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    The Role of Leptospiremia and Specific Immune Response in Severe Leptospirosis - 41598 - 2021 - Article - 94073

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    www.nature,com/scientificreports scientific reports OPEN (@ Oreck en The role of leptospiremia and specific immune response in severe leptospirosis Umaporn Limothai!?", Nuttha Lumlertgul!2*2, Phatadon Sirivongrangson2", Win Kulvichit*®, Sasipha Tachaboon*®, Janejira Dinhuzen*2, Watchadaporn Ch: Sadudee Peerapornratana*?™, Chintana Chirathaworn’, Kearkiat Praditpornsilpa’, Somehai Eiam-Ong’, Kriang Tungsanga? & Nattachai Srisawat!"#! Leptospirosis can cause ahigh mortality rate, especially in severe cases. This multicenter cross- sectional study aimed to examine both host and pathogen factors that might contribute tothe disease severity. A total of217 leptospirosis patients were recruited and divided into two groups of non-severe land severe. Severe leptospirosis was defined by a modified sequential organ failure assessment (mSOFA) score of more than two or needed for mechanical ventilation support or had pulmonary hemorthage or death. We found that leptospiremia, plasma neutrophil gelatinase-associated lipocalin (PNGAL), and interleukin 6 (IL-6) atthe frst day of enrollment (day 1) and microscopic agglutination test (MAT) titer at 7 days after enrollment (days 7) were significantly higher in the severe group than in the non-severe group. After adjustment for age, gender, and the days of fever, there were statistically significant associations of baseline leptospiremia level (OR 1.70, 95%C11.23-2.34, p=0.001), pNGAL (OR9.46, 959% C1 4.20-21.33, p<0.001), and IL-6 (OR 2.82, 95%C11.96-6.07, p<0.001) with the rity. In conclusion, a high laptospiramia, BNGAL, and IL-6 level at baseline were associated with ptospirosis Leptospirosis is one of the most important worldwide zoonosis and is a major public health issue in many coun: tries. The disease is caused by spirochetes from the genus Leplospira and is transmitted by contact of abraded skin or mucous membranes with contaminated rodent urine, water, or soil’. There isan estimation of more than ‘one million cases of severe leptospirosis per year worldwide". Leptospirosis can cause severe multiple organ {allure witha mortality rate as high as 50%”. Severe leptospirosis paicats should receive early recognition and intensive medical care. The pathology of leptospirosis and the factors causing severe leptospirosis ae sill unclear. lst and pathogen factors might play an important role in the pathogenesis of leptospirosis. high leptospi ral load was found to be asoctated withthe severity in several studies, but overall, the results are inconsistent” * Different Leptospira serogroups have different lepospirallipopelysaccharides (LPS) and hemolysin, which are virulence factors and cause adiference in the disease severity". However, the role of specific antibody response to pathogenic leptospites inthe pathogenesis of severe leptospirosis is still unclear Host responses in leptospirosis are complex but important in the pathogenesis of the disease. Contact of the host with the pathogen causes the release of cytokines". The extensive release of cytokines, including inter Jeukin 6 (IL-6), interleukin 1 bets (IL-1), and tumor necrosis factor-alpha (TNF-a), are known asa cytokine [Excellence Center far Critical Care Nephrolagy, Thai Red Crate Society, King Chulalongkorn Memarial Hopital, 1673, Rama 4 Ré,, Lomphini, Pathumwan, Bangkok 10330, Thailand, “Critical Care Nephrology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. *Divsion of Nephrology, Department fof Medicine, Faculty of Medicine, Chulalongkorn ‘University, and King Chulalongkorn Memorial Hospital Bangkok, Thailand. “Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand. ‘Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand. “Academy of Science, Royal Society of Thailand, Bangkok, Thailand. “Tropical Medicine Cluster, Cholslongkorn University, Bangkok, Thailand, Excelence Center for Critical Care Medicine, King Chulalongkorn Memorial Hospital, Bangkok, Thailand. *Center for Critical Cate Nephrology, The CRISMA Center, Department of Critical Cate Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. These authors contributed equally: Umaporn Limothai, Nutthe Lumlertgul and Phatadon Sirivongrangson. “email srisawatsr@ chulaacth SelentifeReports| _(2021):114690 [htpsdo\orgao 2038/5253 10732 ‘ature portfolio wurw.nature.com/scientificreports) storm, Many sds have demonstrate the rol of eytoknes in thecinial manifestations and pathogenesis of leplosptons However there Isincansislncy among ras, probably dc to elerogencous sty desig" Renal biomarkers such as neutophl eatinase-asoiated lipocalin (GAL) ig also be involved In the pathogenesis of leptospirosis. A