PH12018050189A1 - Early diagnosis and prognosis of complicated leptospirosis using molecular markers - Google Patents

Abstract

The present technology relates to using molecular markers in clinical diagnostics, specifically the use of Defensin Alpha 1, also known as DEFA1 gene, for the prognostic marker of complicated leptospirosis in human patients. The technology particularly relates to a process of detecting and quantitating the level of expression of DEFA1 gene in peripheral blood mononuclear cells (PBMCs) taken from human patients, as a means to predict whether their leptospirosis infection will progress into a complicated leptospirosis, specifically whether the patient will experience Weil's syndrome. DEFA1 gene expression was found to be significantly elevated in PBMCs of human patients with severe leptospirosis relative to patients with acute fever and healthy patients. Microarray technology was used to measure the expression of many genes and DEFA1 gene was found to be the only significantly upregulated gene in PBMCs of patients with severe leptospirosis versus healthy controls and patients with uncomplicated febrile cases. Expression of DEFA1 gene can also be detected using a targeted gene expression platform such as the real time quantitative Polymerase Chain Reaction (qPCR) analysis as recommended to make the test relatively simpler and cost-effective.

Description

presenting symptoms in the early onset of leptospirosis. In particular, the present invention aims to provide a diagnostic method that makes use of a host biomarker that is appreciable through laboratory tests even at the early onset of symptoms.

As such, this diagnostic method offers a reliable means for predicting whether the patient's symptoms will progress into Weil's disease, thereby providing accurate technical basis for physicians in making decisions towards effectual triage and providing pre-emptive care.

SUMMARY OF THE INVENTION

The present invention relates to a method involving a molecular marker for early diagnosis and prognosis of complicated leptospirosis comprising the following steps: a) Isolating RNA from a biological sample received for diagnosis; b) synthesizing complementary DNA from the isolated RNA; c) quantifying the RNA relative to reference samples or a comparator healthy control sample; and d) analyzing the results where significantly high levels of the molecular marker indicate severe or complicated leptospirosis.

For better understanding of the present invention and to show how the same may be performed, a preferred embodiment thereof will now be described, by way of non-limiting example only.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a graphical representation of the result of Receiver Operator

Characteristic (ROC) analyses.

Figure 2 shows the probe sequences for DEFA1, DEFA1B and DEFA3 in the lllumina HT-12 annotation list.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the technical features of the present invention of a method involving a molecular marker for early diagnosis and prognosis of complicated leptospirosis will be explained in more detail with reference to a preferred embodiment thereof as shown in the accompanying drawings. The drawings however, are illustrative only and should not be used to limit the scope of the present invention.

A preferred embodiment of the present invention is a method involving defensins as a diagnostic and prognostic marker for complicated leptospirosis, the method comprising the steps of a) separating PBMCs from blood sample received for diagnosis; b) extracting RNA from the PBMCs; c) synthesizing complementary DNA from the RNA samples; d) quantifying in the isolated RNA the levels of any one, or a combination, of the following: DEFA1, DEFA1B or DEFAS3, relative to reference samples or a comparator healthy control sample, using a selected platform or quantifying technique such as, qPCR wherein the primers should cover the nucleic acid sequences specified in the PCR system used; and e) analyzing the results where significantly high levels of any one of or combined DEFA1, DEFA1B or DEFA3 indicate severe or complicated leptospirosis.

Alternatively, mRNA can be extracted from whole blood, or any fraction of the PBMCs. Also, such mRNA is deemed to be sourced from other affected biological fluids, such as urine, sputum and other fluids in the inflamed sites.

Another embodiment of the process is to quantify the RNA using other techniques such as nucleic acid-based microarray technology or other existing techniques such as RNA sequencing or blotting techniques, and lateral flow assay or dipstick to determine the quantity of the DEFA1-3 mRNA or other diagnostic and prognostic marker used.

