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Diagnosing schistosomiasis-induced liver morbidity: implications for global control
Accepted Manuscript Title: Diagnosing schistosomiasis-induced liver morbidity: implications for global control Author: David U. Olveda Marianette Inobaya Remigio M. Olveda Marilyn L. Vinluan Shu-Kay Ng Kosala Weerakoon Donald P. McManus Grant A. Ramm Donald A. Harn Yuesheng Li Alfred K. Lam Jerric R. Guevarra Allen G. Ross PII: DOI: Reference:
S1201-9712(16)31208-5 http://dx.doi.org/doi:10.1016/j.ijid.2016.10.024 IJID 2755
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
16-9-2016 21-10-2016 25-10-2016
Please cite this article as: Olveda DU, Inobaya M, Olveda RM, Vinluan ML, Ng S-K, Weerakoon K, McManus DP, Ramm GA, Harn DA, Li Y, Lam AK, Guevarra JR, Ross AG, Diagnosing schistosomiasis-induced liver morbidity: implications for global control, International Journal of Infectious Diseases (2016), http://dx.doi.org/10.1016/j.ijid.2016.10.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Menzies Health Institute Queensland, Griffith University, Australia; Research Institute for Tropical Medicine, Department of Health, the Philippines; QIMR Berghofer Medical Research Institute, Australia; Faculty of Medicine and Biomedical Sciences, The University of Queensland, Australia; Centre for Tropical and Emerging Global Health Diseases, University of Georgia, USA; Hunan Institute of Parasitic Diseases, W.H.O. Collaborating Center for Research and Control of Schistosomiasis, China;
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David U. Olveda, M.D.1, Marianette Inobaya M.Sc.1,2, Remigio M. Olveda, M.D.2, Marilyn L. Vinluan, M.D.2, Shu-Kay Ng, Ph.D.1, Kosala Weerakoon, M.D.3, Donald P. McManus, Ph.D., D.Sc.3 Grant A. Ramm, Ph.D.3,4, Donald A. Harn, Ph.D.5, Yuesheng Li, M.D, Ph.D.3,6, Alfred K. Lam, M.D., Ph.D.1, Jerric R. Guevarra, B.Sc.,2 & Allen G. Ross, M.D., Ph.D.1†
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Diagnosing schistosomiasis-induced liver morbidity: implications for global control
ABSTRACT
Background. We evaluated the subclinical morbidity due to schistosomiasis in 565 patients
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and assessed for the first time the Enhanced Liver Fibrosis (ELF) test as a potential screening
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tool for disease.
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Methods. At baseline and two years after curative treatment, the prevalence and intensity of
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infection were determined by Kato-Katz thick smear stool examination. The degree of
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hepatic fibrosis was assessed by ultrasound. Non-invasive serum biomarkers of hepatic
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fibrosis were also evaluated.
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Results. The baseline human prevalence and infection intensity were found to be moderately
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high at 34% and 123 eggs per gram respectively. However, hepatic parenchymal fibrosis
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occurred among 50% of subjects, of which 19% had grade II fibrosis and 6% grade III. The
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Enhanced Liver Fibrosis (ELF) marker score and higher tissue inhibitor metalloproteinase
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inhibitor 1 (TIMP-1) and Hyaluronic acid (HA) serum levels correlated with liver fibrosis
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grade. 1 Page 1 of 26
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Conclusions. The findings demonstrated that praziquantel treatment had a short-term impact
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on both the prevalence and intensity of infection but less of an impact on established
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morbidity. Higher TIMP-1 and HA serum levels, and an ELF cut-off score of 8 correlated
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with liver fibrosis grade, thus may assist physicians in identifying individuals at greater risk
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of disease.
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Keywords. Schistosomiasis, morbidity, ultrasound, liver fibrosis, serum makers
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INTRODUCTION
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Schistosomiasis is a major public health problem that afflicts approximately 240 million
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people worldwide1 and causes approximately 70 million disability adjusted life years lost2.
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Preventive chemotherapy has been endorsed and advocated by the World Health
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Organisation (WHO) for the global control of schistosomiasis.3 Since its inception in 1979,
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much optimism has surrounded mass drug administration (MDA) for the worldwide control
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of schistosomiasis, for which praziquantel (PZQ) has served as the cornerstone drug.
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Numerous studies have claimed that preventive chemotherapy (i.e., 40 mg/kg PZQ), given
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once or twice yearly, can significantly reduce the prevalence and intensity of infection and
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control morbidity in the long term.3
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Schistosomiasis was first reported in 1906 in the Philippines. Approximately 865,000 people
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are currently infected and a further 12 million are at risk of infection.3,4 Major endemic foci
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(80%) are in the poorest regions of the Visayas (Samar and Leyte) and Mindanao.3,4 The
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current national control program comprises annual free MDA (40 mg/kg PZQ) in all 2 Page 2 of 26
schistosomiasis-endemic communities with a prevalence of >10%. The Philippines National
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Schistosomiasis Control Program has recently reported that the human prevalence has
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declined to less than 3% nationally5. However, contradictory reports claim the program is
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failing because of poor drug compliance, poor drug coverage, infrequent monitoring and
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evaluation, and rapid reinfection rates.4-6 Moreover, newly published data reported very high
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prevalence rates in both humans and bovines in endemic areas throughout the country.4-6
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There are now advanced schistosomiasis cases and disease-related deaths being reported by
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the National Department of Health for Mindanao, Samar, Leyte, and Oriental Mindoro.4
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Hepatic fibrosis is the major cause of morbidity and mortality among people with chronic
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schistosomiasis. Schistosomiasis-induced liver fibrosis in the field setting is best assessed
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with a portable ultrasound.6 The instrument has been reliable for detecting and assessing the
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degree of parasite-induced liver abnormalities, and for monitoring pathology regression
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following anti-schistosome treatment.6 Despite inter-observer variability related to its use8,
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several studies confirmed its usefulness in evaluating hepatosplenic schistosomiasis in the
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field over the past 30 years.6,9-125 The potential of non-invasive markers to complement the
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imaging assessment of organ morbidity has been studied before.5 Among the most promising
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for assessing schistosome-induced liver fibrosis include hyaluronic acid, collagenous
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proteins, matrix metalloproteinases and intercellular adhesion molecules.5,14,15
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We evaluated the clinical morbidity due to schistosomiasis at baseline and two-years post
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curative treatment among 565 residents residing in a known endemic area in Northern Samar,
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the Philippines. We also assessed for the first time the Enhanced Liver Fibrosis (ELF) test as
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a potential field screening tool for advanced disease.
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METHODS 3 Page 3 of 26
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Study Area
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The study area comprised 18 schistosomiasis endemic barangays in the municipalities of
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Laoang and Palapag, Northern Samar, the Philippines. Residents area are typically poor rice
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farmers with family incomes far below the national average. Over 50% of the population
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lives below the poverty line, with rudimentary water supply, sanitation and hygiene.7 The
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area is non-endemic for malaria but has had an active schistosomiasis control program for
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more than two decades including an MDA program that commenced in 2008. All individuals
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aged 5–65 years were offered free annual treatment (40 mg/kg of praziquantel) in accordance
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with the Department of Health Administrative Order 2007–0015.7
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Study Procedures
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In 2012 we conducted a cross-sectional schistosomiasis survey on approximately 20,000
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individuals in order to determine the prevalence, intensity of infection and morbidity
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associated with the disease (Figure 1).7 The baseline prevalence and intensity of infection
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were determined by Kato-Katz thick smear stool examination. Individuals were asked, over
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the course of a week, to provide two stool specimens from which six 50 g Kato-Katz thick
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smears were prepared on microscope slides according to established methods.20 Slides were
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examined under a light microscope by experienced laboratory technicians, who counted the
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number of S. japonicum eggs per slide. For quality control, 10% of all slides were randomly
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selected and re-examined by a senior microscopist at the Research Institute for Tropical
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Medicine, Manila. S. japonicum egg counts were expressed as eggs per gram (epg) of stool.18
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Intensity of infection was graded according to WHO criteria: light infection (1-99 epg),
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moderate infection (100-399 epg) and heavy infection (≥400 epg).19
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For a more accurate assessment of schistosome-induced hepatosplenic morbidity
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ultrasonographic studies were performed on subset of individuals with symptoms and signs
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suggestive of schistosomiasis. All subjects (n = 736) who reported gastrointestinal, and/or
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neurologic symptoms (ie. fatigue, malaise, abdominal pain, blood per rectum, hematemesis,
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diarrhoea, jaundice, dizziness, headache, and seizures) and/or were clinically assessed to have
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morbidity (i.e., palpable liver and spleen, varices, ascites, etc.) based on physical examination
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were initially selected for the ultrasound investigation. However, only those with baseline
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stool and ultrasound examination results were included in the final analysis (n = 565). The
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degree of hepatic fibrosis was assessed by ultrasound examination using a portable gray-scale
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ultrasonogram equipped with 3 MHz curve array transducer (SONOACE X1, Madison Co.,
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Ltd., Seoul, South Korea). The liver size was measured in millimetres along the mid-sternal
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line (MSL) and mid-clavicular line (MCL) for the left lobe and right lobe, respectively.
