Decreasing Leptospirosis Emergence Through Prognosis and Treatment Optimization (DeLEPTO) Project 1: Preventive Strategies for Early and Late Complications of Leptospirosis
Phase: PHASE2
Conditions: Leptospirosis
Sponsor: National Kidney and Transplant Institute, Philippines

 
 
 
Decreasing Leptospirosis Emergence 
through Prognosis and Treatment 
Optimization (DeLEPTO) Project 1: 
Preventive Strategies for Early and 
Late Complications of Leptospirosis  
 
Study Protocol and Statistical Analysis 
Plan 
 
Version 7  
 
2026-03-17 
 
 

 
Component Project Details 
 
Program Title: Decreasing Leptospirosis Emergence through Prognosis and Treatment 
Optimization (DeLEPTO) 
 
Proposal Title: Preventive strategies for early and late complications of leptospirosis 
 
Project Leader: Romina Danguilan, MD 
 
UP Manila site primary investigator: Jose B. Nevado Jr., MD, PhD 
 
SLH site primary investigator: Rontgene M. Solante, MD, FPCP, FPSMID 
 
Target Start Date: April 1, 2023 ​
​
Target End Date: March 2027 
 
Implementing Agency: National Kidney and Transplant Institute 
 
Co-implementing Agency(ies): San Lazaro Hospital (SLH) 
 
Cooperating Agencies: Institute of Human Genetics, NIH UP Manila 
 
SJREB Protocol Code: SJREB 2024-70 
 
Type of Research: Applied Science 
 
R&D Priority Area and Program (HNRDA): Health: National Unified Health Research Agenda 
 
Select HNRDA Item: Diagnostics Program 
 
Sustainable Development Goal: Good Health and Well-Being 
 
Line-Item Budget 
 
 
1.​ Executive Summary (not to exceed 300 words): an abstract of the project including details on the 
requested budget and the targeted 6 P’s 
 
Leptospirosis is a bacterial disease of severe public health concern in the Philippines. Aware of the 
increasing number of individuals dying from this disease and its impact on society, the National 
Kidney and Transplant Institute (NKTI), San Lazaro Hospital (SLH) and the Institute of Human 
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1 
Source of Fund 
PS 
MOOE 
EO/CO 
Total 
DOST-GIA 
9,613,137.60 
46,168,765.60 
4,570,000.00 
60,351,903.20 
National Kidney and Transplant 
 
15,480,000.00 
 
15,480,000.00 
Institute 
 
 
 
UP National Institutes of Health- 
798,310.08 
 
798,310.08 
Institute of Human Genetics 
 
 
 
Total Direct Cost (incl 
counterpart) 
8,829,118.08 
57,867,400.00 
4,500,000.00 
71,196,518.08 
Total Indirect Cost 
1,582,329.60 
3,781,365.60 
70,000.00 
5,433,695.20 
Overall 
10,411,447.68 
61,648,765.60 
4,570,000.00 
76,630,213.28 

 
Genetics, National Institutes of Health-University of the Philippines Manila (IHG, NIH-UPM) teamed 
up to investigate ways to improve the clinical outcomes of leptospirosis patients. This collaboration 
intends to explore using a serum protein recently identified as a potential prognostic biomarker for 
organ complications in leptospirosis and as a predictor of a patient's response to different preventive 
strategies. Unfortunately, no conclusive evidence yet supports a prognosticator aided intervention. It 
is hoped that valuable insights into the progression of severe leptospirosis could be generated 
through this study. Furthermore, these insights will expand on the currently limited information on 
host responses against leptospirosis and help inform patient management and clinical guidelines. 
Prognostic biomarker-based assays and early interventions will be developed, which could lead to 
shorter hospital stays of patients, reduced healthcare costs for both the patient and the hospital, 
lesser facility and human resources stress, and decreased mortality and morbidity. 
 
A budget of at least 76M Php (including 23% counterpart funding) spread out over three years is 
proposed to implement a clinical study with a high confidence level from which impactful and 
biologically meaningful data can be generated. With those funds, NKTI will be able to explore the 
real potential of prognosticator-guided preventative approaches for severe leptospirosis. In addition, 
because we are targeting the host immune responses during infection, findings from this study may 
be translated into other fatal infectious diseases such as sepsis, dengue, and malaria. 
 
2.​ Project Description (not to exceed 300 words): a formally written declaration of the project and 
its idea and context to explain the goals and objectives to be reached and other relevant information 
that explains the need for the project startup and aims to describe the amount of work planned for 
implementation; refers to a simple explanation or depiction of the project that can be used as Press 
Material. 
 
This study aims to determine the clinical utility of complement factor I (CFI) as a prognosticator in 
patients with complicated leptospirosis without severe pulmonary complications and to determine if 
its guidance to preemptive measures can lead to a reduction in adverse clinical outcomes, 
specifically the occurrence of pulmonary bleeding and acute respiratory distress syndrome (ARDS), 
and mortality. Hence, the results of the study may lead to novel treatment approaches that can be 
readily applied in clinical practice. The decision to provide preemptive non-invasive therapies or 
early intensive care admission could lead to significant breakthroughs in managing the disease. 
 
Within the DeLEPTO program’s vision of developing tools to increase the survival of leptospirosis 
patients, this project will explore the avenue of novel tertiary care. Specifically, the program will look 
into the possibility of CFI repletion using plasma transfusion, cytokine depletion strategies using 
hemoperfusion (HP), and extracorporeal membrane oxygenation (ECMO). It would be interesting to 
see how such interventions could work individually or in a pipeline with other proposed 
interventions. In addition to plasma therapy, ECMO was observed to improve outcomes in severe 
leptospirosis (1). As a secondary endpoint, it would also be interesting to know if CFI can 
prognosticate who will benefit the most from such interventions. 
 
3.​ Significance (not to exceed 300 words): refers to the alignment to national S&T priorities, 
strategic relevance to national development, and sensitivity to Philippine political context, culture, 
tradition, and gender and development. 
 
Many leptospirosis patients still develop serious, costly, and potentially fatal complications. Although 
there are guidelines to manage leptospirosis effectively, these treatment strategies, such as dialysis, 
ICU care, and hemo-respiratory support, incur a relatively high cost for the patient. For example, 
patients developing acute kidney injury requiring dialysis or patients with pulmonary failure requiring 
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mechanical ventilation for lung support will incur an additional cost per week amounting to Php 
24,000.00 for four sessions of hemodialysis or Php 70,000.00 for daily use of a mechanical 
ventilator, and a conservative amount of Php 35,000.00 in ICU stay. This still does not include 
laboratory tests required daily. 
 
Moreover, despite these interventions, the hospital stay and mortality rate of leptospirosis remain 
significant. With the need to properly allocate financial, medical, and human resources, clinicians 
need to have a more efficient and timely way to identify who will develop such complications. There 
needs to be more understanding of how to determine which leptospirosis patients need treatments 
the most, especially preemptive care for secondary prevention and treatment of severe cases. 
 
It is essential to underscore that the results of the study can impact the therapeutic and prognostic 
guidelines of leptospirosis that is prevalent in a tropical country such as the Philippines. 
Prognosticators can help select persons who will benefit most from limited intensive care; these 
therapies would instead be made available for other patients requiring these life-saving treatments. 
The proposed therapy is also a relatively safe and inexpensive plasma transfusion. Plasma is 
usually among the least-utilized blood products in blood banks and is generally discarded. If found 
effective, we may repurpose plasma into a life-saving measure. 
 
4.​ General Objective: 
To evaluate the clinical applicability of selected interventions and the use of complement factor I 
(CFI) gene expression as a prognosticator for developing pulmonary complications in leptospirosis. 
 
5.​ Specific Objectives: 
1)​ To determine the effect of plasma component transfusion in moderate leptospirosis on the 
following outcomes: 
a)​ Mortality 
b)​ Significant pulmonary involvement 
c)​ Refractory hypotension 
d)​ Days to renal recovery 
e)​ Hospital stay 
2)​ To determine the effect of hemoperfusion in moderate to severe leptospirosis on the following 
outcomes: 
a)​ Mortality 
b)​ Significant pulmonary involvement 
c)​ Refractory hypotension 
d)​ Days to renal recovery 
e)​ Hospital stay 
3)​ To verify technologies for measuring CFI gene expression levels in the blood 
a)​ PBMC mRNA expression 
b)​ Blood protein levels 
4)​ To correlate CFI gene expression with the development of clinical complications 
a)​ Concerning the eventual occurrence of the aforementioned complications (controls only) 
b)​ Concerning response to treatment 
 
6.​ Review of Literature: discuss here the following: (a) related research that has been conducted, 
state-of-the-art or current technologies from which the project will take off; (b) scientific/technical 
merit; (c) results of related research conducted by the same Project Leader, if any; (d) Prior Art 
Search; and (e) other relevant materials. 
 
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Leptospirosis as a public health concern 
Leptospirosis is a prevalent zoonotic disease typically affecting tropical and subtropical countries. 
The most affected countries include those in Southeast Asia, including the Philippines (2). It is 
mainly contracted from water contaminated with pathogenic leptospires that enter through an 
interruption in the skin or mucosa of the host. It is typically seasonal, with increased incidence 
during times of flooding. However, it could be sporadic, especially among occupations associated 
with immersion in contaminated water, such as sewage management or farming (3). 
 
Though most cases manifest with flu-like illness, about 5-10% will have a more severe form called 
Weil’s syndrome. In a systematic review published in 2015, it was estimated that there were about 
1.03 million cases and 58,900 deaths due to leptospirosis worldwide (2). According to the PH 
Department of Health, in 2019, there were 3,011 cases, whereas in 2018, there were 5,116. A 
higher mortality rate is expected in the tertiary hospital setting (4). Most fatalities are males aged 
20–49 years (2). In addition to hepatic, renal, and central nervous system (CNS) involvement, which 
are classic complications of leptospirosis, the disease may also be complicated by acute respiratory 
distress syndrome (ARDS) and pulmonary hemorrhage (3,5). In the Philippines, the National Kidney 
Transplant Institute of the Philippines reports that during the 1st three quarters of 2019, there were a 
total of 226 leptospirosis-associated admissions in their hospital alone, with almost 78.3% requiring 
dialysis due to renal complications. 
 
The presentations and complications of leptospirosis 
Due to its protean manifestations and the wide spectrum of its severity, leptospirosis is classified 
presently as mild, moderate, or severe. The following description of the severity of leptospirosis is 
provided in detail by Haake and Levett (5), Barthi et al. (3), and the clinical practice guideline of The 
Leptospirosis Task Force (6). Mild leptospirosis is characterized by stable vital signs, and the 
absence of jaundice, renal failure, and pulmonary complications. Nonspecific symptoms, such as 
fever, headache, myalgia, nonproductive cough, and a maculopapular rash may be present. Over 
90% of mild leptospirosis cases typically resolve within a few days without progression to more 
severe illness. Whereas moderate leptospirosis patients present with unstable vital signs, jaundice, 
and signs of organ dysfunction, such as changes in mental status and oliguria. In contrast, severe 
leptospirosis is characterized by complications, including acute renal failure, episodes of 
hypotension, and the occurrence of gross pulmonary hemorrhage or congestion, herein referred to 
as pulmonary complications. To be more specific, such complication could be described as 
pulmonary injury as indicated by severe dyspnea and/or needing mechanical ventilator support plus 
one of the following in radiographs: patchy alveolar infiltrates in at least 1/3 of the lung area, 
presence of alveolar infiltrates in the chest radiograph, or gross hemoptysis/hemorrhage. However, 
in a clinical setting, pulmonary complications are challenging to evaluate and can be missed early 
on (7). Notably, the reports on the prevalence of pulmonary hemorrhage vary but can go as high as 
39% (8). 
 
Leptospirosis as a detrimental host response 
Weil’s syndrome can be considered as a host response mechanism to leptospiral pathogens. It is 
generally characterized by hyperinflammatory reactions in one or more of the host's organs, such as 
the kidneys, liver, joints, meninges, and other organs (3,5). Molecularly, such responses are 
manifested as increases in blood levels of tumor necrosis factor- alpha (TNF-alpha) or interleukin 6 
(IL-6) (5,9). Such a storm could be due to the loss of modulatory molecules, the brute force of the 
degree of activity of inflammatory pathways, or both. If both, such phenomena could reinforce each 
other. On the one hand, the hyperactivation of pathways, such as the alpha-interferon pathway and 
the Toll-receptor- mediated pathways, is strongly suspected (5). On the other hand, hyperactivity of 
the alternative complement pathway, through the lack of CFI inhibition, is also strongly suspected 
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(Nevado et al. 2019, unpublished). These speculated mechanisms will be the central basis for this 
proposal's depletion and repletion approaches. Since Weil’s syndrome is a host response, intuitively, 
one considers the human host the most practical source of an indicator biomarker. This is pertinent 
to recommendations on the diagnosis and therapy of leptospirosis that advocate the importance of 
early diagnosis and treatment. 
 
Moreover, in leptospirosis, neutrophil extracellular traps are innate mechanisms of the body against 
infection by extruding chromatin from neutrophils to sequester circulating pathogens through a 
process called netosis (10,11). Netosis was observed to be active during leptospirosis (10) and may 
be involved in the detrimental effects of the infection.  
 