Supporting Experimental Studies

With the advent of microarray technology, host biomarker identification and characterization has become more common. The following details are presented in support of the preferred embodiment of the invention where DEFA1 is the molecular marker used for early diagnosis and prognosis of complicated leptospirosis. The method uses DEFA1 gene expression molecules that can be extracted from PBMCs and quantified as a means to distinguish a complicated leptospirosis from a mild form of leptospirosis and from a healthy condition.

A. Human subject recruitment and sample collection

Subjects are invited to join the study only after signing an informed consent forms duly approved by the University of the Philippines Manila Review Ethics

Board (UPMREB), Ospital ng Maynila Medical Center Ethic Board, and San

Lazaro Hospital Research Ethics Review Unit (SLH RERU). All subjects were recruited from the emergency rooms, wards and clinics of the University of the

Philippines-Philippine General Hospital (UP-PGH), Ospital ng Maynila Medical

Center (OMMC) and San Lazaro Hospital (SLH) as these hospitals cater as tertiary care centers for Weil's disease patients in Metro Manila.

A prospective subject was screened first based on his/her clinical presentation and a pre-set inclusion/exclusion criteria. If deemed qualified, the prospective subject (or if unable to do so, by his’her primary caregiver with legal capacity) was invited to join the study; and if amenable, was made to sign an informed consent. The study recruited subjects who are 18-60 years of age. It recruited patients with moderate Weil's syndrome without significant renal failure, pulmonary complications (hemorrhage or severe dyspnea), or refractory hypotension. As much as possible, the blood extraction were from from patients who are receiving initial care from the hospitals. Uncomplicated mild leptospirosis with the presence of accompanying symptoms only— headache, myalgia, maculopapular rash, and with stable vital signs — were not recruited as cases.

After screening using the clinical criteria, the potential participants were further screened by doing a leptospiral microscopic agglutination test (MAT) assay with the following decision algorithm: 1:1600 or higher, for inclusion to “Uncomplicated moderate Weil's syndrome (Cases)”; lower than 1:100, for inclusion to “Uncomplicated acute febrile illness and not leptospirosis (febrile controls)”; and 1:100 to less than 1:1600, were not included, unless they are positive for blood flagellin B (flaB) gene amplification, in which case the flaB- positive samples were included in the cases. Flagellin B is uniquely present in pathogenic leptospira and not in saphrophytic ones.

In addition, to further diagnose leptospirosis, blood samples and urine samples from subjects were to be cultured for leptospira. The leptospiral MAT assay and cultures were done in the Leptospirosis Consortium laboratory in the

College of Public Health, UP Manila. Lastly, additional optional blood extraction were done after about 2-weeks (convalescent phase) after an initial MAT test in cases of non-conclusive MAT results (>1:100 but <1:1600) with a negative leptospiral DNA test or culture from the blood. In this test, a four-fold rise in titer or recent seroconversion was diagnostic of leptospirosis. This repeat MAT was considered as a third confirmatory testing for leptospirosis after an initial single

MAT of 1:1600 or a second test using positive flab amplification. This repeat MAT assay was done in case of non-conclusive testing after the first 2 assays to ensure the study can maximize the number of cases for inclusion.

The cases and diseased controls were recruited from the emergency room complex within 48 hours upon admission. This will insure that the effect of subsequent interventions to the genetic expression of the subjects will be minimized. In addition to the febrile groups, a set of age-matched, sex-matched healthy asymptomatic subjects were recruited for comparison.

B. Diagnosis and Prognosis of Complicated Leptospirosis

1. Isolation of peripheral blood mononuclear cells (PBMCs) from blood samples

Blood samples amounting to 3 ml were transferred into tubes containing

EDTA and kept on ice during transport. Within 2 hours from time of blood sample collection, the peripheral blood mononuclear cells (PBMCs) were isolated. In brief, 3 mL of blood is diluted in equal amount of ice-cold phosphate-buffered saline. In a 15-mL conical tube, the blood is carefully layered on top of 3 mL polysaccharide solution (Lymphoprep; Axis-Shield, Oslo, Norway), and the mixture centrifuged at 800g for 20 min at 4°C. The PBMC interface is collected and kept at -80°C until

RNA extraction. The plasma and PBMC fractions will undergo separate RNA extraction. 2. Total RNA extraction blood cells

The nucleic acid extraction works were done in the molecular genomic laboratory of the Institute of Human Genetics, University of the Philippines Manila.