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Splenic size was also measured in millimetres along the left mid-axillary line (MAL). Hepatic
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fibrosis grading was adopted from the practical guidelines established for ultrasonography by
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the WHO/TDR (1990).20 Ultrasound findings were arrived at by consensus agreement of two
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experienced ultrasonographers. Ten percent of the ultrasound images were independently
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validated by a radiologist in Manila.
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Serum biomarkers of hepatic fibrosis were also evaluated on a random subset of patients. The
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selection of the sample subset from each fibrosis grade category was done using computer
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generated random numbers. Other laboratory investigations were conducted to rule out
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known liver pathology aetiologies (e.g. Hepatitis B and alcoholism). Blood (10 ml) was
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collected from each enrolled subject. Serum was separated from the whole blood and
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refrigerated at 2-8 0C. Collected sera were then transferred to a 40C cooler box and shipped to
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the Research Institute for Tropical Medicine (RITM) clinical laboratory in Manila. Each
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serum sample was divided into equal aliquots. Half of the serum specimens were used for
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liver function and hepatitis B surface antigen (HBsAg) testing and the other serum half was
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kept at -800C for fibrosis marker assays. Hepatitis C testing was excluded due to the very
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low prevalence (<3%) reported for rural remote communities in the Philippines. Liver
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function (including tests for hepatocellular injury, parenchymal damage, capacity for protein
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synthesis, and cholestasis) and HBsAg tests were performed on the samples using an
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automated machine (A15, Biosystem, S.A. Costa Brava, Barcelona, Spain) and EIA
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commercial kits (MonalisaBiorad, France), respectively. The serum samples for fibrosis
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marker testing were transferred to Siemens, Australia who evaluated hyaluronic acid (HA),
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procollagen type III (PIIINP), and tissue inhibitor of metalloproteinases (TIMP-1) serum
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levels using the Enhanced Liver Fibrosis (ELF) test.21 The ADVIA Centaur CP XP
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immunochemical analyser was used according to the manufacturer’s instructions. The ELF
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score was calculated directly by the instrument using the following equation:
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ELF score = 2.278 + 0.851 ln(CHA) + 0.751 ln(CP3NP) + 0.394 ln(CTIMP-1)
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The sICAM-1 serum levels were measured at QIMR Berghofer Medical Research Institute
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using a separate ELISA kit (R&D systems, Minneapolis city, Minnesota, USA).
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Study Oversight
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Ethical consent for the study was obtained from the ethical review boards of the Department
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of Health in the Philippines (Institutional Review Board Number 2012-13-0), Griffith
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University, Australia and QIMR Berghofer Medical Research Institute, Australia. Written
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informed consent was obtained from each individual or for those aged <15 years from their
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parents/ legal guardians. Stool-positive subjects were treated orally with 60 mg/kg PZQ in
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two divided doses, followed by another treatment of the same dose two weeks later. Stool-
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negative individuals were treated with a single oral dose of 40mg/kg.
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Statistical Analysis
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FoxPro (version 6.0) was used for double entry of data, and data were cross-checked and
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analyzed with SAS, version 9.4. Statistical analysis was performed using STATA version 11.
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Data were summarized as frequencies and mean ± SD as appropriate. All categorical
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variables were evaluated using Chi-Squared test. Wilcoxon Signed Ranks and McNemar’s
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tests were used for paired analysis of infection intensity and prevalence respectively across
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different age groups. With regards to treatment impact on infection intensity, S. japonicum
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egg count + 1 was log-10 transformed to bring observed distributions closer to normality.
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The Kruskall-Wallis was used to detect significant differences in the level of serum marker
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types across fibrosis grades. Receiver operator characteristics (ROC) analysis were
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performed to assess the clinical utility of the three serum fibrosis markers (HA, TIMP-1, and
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PIIINP), both individually, as well as their combined quantitative results (ELF score) in
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identifying subjects with high grade liver fibrosis (grade II-III). A significance level of 5%
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was used for statistical inference.
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RESULTS 7 Page 7 of 26
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Study Descriptors
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Fifty-three per cent of the study sample (n = 565) was male, with a mean age of 39.4 years
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(95% CI, 38-41 years). Most were poor rice farmers with a high degree of exposure. There
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was no statistical difference in the sex profile of the sample. However, most of the study
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participants belonged to age groups of 36-45 years (24%) and 46-55 (27%) years,
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respectively. Only 1.0% of the population reported having been diagnosed with hepatitis B;
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however, 38.4% drank alcohol moderately (consumed 1-2 drinks per day).
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Prevalence, intensity and morbidity outcomes
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The study communities have participated in an active schistosomiasis program for the past 20
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years. However, the prevalence of S. japonicum infection among the pre-treatment cohort
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subjects (n = 565) was 34% (Figure 2). Males had a higher prevalence of infection (46%)
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than females (20%). S. japonicum infection was more prevalent (49%) among subjects aged
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less than 20 years of age. The mean intensity of infection was 123.1 epg (95% CI; 78.9–
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167.3epg) (Figure 2). Twenty-five percent had light intensities of infection, 6% moderate
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intensities of infections, and 2% heavy infections. Hepatic parenchymal fibrosis occurred
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among 50% of subjects, of which 19% had grade II fibrosis and 6% grade III (Table 1). The
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fibrosis prevalence in males and females were 52.7% (298/565) and 47.3% (267/565),
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respectively. The degree of liver fibrosis correlated significantly (P = 0.0002) with infection
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intensity. The left and right hepatic lobe lengths increased with increasing fibrosis grade. The
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mean left lobe lengths of those with grade II (mean length = 93.7 mm; 95% CI, 92.5-94.8
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mm) and III (mean length = 98.4 mm; 95% CI, 96.4–100.4 mm) fibrosis were significantly
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larger (P = 0.0001) than those without fibrosis (mean length = 87.7 mm; 95% CI 86.4–88.9
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mm). However, only the mean length of the right lobe of individuals with grade III fibrosis
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(122.3 mm; 95% CI 120.7–124.0 mm) was significantly (P= 0.04) larger compared with
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those lacking fibrosis. Likewise, the mean length of the spleen increased with increasing
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degree of fibrosis, although not significantly (P = 0.21).