Limitations of the current management of leptospirosis 
The present management of leptospirosis is mainly clinical and supportive (5). The cornerstone of 
the current management of leptospirosis heavily relies on antibiotics, supportive treatment, and 
alleviation of symptoms. At the start of suspicion of the conditions, antibiotics such as beta-lactams 
(penicillin G, ampicillin, and ceftriaxone), doxycycline, and azithromycin are typically administered. 
These are accompanied by supportive interventions, such as intravenous fluids, electrolyte 
correction, inotropic support, ventilatory and renal replacement support if applicable, and nutritional 
support. Supportive treatment includes medications to alleviate symptoms such as fever, headache, 
and bodily pain, as well as to manage bleeding manifestations. In general, the approaches are 
reactive and still need further refinement, as reflected by the persistently significant mortality in 
complicated conditions. 
 
Importance and challenges of early intervention in leptospirosis 
Management at the early phase of the disease seems to lower the risk of complications and fatality 
(12), and early diagnosis and treatment remain the most practical public health strategy to deal with 
leptospirosis outbreaks. This is particularly important in pulmonary complications because of the 
associated increased mortality, consistently observed to be typically more than 50% in cases of 
acute lung injury and more than 80% in the presence of pulmonary hemorrhage (8,13,14,15,16,17). 
Early diagnosis and prognosis of leptospirosis present a significant challenge even in tertiary 
hospitals because both remain heavily dependent on clinical parameters, such as the presence of 
jaundice or low urine output, which are mainly concurrent with the severe state of patients. 
 
Contrary to expectations, clinical and laboratory parameters for leptospirosis do not help predict the 
development of complications. Instead, laboratory testing of biomolecules, such as high creatinine 
levels and blood electrolyte levels to indicate renal failure, low partial thromboplastin time, aspartate 
aminotransferase or alanine aminotransferase to indicate liver failure, and the derangement of blood 
gases to indicate lung failure, are used to detect the presence of end-organ complications 
(16,18,19,20). Other generic predictors include blood cell counts and gender (21). However, all the 
parameters mentioned above occur during the phase wherein end-organ damage is apparent and 
may have less impact on preventing complications. 
 
Current practices in prognosticating leptospirosis 
Clinical parameters are currently the most supported means of prognosticating leptospirosis. 
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 likely already late manifestations and could be less reliable in the crucial 
clinical decision-making. This is not surprising as most evidence was obtained from clinical studies 
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with minimal molecular data. For example, a study on patients with evident acute lung injury 
concluded that hemodynamic disturbance, high serum creatinine level, and elevated serum 
potassium level were determinants of mortality (16,18). Moreover, such a panel of clinical 
determinants typically differs widely across various studies (18,19,23,24). Even the determination of 
the presence of anti- leptospiral immunoglobulins, more commonly detected through IgM 
microagglutination tests and IgG, cannot predict the eventual development of complications in 
leptospirosis, probably because of the nonspecific immune responses of the host at this point of the 
disease history. 
 
The concept of host molecular markers 
Because molecular host changes may occur early during the pathogenesis of diseases, they could 
be better alternatives for the prognosis and diagnosis of diseased conditions. Molecular biomarkers 
are molecules that are taken from an organism to indicate a particular condition. Traditionally, 
diagnostics for infectious diseases mainly rely on biomarkers that originate from the corresponding 
pathogen. While these biomarkers confirm the presence of the pathogen, they do not provide 
physicians with a way to predict whether the patient will develop Weil’s disease. Meanwhile, 
antibody-detecting diagnostics are only capable of diagnosing leptospirosis but not differentiating 
between milder forms and complicated forms. 
 
This underscores the importance of prognostication by host response. Host inflammatory processes 
are supposedly evolution-derived reactions 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 in acute-onset hyperinflammatory responses, inflammation by 
itself has become a cause of disorders that can adversely affect the human host. More likely, the 
detrimental reactions occur as a result of active biomolecular and hyperinflammatory reactions that 
perturb host homeostasis, resulting in disease or even death. In other words, in the case of severe 
leptospirosis, the presence of a pathogen may indicate infection. However, the issue of developing 
complications can be better assessed by the host response. 
 
It is also important to underscore that, in general, molecular markers on infectious diseases 
traditionally rely on the features of the pathogens, such as the amplification of the flagellin B gene 
(flB) in pathogenic Leptospira. However, such markers may only appear in the acute phase and may 
not mean that the patient will go into Weil’s syndrome. Investigations on the use of host biomarkers 
are relatively novel, considering that high throughput technology has become available to countries 
that are greatly affected by infectious diseases. This technology is able to screen a large number of 
candidate markers that can have an impact on the diagnosis and prognosis of diseases. In addition, 
candidate pathways/genesets can be inferred, which in turn can serve as markers themselves and 
may also suggest potential mechanisms of injury. In summary, by considering the host response 
mechanism as a potential source of markers for patients with leptospirosis, the present discovery 
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. However, as in the case of 
uncomplicated leptospirosis, a biomarker that can sufficiently predict complicated leptospirosis from 
the early onset of symptoms has yet to be found. 
 
Complement factor I (CFI) and its possible role in the pathogenesis of leptospirosis 
The gene complement factor I (CFI) codes for an important regulator of the complement system, 
particularly the alternative pathway. It inactivates C4b and C3b and prevents the formation of C3 
and C5 convertase enzymes (24). This leads to the inactivation of the complement system, 
including the formation of the membrane attack complex. Loss-of-function mutations in the gene 
resulting in loss-of-function per se, lack of secretion to the extracellular space, or the lack of 
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expression of the protein has been associated with susceptibility to organ damage via atypical 
hemolytic uremic syndrome (aHUS), and severe recurrent bacterial infections (25,26). The encoded 
protein is a heterodimer composed of a light (L) and a heavy (H) chain from a single peptide 
processed in most cells, including the PBMCs. The H chain is an allosteric inhibitor, and the L chain 
is the catalytic component. Inhibition of complement targets occurs upon binding to its target 
substrate that complexes with an appropriate cofactor that can disrupt the binding of the H chain. 
The exact mechanism of organ injury remains unclear. However, complement activation is 
increased, and C3 levels were found to be decreased in those with decreased CFI activity. A 
possibility of a self-imposed injury by complement overactivity is speculated but needs to be 
substantiated. On the other hand, the resulting lessening of activity could result in dire 
consequences due to the complex interaction of the system with other immunologic processes. In 
summary, it is surmised that the increased complement system may directly contribute to organ 
injury, either as a direct mechanism or a mediator of other immunological processes that result in 
complications. 
 
Concerning leptospirosis, it was observed that pathogenic leptospira are resistant to complement 
attack and may contribute to sustained hyperactivity of the complement system (27,28). The 
bacteria activate the complement system mainly through the alternative pathway, consistent with the 
CFI hypothesis above. In addition to activating the complement system, it was also observed that it 
prevents the modulated inhibition of the complement system by binding and inhibiting factor H, a 
cofactor for CFI. Interestingly, a recent report considers the possibility of severe leptospirosis being 
a variant of HUS (29). Hence, this raises the consideration that Weil’s syndrome is a hemolytic 
uremic syndrome variant due to complement activity. 
 
CFI as a marker for pulmonary complications in leptospirosis 
A study from Nevado and colleagues in 2019, sponsored by the Department of Science and 
Technology (DOST Philippines) and the Philippine Council for Health Research and Development 
(PCHRD), showed a verified association of CFI levels with the eventual development of pulmonary 
complications in leptospirosis. Using PBMC samples from the first 24 hours after admission from 
patients with leptospirosis who have no evident severe organ damage, transcriptional profiling is 
then performed. Microarray values of CFI mRNA levels were lower in cases who eventually 
developed clinically serious pulmonary complications compared with those who did not (4.10 
[95%CI: 3.89-4.30] versus 4.60 [95%CI: 4.50-4.70; unadjusted p<0.01). ROC area is 0.89. The 
levels were only significantly different (which is lower) for those with complications and were even 
lower when compared with healthy participants (4.52 95%CI: 4.45-4.60]; unadjusted p<0.01) and 
with febrile controls (4.44 [95%CI: 4.36-4.53]; unadjusted p<0.05). 
 
Subsequent verification in a separate and independent sample of patients using quantitative 
polymerase chain reaction (qPCR) showed a generally similar and significant trend. Using deltaCq 
values, the mean for those who eventually developed pulmonary complications is significantly 
higher (lower CFI expression compared with those without (15.68 [95%CI: 14.54-16.81] versus 
11.65 [95%CI: 10.72-12.59; unadjusted p<0.01). ROC area is 0.92, with optimum specificity of 
85.42% on 100% sensitivity. 
 
Curious as to the occurrence of most deaths in complicated leptospirosis due to pulmonary 
complications, especially pulmonary hemorrhage, we correlated CFI levels with mortality. Overall, 
50% (3/6) of those with pulmonary complications died. Four deaths occurred, with 3 (75%) having 
pulmonary complications. For all deaths with pulmonary complications, the adverse respiratory 
condition was assessed two days after the determination of the low CFI mRNA levels. In contrast, 
the lone mortality had minimal pulmonary complications and had died two weeks after admission. 
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Noticeably, the CFI level is higher than the other deaths. 
 
Hence, levels of CFI can be used to indicate the eventual occurrence of complicated leptospirosis, 
especially pulmonary complications. The possibility that this molecule is causative suggests that it is 
a potential therapeutic target in the alleviation and prevention of complications. 
 
Proposed interventions in the treatment of complications in leptospirosis 
A.​ Prophylactic plasma transfusion (PPT) for less complicated leptospirosis 
Studies on plasma transfusion in leptospirosis indicate potential benefits in terms of reduction in 
mortality due to complications from leptospirosis. In particular, most studies focused on the stages 
when complications are already present; hence, termed therapeutic plasma exchange (TPE) is also 
an interesting approach as it could be a cost-effective means to replete CFI and remove toxins from 
the body. Fresh frozen plasma has been found to be stable during blood collection. Because most 
people have normal CFI activity (data unpublished), such could explain a potential therapeutic 
application of TPE. Several studies have shown potential promise for the intervention (Table 1). In 
particular, the mid-sized study by Herath et al. (15) and Trivedi et al. (8) showed a more than 60% 
reduction in death in severe leptospirosis using therapeutic plasma exchange despite the 
occurrence of pulmonary hemorrhage. 
 
In the present study, such repletion therapy as a preemptive treatment for less severe leptospirosis 
appears promising in the context of CFI repletion. As pathway analyses of CFI gene expression 
demonstrate the downregulation of the alpha interferon signaling and the upregulation of the 
alternative complement pathway due to the relative lack of intrinsic inhibition that is predictive of the 
development of serious complications (Nevado et al. 2019, unpublished), the study entertains the 
possibility of repletion therapy as a prophylactic means to prevent deteriorations in leptospirosis. 
Thus, to differentiate from prior studies, the present study will look into the benefit of prophylactic 
plasma transfusion (PPT) if administered before the onset of complications. 
 
B.​ Hemoperfusion (HP) and cytokine depletion for complicated leptospirosis 
The hypothesis that certain molecules which are able to drive the cytokine storm may be present is 
plausible. The presence of traditional molecules like TNF-alpha and IL6 (9) could result in 
self-inflicted tissue damage. Also, unique to leptospirosis, the low CFI expression may be a result of 
the presence of certain inhibitors, such as the leptospiral immunoglobulin-like proteins (30). Thus, 
the means to remove injuring molecules in the blood is a possible therapeutic approach. 
 
One feasible approach that is available at the National Kidney and Transplant Institute (NKTI) is 
hemoperfusion (HP). The procedure involves passing the blood of patients ex-vivo to activated 
carbon or resin cartridges to remove potentially detrimental molecules either from the pathogen or 
the host that contribute to the development of tissue injury (31). Mainly utilized in sepsis, some 
report potential benefits on mortality, with a relative risk reduction of 0.88 (1,33). However, 
conclusive evidence, particularly in leptospirosis, is needed. More importantly, there is no known 
evidence for its use as a preemptive strategy. One report suggested the potential use of 
hemosorption (33). 
 
Although there was scarce evidence on the use of hemoperfusion in leptospirosis, its utility in 
hyperinflammatory conditions can be approximated by evidence in sepsis and other severe 
infections. The most similar to our study would be those that will use Jafron HA330. Interestingly, 
the device was found to improve organ function at day seven significantly, and  decreased both ICU 
admissions (24% vs. 57% in control; p=0.02) and 28-day mortality (28% vs. 67% in control; 
p=0.009) in the hemoperfusion group compared to controls in patients with nonpulmonary sepsis 
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with significant lung injury (34). There is also a substantial lowering in mechanical ventilatory time. 
 