The procedure followed is according to the manufacturer's instructions of the miRNeasy minikit (Qiagen, Germantown, MD), but modified with pelleted PBMCs as starting material.

The procedure extracted total RNA and small RNAs for downstream studies. Briefly, after loosening the cells by flicking after thawing for 5 min, 700 pl of QlAzol Lysis Reagent was added to the pellet and mixed by vortexing for 1 min, and homogenized by loading into a Qiashredder column, centrifuged at maximum speed for 2 min. The collected homogenate were incubated at room temperature (RT:15-25°C) for 5 min. Afterwards, 140 pl of chloroform was added to the tube, which is capped and shaken vigorously for 15 s. After incubating at RT for 2-3 min, the sample was centrifuged at 12,000xg at 4°C for15 min. After transferring the aqueous phase into a new 1.5 ml tube, 1.5 volumes of 100% ethanol were added and mixed thoroughly by pipetting. immediately, 700 pl of the sample was transferred, including any precipitate, into an RNeasy Mini spin column in a 2 ml collection tube, and centrifuged at 28000 x g for 15 s at room temperature. The flow-through was discarded, 500 ul Buffer RPE was applied onto the RNeasy Mini spin column, which was centrifuged for 15 s at 28000 x g (210,000 rpm) to wash the column. After discarding the flow-through, 500 pl Buffer RPE was added to the

RNeasy Mini spin column, which was centrifuged for 2 min at 28000 x g . To insure removal of ethanol, the RNeasy Mini spin column was placed into a new 2 ml collection tube, which was centrifuged at full speed for 1 min. The RNeasy Mini spin column was transferred to a new 1.5 ml collection tube and 50 pl RNase-free water was applied directly onto the RNeasy Mini spin column membrane. The sample was centrifuged for 1 min at 28000 x g. Samples were kept at -80°C until further use. RNA quantification and OD260/280 determination were initially done using a ND1000 NanoDrop spectrometer, with downstream processing for

A260:280 at 2.00 to 2.20. Samples were divided in 10 ul aliquots and kept at - 80°C until use. Immediately before use for microarray processing or for cDNA synthesis, RNA samples were quantified and analyzed for degradation using

TapeStation RNA Analysis Kits (Agilent, Sta. Clara, CA, USA). 3. Whole-genome expression profiling using microarray

Samples for the profiling used the RNA from the cells. Using microarray technology, specifically the Illumina HiScanSQ system, gene expression analysis is done following the lllumina's Whole-Genome Gene Expression Direct

Hybridization Assay Human Transcriptome 12 (HT-12) protocol as specified in the manufacturer's manual.

Initially, total RNA extracted from blood samples taken from patients and controls were labeled using sample labeling RNA prep kit to produce biotinylated amplified RNA in preparation for microarray analysis. Following the instructions indicated in the kit, the procedure starts with reverse transcription of 250 ng total

RNA with an oligo(dT) primer bearing a T7 promoter to synthesize single-stranded cDNA. The single-stranded cDNA then undergo second strand synthesis and purification to produce double-stranded DNA template for in vitro transcription.

With T7 RNA Polymerase, in vitro transcription was done to produce amplified 11 biotin-labeled cRNA, which in turn was purified by a supplied cRNA filter cartridge.

The purified cRNA was quantified by Agilent Bionalyzer with Agilent RNA 6000nano kit prior to array hybridization. The cRNA samples were stored at -80°C until microarray processing or up to a maximum of 6 months.

Hybridization begins with loading of normalized cRNA (750ng — 1.5ug) into the beadchips (6-12 samples per chip). These beadchips were loaded into an assembled hybridization chamber and placed in a hybridization oven for an overnight incubation (14-20 hours) at 58°C. Washing was done next at 55°C with high-temp wash buffer for 10 min followed by two room-temp washes using fresh wash buffer with ethanol wash in between. Blocking was done subsequently, followed by Cy3-Streptavidin (Cy3-SA) introduction to allow the binding of the

CyDye to the analytical probes hybridized to the beadchips. Beadchips were washed and dried next prior to imaging, which will be done using the microarray scanner. All intensity data from these images will be calculated and analyzed using the GenomeStudio Software.