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473 subjects had both baseline and follow-up stool examination results, hence, these
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individuals were utilized in the final analysis to determine the impact of PZQ treatment on
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prevalence and intensity of infection. The prevalence dropped significantly two years post-
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treatment, especially among those aged 16-25 years (P = 0.008) and 46-55 years (P = 0.001)
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(Figure 2). Similarly, the intensity of infection across all age groups dropped compared with
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pre-treatment levels (Figure 2). The prevalence decline observed among the 16-25 years age
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group may in part be due to limited exposure to infection. On the other hand, acquired
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immunity to reinfection may partially account for the prevalence drop among those aged 46–
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55 years. However, only subjects aged 46-55 years showed significant decreases in the
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intensity of infection compared with the baseline findings (P = 0.003). The prevalence of
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grade II-III fibrosis remained relatively unchanged at 25.2% (107/424) on follow-up (Table
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1). The data suggests that a two year follow-up period may be too short of a time frame to
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assess disease regression following treatment.
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Liver function tests, hepatitis B, and serum fibrosis markers
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Liver function tests were carried out on a selected subset of patients (n = 136). The total
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number of individuals was derived via age-matched specific grouping resulting in 34 subjects
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per each of the four fibrosis clinical grades. Sixty percent (81/136) of the subjects had normal
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liver function tests with the proportion being uniform across all fibrosis grades. Mild SGPT
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elevation was noted in 1.5% (2/136) of the sample. Alkaline phosphatase and total bilirubin
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result were similarly distributed among the four fibrosis groups. Hepatitis B surface antigen
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testing was done on all 565 subjects. The HBsAg carrier rate was 4.2% (24/565). The carrier
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rates in those with grades I, II and III fibrosis were 1.4% (2/145), 2.8% (3/105) and 2.9%
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(1/34), respectively. The carrier rates in those with fibrosis were actually lower than those
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without fibrosis (6.4%). Thus, the prevalence of both alcohol and chronic hepatitis B-induced
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liver fibrosis were demonstrated to be low in the cohort.
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The serum fibrosis marker levels and infection intensity before and after PZQ treatment were
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also assessed. Only baseline mean TIMP1 levels showed cumulative results with increasing
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intensity of infection (P = 0.001). The baseline and endpoint serum levels of the fibrotic
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markers were also compared with parenchymal grading. The Enhanced Liver Fibrosis (ELF)
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marker score and two of three ELF markers correlated with fibrosis severity. The mean
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baseline and follow-up ELF score correlated significantly with increasing liver fibrosis grade
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(P = 0.002; P = 0.049). Before treatment, baseline TIMP-1 levels were increased in all three
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increasing fibrosis grades (P < 0.001). At endpoint, TIMP-1 levels were also significantly
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higher in grade III compared with grade I (P = 0.034). The HA levels were higher in grade III
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compared with grade 0 (P = 0.031) and grade I (P = 0.0429). The sICAM-1 also correlated
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with fibrosis grade (P = 0.0013).
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Diagnostic performance of ELF
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Table 2 shows the baseline and follow-up ROC curve analysis results of the ELF score and
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individual markers to discern subjects with grade II-III fibrosis (severe) from those with
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grade 0-I fibrosis (mild). The diagnostic accuracy of HA and the ELF score were slightly
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higher than the two other ELF markers assessed based on the area under the curve (AUC) at
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baseline. At follow-up, HA, TIMP-1, and the ELF scores had higher diagnostic accuracy than
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PIIINP. The cut-off values of 30.7 ng/mL for HA (AUC 0.69, P < 0.001) and 9.2 for the ELF
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score (AUC 0.68, P < 0.001) to discriminate grade II-III fibrosis from grade 0-I among 177
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subjects at baseline, have sensitivities of 66% and 64%, specificities of 72% and 71
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respectively. At follow-up, the cut-off values of 26.3 ng/mL for HA (AUC 0.63, P < 0.043),
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183.7 ng/mL for TIMP-1 (AUC 0.62, P < 0.001), and 8.9 for the ELF score (AUC 0.62, P <
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0.004) to discern grade II-III fibrosis from grade 0-I among 180 subjects have sensitivities of
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73%, 73%, and 62%, and specificities of 53%, 51%, and 62% respectively. PIIINP was
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uninformative, having the least AUC for both time points. Figures 3 and 4 illustrate the ROC
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curves for the three individual ELF markers as well as the curves for the ELF score. The ELF
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score and HA showed the highest diagnostic performance in detecting severe schistosome-
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induced liver fibrosis. When gender was considered, no significant difference was observed
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in the cut-off values at baseline and at two years follow-up. Cut-off values of 8.9 and 9.2
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ng/mL were noted for males (baseline: AUC 0.69, P = 0.003; follow-up: AUC 0.62, P =
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0.342) and females (baseline: AUC 0.64, P = 0.226; follow-up: AUC 0.69, P = 0.042)
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respectively, suggesting that the same cut-off value can be utilized for both sexes. However,
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when age was taken into account, the cut-off value at baseline was noted to be higher among
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subjects younger than 18 years of age (9.9 ng/mL, AUC 0.96, P = 0.192) compared with
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those aged 18-40 years (8.9 ng/mL, AUC 0.71, P = 0.087), 41-50 (8.9 ng/mL AUC 0.74, P =
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0.005) and over 50 years of age (9.2 ng/mL, AUC 0.61, P = 0.046). This suggests that age
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may influence the ELF test results. On follow-up, the cut-off values could not be determined
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for the adolescent (<18 years) cohort because very few of these individuals had moderate to
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severe fibrosis. Overall, the mean values of the fibrosis markers seemed to increase across the
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fibrosis grades, except for PIINP (Table 3). But within each grade, there seems to be no
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apparent pattern across intensity of infection. This observation may be due to the small
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sample size within each category.
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DISCUSSION
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We evaluated the level of schistosomiasis prevalence, infection intensity and morbidity
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among 565 subjects residing in eighteen moderately endemic barangays in Northern Samar,
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the Philippines. The study communities have participated in the National Department of
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Health human treatment program for schistosomiasis over the past three decades.
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Nevertheless, the baseline human prevalence and infection intensity were 34% and 123 epg
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respectively. Baseline ultrasonography revealed a high level of schistosomiasis-induced
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morbidity among subjects with signs and symptoms suggestive of schistosomiasis. Eighty-
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nine per cent of the patients had left lobe liver enlargement (≥ 70 mm) and 25% had grade II
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or III liver fibrosis. Splenomegaly (≥ 100 mm) was seen in 13% of the population. Infection
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intensity was significantly (P < 0.0002) correlated with grade III fibrosis. Hepatitis B virus,
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alcoholism and malaria were excluded as possible causes. The results suggest that the
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national control strategy for schistosomiasis (i.e., human treatment with 40 mg/kg PZQ) is
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inadequate in the study area. Low drug coverage (<36%), low drug compliance (<40%), and
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inability of PZQ to prevent re-infection given the zoonotic nature of the disease may have
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contributed to the findings.22
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Two years after baseline chemotherapy (i.e., 40-60 mg/kg PZQ), as expected, a greater
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reduction in both the prevalence and intensity of infection were noted. However, treatment
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had less of an impact on lowering schistosomiasis-induced morbidity. Overall, hepatic
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fibrosis (grades I-III) regressed in only 24.3% of those who received a single treatment and in
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only 19.3% of those who received two PZQ doses. This outcome was similar to that reported
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for S. japonicum morbidity in China.9 The baseline grade II-III fibrosis prevalence was
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25.2%, but remained unchanged two years post-treatment.