Table 1. List of reports on the use of TPE as an intervention for pulmonary complications in severe leptospirosis 
Authors 
Year 
published 
Description of pulmonary 
complication 
Description of TPE 
Survival 
Other remarks 
Herath et al.15 
2019 
Severe pulmonary 
hemorrhagic syndrome 
(SPHS) 
Within 48 hours after 
SPHS 
35/59 
(74.5%) 
Without TPE, 
10.5% (2/19) 
survived 
Ekinci et al.35 
2018 
Diffuse bilateral pulmonary 
infiltrates 
5 courses of plasma 
exchange 
  
Turkey; patient is 
8-years old 
Taylor and 
Karamadoukis36 
2013 
Pulmonary hemorrhage 
Two 4-litre exchanges 
using a combination of 
fresh frozen plasma 
and human albumin 
solution 
1/1 
United Kingdom 
Cerdas-Quesada37 
2011 
Diffuse bilateral pulmonary 
infiltrates 
5 courses of plasma 
exchange 
1/1 
Argentina 
Bourquin et al.38 
2011 
ARDS 
5 sessions of plasma 
exchange 
1/1 
Switzerland 
Trivedi et al.8 
2010 
Pulmonary hemorrhage 
2 cycles of plasma 
exchange, 24 h apart, 
25 ml/kg per cycle 
70/114 
(61%) 
India; Without 
TPE, 17% (5/30) 
survived 
Chen et al.39 
2007 
Pulmonary hemorrhage 
8 courses of plasma 
exchange 
1/1 
Taiwan 
Landini et al.40 
1981 
Severe leptospirosis with 
hemorrhagic manifestations 
Average 2 - 5-3 liters 
per session; frequency 
not indicated 
6 patients 
recovered 
Italy 
 
Aside from resin absorption, a more specific approach is to deplete endotoxin levels using 
polymyxin B or to target hypothetically toxic innate molecules (41). A systematic review of controlled 
studies using the endotoxin-specific polymyxin B-immobilized hemoperfusion showed a significant 
reduction in mortality (0.68. 95% CI 0.51–0.91; P=0.01) with significant differences in the presence 
of sepsis or with an Acute Physiology and Chronic Health Evaluation II (APACHE II) score <25 is 
considered (42). Again, similar to the Huang study, an interesting trend emerged that suggests early 
intervention using hemoperfusion may be more beneficial than late treatment in the preemptive 
management of inflammatory conditions, thus supporting and justifying a preemptive role rather 
than a reactive one. 
 
Besides the HA series, for the cytokine-targeted approach, recent trials on hyperinflammatory 
conditions also commonly used CytosorbTM cartridges. However, most studies showed no 
conclusive benefit if used in patients with more severe conditions. In a meta-analysis of patients with 
inflammatory conditions requiring intensive care, hemoperfusion using CytosorbTM was reported to 
have a significant increase in mortality in the CytosorbTM group compared with control (29% versus 
24%; RR = 1.24 [95% CI, 1.04–1.49; p=0.02) (43). Data on earlier interventions is lacking. 
 
C.​ Extracorporeal 
membrane 
oxygenation 
(ECMO) for severe respiratory compromised 
leptospirosis  as a rescue treatment 
 
ECMO involves the use of a pump that circulates the blood out of the human body to an artificial 
lung that oxygenates the blood and pumps the blood back to the body. Considered a lung by-pass 
means, it was proven to be useful in cases when severe respiratory decompensation occurs. 
Several studies found a promising trend in the use of ECMO in leptospirosis. Several cases of using 
ECMO in leptospirosis showed an encouraging trend towards a decrease in mortality (Table 2; 44). 
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In the Philippines, a case report on the use of ECMO was documented in a leptospirosis-afflicted 
male with severe ARDS and pulmonary hemorrhage (1), resulting in the resolution of complications. 
At the same time, Vandroux et al. (51) reported a 75% survival rate among patients with moderate 
to severe acute respiratory distress syndrome (ARDS) (6 out of 8). 
 
To assess the potential benefit of ECMO in severe pulmonary cases of leptospirosis, an indirect 
approach is warranted. This is because the use of ECMO in leptospirosis was mostly reported in 
small case series with minimal controls. 
 
For the sake of evaluating benefits, the nearest evidence that used controlled comparators had 
been acute respiratory distress syndrome (ARDS) and severe pneumonia. In these studies, the 
potential clinical benefits, such as decreased mortality and hospital stay, are significant. For this 
purpose, a combined analysis of 2 large RCTs, namely, the Conventional Ventilator Support vs 
Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Failure (CESAR) trial and the 
ECMO to Rescue Lung Injury in Severe ARDS (EOLIA) trial comprising 214 ECMO cases and 215 
control was performed (45). Results showed a significant reduction in death (36% vs 48%, p=0.013) 
after a terminal 90-day period. This is despite rescue ECMO administered in 17% of the control 
group. Thus, this finding may serve as a satisfactory justification for the extrapolation of ECMO 
benefits to pulmonary complications of leptospirosis. 
 
Safety concerns on the proposed study interventions 
A.​ Prophylactic plasma transfusion (PPT) 
Although the rates are generally low, adverse events associated with plasma transfusion do exist 
and can be life-threatening. In general, the more common serious events can be grouped into 3: (1) 
transfusion-associated circulatory overload (TACO), (2) transfusion-related acute lung injury 
(TRALI), and (3) anaphylactic reactions (46,47,48). According to the US-based National Healthcare 
Safety Network Hemovigilance Module, from Jan 1, 2013, to 2018, there were a reported 8.34 
million transfusions of any blood component, with 1.25 million transfusions being of plasma. Of 
these, there were about 23,083 adverse reactions, with just 1,340 being serious. Serious TACO, 
TRALI, and anaphylaxis occurred in 353, 44, and 75 cases, respectively, making their cumulative 
prevalence well less than 0.04% (49). Such rarity of serious events is supported by a 7-year 
(2006-2012) review of the International Surveillance of Transfusion- Associated Reactions and 
Events (ISTARE) database, with each individualized adverse event having less than 100 per 
100,000 transfusions, with TACO and TRALI reported in <10 per 100,000 transfusions (50). 
Interestingly, whole blood-derived plasma was noted to be less associated with allergic and 
hypotensive reactions compared with apheresis-derived plasma, as the former will be applied in the 
present study. 
 
Table 2. List of sample reports on the use of ECMO as an intervention for pulmonary complications in severe 
leptospirosis 
 
Authors 
Year 
published 
Description of 
presenting pulmonary 
complication 
Description of ECMO 
Survival 
Other 
remarks 
Chavez et al.1 
2019 
Pulmonary hemorrhage 
 
1/1 
Philippines 
Vandroux et al.51 
2019 
Moderate or severe 
ARDS 
Typically, on the day of 
ARDS, for 5-13 days 
6/8 (75%) 
France 
Cantwell et al.52 
2017 
Pulmonary hemorrhage 
On the day of 
admission, for eight 
days 
1/1 
Chile used a 
double setup 
Umei & Ichiba53 
2017 
ARDS with 
On the day of referral, 
1/1 
Japan 
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endobronchial bleeding 
five days after onset of 
ARDS for 11 days 
Pardinas et al.54 
2017 
ARDS with hemoptysis 
on 
day 2 
On day 2 for 14 days 
1/1 
USA 
Liao et al.55 
2015 
Pulmonary hemorrhage 
On the day of 
hemoptysis, for six 
days 
1/1 
Taiwan 
Kahn et al.56 
2006 
ARDS with 
endobronchial bleeding 
On the day of 
admission (3- 4 days 
after documented 
bleed), for 60 hours 
1/1 
Austria 
Arokianathan et 
al.57 
2005 
Pulmonary hemorrhage 
Provided 
post-hemoptysis for 
183 hours 
1/1 
United 
Kingdom 
 
Other important but less serious events include hemolytic, anaphylactic, septic, febrile, delayed 
hemolytic, and delayed serologic reactions. Fortunately, they all have been observed to occur in 
less than 1% (46,49). 
 
Measures to prevent and manage the occurrence of adverse events in plasma transfusion 
The latest definition of TACO was adopted in 2018 from the collaborative works of the International 
Society of Blood Transfusion (ISBT), International Haemovigilance Network (IHN), and the former 
American Association of Blood Banks (AABB) as an onset of respiratory symptoms within 12 hours 
of blood product transfusion. This may be determined by clinical physical examination, radiographic 
chest imaging, other noninvasive assessments of cardiac function, or a combination. Signs include 
tachycardia, a widened pulse, and rales heard bilaterally. The chest radiographs may show signs of 
congestive heart failure: bilateral infiltrates and an enlarged cardiac silhouette. An elevated 
pulmonary capillary wedge pressure (PCWP) and central venous pressure (CVP) can be observed 
(58). Hence, patients at risk include those with heart failure, renal failure, and those >70 years old. 
However, the most important risk factors are the rate of infusion and the amount of infusion. High 
rates (>300 ml/hr) or high volume (> eight units per day) significantly increase the risk for TACO 
(59,60). According to the 2011 annual prospective United Kingdom-based Serious Hazards of 
Transfusion Report (SHOT), an optimum rate with significantly fewer incidents is <120 ml/hour for 
whole blood (58). Subsequent recommendations to minimize TACO place the infusion at <120 
ml/hour for low-risk and <85 ml/hour for those with risk (age >60, history of congestive heart failure, 
myocardial infarction, chronic renal failure, or chronic lung disease). In addition, furosemide 40 mg 
is recommended to be injected every 6 hours for those aged >60 years old (58). Aside from the use 
of loop diuretics, placing the patient in a seated position, providing supplemental oxygen, and 
prompt consultation with cardiology are also recommended (58,60). 
 
TRALI is another potentially serious side effect. By consensus, the 2019 TRALI definition states that 
it is the presence of an acute onset of hypoxemic respiratory distress during or within 6 hours of a 
blood product transfusion. Specifically, the hypoxemic state is defined as PaO2/FiO2 <300 mm Hg 
or O2 saturation <90% on room air (46,48). Currently, the main notion is that the injury is caused by 
neutrophil-mediated damage to the lungs due to interaction with infused antibodies from the 
transfused blood (47). In 20% of TRALI cases, instead of antibodies, the culprit is suspected to be 
biologically active lipids (BALs). The main risk predictor is the blood being from multiparous women 
who had developed antibodies from previous pregnancy tissues (48). Despite a 5-10% mortality, the 
incidence of such events is very low (61). Due to the rarity of the event, it is decided that patients be 
preferentially provided with plasma exclusively from men or, if from women, who are uni- or 
nulliparous. Monitoring of signs and symptoms and vital signs can be done hourly until 8 hours after 
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the end of transfusion. Management will be mechanical ventilation, if indicated, applying restrictive 
tidal volume and possibly diuretics, fluid restriction, and, in extreme cases, extracorporeal 
membrane oxygenation (46). 
 
Note that both TACO and TRALI are usually observed in the setting of plasma transfusion with high 
volume indications (~8 units or more a day) (62). Thus, as a precaution, a volume of just four units 
per day is then prescribed in this study. 
 
For the present study, FFP will be infused using units two times a day, with each unit infused for 
about 3 hours. This assumes a low-volume (12-15 ml/kg) volume at <85 ml/hour. This range of 
plasma dose is within the recommended range in coagulation factor repletion therapy, as observed 
by Chowdary and colleagues (59). Despite previous studies on TPE in leptospirosis-afflicted 
patients done in critical patients having used high volume exchanges at about 25-30 ml/kg per day 
in exchange procedures (8,16), the volume to be used in the present study is deemed to suffice due 
to the recipients having less serious conditions. The important assumption is that the indicated 
volumes and rates are within the limits for precaution for TACO and TRALI. 
 
The third common serious reaction is the cumulated group of anaphylactic and hemolytic reactions. 
Usually occurring as reactions to blood additives, the clinical manifestations include hypotension, 
tachycardia, nausea, vomiting, abdominal pain, severe dyspnea, pulmonary or laryngeal edema, 
and bronchospasm, usually within 4 hours after transfusion (46). Related to this, hemolytic reactions 
are mainly due to blood ABO/Rh mismatching resulting in increased temperature, increased heart 
rate, chills, dyspnea, chest or back pain, abnormal bleeding or shock, hemoglobinuria, epistasis, 
oliguria or anuria, disseminated intravascular coagulation, and pain or oozing at the intravenous site 
(46). Presently, with routine use of typed matched donors and more advanced blood processing, the 
events are considered rare and, if occurring, rarely fatal, more so in cell-depleted plasma. The 
events are less than 1%, and if these occur, supportive care is generally sufficient, which in extreme 
cases may include respiratory support and/or the use of antihistamines and inotropic drugs (46,47). 
In the present study, to prevent this from occurring, cross-matched plasma will be used as the basis 
for transfusion. 
 
Less serious events may also occur, such as febrile nonhemolytic transfusion reactions, graft versus 
host type reactions, delayed hemolytic reactions, and post-transfusion purpura that usually 
manifests with mild and temporary symptoms. However, these are usually less than 1% in incidence 
and are given supportive treatment (47,48). 
 
To minimize the occurrence of these adverse events, hemovigilance guidelines were made and  
published, more notably by the United States Biovigilance Network and International 
Haemovigilance Network (IHVN) (48). Several procedures were suggested to lessen the occurrence 
of the events. Clinically, blood transfusion cessation is recommended in the presence of clinical 
signs and symptoms that include fever, chills, respiratory distress, hypo/hypertension, skin 
manifestations (redness, edema, urticaria), hemoglobinuria, nausea/vomiting, oliguria/anuria, or 
anaphylaxis that is suspiciously associated with transfusion. Recommended standard laboratory 
workup after an event includes clerical checks (bag, label, patient sample), repeat ABO testing on 
posttransfusion samples, visual checks of the physical product unit, and direct antiglobulin test. 
 
Locally, the monitoring of patients during transfusion can be guided using the guidelines set by the 
NKTI Blood Bank (63). Here, the following symptoms will require stoppage of the transfusion (as 
adapted from the proposed guidelines of the NKTI Blood Bank): (1) fever with or without chills; (2) 
shaking chills (rigors) with or without fever; (3) pain at the infusion site or in the chest, abdomen, or 
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flanks; (4) blood pressure changes, usually acute, either hypertension or hypotension; (5) 
respiratory distress, including dyspnea, tachypnea, wheezing, or hypoxemia; (7) skin changes, 
including urticaria, pruritus, flushing, or angioedema; (8) nausea with or without vomiting; (9) 
darkened urine; (10) bleeding or other manifestations of a consumption coagulopathy; and (11) 
other signs or symptoms for which the attending physician determines warrants the discontinuation 
of infusion (such as gastrointestinal symptoms). 
 