C. Data Analyses 1. Preliminary analyses of molecular data

A total of 70 peripheral blood mononuclear cell (PBMC) samples underwent microarray processing using the Illumina HT12 Direct Hyb gene expression chips.

The raw data output were analyzed using the GenomeStudio gene expression module. Of these, 18 are from leptospirosis cases, 25 from febrile controls and 27 from healthy controls (not yet necessarily age- and sex-matched). The gene expression data were normalized using cubic spline adjustments. 2. Assessment of the validity of the procedures

The control summary of specific parameters is evaluated and all parameters show satisfactory implementation of the procedures. In addition, the variations among beadchips and samples indicated significantly low noise and background signals, with the signals from housekeeping genes are significant.

Such variations are accounted for in the normalization process. 3. Differential gene expression among different groups

Differentially expressed genes were determined using a cut-off of p<0.001 using student t-test comparing 2 different groups. The genes are presented as input to STRING: (https:/string-db.org/) to determine pathway enrichment and analyses using a cut-off of false discovery rate (FDR) <0.001 (except in scenario where clarificatory description is deemed helpful) , with 0.40 confidence, with interactions inferred from data in literature, co-expression and experimental. 4. Leptospirosis versus healthy controls

There are 1,066 downregulated genes and 148 upregulated in the leptospirosis group compared with the healthy control group. A list of the top expressed genes is provided in Table 1.

Table 1. List of top expressed genes in leptospirosis group compared with healthy control group

Upregulated in Downregulated in

To provide biological insights, pathway analyses was done. Pathway enrichment analyses shows that the set of differentially expressed genes significantly overrepresents in positive regulation of immune system response as biological process, and external side of plasma membrane and azurophil granule lumen as cellular components, strongly suggesting an immune-mediated response involving cellular receptors resulting to the activation of innate immunity, such as the defensins.

It is surprising that the defensin expression was detected in PBMCs in the subacute phase of leptospirosis, a finding that may imply that the cells, in conjunction or not to the neutrophils, contribute to the organ injury manifested during Weil's syndrome. 5. Leptospirosis versus febrile controls

As expected, in contrast with its comparison with controls, the leptospirosis groups have significantly less differentially expressed genes compared with febrile controls (15 downregulated and 1 upregulated in leptospirosis group, Table 2), perhaps reflecting the common inflammatory mechanisms activated in both case groups. Thus, because of the limited number of inputted genes, no significant pathway was obtained from analyses. Nevertheless, of high interest, defensin 1 gene (DEFAT1) is the only significantly associated upregulated gene in the leptospirosis group. With more than 10 times the expression in the cases, this may be a potential gene that may explain the unique symptomatology of Weil's syndrome.

Table 2. List of top expressed genes in leptospirosis group compared with febrile control group

Upregulated in | Downregulated

T=

Tm [my [sem wm ew ew toc eS 6. Relative quantification of DEFA1 in severe leptospirosis versus controls

Comparison of means of the DEFA1 levels from PBMCs show a significantly higher level in severe leptospirosis compared with febrile uncomplicated cases and healthy controls (Table 3 a, b).

Table 3 a. Comparison of means of DEFA1 levels among groups

DEFA1 level, Mean FU

Te b. Multiple comparisons among groups

Dunn's multiple | Mean rank |Significant?| Adjusted P comparisons difference Value

Healthy vs. >0.9999

CT

Healthy vs. -26.62 Yes <0.0001 ss | | 7

Febrile vs. -31.89 Yes <0.0001 os

Receiver operator characteristic (ROC) analyses to determine the potential of using DEFA1 as a marker to indicate severe leptospirosis was done. ROC analyses results show a high area under the curve of 0.923 with p<0.001 (see

Figure 1). An optimum sensitivity/specificity of 94.12%/ 83.33 is shown.