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morbidity despite standard treatment will need a longer treatment course at a higher dose
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(i.e., 60-80 mg/kg) to reverse fibrosis and improve clinical outcome. Approximately 10% of
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residents in S. mansoni and S. japonicum endemic areas develop severe hepatosplenic
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disease.23-25 These observations have been partly explained by genetic factors regulating
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disease development.23-25
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Individuals with significant
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Liver fibrosis is a complex process involving production and deposition of insoluble
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components that comprise the extracellular matrix (ECM).9 Quantitative and qualitative ECM
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changes in liver fibrosis can be measured in blood or urine using indirect and direct
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biomarkers.5 These biomarkers of hepatic matrix metabolism have also been drawn together
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into multi-parametric scores to produce promising results.21,26 The Enhanced Liver Fibrosis
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(ELF) test is an ECM marker set comprising an algorithm of three direct liver fibrosis
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markers (HA, TIMP-1, and PIIINP). The ELF test was one of the first commercially available
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serum fibrosis tests21, and its diagnostic potential has been explored in identifying and staging
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liver fibrosis among patients with hepatitis C viral infection21,27, alcoholic liver disease26, and
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non-alcoholic fatty liver disease (NAFLD).28 The test is said to be comparable with
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FibroScan (transient elastography)29,30 and more accurate or reproducible than simple panels
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(such as aspartate aminotransferase to platelet ratio and Fibrosis-4) in detecting advanced
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fibrosis.31
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Our study assessed four promising direct biomarkers (HA, PIIINP, TIMP-1, and sICAM-1)
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based on their performance in previous studies.5,9,14,15 To the best of our knowledge, this is
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the first time that an ELF score was deployed in the evaluation of liver fibrosis due to
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schistosomiasis. The best correlation between serum marker level and ultrasonographic
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fibrosis grading was demonstrated with TIMP-1. The TIMP-1 levels increased as the degree
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of fibrosis increased both at baseline and at endpoint. The marker levels also decreased in all
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fibrosis grade categories at endpoint (after treatment) suggesting the TIMP-1 serum levels
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can accurately assess liver fibrotic disease due to schistosomiasis. Also, TIMP-1 serum levels
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correlated positively with intensity of infection, suggesting that the marker may also respond
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to inflammatory reactions to eggs deposited in the liver. HA and sICAM-1 also showed some
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correlation but not as significant as shown for TIMP-1. Moreover, the ROC analysis revealed
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that HA as an individual marker showed the highest sensitivity and specificity in discerning
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higher fibrosis grades from the lower grades. The diagnostic performance of the ELF score
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was equally as useful as that shown for HA alone. However, the AUC values generated using
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both the ELF score and serum biomarkers individually indicated only moderate
353
discrimination between grades II-III and 0-I fibrosis (i.e., AUC’s 0.6-0.7).33 Moreover,
354
consistent with other studies, age should be considered as a factor when generating ELF
355
scores.21 An unadjusted ELF score may misclassify normal individuals younger than 18 years
356
of age as having moderate-to-severe schistosome-induced liver fibrosis. If the diagnostic
357
value of the ELF test can be improved as a reliable stand-alone test and suitable for field
358
application, then it could potentially deployed as a component of the diagnostic and treatment
359
algorithm for S. japonicum infected patients (Figure 5).
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360 361
CONCLUSIONS
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14 Page 14 of 26
We demonstrated a high level of schistosomiasis-induced morbidity among subjects with
364
signs and symptoms suggestive of the disease in a moderately endemic area of the Philippines
365
that has been under active community-based chemotherapy for over three decades. Hepatic
366
fibrosis, the main cause of hepato-splenic schistosomiasis, takes time to reverse with
367
chemotherapy. Individuals with hepato-splenic schistosomiasis should be identified and
368
treated as early as possible to prevent disease progression and possible death. Serum fibrosis
369
markers, particularly
370
may potentially guide program managers on where to direct limited health-care resources
371
for schistosomiasis control. More specifically, if made affordable for field application, the
372
ELF test may assist clinicians in assessing patients with heavy intensities of infection (>400
373
epg) and/or with physical signs of advanced disease (e.g. MCL > 3) in determining their risk
374
of severe liver fibrosis.
375 376 377
Acknowledgments: We thank the Australian National Health and Medical Research Council for providing financial support for our research.
378 379 380
Financial support: Our work is supported by the Australian National Health and Medical Research Council. Professor McManus is a Senior Principal Research Fellow with the Australian National Health and Medical Research Council.
381 382
Potential conflicts of interest: All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential.
383 384
Conflicts of Interest: Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
and
HA
levels,
and
ELF
scores
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TIMP-1
above
8
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Bockarie MJ, Kelly-Hope LA, Rebollo M, Molyneux DH. Preventive chemotherapy as a strategy for elimination of neglected tropical parasitic diseases: endgame challenges. Philosophical transactions of the Royal Society of London Series B, Biological sciences 2013;368:20120144. King CH, Dangerfield-Cha M. The unacknowledged impact of chronic schistosomiasis. Chronic Illness 2008;4:65-79. Inobaya MT, Olveda RM, Tallo V, McManus DP, Williams GM, Harn DA, et al. Schistosomiasis mass drug administration in the Philippines: lessons learnt and the global implications. Microbes and infection / Institut Pasteur 2015;17:6-15. Olveda RM, Tallo V, Olveda DU, Inobaya MT, Chau TN, Ross AG. National survey data for zoonotic schistosomiasis in the Philippines grossly underestimates the true burden of disease within endemic zones: implications for future control. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 2016. Olveda DU, Olveda RM, McManus DP, Cai P, Chau TN, Lam AK, et al. The chronic enteropathogenic disease schistosomiasis. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 2014. Olveda DU, Olveda RM, Lam AK, Chau TN, Li Y, Gisparil AD, 2nd, Ross AG. Utility of Diagnostic Imaging in the Diagnosis and Management of Schistosomiasis. Clinical microbiology (Los Angeles, Calif) 2014;3. Olveda DU, Li Y, Olveda RM, Lam AK, Chau TN, Harn DA, et al. Bilharzia: Pathology, Diagnosis, Management and Control. Tropical medicine & surgery 2013;1. Doehring-Schwerdtfeger E, Kaiser C, Franke D, Kardorff R, Ali QM, Abdel-Rahim IM. Inter-observer variance in ultrasonographical assessment of Schistosoma mansoni-related morbidity in young schoolchildren. Acta tropica 1992;51:85-8. Li YS, Sleigh AC, Ross AG, Li Y, Williams GM, Tanner M, McManus DP. Two-year impact of praziquantel treatment for Schistosoma japonicum infection in China: reinfection, subclinical disease and fibrosis marker measurements. Transactions of the Royal Society of Tropical Medicine and Hygiene 2000;94:191-7. Marinho CC, Voieta I, Azeredo LM, Nishi MP, Batista TS, Pereira AC, et al. Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas. Memorias do Instituto Oswaldo Cruz 2006;101 Suppl 1:317-21. Ruiz-Guevara R, de Noya BA, Valero SK, Lecuna P, Garassini M, Noya O. Clinical and ultrasound findings before and after praziquantel treatment among Venezuelan schistosomiasis patients. Revista da Sociedade Brasileira de Medicina Tropical 2007;40:505-11. Pinto-Silva RA, Queiroz LC, Azeredo LM, Silva LC, Lambertucci JR. Ultrasound in schistosomiasis mansoni. Memorias do Instituto Oswaldo Cruz 2010;105:479-84. Bataller R, Brenner DA. Liver fibrosis. The Journal of clinical investigation 2005;115:209-18. Ismail KA AS, Elleboudy NAF. Serum Hyaluronic Acid (HA) and Soluble Intercellular Adhesion Molecule-1(Sicam-1) as Non-Invasive Markers of Liver Fibrosis in Viral Hepatitis, Schistosomiasis mansoni and Co-infected Patients. J Bacteriol Parasitol 2012;3.