Risk-benefit justification for research investigation on plasma transfusion in leptospirosis 
Because of the precautions mentioned above (blood screening, cross-matching, <6 units/day, at 
<80 ml/hour), the estimated risk for adverse events should be at most <5%, with the serious events 
to be <1%. If the estimated benefit is placed conservatively at a 30% reduction in death (which is 
objectively much lower than the >60% reduction predicted by both Herath et al. (15) and Trivedi (8), 
the projected benefits still greatly outweigh the risks involved; thus, justifying the implementation of 
the study. 
 
B.​ Hemoperfusion (HP) 
Hemoperfusion has been used as a depletion procedure in order to lessen the concentration of 
detrimental molecules that can lead to adverse outcomes. In particular, this treatment has been 
attempted in severe conditions, such as sepsis, kidney and liver failure, drug overdose, and 
poisoning (41). 
 
As part of the procedures, adverse events related to vascular access used during hemoperfusion 
may occur, such as bleeding, pain, and infections. This is more likely due to the use of 
anticoagulation that prevents blood from clotting, especially while it is outside the body. Although 
optional, anticoagulation may be recommended to protect the patient from developing clots that may 
lead to thrombosis (42). 
 
The most important consideration in increasing the material-related safety concern of 
hemoperfusion is the improvement of material biocompatibility. From the initial use about a century 
ago of aluminosilicates (zeolites) and charcoal, then to organic polymer ion exchange resin, and 
presently to synthetic porous polymers (e.g., styrene or acrylic acid-based), biopolymers have 
greatly improved in terms of safety, resulting in designs with generally minimal risk (42). Previous 
concerns related to biocompatibility include cytotoxicity due to released matter from the sorbents, 
the abnormal and possibly detrimental induction of intrinsic mechanisms, such as the coagulation or 
complement pathways, the destruction of cells resulting in leukopenia, thrombocytopenia or similar 
effects, or even the depletion of unwanted molecules resulting to loss of homeostasis (64). 
 
Another important feature of present-day designs is that they minimize the direct contact of sorbent 
material with the cells of the blood and/or the use of various sorbent coatings to minimize such 
inductive interactions (42). In general, present innovations in hemoperfusion (material, 
specific/limited affinity, different pore sizes, membrane choices, biocompatibility) have resulted in 
more selective depletion compared with plasma exchanges and plain hemofiltration. The latter has 
concerns about the removal of potentially beneficial molecules, such as clotting factors, albumin, 
antibiotics, and protective antibodies. In sum, because of newer materials and filter designs, the 
incidence of long-term or serious reduction in blood cell levels is considered minimal. 
 
As the safety of hemoperfusion depends largely on materials used in consumables and in those 
associated with the procedures themselves, we will limit the adverse events unique to Jafron 
HA330, which is an inorganic resin-based polystyrene sorbent-containing hemoperfusion used in 
the present study. 
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Most reports on the use of Jafron HA330 had reported only minor adverse events, with minimal 
serious events that were no more than comparable to either a historical reference or a parallel 
control. In fact, several studies on a total of hundreds of patients showed no serious adverse events, 
except for occasional fever and rashes that cannot be differentiated from controls (64). Bleeding and 
thrombocytopenia, if noted, were transient and did not result in emergency intervention. In addition, 
in 1 case series, using a related Jafron HA280 that differs only in resin loading capacity with HA330, 
5 cases in 41 had adverse effects after hemoperfusion: 2 with nausea and vomiting, 2 with 
hypotension, and 1 with skin bruising after the perfusion (65). 
 
Reiterating, the present study will use a resin-based Jafron HA330 ultrafiltration/absorbent system. 
In brief, the sorbent part has hydrophobic properties that facilitate the adsorption of molecules that 
come into contact with it. Previous versions of this approach used charcoal. However, years of 
research have led to newer and presumably safer materials, such as the present non-ionic 
macroporous resin. The resins are similar to charcoals as they are made of micro-sphere 
agglomerates, which adsorb toxins on their surface. Presently, most nonspecific resin-based 
hemoperfusion, such as Jafron HA330 which makes use of styrene-divinylbenzene-based 
copolymers, are generally used in clinical practice (64). The Jafron HA330 (resin loading capacity = 
330 mL) is indicated for the depletion of cytokines, complements, free hemoglobin, and so on, as it 
has a pore size range from 500 Da to 60 kDa. It is sterilized using irradiation. 
 
Measures to prevent the occurrence of adverse events in hemoperfusion 
To prevent the occurrence of adverse events during hemoperfusion, several measures can be 
implemented. Patients with ongoing bleeding or with a platelet count below 80,000 will not undergo 
hemoperfusion. Due to assumed hepatic dysfunction, heparinization will be deferred unless the 
attending physician orders. The patients will be monitored at least hourly during hemoperfusion until 
2 hours after the procedure, using the following parameters: vital signs, fever, rashes, and bleeding 
(e.g. bruising, hemoptysis, or signs of gastrointestinal bleeding, etc.). 
 
Measures to address adverse events if they happen after hemoperfusion 
In the occurrence of common problems, mitigation measures may help in the management. For all 
suspicious cases, hemoperfusion should be stopped. For mild cases, supportive therapy with 
assurance is indicated. Paracetamol can be prescribed for fever and antihistamine for rashes. For 
more serious cases, specialized treatment should be provided, such as the transfusion of fresh 
frozen plasma and/or packed red blood cells for significant bleeding. 
 
Risk-benefit justification for research investigation on hemoperfusion in leptospirosis 
Overall, the hemoperfusion procedure has risks compared with the standard of care but with 
substantial possible benefits in hyperinflammatory conditions. Objectively, in preemptive designs, 
we can assume that a conservative estimate of relative risk reduction of 30% (which is less than the 
actual preemptive estimate of >60% in the study of Huang et al. (34)), the projected benefit can be 
justified to outweigh the risk, which is projected to be minimal with <1% reported rate for a serious 
adverse event. 
 
C.​ Extracorporeal membrane oxygenation (ECMO) 
Considering that the present study will use the veno-venous ECMO (VV-ECMO) because of the 
primarily pulmonary nature of leptospirosis-associated lung complications and because such 
procedure will be used in the study, we will limit the discussion of ECMO adverse events to such, 
while giving proper regards to adverse events common with veno-arterial ECMO (VA-ECMO) 
Although VV-ECMO has been considered an important life-saving procedure in patients who need 
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pulmonary support, it is still an extraordinary measure with expected adverse effects, some of which 
are potentially debilitating or even fatal. Nonetheless, with proper expertise and equipment, the 
overall prevalence is generally low. In a review of the post-marketing surveillance data from the 
Food and Drug Administration (FDA) Manufacturer and User Facility Device Experience (MAUDE) 
database from January 2009 to March 2019, Khalid and his colleagues (66) reported the most 
common adverse events. The most common patient-related adverse events were hemodynamic 
decompensation of patients, death, atrial perforation, and bleeding. Other less common 
complications noted were infection and air embolism. The most commonly reported mechanical 
failure modes were in the following circuit components: mechanical pump membrane oxygenator 
and access cannulae. However, it is important to note their inferences. First, the total reported 
adverse event is just 82 out of the estimated >100,000 ECMOs being monitored. Although this may 
imply a low rate of complications, post-marketing registries have been noted to be notorious for 
underestimating the true rate of complications. Despite this, it is fair to assume that this 
extraordinary procedure is relatively safe but needs to be done under the strictest of indications. It is 
also important to underscore that due to the critical or serious condition of the patients, it is difficult 
to discern if death or hemodynamic instability is due primarily to ECMO. 
 
In a pooled analysis of 20 studies encompassing 1,866 patients with cardiogenic indications, 
VA-ECMO has been observed to be associated with renal replacement therapy in 6.0% and major 
or significant bleeding in 40.8%. Other reported adverse events include lower extremity ischemia, 
fasciotomy 
for 
compartment 
syndrome, 
lower 
extremity amputation, stroke, neurologic 
complications, acute kidney injury, thoracotomy for bleeding or tamponade in postcardiotomy 
patients, and significant infection (67). However, if such adverse events were due mainly to the 
arterial access/complication or due to the primary disease, it is not clearly indicated. 
 
Due to the foresight that ECMOs are complicated with the need for specialized and yet diverse 
expertise, the Extracorporeal Life Support Organization (ELSO) has recommended the 
establishment of a multidisciplinary team to handle cases (68). Such an approach has resulted in 
significant reductions in mortality, hospital stay, and adverse events in pulmonary cases undergoing 
VV- ECMO (69). 
 
Measures to prevent and manage the occurrence of adverse events in ECMO 
For VV-ECMO, the most anticipated serious complication is bleeding. As intrinsic coagulation 
derangements and the use of anticoagulation is mostly required, such adverse reactions are 
expected. In a study by Popugaev et al. (70), the coagulation derangements are multifactorial and 
may need to be assessed before, during, and even after the procedure. Coagulation testing should 
be done and corrected to acceptable levels prior to interventions and during the management of 
bleeding. The key is to maintain hemostasis and cardiorespiratory stability during the ECMO 
process. 
 
Another preventable complication is infection. As in any invasive procedure, standard care requires 
the use of aseptic procedures and proper wound care, as well as the judicious use of antibiotics and 
antiseptics. Infections should be treated aggressively and preferably guided by culture and 
sensitivity. Historically, the rate of infection at the wound site has been minimal in VV-ECMO (66). 
 
Lastly, as for hemodynamic instability, it is important to point out that both ECMO and the patient’s 
condition, or both, may contribute to its occurrence. However, in most cases, such etiology is hard to 
establish, especially the contribution of ECMO. With prudence, perhaps the justification of likely 
benefit compared to harm will justify only the application of ECMO to those who are deemed to 
require such to save their lives. 
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Risk-benefit justification for research investigation on ECMO in leptospirosis 
Using only the strictest inclusion criteria of severe pulmonary hemorrhage, the use of ECMO can be 
justified in leptospirosis. In the presence of severe pulmonary hemorrhage, an expected mortality of 
50-80% is observed. ECMO has been observed to reduce mortality by 25% in ARDS (68). Thus, 
with this potential clinical benefit with an expected rate of serious adverse events at <10%, the 
present study can argue that investigating ECMO is, therefore, justifiable in ARDS-like leptospirosis- 
associated pulmonary complications. 
 
7.​ Methodology: Discuss here the following: (a) variables or parameters to be measured and 
evaluated or analyzed; (b) treatments to be used and their layout; (c) experimental procedures and 
design; (d) statistical analysis; (e) evaluation method and observations to be made, strategies for 
implementation. (Conceptual/Analytical framework). 
 
Study design, participants, and comparators 
The present study is the first of the four-project Decreasing Leptospirosis Emergence through 
Prognosis and Treatment Optimization (DeLEPTO) Program. It will focus on clinical studies on 
possible clinical applications that can be used to prognosticate and treat leptospirosis. Project 2 will 
involve the construction of cell culture models, including epithelial endothelial cells and immune 
cells. This will serve as test models for leptospira supernatant-activated and patient-derived plasma. 
With these test models, plasma derangement based on pathologic (hyperinflammatory) findings 
from the plasma of patients with leptospirosis and corresponding interventions for plasma calibration 
will also be evaluated. Project 3 will involve in vitro (using modified human RBC and endothelial cell 
culture) and in vivo (using golden Syrian hamsters) testing of candidate interventions or therapeutic 
strategies using blood plasma. Findings from these studies will also be tested in cell culture models 
developed in Project 2. Lastly, Project 4 will be done in parallel with Project 1, which will aim to 
develop a fast point-of-care device that will assess CFI gene expression in patients. Microfluidics 
and digital droplet PCR (ddPCR) technologies will be utilized to create this device. Initial patient 
samples and subsequent pilot scale testing will be done with the assistance of NKTI in Project 1. 
 
Of note, Project 1 will not be dependent on the samples and the results of the other projects in the 
program but will provide them with vital clinical data and biological samples. In turn, the other 
projects, among their numerous works, will investigate how CFI is involved in the development of 
complications to leptospirosis and rationalize the treatment mechanism (e.g., plasma transfusion). 
The results of the other projects will be used to investigate the possible mechanisms of CFI. Further, 
conditions will be optimized for the digital droplet PCR (ddPCR) under Project 4 (LIGTAS), a 
technology developed by Dr. Pobre and his team at the DLSU. The ddPCR platform has many 
advantages compared with the usual qPCR, such as portability and accessibility, shorter processing 
time, reduced patient sample and reagent volume requirements, and may be more cost-effective in 
the long run. 
 
The project is composed of 4 objectives, with the third and fourth having sub-objectives, hereon 
referred to as Objectives 1, 2, 3a, 3b, 4a, and 4b (see the section on Objectives). 
 
All subjects will be recruited from the emergency rooms, wards, and clinics of the National Kidney 
and Transplant Institute (NKTI) and San Lazaro Hospital (SLH), as these hospitals serve as tertiary 
care centers for Weil’s disease patients in Metro Manila. Patient recruitment will be conducted upon 
approval of the Single-Joint Research Ethics Board (SJREB). Participants will be recruited only after 
signing an informed consent form. In the event that the patient is not physically or mentally capable 
of giving consent, a legally authorized representative or guardian may provide consent on their 
behalf. Only trained research personnel can and will interact with patients or their legal 
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representatives to be included in the study. The screened patients will be respectfully asked to join 
the study by explaining carefully the details of the study, especially the responsibilities and rights of 
the participants, as well as answering their concerns. Patients will be given adequate time to decide 
within the study constraints. 
 