Because of the high sequence similarity of DEFA1, DEFA1B and DEFA3 in humans, the transcripts being quantified is a mixture of the three, and thus can be theoretically referred to as DEFA1-3 (see Figure 2).

Preferably, the RNA samples for qPCR has sufficient purity with a

A260:280 of 2.00 to 2.20 or an RNA integrity number of 7 or above. A one-step run or a typically 2-step qPCR run can be done.

D. Summary and Conclusion

Mortality and complications due to leptospirosis are due to a hyperinflammatory response that is likely dependent on the host; thus a host response indicator is presented. Because of the counterintuitive decrease of HNP level in the blood, probably due to tissue sequestration, the protein level may not be a reliable marker. The use of DEFA transcripts from the non-neutrophil sources is recommended, because these are more like induced than constitutive to reflect abnormal response, as our findings show. In particular, we have demonstrated that trnascripts from peripheral blood mononuclear cells (PBMCs) discriminate the complicated cases from other comparator groups.

Conclusive evidence has been provided hereby that diagnosing and predicting complicated leptospirosis in humans is possible and indeed successful with the present technology utilizing DEFA1 as a molecular marker.

Although known to be an early response molecule, the inventors of the present technology found that the expression of the DEFAs/HNPs are significantly higher in complicated leptospirosis. Because Weil's syndrome typically occurs 1 to 2 weeks after an initial onset of fever for leptospirosis, this fact is considered counterintuitive but forms as the basis for considering DEFA1 as a molecular marker for complicated leptospirosis. Compared with other acute febrile illness, the level of DEFA1 (which represents DEFA1-3 in the assay) is very high and can discriminate complicated cases from febrile controls and healthy controls with high accuracy.

The invention has several advantages over existing diagnostic/prognostic methods. First, it does not presume that the presence of leptospirosis can cause complications (about 95% do not). What it does is indicate an ongoing detrimental host response id occuring and may further occur, which is crucial in the decision- making of healthcare providers in preempting such condition that can result in lesser mortality, less cost of care and effective triage of patients.

It is intended that the foregoing description be only illustrative of the present invention and that the present invention is limited only by the hereinafter appended claims. oe

EARLY DIAGNOSIS AND PROGNOSIS OF COMPLICATED LEPTOSPIROSIS

USING MOLECULAR MARKERS

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of molecular diagnostics and prognostics. More particularly, it relates to the use of gene expression molecule for early diagnosis and prognosis of complicated leptospirosis or Weil's syndrome.

BACKGROUND OF THE INVENTION

Leptospirosis, the most widespread zoonotic disease in the world, results from humans and animals getting infected with pathogenic spirochetes; most commonly from Leptospirosis interrogans. Leptospirosis, severe infection due to Leptospira interrogans, is a potentially lethal disease that causes multiple organ failure. In addition to hepatic, renal, and central nervous system (CNS) involvement, which are classic complications of leptospirosis, the disease may also be complicated by adult respiratory distress syndrome (ARDS). It is mainly contracted from contaminated water that enters through an interruption in the skin or mucosa of the host.

Patients of leptospirosis usually present with flu-like illness and generally recover from it through antibiotic treatment. Meanwhile, about 5% will have a more severe form referred to as Weil's syndrome or Weil's disease, with life threatening complications such as severe acute renal failure and pulmonary hemorrhage, which sometimes results to greater than 50% mortality when left untreated.

Early diagnosis and treatment remains as the most practical public health strategy to deal with leptospirosis outbreaks, which usually occur after a flooding.

However, early diagnosis of leptospirosis presents a big challenge even in tertiary hospitals because diagnosis remains heavily dependent on clinical parameters,

such as the presence of jaundice or low urine output. Moreover, laboratory testing of biomolecules is integral to clinical management of the disease.