previous study showed that plasma (p)NGAL and urine (u) NGAL were certelated with acute Key inary (AKI) in pallens wth leptospirosis The NGAL is exetd by activated ‘eatrophlsnresponse to bacterial infections, bu the roles NGAL in he pathogenest a leptospirosis ae Sill unexplored ‘The esl from previously mentioned tudes showed tha! the pathology ofleptospiross remain ucla ‘This prospective cos acctional study simed to examin both hoa and pathogen actors thal might be one wih te duce seventy, including eptonpremia levels, a specic anthody fesponse evaluate inter ofthe Incroscopi agglutination et (IAT) er, pNGAL and II Methods Ethics statement. The study vas conducted in compliance with the Declaration of Helsinki and the prin- ciples of Good Clinical Practice. ll patents had given written informed consent. The study was approved by the Institutional Review Board of Faculty of Medicine, Chulalongkorn University, and the Institutional Review Board of Ministry of Public Health of Thailand (IRB no. 521/62) ‘Study design and participant selection. this multicenter cross-sectional study carried out in a cohort of patients with leptospirosis was conducted in 15 hospitals in Sisaket province Thailand, from November 2015, to December 2017. The list ofthe hospitals is provided inthe acknowledgment section. Clinical suspicions for leptospirosis were a high fever (body temperature higher than 38 °C), severe myalgia, and history of exposure to reservoir animals or flood water. The inclusion criteria were (1) patients older than 18 years, (2) admitted in participating hospitals, and (3) patients were confirmed to have leptospirosis by being positive in one of the standard techniques (MAT, direct culture, and quantitative (GPCR). We excluded the patients who suiTered from other known infectious diseases. Blood samples were collected on the fist day of enrollment (the frst day ofthe clinical suspicious leptospirosis) and day 7 after enrollment. Samples were stored at 80°C until analyzed Allpatients received a broad-spectrum antibiotic such 3rd generation cephalosporin and doxycycline within the first hour of recognizing leptospirosis according tothe sepsis-3 guideline Outcomes, The outcome of interest was severe leptospirosis We defined the patients as severe if the patient died, needed mechanical ventilation support, had pulmonary hemorrhage, or had atleast one organ failure. We defined organ failure using a modified sequential organ failure assessment (mSOFA) score of more than two (which included coagulation, iver, cardiovascular and renal) Exposures. Exposures of interest were the level of leptospiremia, highest MATT titers, NGAL. and IL-6 level, Other covariates that might afect the outcome were sex, age, day of fever until enrolment, an antibiotic treat ment ‘The level of leptospiremia was measure by qPCR, as previously described", Briefly, total DNA was extracted from 200 ul of the whole blood sample using a High Pure PCR Template Preparation kit (Roche Diagnostics, Germany) with 50 ul elution buifer. Pre-validated specitic primers and TagMan probe targeting lipL2 from Stoddard RA etal! were used in th CR prodiet size was 242 bp. The two primers used were as follows 45F primers (5' AAG CATT TAC CGC 1G3) and 286R primers (5° GAA CIC CCA TT T CAG CGA 3’) and Probe 189P (FAM-5'-AA AGC CAG GAC AAG CGC .CG-3'-QS¥). The qPCR reactions were performed in a final volume of 20 corresponding to 5 ul of genomic DNA and 15 ul of reaction mix containing, the 10 ul SsoAdvanced Universal Probe Supermixs (Biorad Laboratories, USA) part aumber 64182275, provid ing final concentrations of 10 wM of each primer and 10 uM of the FAM-QSY labeled probe. A no-template control (NTC) that contained al the above reagents was also included to detect the presence of contaminating DNA. Amplification and fluorescence detection were conducted in the SlepOnePlas Real-Time PCR Systems (Applied Biosystems, USA) with a program of 40 cycles, each cycle consisting of 95°C for 15 sand 60°C for one ‘minute. The qPCR reactions were performed in duplicae. A negative result was assigned where no amplification ‘occurred, Le. the threshold cycle (Ct) value was greater than 40 cycles. “The MAT was performed as previously described ~" using the standard protocol ofthe World Health Organt zation (WHO) guideline”. A postive MAT result defined as having a MAT titer of 1:400 in single sample or four-fold rising in paired samples. The pNGAL levels were measured by the quantikine human lipacalin-2/NGAL. immunoassay (Catalog nam ber: DLCN20, R&D Systems, Minneapolis, MN, USA) according to the manufacturer instructions. ‘The level of serum IL-6 was measured by a chemiluminescence method using the Elecsys IL-6 kit (Roche Diagnostics GmbH, Mannheim, Germany) with an analytical sensitivity of 1.5 pe/ml following the manufac turer instructions. Direct