For Objectives 1 and 2, the design would be open-label controlled trials. Objective 1 participants 
would be moderate and severe leptospirosis without hypotension (or those indicated for 
vasopressor support) AND pulmonary complications (Table 3). These participants will be recruited 
from both the National Kidney and Transplant Institute and San Lazaro Hospital as part of the 
multi-center collaboration. They will be randomized to receive either prophylactic plasma component 
therapy with conventional treatment (PPTTRT) or conventional treatment alone (PPTCONV). For 
Objective 1 (plasma transfusion), the decision to exclude those with hypotensive episodes is to 
allocate the patients for Objective 2 (hemoperfusion), where initial evidence had shown significant 
benefit for those with hypotension (Danguilan et al. 2022, unpublished). According to that study, a 
calculated 0% (0/18) mortality in HP compared with 36.84% (7/19) in the standard of care was 
observed. Moreover, Objective 1 may have overt acute kidney failure. As CFI level is a marker for 
the occurrence of respiratory failure, not renal failure, evidence suggests that plasma intervention 
works on severe leptospirosis (even those with overt renal failure) in preventing pulmonary 
hemorrhage. Thus, the study will hereon include patients with overt renal failure as long as there is 
no pulmonary hemorrhage. 
 
Objective 2 will involve patients with moderate to severe leptospirosis (Table 3) and will be 
randomized to receive either hemoperfusion treatment with conventional treatment (HPTRT) or 
conventional treatment alone (HPCONV). These participants will be recruited from both the National 
Kidney and Transplant Institute and San Lazaro Hospital. In addition to clinical characterization, the 
baseline blood levels of TNF- alpha, IL6, hsCRP, procalcitonin, and CFI (for Objectives 3 and 4) will 
be measured. Baseline and repeat apoptotic assays will also be performed on the blood samples. 
Proteins will be extracted from the hemoperfusion cartridges and quantified. Outcomes to be 
observed will be mortality, end-organ complications, need for emergency procedures, and hospital 
stay. 
 
Objective 3 will obtain data pertinent to the blood CFI levels of all participants, either in the form of 
PBMC RNA levels (Objective 3a) or whole blood protein levels (Objective 3b). The assays would be 
optimized quantitative PCR (qPCR, also known as real-time PCR or RT-PCR). The two sets will be 
compared for head-to-head correlation and will be optimized as needed depending on observed 
problems. The process should produce normalized values for the tests with optimized conditions. 
Although the qPCR has been optimized to measure CFI expression levels, there is a need to verify 
our conditions in a different laboratory (NKTI setting) and validate these findings in the clinical 
setting. 
 
Objective 4 aims to correlate CFI gene expression with the eventual occurrence of clinical 
complications and outcomes. The purpose of this objective involving CFI is to validate if CFI can be 
used to predict the occurrence of complications. Although such applications may identify who might 
benefit from interventions such as therapeutic plasma exchange or hemoperfusion, at present, the 
main aim of the study is to establish the correlation of CFI levels; it will not be used to determine the 
type of intervention the patients will receive. It will be divided into two sub-objectives. To assess the 
potential of CFI as a prognosticator, Objective 4a will compare the CFI values of the patient who will 
eventually have clinical outcomes (death, refractory hypotension, significant organ damage, and 
prolonged hospital stay) to those who did not upon hospital stay. The patients who belong to the 
controls (PPTCONV or HPCONV) will be included in the analyses. On the other hand, Objective 4b 
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will compare the CFI values of patients who either responded to PPT or HP. The CFI values will be 
divided into high (within mean + 2SD of normative value) (CFIHIGH) or low (CFILOW). Subgroup 
analyses of the effects of the treatment will be compared in the two subsets. Convalescent CFI gene 
expression will also be determined to see whether the patients were genetically predetermined to 
develop pulmonary complications (if CFI expression remains low) or whether the reduction in CFI 
levels was lepto-induced (return to normal levels of CFI). 
 
An overview of the study design is provided in the schema in Figure 1. When a participant is 
recruited in the study as having leptospirosis without significant pulmonary complications, he/she 
will be assigned to either Objective 1 (Prophylactic Plasma Transfusion) or Objective 2 
(Hemoperfusion) then to be randomized into the treatment group (PPT TRT or HP TRT) or control 
group (PPT CONV or HP CONV) and will receive their assigned intervention. Patients can 
deteriorate from the particular objective they are assigned to and the attending physician may 
employ any rescue treatment, such as Extracorporeal Membrane Oxygenation (ECMO), that he/she 
may deem necessary. However, for the study, ECMO will only be employed in NKTI. 
 
 
 
Figure 1. Overview of the experimental designs and objectives of the study 
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Human subject recruitment 
Subjects will be invited to join the study only after signing an informed consent form (Appendices A 
and B) duly approved by the ethics review board of the Single-Joint Research Ethics Board 
(SJREB). 
 
All subjects will be recruited from the emergency rooms, wards, and clinics of the National Kidney 
and Transplant Institute (NKTI), San Lazaro Hospital (SLH), and other hospitals that admit a 
significant number of leptospirosis cases as these hospitals serve as tertiary care centers for Weil’s 
disease patients in Metro Manila. These hospitals have wards, rooms, and intensive care units and 
have specialized set-ups for tertiary care to address complicated leptospirosis. 
 
Inclusion and exclusion criteria 
The inclusion and exclusion criteria for the cases are indicated in Table 3. The definitions of the 
outcomes of interest are summarized in Table 4. 
 
Note that from version 4 onwards, the P/F ratio for inclusion for Objective 1 was increased from 200 
to 300. As the study looks into the aspect of plasma transfusion in preventing pulmonary 
hemorrhage and overt respiratory failure, a higher starting P/F ratio of 300 is proposed rather than 
200, which may be clinically late already. 
 
The study will not discriminate against COVID-19 infection. While the effect of COVID-19 infection 
on CFI levels is not yet known, it would be interesting to note how the co-infection will affect CFI 
levels through a post-hoc analysis. 
 
Withdrawal criteria 
A participant will be withdrawn under the following conditions: (1) if s/he withdraws from the study 
for whatever reason, (2) if the samples are not feasible for testing, and (3) if the study is terminated 
prematurely. His/her participation in this study is voluntary, and s/he can withdraw anytime, for any 
reason. This will not affect his/her treatment provided by the participant’s doctors. In case s/he 
withdraws permission, s/he will be withdrawn from the study, his/her record will be deleted from the 
database, and the sample collected from him/her will be properly disposed of. 
 
Table 3. Inclusion and exclusion criteria of participants in the study 
Objectives 
Inclusion 
Exclusion 
General 
●​ Subjects with acute fever (38ºC for 
at least two days) and at least one 
of the following: myalgia, jaundice, 
headache, meningeal irritation, 
oliguria, conjunctival suffusion, 
●​ Who have a microscopic 
agglutination test (MAT) that 
indicates a single serum sample 
MAT titer ≥1:400, 
●​ Or a positive result for the latex 
agglutination test or a repeat test 
after seven days, 
●​ Or a positive result for Leptospira 
IgG/IgM lateral flow 
●​ Previous diagnosis of chronic 
kidney disease or on maintenance 
dialysis 
●​ Previous diagnoses of diseases 
associated with hemoptysis, such 
as bronchiectasis 
●​ Blood dyscrasias, malignancy, 
severe heart disease, HIV, cavitary 
PTB, Cirrhosis by ultrasound, 
severe malnutrition (Wt. < 35kg) 
●​ Post cardiac arrest or those with 
GCS <8 at present. Subject has had 
chest compressions or CPR. 
●​ Pregnancy 
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immunochromatographic test (ICT) 
or a repeat test within 3-14 days 
after the baseline test, 
●​ Or a positive result for Leptospira 
polymerase chain reaction (PCR), 
●​ Or a positive blood culture of 
leptospira WITHOUT the 
complication specified in a 
subgroup of interest, 
●​ Should be 18-60 years old 
Objective 1 (PPT) 
●​ As in General 
●​ Not requiring ventilator support 
(see Case Definitions in Table 4) 
●​ As in General 
●​ Requiring emergent dialyses (see 
Case Definitions in Table 4) 
●​ Significant lung pathology as 
defined by P/F ratio <300, or 
obvious respiratory distress 
●​ Presence of severe neurological 
symptoms 
●​ Hypotension (or need for 
vasopressor support) 
●​ Ongoing hemodynamic instability 
Objective 2 (HP) 
●​ As in General, plus: 
●​ Dialysis Requiring Acute Kidney 
Injury. Defined as KDIGO Acute 
Kidney Injury Stage 3 or requiring 
renal replacement therapy to 
correct intractable acidosis, 
electrolyte abnormality, or over 
uremic encephalopathy or 
pericarditis 
●​ Vasopressor Requiring – The 
subject must have received 
intravenous fluid resuscitation of a 
minimum of 30ml/kg within 24 
hours of eligibility and still with 
hypotension (blood pressure less 
than 90/60, MAP < 65) requiring 
vasopressor support 
●​ SOFA SCORE less than 15 
●​ As in General 
Rescue Treatment 
Inclusion 
Exclusion 
ECMO 
●​ As in General, plus: 
●​ A Murray score of ≥ 2.75 
●​ As in General 
 
Table 4. Definitions of clinical outcomes and complications 
Outcomes 
Definition 
Objective 1 (PPT) 
 
Mortality 
Death occurring to be related to the natural course of the present condition of 
leptospirosis or its complication, but not more than two weeks upon discharge 
by attending physician after being assessed as well, recovered, or the like. 
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Leptospirosis with 
significant pulmonary 
involvement 
As in leptospirosis (Weil’s syndrome) plus evidence of pulmonary injury as 
indicated by (1) the need for mechanical ventilator support OR (2) P/F ratio 
<200 OR (3) gross hemoptysis, (4) chest x-ray result consistent with 
leptospirosis-related pulmonary changes 
Refractory hypotension 
Occurrence of systolic blood pressure less than 90 mm Hg, OR mean arterial 
pressure less than 65 mm Hg, OR a decrease of 40 mm Hg in systolic blood 
pressure compared to baseline: unresponsive to crystalloid ﬂuid challenge of 20 
to 40 mL/kg OR requiring vasopressor support 
Leptospirosis with 
significant renal 
involvement 
As in leptospirosis (Weil’s syndrome), plus evidence of severe acute kidney 
injury as indicated by the need for emergency dialysis due to intractable 
acidosis, hyperkalemia, uremic encephalopathy or pericarditis 
Hospital stay 
To be computed from the date of admission to the date of discharge. Mortality or 
discharge against medical advice will be penalized with a maximum stay of at 
least 30 days. 
Objective 2 (HP) 
 
Mortality 
Death occurring to be related to the natural course of the present condition of 
leptospirosis or its complication, but not more than two weeks upon discharge 
by an attending physician after being assessed as well, recovered, or the like. 
Need for renal 
replacement therapy 
Number of days requiring dialysis during hospital stay 
Need for inotropic 
support 
Number of days requiring inotropic support to attain MAP >65 
Number of days requiring each inotrope 
Need for emergent 
invasive respiratory 
support 
Inability to maintain Sp02 >92% on maximum non-invasive respiratory support 
Need for ECMO 
Attainment of a Murray score ≥ 2.75 or PaO2/FiO2 ratio of less than 200 
Hospital stay 
To be computed from the date of admission to the date of discharge. Mortality or 
discharge against medical advice will be penalized with a maximum stay of at 
least 30 days. 
ICU days 
Number of days patient is required to stay in ICU (date out of ICU will be date 
ordered by the attending physician) 
 
A prospective subject will be screened first based on his/her clinical presentation (Appendix C). If 
deemed qualified, the prospective subject (or if unable to do so by his/her primary caregiver with 
legal capacity) will be invited to join the study and, if amenable, will be made to sign an informed 
consent. The study will recruit subjects who are 18-60 years of age. It will recruit patients with 
manifestations of leptospirosis, as defined in Table 2, for the specified groups of interest. As much 
as possible, the blood extraction will be from patients who are receiving initial care from the 
hospitals. After screening using the clinical criteria, infection with leptospirosis will be confirmed by 
doing a Leptospira IgG/IgM lateral flow immunochromatographic test (ICT) to be performed in the 
diagnostics laboratory of NKTI or SLH, and/or the leptospiral microscopic agglutination test (MAT) 
assays in an accredited institution. 
 
Typically, ICT has the shortest turn-around time, and a positive result becomes the basis for 
inclusion. For initially ICT negative patients, a repeat ICT will be performed and if positive, the 
patient will be included in the study. Treatment will be initiated and continued while waiting for the 
results of the repeat ICT, MAT, LAT, PCR, and/or blood culture. If all test results are negative, the 
patient will then be withdrawn (see withdrawal criteria) from the study, and their data will not be 
included for analysis. However, their treatment will still continue as their attending physician sees fit. 
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By definition, the NKTI or SLH Lab services will include procalcitonin and hsCRP assays, while 
patient tests will consist of routine clinical laboratory tests, like CBC, chemistry, leptospirosis tests, 
and urinalysis. 
 
Baseline CFI assays will be done on blood collected at most 24 hours after admission. Cytokines 
will be tested once the inclusion/exclusion criteria are met according to the specific objective. Data, 
including general information, clinical data, clinical test results, and outcomes, will be collected in a 
case report form (CRF; Appendix D). In addition to clinical characterization, the baseline blood 
levels of TNF-alpha, IL-6, CFI, procalcitonin, and hsCRP (for Objective 2) will be measured on 
enrollment. 
 