Aside from early diagnosis, another issue is prognostication. Wiel's syndrome, the severe form of leptospirosis, is a good example of a detrimental host response. Host inflammatory processes are supposedly evolution-derived reaction designed to protect an organism from injury. However, through various phenomena, such as the co-evolution of the pathogens to evade or the persistence of genetic derangements resulting to adult-onset hyperinflammatory responses, inflammation by itself has become a cause of disorders that can adversely affect the human host.

The detrimental reactions occur as a result of active biomolecular and hyperinflammatory reactions that can perturb host homeostasis resulting to disease, or even death. In other words, the presence of a pathogen may indicate infection, but the issue of developing complications can be better assessed by host response in this case.

To date, available laboratory tests for monitoring the prognosis, or the likely course of the leptospirosis disease, in a patient lack the capacity to predict complications mainly because many of the laboratory criteria used to diagnose complicated leptospirosis become clinically appreciable during the end-stage of organ damage and at a time when clinical management often becomes too difficult because of the severity of complications. High creatinine levels to indicate renal failure, low partial thromboplastin time, aspartate aminotransferase, or alanine aminotransferase to indicate liver failure and low oxygen levels to indicate lung failure, positively indicate complicated leptospirosis and these occur during the phase wherein end-organ damage is obvious, and is not particularly useful towards prevention of complicated leptospirosis.

As predictors of complications, an early manifesting biomarker can be crucial in the secondary prevention and proper triage of patients. Though there are clinical prognosticators, such as dyspnea, oliguria, presence of pulmonary rales, hyperkalemia, abnormal serum creatinine, leukocytosis, thrombocytopenia, elevated bilirubin, hypotension, arrhythmia, acute respiratory distress syndrome, pulmonary hemorrhage and altered mental status, these are already late manifestations and could be less reliable in the crucial clinical decision-making.

Thus, by looking at the perspective of the host, molecular cues and pathways can be perceived as potential signatures that can be of use to diagnose, prognosticate or even be targeted for therapy.

Currently, diagnostic biomarkers for infectious diseases mainly use biomarkers that originate from the pathogen. While these biomarkers confirm presence of the pathogen, it does not give doctors a way to predict whether the patient will develop Weil's disease as is in the case of the amplification of the flagellin B (fIB) gene of pathogenic leptospira. Meanwhile, IgM and IgG-based diagnostic processes are only capable of diagnosing leptospirosis but not differentiating between a mild form from its complicated form.

Weil's syndrome or Weil's disease is a host response mechanism to infectious pathogens. It is generally characterized by hyperinflammatory reaction in one or more of the organs of the host, either the kidneys, liver, joints, meninges and/or other organs. If not treated, hepatorenal failure results and possibly death.

Since Weil's syndrome is a host response, intuitively one looks to the human host as the most practical source of biomarker.

In US 20030027176 A1, P. Dailey discloses a method for determining the type of an infectious pathogen in a patient who is suspected to be suffering from an infectious pathogen. The method involves first measuring the amounts of a plurality of markers in a body fluid sample of the patient. The markers of interest are produced by the patient as part of that patient's innate immune response to the presence of the infectious pathogen and are indicative of the type of the infectious pathogen in the patient. Next, a marker profile is identified based on the measured amounts of the plurality of markers. Finally, if the marker profile is indicative of an infection, then the type of infectious pathogen within the patient is determined from the marker profile. In preferred embodiments, any individual marker is either an mRNA or a protein.

Maximow et al. disclose in W02014125164A1 methods for monitoring the development of and for treatment of ARDS in a patient. The method for monitoring the development of ARDS is based on comparing the level or activity of the biomarkers obtained in a sample drawn at a later point of time to the levels or activities of the same biomarkers in a sample drawn at a previous point of time. A favourable change in the level or activity of a certain biomarker represents a regression of the disease (recovery of the patient), and, conversely, an adverse change in the level or activity of a certain biomarker represents a worsening of the disease. If, for example, the level or activity for one or more of the biomarkers monitored discontinues to show a favourable change or starts to show an unfavourable change, the treatment of the patient is enhanced by administering a therapeutically active agent useful in the treatment of ARDS.