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Hou XY, Ellis MK, McManus DP, Wang YY, Li SD, Williams GM, Li YS. Diagnostic value of non-invasive bio-markers for stage-specific diagnosis of hepatic fibrosis in patients with advanced schistosomiasis japonica. International journal for parasitology 2011;41:325-32. Olveda R, Daniel B, Ramirez D, Aligui G, Acosta L, Fevidal P, et al. Schistosomiasis Japonica in the Philippines: The Long-Term Impact of Population-Based Chemotherapy on Infection, Transmission and Morbidity. The Journal of Infectious Diseases 1996;174:163-72. Baird JK, Bangs MJ, Maguire JD, Barcus MJ. Epidemiological measures of risk of malaria. Methods in molecular medicine 2002;72:13-22. Katz N, Chaves A, J P. A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Rev Inst Med Trop Sao Paulo 1972;14:397-400. Olveda R, Tiu E, Fevidal PJ, De Veyra FJ, Icatlo FJ, Domingo E. The Relationship of Prevalence and Intensity of Infection with Morbidity in Schistosomiasis Japonica: A Study of Three Communities in Leyte, Philippines. American Journal of Tropical Medicine and Hygiene 1983;32:1312-21. Niamey Working Group 2000. Ultrasound in schistosomiasis. A practical guide to the standardized use of ultrasonography for the assessment of schistosomiasis-related morbidity. World Health Organization/TDR/SCH/ULTRASON/document. Geneva, Switzerland. Lichtinghagen R, Pietsch D, Bantel H, Manns MP, Brand K, Bahr MJ. The Enhanced Liver Fibrosis (ELF) score: normal values, influence factors and proposed cut-off values. Journal of hepatology 2013;59:236-42. Ross AG, Olveda RM, Acosta L, Harn DA, Chy D, Li Y, et al. Road to the elimination of schistosomiasis from Asia: the journey is far from over. Microbes and infection / Institut Pasteur 2013;15:858-65. Dessein A, Chevillard C, Arnaud V, Hou X, Hamdoun AA, Dessein H, et al. Variants of CTGF are associated with hepatic fibrosis in Chinese, Sudanese, and Brazilians infected with schistosomes. The Journal of experimental medicine 2009;206:2321-8. Blanton RE, Salam EA, Ehsan A, King CH, Goddard KA. Schistosomal hepatic fibrosis and the interferon gamma receptor: a linkage analysis using single-nucleotide polymorphic markers. European journal of human genetics : EJHG 2005;13:660-8. Dessein AJ, Marquet S, Henri S, El Wali NE, Hillaire D, Rodrigues V, et al. Infection and disease in human schistosomiasis mansoni are under distinct major gene control. Microbes and infection / Institut Pasteur 1999;1:561-7. Rosenberg WM, Voelker M, Thiel R, Becka M, Burt A, Schuppan D, et al. Serum markers detect the presence of liver fibrosis: a cohort study. Gastroenterology 2004;127:1704-13. Parkes J, Guha IN, Roderick P, Harris S, Cross R, Manos MM, et al. Enhanced Liver Fibrosis (ELF) test accurately identifies liver fibrosis in patients with chronic hepatitis C. Journal of viral hepatitis 2011;18:23-31. Nobili V, Parkes J, Bottazzo G, Marcellini M, Cross R, Newman D, et al. Performance of ELF serum markers in predicting fibrosis stage in pediatric nonalcoholic fatty liver disease. Gastroenterology 2009;136:160-7. Friedrich-Rust M, Rosenberg W, Parkes J, Herrmann E, Zeuzem S, Sarrazin C. Comparison of ELF, FibroTest and FibroScan for the non-invasive assessment of liver fibrosis. BMC Gastroenterol 2010;10:103. EASL-ALEH Clinical Practice Guidelines: Non-invasive tests for evaluation of liver disease severity and prognosis. Journal of hepatology 2015;63:237-64.
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17 Page 17 of 26
31.
32.
33.
Fagan KJ, Pretorius CJ, Horsfall LU, Irvine KM, Wilgen U, Choi K, et al. ELF score >/=9.8 indicates advanced hepatic fibrosis and is influenced by age, steatosis and histological activity. Liver international : official journal of the International Association for the Study of the Liver 2015;35:1673-81. Yoo EJ, Kim BK, Kim SU, Park JY, Kim do Y, Ahn SH, et al. Normal enhanced liver fibrosis (ELF) values in apparently healthy subjects undergoing a health check-up and in living liver donors in South Korea. Liver international : official journal of the International Association for the Study of the Liver 2013;33:706-13. Hosmer DW, Lemeshow S. Applied Logistic Regression. 2nd ed: John Wiley & Sons, Inc.; 2000.
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483 484 485 486 487 488 489 490 491 492
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18 Page 18 of 26
494 N= 20,000 Eligible residents aged 5-65 years from 22 barangay
cr
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n = 18,221 Completed individual medical questionnaire
n = 58 Individuals without head of hous e questionnaire
n = 10,435 (57%) Com plete stool data
n = 7,786 Incomplete stool data
an
us
n = 18,163 (99%) Indivduals with head of house ques tionnaire
ed
M
n = 6,074 (33%) Physical exam ination
pt
n = 736 (4%) Ultras ound examination
496 497 498 499 500
n = 171 incomplete stool data
n = 424 Ultrasound Follow-up + Stool Follow-up
n = 141 Incom plete stool data
Ac ce
495
n = 565 Ultrasound exam ination + Baseline stool exam ination
Figure 1. Study profile and compliance among 18,221 residents from 22 schistosomiasisendemic barangays in Palapag and Laoang, Northern Samar, the Philippines. All inhabitants aged 5–65 years were invited to participate in the questionnaire and provide 2 stool samples for parasitological examination. A subset of patients (n = 736) were selected for ultrasound investigations.
19 Page 19 of 26
ip t cr us Figure 2. Prevalence and intensity of infection in subjects with S. japonicum by age before (2012) and after (2015) two years of praziquantel treatment (i.e., 40-60 mg/kg; n = 473).
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502 503 504
an
501
505
pt
508
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507
ed
506
20 Page 20 of 26
508
513 514 515 516
pt
511 512
Figure 3. Baseline ROC curves (n = 177) for serum concentrations of ELF, HA, PIIINP, and TIMP-1 for differentiating grade II-III liver fibrosis from grade 0-I fibrosis.
Ac ce
510
ed
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509
517 518 519
21 Page 21 of 26
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Figure 4. Two year post-treatment follow-up ROC curves (n = 180) for serum concentrations of ELF, HA, PIIINP, and TIMP-1 for differentiating grade II-III liver fibrosis from grade 0-I fibrosis.
pt
521 522 523
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520
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525
Patient with heavy intensity of infection ( > 400 epg) and/or liver enlargment (MCL .> 3 cm)
Grade II/III liver fibrosis unlikely Refer to local treament and follow-up
Grade II/III liver fibrosis likely Refer for ultrasound as sessment
an M
529 530
Ultras ound positive
Refer to Gastroenterologist
Ac ce
527 528
pt
ed
Ultras ound negative
526
cr
ELF score > 8
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ELF Score < 8
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Referral for ELF Test
Figure 5: Algorithm for the recommended diagnosis and management of severe hepatic fibrosis (grade II-III) due to gastrointestinal schistosomiasis infection.
531 532
23 Page 23 of 26
532
Table 1. Dynamics of schistosomiasis liver parenchyma grading before and after praziquantel treatment in a cohort of 424 subjects from Northern Samar, Philippines from 2012 – 2015.
Parenchyma Grading 2012
Parenchyma Grading 2015
Total (N)
Total (%)
Progressed (%)
Regressed (%)
Grade 1
Grade 2
Grade 3
Normal
156
54
3
0
213
50.24
Grade 1
40
40
17
7
104
24.53
Grade 2
8
17
33
22
80
18.87
27.5
31.3
Grade 3
2
1
14
10
27
6.37
0.0
63.0
Total (n)
206
112
67
39
424
103
82
535
0.0
23.1
38.5
cr
26.8
100
an
536
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Normal
us
533 534
537
Ac ce
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ed
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538
24 Page 24 of 26
538 539 540 541 542
Table 2. Area under the curve (AUC), and diagnostic sensitivities and specificities for ELF score cut-off values and individual serum fibrotic markers at baseline and two years after treatment using receiver operating analyses to distinguish between severe fibrosis (grade IIIII) from low grade fibrosis (grade 0-I).