For Objectives 1 and 2, within the first 24 hours of admission, routine laboratory test and imaging 
data will be expectedly collected as ordered (or else, will be ordered by the project) that include 
complete blood count, hemoglobin, hematocrit, creatinine, blood urea nitrogen, serum potassium, 
chest radiograph, arterial blood gases, protime and partial thromboplastin time, as well as the 
leptospirosis latex agglutination test and microagglutination tests. Three sets of 4-ml blood samples 
for CFI assays, pre- and post-transfusion, defined as taken within 24 hours before and after the first 
and last unit transfused, respectively. Other tests ordered by the attending physician during the 
hospital stay will be collected. 
 
For Objective 2, an additional two sets of 10-ml blood samples for apoptotic assays will be extracted 
from patients pre- and post-transfusion. Hemoperfusion cartridges of HPTRT patients will also be 
stored for protein extraction and quantification. These specimens will be sent out to DeLEPTO 
Project 2 staff stationed at University of the Philippines - Diliman (UPD). 
 
Additionally, for all procedures, additional imaging and/or tests and monitoring parameters will be at 
the discretion of the attending physicians. They should be considered as part of the standard of 
care. 
 
The participants will be randomized for inclusion in Objective 1 or 2 using a preset assignment in 
which the assigner is blinded. 
 
Sample size calculations 
The minimum computed sample sizes are provided in Table 5. 
 
For Objective 1, a computed sample size of at least 17 per group (PPTTRT and PPTCONV) was 
calculated based on the following assumptions: using an independent open-label randomized 
controlled trial, using hospital stay as a primary outcome, a reduction mean of at least three days in 
PPTTRT compared with PPTCONV with a standard deviation of 3 days, a one-tailed α=0.05; power 
of 0.80; and a 1:1 case-control ratio. If this sample size takes into consideration adjustments for 
Objective 4b, in which a minimum sample size should be adequate to assess the effect of low CFI 
subpopulation (estimated at 12% of the total population), then the estimated sample size would 
become 142 per group. 
For Objective 2, a computed sample size of at least 197 leptospirosis patients for hemoperfusion 
treatment and 197 patients to undergo standard treatment was calculated based on the latest 
preliminary data from NKTI showing a 42% risk reduction in mortality among those on 
hemoperfusion and ECMO. For moderate leptospirosis with an estimated 15% mortality among 
those on standard care (usual mortality is 5%-15%), the 42% risk reduction translates to 6.3% 
mortality among those on HP and ECMO. Other assumptions used are alpha of 0.05, power of 0.80, 
and 1:1 exposed/unexposed. 
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Taking into account the limited capacity (in terms of equipment and expertise) of NKTI, the number 
of ECMO procedures as a rescue treatment to be observed will only be 16. This will be the largest 
case series to describe the potential benefit of ECMO as a life-saving procedure in leptospirosis. 
 
Table 5. Estimated sample size of planned clinical groups in the study 
Groups 
Description 
Estimated Sample 
Size 
PPTTRT 
Cases for prophylactic plasma transfusion 
142 
PPTCONV 
Controls for prophylactic plasma transfusion 
142 
HPTRT 
Cases for hemoperfusion 
197 
HPCONV 
Controls for hemoperfusion 
197 
TOTAL  
678 
ECMO 
Cases that will undergo ECMO as a rescue treatment 
16 
 
Plasma transfusion 
This is for Objective 1 of the project. The assignment of treatment groups will be decided upon 
recruitment by a random picking of envelopes. Plasma transfusion will commence within 24 hours 
after inclusion in the study as long as the patient remains eligible. As the study considers overt renal 
failure as an inclusion in Objective 1 (plasma transfusion), the hemodialysis for the plasma 
transfusion group will proceed as a preemptive procedure to prevent plasma-induced overload. As 
for the controls, hemodialysis will depend on the attending physician's decision as part of the 
standard of care. 
 
Participants in the plasma transfusion group will receive transfusion if the PBMC CFI qPCR deltaCT 
is found to be at least 25 or more; this was based on the preliminary data gathered by the research 
team with a mean of 23 using the present set of primers. The previous study of Nevado et al. 
(unpublished) indicated that all patients with eventual pulmonary complications had a PBMC CFI 
qPCR deltaCT of a mean of 15.68 with a range from 14.79 to 17.04 and a standard deviation of 
0.91. The control has a mean deltaCT of 12.41 with a standard deviation of 0.42. 
 
ABO/Rh-type compatible fresh frozen plasma (FPP) units will be thawed to 37° prior to 
administration. Plasma transfusion will be administered intravenously, 1 unit for 4 hours every 12 
hours for two consecutive days. There will be two consecutive days for the transfusion for a total of 
4 units. For practical reasons, the FFP units should be processed and procured through 
DOH-accredited blood banks specifically from NKTI and SLH Blood Banks, which are properly 
screened for pathogens, typed, and clinically cleared for use. This will allow fresh frozen plasma 
readily available for both sites and can have the option to have it transported when needed. 
 
Buffer stocks of FFPs will be stored in -80 freezers designated for the project at any time and will be 
constantly refilled daily if needed. Different blood type-specific FFPs will be stored to accommodate 
at least 12 patient-regimen, with 6 being type O and 2 each for types A, B, and AB. Due to a lack of 
availability, the project will not stock Rh- FFPs. 
 
During the transfusion, parameters will be closely monitored to ensure the safety of the 
interventions. The following symptoms will require stoppage of the transfusion (as adapted from the 
transfusion reaction guidelines of the NKTI Blood Bank: (1) fever with or without chills; (2) shaking 
chills (rigors) with or without fever; (3) pain at the infusion site or in the chest, abdomen, or flanks; 
(4) blood pressure changes, usually acute, either hypertension or hypotension; (5) respiratory 
distress, including dyspnea, tachypnea, wheezing, or hypoxemia; (7) skin changes, including 
urticaria, pruritus, flushing, or angioedema; (8) nausea with or without vomiting; (9) darkened urine; 
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(10) bleeding or other manifestations of a consumption coagulopathy; and (11) other signs or 
symptoms for which the attending physician determines warrants the discontinuation of infusion 
(such as gastrointestinal symptoms) (63). 
 
In addition to the pre-treatment CFI serum ELISA, post-treatment serum CFI ELISA will be 
performed for those who were plasma transfused within 3 hours after the last transfusion. Serum 
CFI ELISA will also be performed on admission and on day 3 or 4 of the control group (PPTCONV). 
 
Hemoperfusion (HP) 
The HP procedure will follow the standard procedure of NKTI using Jafron HA330 hemoperfusion 
cartridge. First, an internal jugular catheter is attached to the patient. Alternatively, an arteriovenous 
fistula or arteriovenous graft may be placed on the patient. The patient will then be hooked to a 
hemodialysis machine. Blood pump speed will be set to 150-200mL/min, and HP will last for 2 to 2.5 
hours. Whole blood will flow through the sorbent HA330 cartridge and back to the patient. 
Anticoagulation is not necessary due to the short treatment time. Hemoperfusion will be repeated 
after 12-24 hours for at least three days. 
 
For cytokine assays, ELISA will be performed for serum TNF-alpha quantification as serviced by the 
Molecular Research and Diagnostic Unit (MRDU) of IHG NIH-UPM. Immunoturbidimetric assay for 
hsCRP, fluorescent immunoassay for IL-6, and chemiluminescent microparticle immunoassay 
(CMIA) for procalcitonin will be serviced by NKTI and SLH Medical Laboratories.  
 
Blood samples for sendout to UPD will undergo the following assays: Annexin propidium iodide (PI) 
assay for apoptosis; 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay for 
general cytotoxicity; Lactate dehydrogenase (LDH) release assay for cell lysis; and Membrane 
attack complex (MAC) formation assay for complement attack.  
 
Hemoperfusion cartridges for sendout to UPD will undergo the following processes: Ammonium 
sulfate extraction for protein concentration; Polyacrylamide gel electrophoresis (PAGE) or NanoDrop 
spectrophotometer for protein quality assessment; and Bicinchoninic acid (BCA) assay for protein 
quantification. 
 
Pre-HP assays will be done upon recruitment, and post-HP assays will be done within 3 hours after 
the 3rd HP session. For the control group (HPCONV), cytokine assays will be done upon 
recruitment and on day 2 or 3 of hospitalization. 
 
Extracorporeal membrane oxygenation (ECMO) 
The procedure is adapted from Lee et al. (80). A veno-venous ECMO (VV ECMO) will be applied to 
the patients by aseptically inserting a venous cannula into the femoral veins. The patients will be 
hooked to an ECMO machine. For anticoagulation, patients without significant bleeding or vascular 
intervention will be managed with an activated clotting time set at 140‒180 sec by 800‒1000 U/h of 
heparin. Otherwise, the heparin will be titrated to maintain a partial thromboplastin time of 60‒80 
sec. 
 
ECMO flow will be maintained at a mean blood pressure of >60 mm. SaO2 will be maintained at 
>90% with a flow of 3.5‒4.5 L/min. Hematocrit will be maintained at >35% and platelets 
>50000‒100000/mL. Transfusions will be done when necessary. ECMO will be weaned when 
arterial blood gas analysis revealed pH 7.35‒7.45, PaO2 >80 mm Hg, and PCO2 <45 mm Hg under 
the following conditions: a gas blender FiO2 of 0.21, sweep gas of 0 L/min at an ECMO flow of 2 
L/min, and the ventilator mode (if applicable) set to an FiO2 of 0.6, a tidal volume of 6 mL/kg, a 
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PEEP of 8 cmH2O, and an RR of 12‒16/min for VV ECMO or 3 L/min of O2 via nasal prong with 
awakening ECMO patients. 
 
Sample collection and total RNA extraction from peripheral blood mononuclear cells 
Blood extractions explicitly for the initial qPCR and ELISA for CFI and cytokine levels will be 
performed immediately upon recruitment, within 24 hours upon admission. Baseline levels of 
TNF-alpha, IL6, procalcitonin, and hsCRP will be taken from participants in Objective 2. The assay 
will be repeated within 24 hours after the third session of hemoperfusion. Also, blood CFI protein 
determination will be repeated within 24 hours after plasma transfusion in participants in Objectives 
1. In addition, a random sample of 20 participants in the study will be selected for blood extraction 
upon discharge to assess CFI protein levels in the convalescent phase of infection in patients with 
initial low CFI levels. RNA extraction, qPCR, and ELISA procedures will be done at the NKTI, SLH, 
or the Microarray Core Laboratory of the Institute of Human Genetics, National Institutes of 
Health-University of the Philippines-Manila (IHG, NIH-UPM). Random sampling of blood for CFI 
assay upon discharge will demonstrate the effect of convalescence on the level of the CFI upon 
recovery. This will give us an idea of whether CFI levels are affected by the presence of infection or 
an inherent deficiency. 
 
For RNA extraction and serum processing, whole blood amounting to about 4 ml (4 ml with 
anticoagulant for RNA extraction and serum processing) will be collected. Samples for qPCR 
analysis will be stored in EDTA-containing blood tubes, and the samples for ELISA will be stored in 
blood tubes without additives. Blood tubes will be stored immediately in ice-cold containers. The 
other whole blood aliquot collected in the tube without additives will be centrifuged in order to obtain 
serum. The serum samples will then be stored at -80°C until ELISA is performed. Peripheral blood 
mononuclear cells (PBMCs) will be isolated from whole blood collected in the EDTA-containing 
blood tube within 2 hours after extraction through Ficoll density gradient centrifugation using 
Lymphoprep (Stemcell Technologies, Vancouver, Canada). Isolated PBMCs will be temporarily 
stored at -80°C until RNA extraction. 
 
RNA extraction of the PBMC will be performed using the QIAmp RNA Blood Minikit (Qiagen, 
Germantown, MD) according to the manufacturer’s instructions. Briefly, the PBMC pellets will be 
mixed with 600 μl of buffer RLT, which will be placed in a QIAshredder spin column in a 2 ml 
collection tube that will be spun at maximum speed for 2 min. To the homogenized sample, 600 μl of 
70% ethanol will be added and mixed by pipetting. The samples will be placed in a new QIAamp 
spin column and centrifuged for 15 seconds at >8000 x g. After discarding the eluate, the samples 
will be added with 700 μl Buffer RW1, centrifuged for 15 s at >8000 x g, and the eluates will be 
discarded. Two sets of washings with Buffer RPE will be done with successive centrifugations at 
>8000 x g for 15 sec and at full speed for 3 min. In a new 2-ml tube, the samples will be further 
centrifuged at full speed for 1 min. The RNAs will be eluted by two successive applications of 40 μl 
of RNase-free water with centrifugation at >8000 x g for 1 min. RNA quantification and OD260/280 
determination will be done using an ND1000 NanoDrop spectrometer. Samples will be kept at -80°C 
until use. 
 
