infected men who have sex with men1. In our previous study2, majority of syphilis positive patients with high rapid plasma reagin (RPR) titers are male co-infected with HIV. Co-infected individuals may experience more aggressive, atypical secondary syphilis, and fast progression to neurosyphilis. Syphilis and HIV co-infection involves complex host-pathogen interaction that may include profound alteration in host metabolic state. Metabolomic study provides system-level functional read-out of biological processes by capturing downstream biochemical changes reflecting immune activation, tissue injury, microbial nutrient utilization, and treatment effect, thereby enabling comprehensive characterization of metabolic perturbation associated with the infection and systemic inflammation. However, there are very limited metabolomic studies on syphilis infection, and to the best of our knowledge, no metabolic study on syphilis and HIV co-infection. Existing studies suffer from small sample sizes and lack of patient clinical information3.
OBJECTIVES:
Our central hypothesis is that syphilis and HIV coinfection causes system-level perturbation, including metabolic signatures and pathways, compared to healthy controls. Therefore, we aim to compare the metabolites of syphilis+/HIV+ patients to the syphilis-/HIV- controls, and perform differential metabolites pathway enrichment analysis. In addition, we will compare syphilis+/HIV+ cohort with the syphilis-/HIV+ cohort to assess the metabolic alterations specifically associated with syphilis after controlling for the background effects of HIV. Third, we will evaluate the metabolic impact of HIV infection alone by comparing the syphilis-/HIV+ cohort with the syphilis-/HIV- controls.
METHODS:
Remnant serum samples were collected from 424 male patients who were ordered routine syphilis serologic testing. After excluding patients who had end-stage renal failure, liver failure, sepsis, and other critical diseases, 365 samples were included in data analysis, divided into 3 cohorts: 1) syphilis+/HIV+ (n=92), syphilis-/HIV+ (n=109) and syphilis-/HIV- (n=164). Samples were analyzed using a targeted metabolomics workflow on Agilent 1290 Infinity II HPLC coupled with 6546 quadrupole time-of-flight high-resolution mass spectrometry. InfinityLab Poroshell 120 HILIC-Z column (2.1 × 150 mm, 2.7 µm) was used, with mobile phase A of water with 20 mM ammonium acetate (pH 9.3) and mobile phase B of 100% acetonitrile. Total run time was 23 min for each sample. NIST standard reference material 1950 was used as the system validity testing, and precision of metabolite signal levels in the pooled quality-control was closely monitored. Raw data acquired from MassHunter Profinder software was pre-processed by normalization (total ion chromatogram count and protein concentration), log2 transformation, outlier cleanup, and missing value imputation. Combat batch correction was performed to reduce batch-to-batch variation. For data analysis, on one hand, univariate analysis was performed using the Mann-Whitney U test with a fold change criterion. On the other hand, LASSO logistic regression model was employed to select discriminatory metabolites that help separate the two classes. In addition, differential metabolite pathway enrichment analysis was carried out to identify perturbed pathways in syphilis/HIV co-infection as well as syphilis infection alone.
RESULTS:
A review of patient demographic and clinical characteristics demonstrated no significant difference in race, BMI, percentages of cancer, heart disease, renal failure and hyperlipidemia between the three cohorts. Additionally, there was no significant difference in age, percentage of diabetes between the syphilis/HIV co-infected group and controls. Comparison of the syphilis+/HIV+ and syphilis-/HIV- groups revealed 27 unique metabolites (FDR adjusted p<0.05 and fold change >1.25 or <0.8) by both positive and negative modes. Top-ranked differential metabolites were mainly involved in perturbed amino acid metabolism and increase host stress-response/steroid metabolism. Cysteine and methionine metabolism pathway and valine, leucine, and isoleucine biosynthesis and degradation pathways were the top-ranked pathways associated with co-infection. In addition, LASSO regression model selected 89 metabolites that contributed to the discrimination of the two groups, and the model was able to separate the syphilis/HIV co-infection from healthy controls with an area under the ROC curve with cross validation mean and standard deviation of 0.87 +/- 0.05. Comparison of syphilis+/HIV+ group and syphilis-/HIV+ group revealed 25 significantly differential metabolites, indicating these metabolites are associated with syphilis after controlling for the background effects of HIV infection. Cysteine and methionine metabolism pathway was again the top-ranked pathway associated with syphilis infection. Finally, the comparison of syphilis-/HIV+ group and syphilis-/HIV- controls revealed only 7 significantly differential metabolites, however, no significant pathways were identified, partly because most HIV positive cases in our dataset were well-controlled HIV rather than advanced uncontrolled HIV.
CONCLUSIONS:
Syphilis/HIV co-infection is associated with substantial metabolic perturbation compared to the negative control group. Co-infection is associated with multiple biologically interesting, high-coverage pathways, particularly involving amino acid metabolism and stress-responses/steroid metabolism. Cysteine and methionine metabolism is the top-ranked pathway associated with syphilis infection after controlling for the background effect of HIV infection.
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= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