543
0.68 0.69 0.56 0.65
<0.001 <0.001 0.515 <0.001
64 66 61 63
0.62 0.63 0.57 0.62
0.004 0.043 0.475 0.001
62 73 58 73
9.2 30.7 10.2 253.6
62 53 57 51
8.9 26.3 9.4 183.7
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ed
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544
71 72 51 68
Cut-off
ip t
Sensitivity Specificity
cr
P
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AUC
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Fibrosis Marker Baseline (n=177) ELF Hyaluronic Acid PIINP TIMP1 Follow-up (n=180) ELF Hyaluronic Acid PIINP TIMP1
25 Page 25 of 26
544
Table 3. Mean fibrosis marker value by intensity of infection and fibrosis grade.
545
Intensity of Infection
n
ELF
HA
PIINP
TIMP
Negative
68
8.8
31.6
12.1
199.8
Mild
10
8.9
23
14.7
252.5
Moderate
2
8.5
21.4
8.2
278.1
Heavy
0
cr
Grade 1 0
Mild
7
8.8
23.2
13.7
295.3
Moderate
4
9
27.3
13.6
290.1
Heavy
1
9.4
12
27.6
594.5
Negative
14
9.3
41.1
Mild
17
9.1
46.5
Moderate
4
9.4
Heavy
1
7.7
Mild
546 547
an
M 10.3
274.6
10
241.1
5.6
10.5
277.1
pt
8
294.6
59.2
85.5
21.4
302.8
10
Ac ce
Negative
12.3
ed
Grade 2
us
Negative
Grade 3
ip t
Grade 0
10
9.6
64.6
11.2
279.5
Moderate
3
10.2
76.2
14.2
370.4
Heavy
2
8.7
24.1
10.3
457.8
548
26 Page 26 of 26
S1201-9712(16)31208-5 http://dx.doi.org/doi:10.1016/j.ijid.2016.10.024 IJID 2755
To appear in:
International Journal of Infectious Diseases
Received date: Revised date: Accepted date:
16-9-2016 21-10-2016 25-10-2016
Please cite this article as: Olveda DU, Inobaya M, Olveda RM, Vinluan ML, Ng S-K, Weerakoon K, McManus DP, Ramm GA, Harn DA, Li Y, Lam AK, Guevarra JR, Ross AG, Diagnosing schistosomiasis-induced liver morbidity: implications for global control, International Journal of Infectious Diseases (2016), http://dx.doi.org/10.1016/j.ijid.2016.10.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Menzies Health Institute Queensland, Griffith University, Australia; Research Institute for Tropical Medicine, Department of Health, the Philippines; QIMR Berghofer Medical Research Institute, Australia; Faculty of Medicine and Biomedical Sciences, The University of Queensland, Australia; Centre for Tropical and Emerging Global Health Diseases, University of Georgia, USA; Hunan Institute of Parasitic Diseases, W.H.O. Collaborating Center for Research and Control of Schistosomiasis, China;
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1. 2. 3. 4. 5. 6.
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David U. Olveda, M.D.1, Marianette Inobaya M.Sc.1,2, Remigio M. Olveda, M.D.2, Marilyn L. Vinluan, M.D.2, Shu-Kay Ng, Ph.D.1, Kosala Weerakoon, M.D.3, Donald P. McManus, Ph.D., D.Sc.3 Grant A. Ramm, Ph.D.3,4, Donald A. Harn, Ph.D.5, Yuesheng Li, M.D, Ph.D.3,6, Alfred K. Lam, M.D., Ph.D.1, Jerric R. Guevarra, B.Sc.,2 & Allen G. Ross, M.D., Ph.D.1†
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3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
__________________________________________________________________________________
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2
Diagnosing schistosomiasis-induced liver morbidity: implications for global control
ABSTRACT
Background. We evaluated the subclinical morbidity due to schistosomiasis in 565 patients
M
1
and assessed for the first time the Enhanced Liver Fibrosis (ELF) test as a potential screening
23
tool for disease.
ed
22
24
Methods. At baseline and two years after curative treatment, the prevalence and intensity of
26
infection were determined by Kato-Katz thick smear stool examination. The degree of
27
hepatic fibrosis was assessed by ultrasound. Non-invasive serum biomarkers of hepatic
28
fibrosis were also evaluated.
Ac ce
pt
25
29 30
Results. The baseline human prevalence and infection intensity were found to be moderately
31
high at 34% and 123 eggs per gram respectively. However, hepatic parenchymal fibrosis
32
occurred among 50% of subjects, of which 19% had grade II fibrosis and 6% grade III. The
33
Enhanced Liver Fibrosis (ELF) marker score and higher tissue inhibitor metalloproteinase
34
inhibitor 1 (TIMP-1) and Hyaluronic acid (HA) serum levels correlated with liver fibrosis
35
grade. 1 Page 1 of 26
36
Conclusions. The findings demonstrated that praziquantel treatment had a short-term impact
38
on both the prevalence and intensity of infection but less of an impact on established
39
morbidity. Higher TIMP-1 and HA serum levels, and an ELF cut-off score of 8 correlated
40
with liver fibrosis grade, thus may assist physicians in identifying individuals at greater risk
41
of disease.
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Keywords. Schistosomiasis, morbidity, ultrasound, liver fibrosis, serum makers
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Word count: Abstract = 197; Text = 3,664
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42 43 44 45 46 47 48 49 50
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37
INTRODUCTION
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Schistosomiasis is a major public health problem that afflicts approximately 240 million
53
people worldwide1 and causes approximately 70 million disability adjusted life years lost2.
54
Preventive chemotherapy has been endorsed and advocated by the World Health
55
Organisation (WHO) for the global control of schistosomiasis.3 Since its inception in 1979,
56
much optimism has surrounded mass drug administration (MDA) for the worldwide control
57
of schistosomiasis, for which praziquantel (PZQ) has served as the cornerstone drug.
58
Numerous studies have claimed that preventive chemotherapy (i.e., 40 mg/kg PZQ), given
59
once or twice yearly, can significantly reduce the prevalence and intensity of infection and
60
control morbidity in the long term.3
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61 62
Schistosomiasis was first reported in 1906 in the Philippines. Approximately 865,000 people
63
are currently infected and a further 12 million are at risk of infection.3,4 Major endemic foci
64
(80%) are in the poorest regions of the Visayas (Samar and Leyte) and Mindanao.3,4 The
65
current national control program comprises annual free MDA (40 mg/kg PZQ) in all 2 Page 2 of 26
schistosomiasis-endemic communities with a prevalence of >10%. The Philippines National
67
Schistosomiasis Control Program has recently reported that the human prevalence has
68
declined to less than 3% nationally5. However, contradictory reports claim the program is
69
failing because of poor drug compliance, poor drug coverage, infrequent monitoring and
70
evaluation, and rapid reinfection rates.4-6 Moreover, newly published data reported very high
71
prevalence rates in both humans and bovines in endemic areas throughout the country.4-6
72
There are now advanced schistosomiasis cases and disease-related deaths being reported by
73
the National Department of Health for Mindanao, Samar, Leyte, and Oriental Mindoro.4
us
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66
74
Hepatic fibrosis is the major cause of morbidity and mortality among people with chronic
76
schistosomiasis. Schistosomiasis-induced liver fibrosis in the field setting is best assessed
77
with a portable ultrasound.6 The instrument has been reliable for detecting and assessing the
78
degree of parasite-induced liver abnormalities, and for monitoring pathology regression
79
following anti-schistosome treatment.6 Despite inter-observer variability related to its use8,
80
several studies confirmed its usefulness in evaluating hepatosplenic schistosomiasis in the
81
field over the past 30 years.6,9-125 The potential of non-invasive markers to complement the
82
imaging assessment of organ morbidity has been studied before.5 Among the most promising
83
for assessing schistosome-induced liver fibrosis include hyaluronic acid, collagenous
84
proteins, matrix metalloproteinases and intercellular adhesion molecules.5,14,15
M
ed
pt
Ac ce
85
an
75
86
We evaluated the clinical morbidity due to schistosomiasis at baseline and two-years post
87
curative treatment among 565 residents residing in a known endemic area in Northern Samar,
88
the Philippines. We also assessed for the first time the Enhanced Liver Fibrosis (ELF) test as
89
a potential field screening tool for advanced disease.
