Development and validation of IVDMIA as a novel biomarker of early sepsis using metabolomics approach

Abstract

Background: Sepsis is a syndrome that is influenced by pathogens and host factors and is characterized by an aberrant or dysregulated host response and organ dysfunction. Sepsis is the primary cause of death from infection, especially if not recognized and treated promptly. Its recognition mandates urgent attention. However the underlying mechanisms of sepsis are not completely understood. Therefore, a multimarker strategy may be helpful for improving the understanding of the complex pathogenesis of sepsis and its evolution, and especially for facilitating early risk stratification and implementing personalized therapies. The use of emerging metabolomics tools is particularly promising for the diagnosis of complex and heterogeneous conditions such as sepsis and septic shock. Methods: Several different biomarkers are known to aid the diagnosis of sepsis and are used as severity or outcome indexes for patients with sepsis. For the application of metabolomics to sepsis research, we recruited individuals and categorized them in three groups – normal healthy group, systemic inflammatory response syndrome (SIRS) group, and sepsis group. We assayed the serum levels of amino acids and the respective metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and analyzed the result to screen promising biomarkers of sepsis. Lastly, an In Vitro Diagnostic Multivariate Index Assay (IVDMIA) was developed by formulating the indexes using the amino acids selected previously. Results: Principle component analysis (PCA) was performed using the results of metabolomics research. Based on the score plot and loading plot, we confirmed that the three aforementioned groups were distinguishable and formed clear clusters. We could also infer that amino acid derangements exist in patients with sepsis, and that these metabolomic derangements could be employed as biomarkers using LC-MS. Based on the PCA results, we selected the candidate amino acids that could be used in IVDMIA. We selected four index amino acids (kynurenine (KYN), tryptophan (TRP), phenylalanine (PHE), and arginine (ARG)) and one ratio (KT ratio). We developed various formulas using the five variables mentioned. After assessing the sensitivity, specificity, accuracy, and area under the ROC curve (AUC) from each formula, the formula with the highest performance was selected for developing the IVDMIA for sepsis. The generated IVDMIA was subjected to validation processes, which included performance comparison with preexisting sepsis markers such as White Blood Cell (WBC), C-Reactive Protein (CRP), and procalcitonin (PCT). The selected IVDMIA formula is as follows: ARG  (-0.0513) + PHE  0.0642 + KT ratio  27.6591 - 5.4765. We observed that the developed IVDMIA had similar or better potential than procalcitonin as a sepsis marker. Conclusions: This study demonstrated that the amino acid composition of the body in patients with sepsis differs significantly from those in normal individuals or in patients with SIRS. We developed an IVDMIA specific for sepsis and validated it. The IVDMIA developed in this study can be used to diagnose sepsis in patients. Small-scale or point-of-care testing MALDI-TOF can be used instead of LC-MS/MS for amino acid analysis, which improves the applicability of the test. It is expected to be ready for clinical practice in the near future. In addition, the variations in the concentrations of amino acids we assessed improved our understanding of metabolic alterations in sepsis. 배경: 패혈증은 미생물에 감염되어 전신에 걸친 염증 반응이 나타나는 상태이며, 이러한 상태가 지속되거나 악화하여 다발성 장기 부전을 거쳐 사망에 이를 수 있는 주요한 질환이다. 그러나 패혈증의 병태생리학이나 대사 작용에 대해서는 알려진 바가 많지 않다. 패혈증은 다양하고 복잡한 특징을 지니기 때문에 각 경우에 맞는 적절한 진단과 치료가 필요하지만, 맞춤 의학이 발전하는 최근에도 이를 패혈증에 적용하기는 쉽지 않은 것이 현실이다. 따라서 여러 가지의 바이오마커를 병합하여 이용하는 방안인 체외진단다지표분석 (In Vitro Diagnostic Multivariate Index Assay, IVDMIA)이 제기되었고, 본 연구에서는 Metabolomics 기법을 비용한 패혈증 환자에서의 새로운 조기 바이오마커의 개발 및 검증이 시행되었다. 방법: 연구에 동의한 건강검진 대상자, 전신염증반응증후군 환자, 패혈증 환자의 세 그룹 참여자들로부터 검사 후 잔여 혈청을 획득하였다. 이 검체들을 대상으로 액체 크로마토그래피 질량 분광법을 이용해 Metabolomics를 시행하였고, 그 결과를 주성분 분석 통계법으로 분석하였다. 분석 결과를 통해 패혈증에서의 체외진단다지표분석에 이용할 패혈증 조기 마커들을 선정하였고, 이들을 적용해 패혈증 지표를 생성하였다. 결과: 세 그룹에 대해 시행한 Metabolomics결과 분석을 거쳐 kynurenine, tryptophan, KT ratio, phenylalanine, 그리고 arginine 등 5개의 아미노산 및 그 계산치를 패혈증 바이오마커로 선정하였고, 선정된 바이오마커들을 이용해 패혈증에서의 체외진단 다지표분석을 거쳐 임상에서 사용이 용이한 패혈증 지표-IVDMIA-를 만들어내었다. 생성된 패혈증 지표는 실제 환자 그룹에 적용되어 진단, 중증도, 예후 평가 및 예측 등에서 그 임상적 성능이 검증되었다. 결론: 본 연구는 Metabolomics 기법을 통해 패혈증 환자에서 아미노산 구성을 포함한 신진대사가 유의하게 교란됨을 입증하였으며, 혈청의 아미노산 분석을 통해 패혈증에 유의한 아미노산들을 발굴하였다. 이를 기반으로 패혈증 지표가 생성되었고 그 성능이 검증되어 임상에서 유용하게 쓰일 것으로 기대된다.