In" US20140154711A1, A. Daily et al. disclose non-invasive methods of detecting and providing an early diagnosis of cancer, in particular breast cancer.

The methods include obtaining a sample, suitably an ocular wash sample, from a subject and determining the level of [alpha]-Defensin 1, [alpha]-Defensin 2 and/or [alpha]-Defensin 3 in the sample. Increased levels of any of these proteins as compared to a negative contro! or non-cancerous subject are indicative of cancer in the subject from which the sample was obtained.

Defensins are molecule markers of note, being used both in diagnosis and therapy. Defensins are very potent molecules which help in the immediate attenuation of infections, as the body awaits for the adaptive immunity to activate.

These molecules are short peptides which feature 3 Cys-Cys bonds in its amphiphatic and cationic structure. They have multiple functions, from a wide spectrum of antimicrobial to immunomodulatory effects, being part of innate immunity response milieu.

:

The so-called alpha defensins (DEFAs) belong to one of 2 groups of defensins in humans that can be distinguished by their unique Cys-Cys bond patterns and amino acid sequence. Among them and of particular interest are the so called human neutrophil peptides 1, 2 and 3 (HNP1, HNP2, HNP3) which only s differ by 1-2 amino acids, and are coded in humans by the genes defensin alpha 1 (DEFA1), defensin alpha 1B (DEFA1B) and defensin alpha 3 (DEFA3). Another

DEFA, DEFA4, has less sequence similarity with DEFA1-3, and is expressed in a lesser degree. DEFA5 and DEFA6 are primarily expressed in epithelial and

Pannet cells in intestines. HNPs are mainly expressed in neutrophils and are 10 stored in the azurophilic granules for secretion in target sites or to fuse to vesicles containing ingested pathogens from antigen-presenting cells. Besides the neutrophils, other cells that store the HNPs are the cytotoxic killer cells and the monocyte/macrophage group. 15 With the advent of nucleic acid-based microarray technology, generating gene expression profiles has somewhat spurred the growth of host biomarker research. However, as in the case of complicated leptospirosis, a biomarker that can sufficiently predict complicated leptospirosis from the early onset of symptoms has yet to be found. 20

Since molecular host changes occur early during the pathogenesis of diseases, the present technology incorporates host expression data for diagn c purposes in carrying out a timely and effective response against leptospirosis. By considering the host response mechanism as a potential source of 25 prognosticators for patients, the present technology can help in the early diagnosis and in the prediction of complications, as well as in the triage of patients who are at-risk for the complications of leptospirosis.

OBJECTIVE OF THE INVENTION

30

It is the object of the present invention to provide a diagnostic method which can distinguish complicated leptospirosis from other uncomplicated febrile illnesses and the healthy condition, as well as from other diseases with similar

Claims (8)

1. A method involving a molecular marker for early diagnosis and prognosis of complicated leptospirosis comprising the steps of a) isolating RNA from a biological sample received for diagnosis; b) synthesizing complementary DNA from the isolated RNA, ¢) quantifying the molecular marker in the isolated RNA relative to reference samples or a comparator healthy control sample; and d) analyzing the results where significantly high levels of the molecular marker indicate severe or complicated leptospirosis.

2. The method according to Claim 1 wherein the biological sample is PBMCs (peripheral blood mononuclear cells), the molecular marker in the RNA that is quantified is DEFA1, and quantifying is done using qPCR.

3. The method according to Claim 1 wherein the biological sample is PBMCs.

4. The method according to Claim 1 wherein the molecular marker in the RNA that is quantified is DEFA1.

5. The method according to Claim 1 wherein the RNA isolated is mRNA.

6. The method according to Claim 1, wherein quantifying is done using a platform or quantifying technique selected from the group of qPCR, microarray, lateral flow assay, dipstick, and RNA sequencing.

7. Use of DEFA1 in the method according to Claim 1 or Claim 2 for diagnosis or prognosis of leptospirosis in biological samples from humans.

8. Use of DEFA1 in the method according to Claim 1 or Claim 2 for diagnosis or prognosis of leptospirosis in biological samples from animals.