90
METHODS 3 Page 3 of 26
91 92
Study Area
93
The study area comprised 18 schistosomiasis endemic barangays in the municipalities of
95
Laoang and Palapag, Northern Samar, the Philippines. Residents area are typically poor rice
96
farmers with family incomes far below the national average. Over 50% of the population
97
lives below the poverty line, with rudimentary water supply, sanitation and hygiene.7 The
98
area is non-endemic for malaria but has had an active schistosomiasis control program for
99
more than two decades including an MDA program that commenced in 2008. All individuals
100
aged 5–65 years were offered free annual treatment (40 mg/kg of praziquantel) in accordance
101
with the Department of Health Administrative Order 2007–0015.7
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us
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94
104
Study Procedures
ed
103
M
102
In 2012 we conducted a cross-sectional schistosomiasis survey on approximately 20,000
106
individuals in order to determine the prevalence, intensity of infection and morbidity
107
associated with the disease (Figure 1).7 The baseline prevalence and intensity of infection
108
were determined by Kato-Katz thick smear stool examination. Individuals were asked, over
109
the course of a week, to provide two stool specimens from which six 50 g Kato-Katz thick
110
smears were prepared on microscope slides according to established methods.20 Slides were
111
examined under a light microscope by experienced laboratory technicians, who counted the
112
number of S. japonicum eggs per slide. For quality control, 10% of all slides were randomly
113
selected and re-examined by a senior microscopist at the Research Institute for Tropical
114
Medicine, Manila. S. japonicum egg counts were expressed as eggs per gram (epg) of stool.18
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4 Page 4 of 26
115
Intensity of infection was graded according to WHO criteria: light infection (1-99 epg),
116
moderate infection (100-399 epg) and heavy infection (≥400 epg).19
117
For a more accurate assessment of schistosome-induced hepatosplenic morbidity
119
ultrasonographic studies were performed on subset of individuals with symptoms and signs
120
suggestive of schistosomiasis. All subjects (n = 736) who reported gastrointestinal, and/or
121
neurologic symptoms (ie. fatigue, malaise, abdominal pain, blood per rectum, hematemesis,
122
diarrhoea, jaundice, dizziness, headache, and seizures) and/or were clinically assessed to have
123
morbidity (i.e., palpable liver and spleen, varices, ascites, etc.) based on physical examination
124
were initially selected for the ultrasound investigation. However, only those with baseline
125
stool and ultrasound examination results were included in the final analysis (n = 565). The
126
degree of hepatic fibrosis was assessed by ultrasound examination using a portable gray-scale
127
ultrasonogram equipped with 3 MHz curve array transducer (SONOACE X1, Madison Co.,
128
Ltd., Seoul, South Korea). The liver size was measured in millimetres along the mid-sternal
129
line (MSL) and mid-clavicular line (MCL) for the left lobe and right lobe, respectively.
130
Splenic size was also measured in millimetres along the left mid-axillary line (MAL). Hepatic
131
fibrosis grading was adopted from the practical guidelines established for ultrasonography by
132
the WHO/TDR (1990).20 Ultrasound findings were arrived at by consensus agreement of two
133
experienced ultrasonographers. Ten percent of the ultrasound images were independently
134
validated by a radiologist in Manila.
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pt
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135
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118
136
Serum biomarkers of hepatic fibrosis were also evaluated on a random subset of patients. The
137
selection of the sample subset from each fibrosis grade category was done using computer
138
generated random numbers. Other laboratory investigations were conducted to rule out
139
known liver pathology aetiologies (e.g. Hepatitis B and alcoholism). Blood (10 ml) was
5 Page 5 of 26
collected from each enrolled subject. Serum was separated from the whole blood and
141
refrigerated at 2-8 0C. Collected sera were then transferred to a 40C cooler box and shipped to
142
the Research Institute for Tropical Medicine (RITM) clinical laboratory in Manila. Each
143
serum sample was divided into equal aliquots. Half of the serum specimens were used for
144
liver function and hepatitis B surface antigen (HBsAg) testing and the other serum half was
145
kept at -800C for fibrosis marker assays. Hepatitis C testing was excluded due to the very
146
low prevalence (<3%) reported for rural remote communities in the Philippines. Liver
147
function (including tests for hepatocellular injury, parenchymal damage, capacity for protein
148
synthesis, and cholestasis) and HBsAg tests were performed on the samples using an
149
automated machine (A15, Biosystem, S.A. Costa Brava, Barcelona, Spain) and EIA
150
commercial kits (MonalisaBiorad, France), respectively. The serum samples for fibrosis
151
marker testing were transferred to Siemens, Australia who evaluated hyaluronic acid (HA),
152
procollagen type III (PIIINP), and tissue inhibitor of metalloproteinases (TIMP-1) serum
153
levels using the Enhanced Liver Fibrosis (ELF) test.21 The ADVIA Centaur CP XP
154
immunochemical analyser was used according to the manufacturer’s instructions. The ELF
155
score was calculated directly by the instrument using the following equation:
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ELF score = 2.278 + 0.851 ln(CHA) + 0.751 ln(CP3NP) + 0.394 ln(CTIMP-1)
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The sICAM-1 serum levels were measured at QIMR Berghofer Medical Research Institute
160
using a separate ELISA kit (R&D systems, Minneapolis city, Minnesota, USA).
161 162
Study Oversight
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6 Page 6 of 26
Ethical consent for the study was obtained from the ethical review boards of the Department
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of Health in the Philippines (Institutional Review Board Number 2012-13-0), Griffith
166
University, Australia and QIMR Berghofer Medical Research Institute, Australia. Written
167
informed consent was obtained from each individual or for those aged <15 years from their
168
parents/ legal guardians. Stool-positive subjects were treated orally with 60 mg/kg PZQ in
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two divided doses, followed by another treatment of the same dose two weeks later. Stool-
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negative individuals were treated with a single oral dose of 40mg/kg.
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Statistical Analysis
an
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FoxPro (version 6.0) was used for double entry of data, and data were cross-checked and
175
analyzed with SAS, version 9.4. Statistical analysis was performed using STATA version 11.
176
Data were summarized as frequencies and mean ± SD as appropriate. All categorical
177
variables were evaluated using Chi-Squared test. Wilcoxon Signed Ranks and McNemar’s
178
tests were used for paired analysis of infection intensity and prevalence respectively across
179
different age groups. With regards to treatment impact on infection intensity, S. japonicum
180
egg count + 1 was log-10 transformed to bring observed distributions closer to normality.
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The Kruskall-Wallis was used to detect significant differences in the level of serum marker
182
types across fibrosis grades. Receiver operator characteristics (ROC) analysis were
183
performed to assess the clinical utility of the three serum fibrosis markers (HA, TIMP-1, and
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PIIINP), both individually, as well as their combined quantitative results (ELF score) in
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identifying subjects with high grade liver fibrosis (grade II-III). A significance level of 5%
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was used for statistical inference.
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RESULTS 7 Page 7 of 26
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Study Descriptors
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Fifty-three per cent of the study sample (n = 565) was male, with a mean age of 39.4 years
193
(95% CI, 38-41 years). Most were poor rice farmers with a high degree of exposure. There
194
was no statistical difference in the sex profile of the sample. However, most of the study
195
participants belonged to age groups of 36-45 years (24%) and 46-55 (27%) years,
196
respectively. Only 1.0% of the population reported having been diagnosed with hepatitis B;
197
however, 38.4% drank alcohol moderately (consumed 1-2 drinks per day).