Quantitative polymerase chain reaction 
Reverse transcription will be performed using an iScriptTM cDNA synthesis kit (Biorad). Each 
reaction will contain 1 μl reverse transcriptase, 4 ul reaction buffer, and 200 ng of RNA sample, to 
be added to nuclease-free distilled water and appropriate primers for a total of 20 μl samples. All 
preparations will be performed on ice. Primers will be designed to have at least one of a primer pair 
per segment of interest to bridge across exon-exon borders to lessen the possibility of genomic 
amplification. Reaction conditions will be 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, and 4°C 
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onwards. Quantitative PCR will use the SsofastTM EvaGreen kit (Biorad). One reaction mix will 
contain 2 μl cDNA from the previous reverse transcription samples, 2 μl each of 5 mM forward and 
reverse primers, and nuclease-free water for a total of 20 ul. Quantitative PCR conditions to be used 
will be as follows: 95°C for 5 min, with 39 cycles consisting of 95°C for 45 sec, chosen annealing 
temperature for 45 sec, 72°C for 45 sec, read at 78°C, and 95°C for 45 sec. Melting curve 
determination will range from 72°C to 95°C with 0.2°C increments. The primers that will be used for 
the CFI qPCR are CFI forward, 5′- ACACAGAAAGCAGATTCTCCA–3’, and CFI reverse, 
5′-ACAATGTGCAGCAGTCAGAA-3’, with an annealing temperature of 58°C. The 18S mRNA will 
serve 
as 
the 
internal 
control 
with 
the 
following 
sequences: 
18S 
forward 
5’-GCTTAATTTGACTCAACACGGGA-3’​
and 
18S 
reverse 
5’-AGCTATCAATCTGTCAATCCTGTC-3’ (71). 
 
Enzyme-linked immunosorbent assay 
The assay will utilize an in vitro competitive ELISA for CFI (Abcam, HK, catalog no. ab195460). The 
96-well plate is precoated with anti-CFI antibody. Sera will be prepared from blood samples that 
would be clotted for not more than 3 hours and centrifuged at 3000g for 5 min. Sera will be stored at 
-80°C until use. 
 
On the day of assay, the sera will be diluted at 20x with PBS as per the instruction of the 
manufacturer. Next, 25 μL of CFI standard (as supplied with dilution ranging from 0 [blank] to 24 
μg/mL) or sample per well was added, together with 25 μL of 1X biotinylated CFI to each well 
(supplied) and incubated for 2 hours at room temperature. After washing with 200 μL of supplied 1X 
Wash Buffer, 50 μL of 1X SP Conjugate (supplied) was added to each well and incubated for 30 
minutes. After washing, 50 μL of Chromogen Substrate (supplied) was added per well and 
incubated for 10 minutes. Fifty μL of Stop Solution (supplied) was added to each well. The plate was 
read at 450 nm. 
 
For cytokine assays, sandwich ELISA with standards will be performed for TNF-alpha, 
quantification. 
 
Outcomes and follow-ups 
For Objectives 1 and 2, 5 sets of 4-ml sera will be taken before and after the transfusion and/or 
hemodialysis/hemoperfusion to determine blood levels of CFI, and other cytokines that may be 
deemed relevant for monitoring. 
 
For Objectives 1 and 2, the patients will be followed up during their hospital stay or until the 30th 
day post-admission. Prolonged hospital stay will be an independent outcome from multi-organ 
failure, which has a separate definition. However, in the presence of both outcomes, patients with 
prolonged hospital stays will be interpreted as having multi-organ failures in hierarchical outcome 
designation in composite analyses (1 outcome per participant). The separate designation for 
prolonged hospital stay is in recognition that it can be due to other causes that may lead to such 
outcome, including nosocomial infections, interventional complications, and disease deterioration. A 
daily monitoring form was incorporated to record the progress of the participants (Case Report 
Form; Appendix D); it contains the following information: the dose and time of administration of the 
administered plasma, hemoperfusion, and ECMO intervention; daily monitoring of signs and 
symptoms associated with the plasma, hemoperfusion, and ECMO intervention; the list of drugs 
used, their doses and schedule; and the outcomes of interest as stipulated in this protocol (mortality, 
with significant pulmonary involvement, with significant renal involvement, and hospital stay). 
 
The participant is expected to participate during his/her hospital stay or until the 30th day after 
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inclusion in the study, whatever is shorter. 
 
Statistical analyses 
For Objectives 1 and 2, we will determine differences in categorical outcomes (mortality, presence of 
shock) using chi-square tests and continuous variables (number of hospital days, number of days in 
dialysis) using the Student’s T-test. The time-to-event analysis will be done using Kaplan-Meier 
curves if applicable. 
 
For Objective 2, interim analyses will be done at every quartile (50th, 99th, and 148th sample for 
each group is completed) of the initial estimated sample size (198 per group), with significance set 
at p<0.01. 
 
According to the latest study on the use of HP on severe leptospirosis by Danguilan et al. (2022, 
unpublished) as a basis, a calculated 0% (0/18) mortality in HP compared with 36.84% (7/19) in the 
standard of care was observed. The present study will serve as validation. Hence, in a more 
conservative scenario of 2% mortality in controls and 30% in cases, the minimum sample size to 
attain significance at p<0.01 at minimum power of 90% is 48 per group. If this is observed, the study 
group will report to the ethics committee and will request for study pre-termination. Similar 
assumptions will be applied to Objective 1. 
 
For those that undergo ECMO as a rescue treatment, descriptive analytics will be done, indicating 
means and ranges of hospital stay, number of mortalities, and complications. 
 
For Objective 3, correlation of values of qPCR results to ELISA data will be performed using 
Pearson correlation (r2 of 0.80 or higher will be considered highly correlated). Test for concordance 
using Kendall’s W may be done. 
 
For Objective 4, receiver operator characteristic analyses for qPCR and ELISA will be compared for 
the eventual development of adverse outcomes using the Wilson-Brown method. Percentage values 
will be used. An area under the curve of at least 0.80 is considered significant. Data will be analyzed 
using the deltaCT method. The means and standard deviation will be analyzed using ANOVA or 
student t-test (for parametric comparison), Kruskal-Wallis test or Wilcoxon test (for nonparametric 
comparison), or the Chi-square or Fisher exact tests for association with categorical variables 
depending on the quantitative or qualitative nature, respectively, of the clinical outcomes. A p-value 
of <0.05 is considered significant. Sensitivity, specificity, and predictive values will be computed for 
each pair of relevant tests and outcomes, such as mortality, end-organ failure, shock, and hospital 
stay. 
 
The data of the patients will be encoded and summarized in a Microsoft Excel sheet and analyzed 
using GraphPad Prism v10. 
 
Biosafety considerations 
All biological samples from infected subjects of objectives 1 and 2 will be processed under a 
biosafety cabinet, at least of type 2A, with researchers donning proper PPEs. All disposed samples 
and containers will undergo decontamination with 10% hypochlorite and autoclaving in a sealed 
container prior to disposal in a biohazard container under 121°C (250°F) for around 15–20 min prior 
to disposal as biohazard waste. 
 
Ethical considerations 
The project leader, co-investigators, and designated clinical research assistants are responsible for 
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the recruitment of human subjects. All research staff will undergo training on Good Clinical Practice 
(GCP) prior to interviewing patients. Patient recruitment will be conducted upon approval of the 
ethics review board of the Single-Joint Research Ethics Board (SJREB). Participants from the 
emergency rooms, wards, and clinics of the NKTI and SLH will be recruited only upon willful signing 
of an informed consent form.  In the event that the patient is not physically or mentally capable of 
giving consent, a legally authorized representative or guardian may provide consent on their behalf. 
 
The primary investigator or designated clinical research assistant will provide the necessary 
information about the study and will answer questions from the potential participant. It is important to 
ensure that the participant will read and understand the contents of the Informed Consent, or if the 
participant desires, the research assistant will read and explain the contents of the ICF. 
 
Only trained research personnel can and will interact with patients or their legal representatives to 
be included in the study. The screened patients will be respectfully asked to join the study by 
explaining carefully the details of the study, especially the responsibilities and rights of the 
participants, as well as answering their concerns. Patients will be given adequate time to decide 
within the study constraints. 
 
Medical information, if warranted, will be obtained from medical records, including age, sex, 
address, case number, laboratory data, and clinical metadata. To gain access to medical records, a 
request letter will be sent to the medical director of NKTI or SLH, where recruitment will take place. 
Further, the informed consent will also stipulate permission from the participant to access his/her 
medical records. Only upon the provision of official permission to access medical records and the 
signing of the informed consent by the participant will the records be scrutinized and their relevant 
data abstracted to study forms and databases. Medical records can only be accessed by 
investigators and research assistants of the study, and data from the records will be treated with 
utmost confidentiality. 
 
Several measures will protect the data confidentiality and privacy of the participants. All records or 
personal information related to the study will be kept confidential. These will be coded and kept in 
secured/locked cabinets and password-protected computers. Any genetic, clinical, or personal 
information directly traceable to the participant will not be released to others, including family 
members, without written consent from him/her. All samples will be kept in freezers, with the key 
kept by authorized personnel only. 
 
The research assistant will also inform the participants that the samples may be stored for at most 
five years from the end of the study and may be used for future studies upon his/her consent. This is 
stated in the informed consent form, and corresponding tickboxes are used to indicate whether or 
not the participant provided future use consent. Biological samples will be stored in -20°C to -80°C 
freezers and will be kept at most for five years either at the National Kidney and Transplant Institute 
(NKTI), San Lazaro Hospital (SLH) or at the Institute of Human Genetics, NIH-UP Manila. The 
stored samples may be used for future studies only upon the consent of the participant and provided 
that the identifying data will be removed from the samples. If the participant did not give his/her 
future use consent, s/he can still participate in the present study as long as informed consent is 
provided for the present study. In this case, samples will only be used for the present study and will 
be properly disposed of 1 year after this study. 
 
However, the results of the study may be presented at scientific or medical meetings and 
conferences or published in scientific articles. To maintain privacy, there will be no mention of the 
name or any information of the participants that can be traced directly to him/her. Aside from project 
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investigators and staff of the current, the study monitor(s), auditor(s), the institutional ethics review 
board of NKTI, UP Manila, SLH, SJREB and regulatory authorities will be granted direct access to 
the participants’ medical records for purposes only for verification of clinical trial procedures and 
data. This also applies to future research/es in which the data from the participants are to be used. 
 
The participants have the right to know the full results of this study, and they can request the results 
of the study. Moreover, though they cannot have direct access to their records, they still maintain 
their right to know data relevant to them. 
 
The participants or their legal representatives will be informed if there are changes or amendments 
to the information they supplied in this study. 
 
As the study assists standard of care testing and treatment for all participants, there will be a direct 
benefit for the patients. However, the results of the study are still of unknown significance, given the 
current scientific knowledge on leptospirosis. Thus, the participants are also encouraged to 
communicate anytime with the primary investigator concerning the results of the study and their 
impact on their health. 
 
All expenses directly related to the use of fresh frozen plasma for the transfusion procedure under 
Objective 1 will be covered by the project budget. For patients under Objective 2, the study will 
cover the expenses for a maximum of three hemoperfusion cartridges. Inflammatory marker blood 
tests, such as Interleukin-6 (IL-6), High-sensitivity C-reactive protein (hsCRP), Procalcitonin, and 
TNF-alpha, will also be covered for patients under Objective 2.  For patients that will undergo ECMO 
as a rescue treatment, the study will cover the expenses for the ECMO device, cannulae, and 
insertion kits used in the procedure. The study will also cover the expenses for the following 
interventions for all patients, regardless of treatment assignment: Intravenous fluids and 
medications up to Php 2000.00 and standard-of-care laboratory tests up to Php 2000.00 for 
participants without other available sources or medical insurance (such as PhilHealth), as 
applicable.  
 
The study will still cover the expenses mentioned above for initially ICT negative patients while 
waiting for the results of the repeat ICT, MAT, LAT, PCR, and/or blood culture. 
 
Each participant will receive a Php 1000 monetary remuneration for his/her participation in the study 
and Php 2000 for intravenous fluids and medicine. If needed, he/she is still expected to spend for 
transportation on follow-up. Such expenses will not be directly reimbursed, but the study will provide 
compensation during follow-up to cover food, transportation, and the possible loss of income. 
Furthermore, s/he will be supported for the following tests, if needed: test for leptospirosis, serum 
creatinine, blood urea nitrogen, chest X-ray, complete blood count with differential count, and 
urinalysis. If s/he prefers, health information regarding leptospirosis will be offered. The remaining 
expenses will be sourced from the participant. The DeLEPTO Project 1 Project Development Officer 
III is responsible for reviewing, approving, and monitoring the coverage of all expenses. This 
process is coordinated with the project's clinical team, who will ensure that any intervention aligns 
with the project objectives and the patient's classification. 
 
There will be risks involved in the conduct of the study. There is minimal risk associated with blood 
extraction provided that the subjects are hemodynamically stable, without respiratory distress, and 
have a hemoglobin level of not less than 90 g/li. These include bleeding from the site of injection, 
pain, local reaction to the tape or cotton, and infection. For patients undergoing plasma transfusion, 
there may be adverse effects, such as difficulty in breathing, itchiness, rashes, swelling, lowering of 
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blood pressure, and faster or slower beating of the heart. For patients undergoing hemoperfusion, 
there may be some events such as bleeding in wound sites and other parts of the body, low blood 
pressure and palpitations, as well as temporary lowering of platelet count that may need monitoring. 
For patients undergoing ECMO, there may be bleeding in wound sites and other parts of the body, 
sudden changes in blood pressure and heart rate, and infection in areas where the body is attached 
to the machines. Moreover, there is also the possibility of the machine having some failure. In most 
instances, most side effects are mild, and serious occurrences are less than 5%, with the potential 
benefit projected to be more than the risk of harm for the patients’ condition. 
 
Suppose there is a persistence of symptoms (such as rashes and itchiness), sudden difficulty of 
breathing, new onset of pain, severe bleeding that will not stop with gentle pressure, and worsening 
fever or weakness. In that case, it is advised that Dr. Romina Danguilan of NKTI and Dr. Rontgene 
Solante of SLH be consulted. The participant can consult any emergency room/clinic/doctor 
(preferably NKTI or SLH) if any of the above occurs. Less serious adverse events can be consulted 
through out-patient consultation in clinics. A portion of the project fund is allocated to support 
affected participants in the event of hospitalization and/or required treatment resulting from 
study-related adverse events or complications. 
 