culture of leptospires. We performed clirect culture of leptosptes by adding one drop of whole blood into 4 ml of liquid Ellinghausen-McCullough-fohnson-Harris (EMH) medium and incubating ths at 29°C for 2 weeks. Leptospira were then abserved and counted under dark-feld microscopy by direct observa tion, Scientife Reports| aozya134830 | hups:idol org 2034)s41598-071-S40732 natureportiolio wurw.nature.com/scientificreports) Patients clinically suspected for leptospirosis (n=330) Exclude (0-113) 100 nomieptosprosis 13 no clinical data Confirmed leptospirosis. (02217) | ‘Non severe group Severe group (271) Statistical analysis. Continuous variables are presented asthe mean one standard deviation (SD) if para meric and asa median and interquartile range (IQR) in case of non-parametric data Categorical variables are presented as numbers and percentages. Comparisonsbetween groups were analyze by thechi-square or Fisher's hac et for categorical varices and by the Mann-Whitney U-test oe Stadents st foe quantitative variables, Logistic regression was used to asess the ORS relating to variables associated with severe leptospirosis. Addi tionally, we checked for mulicllineaity inthe regression models using the valance inflation factor (VIF). and no problem was detected. Al tatistical tests were fwo-sided, and pevalues (105 were considered sigoificat “There were 16 samples on day 1 and 182 samples on day 7 ater enrollment were missing Values of eptospireia die tothe vale below the Fit of detection, While there were missing vals of MAT titer da to the same issue in 207 samples om day 1 and 172 samples on day 7 after encollment. Likewise there were 22 missing vaes for NGAL and 30 missing values for IL-6. The value changes between two-time points (or the pated date on D1 and D7) within-group comparisons wer tested by Wilcoxon signed-rank test. We performed all statistical analyses using the SPSS Version 22 software (SPSS, Chicago TL). and gures were drawn using GraphPad Prism 9 (GraphPad Sofware Inc, California, USA) Results Participant characteristics. Among the 330 patients suspected of leptospirosis, those who were negative ‘upon testing (non-leptospirsis patients) and patients with incomplete data were excluded leaving a total of 217, leptospirosis patients (146 patients in the non-severe group and 71 patients inthe severe group) in this study (Fig. “The characteristics ofthese patients are shown in Table 1. In total, 101 (69.2%) non-severe leptospirosis patients and 45 (30.8%) severe leptospirosis patients had a fever for 3 days or less before the diagnosis of lepto- spiross infection. Compared with the non-severe group, the severe group had a significantly ower body tem: perature, systolic blood pressute, diastolic blood pressure, hemoglobin, platelet count, and serum bicarbonate, ‘but a higher level of serum creatinine, white blood cell cout, toal bilirubin, cite bilirubin, and serum glutamic oaloaceti transaminase. There was no significant difference in terms of the other clinical characteristics. The severe features of leptospirosis are summarized in Supplementary Table onlin. Seven percent died, out 8% were diagnosed with severe liver fallure, ane 14% were dlagnosed with severe renal fallure. Approx mately 5% needed dialysis, 21% had severe coagulopathy, and 7% had a severe cardiovascular system failure The incidence of pulmonary hemorrhage was 8%. There were 12% of patients required mechanical ventilation, Distribution of Leptospira serogroups. Among the 217 study participants, 54 (24.9%) had Leptaspira agglutinaing antibodies, as determined by the presence of MAT tier of 21400 ina single sample or four-fold ‘sein paired samples. The most common serogroup was Shermani, followed by Australis and Louisaina. The serogroups of leptospirosis are summarized in Fie, 2. ‘The level of leptospiremia, MAT titer, PNGAL, and IL-6 in relation to the severity of leptos} rosis. On the fist day of enrollment (day), the median level of leptospiremia was significantly higher in the severe group than the non-severe group (1440 copies/ml vs. 420 copies/ml), as shown in Fig. 3A. Moreover, the level of pNGAL (Fig, 3C) was aso significantly higher inthe severe group than in the non-severe group (512 ng ‘ml vs, 194 ng/ml). Compared with the non-severe group, the severe group also had a significantly higher IL-6 level (323 pg/ml vs. 40 pg/ml, Fig. 3D). Leptospira agglutinating antibodies were detected (MAT tter > 1400) In Scientife Reports| aozya134830 | hups:idol org 2034)s41598-071-S40732 ‘natureportilio wurw.nature.com/scientificreports) Mae gale. 0 0) 188) 59651) 9670 syns ime, SD) Tea07) Tae a0 Fever dys mean TOR) 25040 3020.40 02100 OSs 008) 71693) Eos) aver moe a) 10612) 208 Body temperate (mean SD) [S84 (LD) 3) oon SH mig edn 1 ENED) Toto wenn) |

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