A participant will be withdrawn under the following conditions: (1) if s/he withdraws from the study 
for whatever reason, (2) if the samples are not feasible for testing, and (3) if the study is terminated 
prematurely. His/her participation in this study is voluntary, and s/he can withdraw anytime, for any 
reason. This will not affect his/her treatment provided his/her doctors. In case s/he withdraws 
permission, s/he will be withdrawn from the study, his/her record will be deleted from the database, 
and the sample collected from him/her will be properly disposed of. 
 
Considering the risk associated with leptospiral infection, while handling infected samples, 
precautions will be taken to protect the research staff and other people s/he will interact with. First, 
research personnel will wear recommended protective personnel equipment (PPEs) at all times in 
compliance with institutional protocols. Staff who will be facing participants should have training on 
integrating with patients, donning and doffing procedures of PPEs, and sample handling and 
disposal. Budget will be allocated for testing for leptospirosis using latex agglutination tests. In case 
of hospitalization, a budget will be allocated for emergent care, in addition to the universal health 
care insurance by the government. 
 
Dr. Nevado declares a potential conflict of interest as he has a patent for a CFI quantification kit for 
the prognostication of leptospirosis for its pulmonary complications. Otherwise, Dr. Danguilan, Dr. 
Chavez, Dr. Arakama, Dr. Solante, Dr. Sayo-Abungan, Dr. Verona, Dr. Trifalgar-Arches and Dr. Lee 
do not have any conflict of interest to declare. Drs. Chavez, Arakama, Solante, Sayo-Abungan, 
Verona, Trafalgar-Arches and Danguilan may serve as both investigators and the participants’ 
attending physicians, but Dr. Nevado and Dr. Lee will not. 
 
8.​ Theoretical Framework: Description of the theory on why the research problem exists. It can also 
be a diagram; just put the image here  
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9.​ Expected Outputs (6Ps) 
a.​ Publication: published aspect of the research, or the whole of it, in a scientific journal or 
conference proceeding for peer review or in a popular form 
This project will hopefully produce data on the clinical effectiveness of prophylactic plasma 
transfusion, hemoperfusion, and ECMO for leptospirosis. In addition, it can provide evidence 
of the use of CFI as a prognostic biomarker and of the different preemptive medical 
procedures that might prevent the development of complications in leptospirosis. Insights 
gained from this data will be published in at least one (1) peer-reviewed indexed international 
journal.  
 
b.​ Patent: proprietary invention or scientific process for potential future 
The investigators are currently applying for a patent for CFI as a potential prognostic marker 
for pulmonary complications in leptospirosis and related technologies. For now, no additional 
patents will be sought. 
 
c.​ Product: invention with a potential for commercialization 
Quantitative diagnostic and prognostic kits in the form of optimized qPCR and possible 
ELISA kits for the early risk assessment of leptospirosis will hopefully be developed. 
 
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d.​ People Service: people or groups of people who receive technical knowledge and training 
This project will provide opportunities for pulmonary fellows to be trained on pre-emptive 
medical procedures such as ECMO and HP. Medical technologists in hospitals could also 
benefit from learning qPCR and ELISA techniques that will be important in assessing CFI 
gene expression levels. 
 
e.​ Place and Partnership: linkage forged because of the study 
A multi-site approach will be implemented in this project, with NKTI and San Lazaro Hospital 
responsible for the clinical aspects of the project, and IHG-NIH leading the molecular tests 
and analysis. In addition, as part of the DeLEPTO program, these institutions will also be in 
close collaboration with labs from UP Diliman and UP Manila, which are responsible for the 
basic science and pre-clinical aspects of the program. 
 
f.​ Policy: science-based policy crafted and adopted by the government or academe as a result 
of the study 
The clinical insights to be gained from this study will aid in informing clinical guidelines for 
the improved management of leptospirosis patients. 
 
10.​ Potential Outcome: refers to the result that the proponent hopes to deliver three (3) years after 
the successful completion of the project 
The procedure for ELISA and qPCR were mentioned in the methods. The study will generate 
quantitative prognostic kits in the form of optimized qPCR and possibly ELISA kits for the early risk 
assessment of leptospirosis, especially those who could develop pulmonary complications; these 
will serve as prototypes of the kits. This may be included in hospital and clinic routines to provide 
aid in admission and preemptive treatment. In addition, the development of digital droplet PCR kits 
is the foremost objective of Project 4-LIGTAS, to be done in close coordination with Project 1. 
 
The investigators will be able to deliver the results as long as the expected number of patients are 
admitted as planned. The NKTI is a renal referral center for leptospirosis patients with kidney 
failure requiring dialysis. San Lazaro Hospital is a tertiary care center which receives leptospirosis 
patients of varying severity even during off-seasons. Thus, as referral centers in the middle of 
Metro Manila, they have the catchment areas for the specific disease that is the focus of the study. 
 
11.​ Social Impact: refers to the effect or influence of the project on the reinforcement of social ties 
and the building of local communities. 
Leptospirosis is a devastating disease affecting the financially disadvantaged population. The male 
breadwinner of families is usually affected, and their death causes families extreme 
dysfunctionality. Investing in innovative treatments that can lead to saving the lives of these 
patients is evidence that the government considers the preservation of their health important to the 
community. 
 
12.​ Economic Impact: refers to the effect or influence of the project on the commercialization of its 
products and services, improvement of the competitiveness of the private sector, and local, 
regional, and national economic development 
Leptospirosis has a very high mortality rate. Therapies aimed at lowering the mortality from 
leptospirosis from innovative therapies ultimately lead to the preservation of the workforce and a 
positive impact on the economy. 
 
Meanwhile, the kits may be produced by biotechnology companies that specialize in medical 
testing. With the prevalence of reported leptospirosis of about 5,000 a year and nearly three times 
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that number of suspected cases, the potential coverage could be estimated at 20,000 patients who 
are admitted to large hospitals capable of HP or ECMO. 
 
13.​ Target Beneficiaries: Refers to groups/persons who will be positively affected by the conduct of 
the project. 
a)​ The Filipino patients who can directly benefit from early prognostication and the 
companion early treatment; 
b)​ The clinicians, who can benefit from potential products and can help decide on the proper 
treatment of their patients; 
c)​ The health institutions, particularly the hospitals, in their wise allocation of resources and 
workforce to treat higher-risk individuals; 
d)​ The basic science researchers in both private and public sectors, as they further their 
technologies to develop better testing as well as create preemptive innovations, not only 
for leptospirosis but for other similar conditions as well (such as dengue and sepsis). 
e)​ The biological/medical industry, as any product related to this study, could be exploited 
economically and socially for the benefit of the global audience. 
 
14.​ Sustainability Plan: refers to the continuity of the project or how it shall be operated amidst 
financial, social, and environmental risks. 
The main challenge is the inherent seasonal occurrence of leptospirosis. By involving NKTI and 
SLH, which typically have more than 20 patients with leptospirosis even in off-season, the project 
has a good possibility of completion. 
 
Another challenge is the logistics of the hospital. Seemingly, this can be overcome by the backup 
facilities in NKTI and SLH, such as the qPCR machines and freezers that had been operational 
before due to the COVID-19 pandemic. These, as well as the newly established research center, 
may serve as counterpart facilities during the implementation. 
 
15.​ GAD Score: refers to the result of accomplishing GAD checklists for project monitoring and 
evaluation/project management and implementation) to highlight the contribution of the project in 
the achievement of the objectives of Republic Act 7192, “Women in Development and Nation 
Building Act,” interpreted as gender-responsive gender-sensitive, has promising GAD concepts, or 
GAD is invisible. 
 
GAD score = 15.49 
 
16.​ Limitations of the Project: refers to restrictions or constraints in the conduct of the project. 
Since protocol development was jointly prepared by consultants of the NKTI and the other 
members of the DeLEPTO team, there are no major constraints foreseen. The objective of the 
study will be clearly explained to possible participants. 
 
17.​ Risk: refers to an uncertain event or condition that its occurrence has a negative effect on the 
project. 
Suppose patients who are affected by floods are given timely prophylaxis. In that case, this will 
result in a decreased number of patients who will develop complications from leptospirosis. This is 
considered an ideal outcome for patients. However, this will also result in a decrease in patient 
enrollment, and in turn will lead to a longer recruitment period for the study. 
 
18.​ Assumption: refers to an event or circumstance that its occurrence will lead to the success of the 
project 
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The rainy season every year starts in June, and if there has been no substantial improvement in 
the drainage system of Metro Manila’s streets, then floods are to be expected. If those who are 
exposed to floods are not provided with timely prophylaxis then they are at high risk for developing 
leptospirosis. Patients exhibiting symptoms of leptospirosis are brought to SLH, while patients who 
develop renal failure from leptospirosis are usually brought to the NKTI for dialysis. These patients 
are the participants in the present study. 
 
19.​ Literature Cited 
 
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20.​ Line-Item Budget 
 
(Find attached LIB form) 
 
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21.​ List of Personnel 
Position/ 
Designation 
Salary 
Grade 
Number of 
Positions 
Duration 
Percent Time 
Devoted to 
Project 
Responsibilities 
Science 
Research 
Specialist II 
16 
5 
48 
months 
100% 
1.​ Provide hands-on 
assistance on the 
experiments; 
2.​ Report progress of the 
study to the project leader; 
3.​ Present the results of the 
study in meetings; and 
4.​ Regularly attend meetings 
for feedback relevant to 
the experiments 
Project 
Development 
Officer III 
18 
1 
48 
months 
100% 
1.​ Establish/organize the 
schedules of meetings 
and representation 
relevant to the projects 
2.​ Prepare the administrative 
needs necessary for 
continuing operation of the 
projects 
3.​ Coordinate the smooth 
flow of acquisition and 
procurement of supplies 
and equipment of the 
project 
4.​ Prepare and archive 
documentary requirement 
for administrative 
purposes, such as 
financial reports, progress 
reports, annual reports, 
attendance sheets, and 
worksheets 
5.​ Liaise with sponsors and 
various stakeholders and 
collaborators 
6.​ Record and document 
relevant communications 
7.​ Assist in other 
administrative concerns as 
deemed necessary 
Project 
Leader 
Hono 
1 
48 
months 
15% 
1.​ Directly provide 
supervision to the 
research staff; 
2.​ Submit progress and 
financial reports to 
research sponsors 
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regularly; 
3.​ Coordinate activities of 
various 
collaborators/centers 
involved in the study; 
4.​ Keeps records and 
backup files for the study; 
5.​ Ensure that guidelines are 
followed in their respective 
projects, including the 
consent forms; 
6.​ Organize meetings and 
presentations pertinent to 
the study; 
7.​ Ensure proper training of 
research staff; 
8.​ Approve final write-ups 
prior to publication. 
9.​ Analyze and interpret 
results; 
10.​Actively participate in the 
publication and protection 
of data/intellectual 
property. 
Project Staff 
Level III 
Hono 
6 
48 
months 
15% 
1.​ Assists the research staff; 
2.​ Assist in sample 
collection; 
3.​ Keeps records and 
backup files for the study; 
4.​ Ensure that guidelines are 
followed in their respective 
projects, including the 
consent forms; 
5.​ Organize meetings and 
presentations pertinent 
to the study; 
6.​ Approve final write-ups 
prior to publication. 
S&T 
Consultant 
Hono 
2 
48 
months 
15% 
Refer patients for 
consideration for recruitment 
 
22.​ List of Official Project Team Members 
 
Title 
First Name 
Middle 
Name 
Last 
Name 
Sex 
Expertise 
Institutional 
Affiliation 
Role 
Dr. 
Romina 
A.  
Danguilan 
F 
Nephrology 
NKTI 
Project 
Leader 
Dr. 
Joselito 
R. 
Chavez 
M 
Pulmonology 
NKTI 
Project 
Staff 
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Dr. 
Jose 
B. 
Nevado 
M 
Internal 
Medicine/ 
Molecular 
Biology and 
Biotechnology 
IHG-NIH 
Project 
Staff 
Dr. 
Mel-hatra 
I.  
Arakama 
F 
Nephrology 
NKTI 
Project 
Staff 
Dr. 
Rontgene 
M. 
Solante 
M 
Infectious 
Diseases 
SLH 
Project 
Staff 
Dr.  
Ana Ria 
 
Sayo- 
Abungan 
F 
Infectious 
Diseases 
SLH 
Project 
Staff 
Dr.  
Jeffrey 
A. 
Verona 
M 
Infectious 
Diseases 
SLH 
Project 
Staff 
Dr. 
Jaime 
D. 
Trifalgar- 
Arches 
F 
Infectious 
Diseases 
SLH 
Project 
Staff 
Dr. 
Nathaniel 
 
Lee 
M 
Infectious 
Diseases 
SLH 
Project 
Staff 
 
23.​ List of Equipment for Project 
Name of Equipment 
Existing Equipment 
in the Agency 
To be purchased 
Justification for 
Purchase 
Qualitative PCR 
machine 
0 
1 
For reverse 
transcription and 
melting curve analysis 
Ultra Low Freezer 
None for the project 
1 
For storage of patient 
samples 
ELISA Reader 
1 (in the cooperating 
agency, MRDU lab of 
IHG NIH UPM) 
0 
Needed to perform 
and detect signals 
from ELISA kits 
Nanodrop 
Spectrophotometer 
0 
1 
For RNA 
quantification and OD 
260/280 determination 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX A 
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