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Prevalence, intensity and morbidity outcomes
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The study communities have participated in an active schistosomiasis program for the past 20
202
years. However, the prevalence of S. japonicum infection among the pre-treatment cohort
203
subjects (n = 565) was 34% (Figure 2). Males had a higher prevalence of infection (46%)
204
than females (20%). S. japonicum infection was more prevalent (49%) among subjects aged
205
less than 20 years of age. The mean intensity of infection was 123.1 epg (95% CI; 78.9–
206
167.3epg) (Figure 2). Twenty-five percent had light intensities of infection, 6% moderate
207
intensities of infections, and 2% heavy infections. Hepatic parenchymal fibrosis occurred
208
among 50% of subjects, of which 19% had grade II fibrosis and 6% grade III (Table 1). The
209
fibrosis prevalence in males and females were 52.7% (298/565) and 47.3% (267/565),
210
respectively. The degree of liver fibrosis correlated significantly (P = 0.0002) with infection
211
intensity. The left and right hepatic lobe lengths increased with increasing fibrosis grade. The
212
mean left lobe lengths of those with grade II (mean length = 93.7 mm; 95% CI, 92.5-94.8
213
mm) and III (mean length = 98.4 mm; 95% CI, 96.4–100.4 mm) fibrosis were significantly
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8 Page 8 of 26
larger (P = 0.0001) than those without fibrosis (mean length = 87.7 mm; 95% CI 86.4–88.9
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mm). However, only the mean length of the right lobe of individuals with grade III fibrosis
216
(122.3 mm; 95% CI 120.7–124.0 mm) was significantly (P= 0.04) larger compared with
217
those lacking fibrosis. Likewise, the mean length of the spleen increased with increasing
218
degree of fibrosis, although not significantly (P = 0.21).
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473 subjects had both baseline and follow-up stool examination results, hence, these
221
individuals were utilized in the final analysis to determine the impact of PZQ treatment on
222
prevalence and intensity of infection. The prevalence dropped significantly two years post-
223
treatment, especially among those aged 16-25 years (P = 0.008) and 46-55 years (P = 0.001)
224
(Figure 2). Similarly, the intensity of infection across all age groups dropped compared with
225
pre-treatment levels (Figure 2). The prevalence decline observed among the 16-25 years age
226
group may in part be due to limited exposure to infection. On the other hand, acquired
227
immunity to reinfection may partially account for the prevalence drop among those aged 46–
228
55 years. However, only subjects aged 46-55 years showed significant decreases in the
229
intensity of infection compared with the baseline findings (P = 0.003). The prevalence of
230
grade II-III fibrosis remained relatively unchanged at 25.2% (107/424) on follow-up (Table
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1). The data suggests that a two year follow-up period may be too short of a time frame to
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assess disease regression following treatment.
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Liver function tests, hepatitis B, and serum fibrosis markers
235 236
Liver function tests were carried out on a selected subset of patients (n = 136). The total
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number of individuals was derived via age-matched specific grouping resulting in 34 subjects
238
per each of the four fibrosis clinical grades. Sixty percent (81/136) of the subjects had normal
9 Page 9 of 26
liver function tests with the proportion being uniform across all fibrosis grades. Mild SGPT
240
elevation was noted in 1.5% (2/136) of the sample. Alkaline phosphatase and total bilirubin
241
result were similarly distributed among the four fibrosis groups. Hepatitis B surface antigen
242
testing was done on all 565 subjects. The HBsAg carrier rate was 4.2% (24/565). The carrier
243
rates in those with grades I, II and III fibrosis were 1.4% (2/145), 2.8% (3/105) and 2.9%
244
(1/34), respectively. The carrier rates in those with fibrosis were actually lower than those
245
without fibrosis (6.4%). Thus, the prevalence of both alcohol and chronic hepatitis B-induced
246
liver fibrosis were demonstrated to be low in the cohort.
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The serum fibrosis marker levels and infection intensity before and after PZQ treatment were
249
also assessed. Only baseline mean TIMP1 levels showed cumulative results with increasing
250
intensity of infection (P = 0.001). The baseline and endpoint serum levels of the fibrotic
251
markers were also compared with parenchymal grading. The Enhanced Liver Fibrosis (ELF)
252
marker score and two of three ELF markers correlated with fibrosis severity. The mean
253
baseline and follow-up ELF score correlated significantly with increasing liver fibrosis grade
254
(P = 0.002; P = 0.049). Before treatment, baseline TIMP-1 levels were increased in all three
255
increasing fibrosis grades (P < 0.001). At endpoint, TIMP-1 levels were also significantly
256
higher in grade III compared with grade I (P = 0.034). The HA levels were higher in grade III
257
compared with grade 0 (P = 0.031) and grade I (P = 0.0429). The sICAM-1 also correlated
258
with fibrosis grade (P = 0.0013).
260
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Diagnostic performance of ELF
261 262
Table 2 shows the baseline and follow-up ROC curve analysis results of the ELF score and
263
individual markers to discern subjects with grade II-III fibrosis (severe) from those with
10 Page 10 of 26
grade 0-I fibrosis (mild). The diagnostic accuracy of HA and the ELF score were slightly
265
higher than the two other ELF markers assessed based on the area under the curve (AUC) at
266
baseline. At follow-up, HA, TIMP-1, and the ELF scores had higher diagnostic accuracy than
267
PIIINP. The cut-off values of 30.7 ng/mL for HA (AUC 0.69, P < 0.001) and 9.2 for the ELF
268
score (AUC 0.68, P < 0.001) to discriminate grade II-III fibrosis from grade 0-I among 177
269
subjects at baseline, have sensitivities of 66% and 64%, specificities of 72% and 71
270
respectively. At follow-up, the cut-off values of 26.3 ng/mL for HA (AUC 0.63, P < 0.043),
271
183.7 ng/mL for TIMP-1 (AUC 0.62, P < 0.001), and 8.9 for the ELF score (AUC 0.62, P <
272
0.004) to discern grade II-III fibrosis from grade 0-I among 180 subjects have sensitivities of
273
73%, 73%, and 62%, and specificities of 53%, 51%, and 62% respectively. PIIINP was
274
uninformative, having the least AUC for both time points. Figures 3 and 4 illustrate the ROC
275
curves for the three individual ELF markers as well as the curves for the ELF score. The ELF
276
score and HA showed the highest diagnostic performance in detecting severe schistosome-
277
induced liver fibrosis. When gender was considered, no significant difference was observed
278
in the cut-off values at baseline and at two years follow-up. Cut-off values of 8.9 and 9.2
279
ng/mL were noted for males (baseline: AUC 0.69, P = 0.003; follow-up: AUC 0.62, P =
280
0.342) and females (baseline: AUC 0.64, P = 0.226; follow-up: AUC 0.69, P = 0.042)
281
respectively, suggesting that the same cut-off value can be utilized for both sexes. However,
282
when age was taken into account, the cut-off value at baseline was noted to be higher among
283
subjects younger than 18 years of age (9.9 ng/mL, AUC 0.96, P = 0.192) compared with
284
those aged 18-40 years (8.9 ng/mL, AUC 0.71, P = 0.087), 41-50 (8.9 ng/mL AUC 0.74, P =
285
0.005) and over 50 years of age (9.2 ng/mL, AUC 0.61, P = 0.046). This suggests that age
286
may influence the ELF test results. On follow-up, the cut-off values could not be determined
287
for the adolescent (<18 years) cohort because very few of these individuals had moderate to
288
severe fibrosis. Overall, the mean values of the fibrosis markers seemed to increase across the
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11 Page 11 of 26
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fibrosis grades, except for PIINP (Table 3). But within each grade, there seems to be no
290
apparent pattern across intensity of infection. This observation may be due to the small
291
sample size within each category.
292
DISCUSSION
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We evaluated the level of schistosomiasis prevalence, infection intensity and morbidity
296
among 565 subjects residing in eighteen moderately endemic barangays in Northern Samar,
297
the Philippines. The study communities have participated in the National Department of
298
Health human treatment program for schistosomiasis over the past three decades.
299
Nevertheless, the baseline human prevalence and infection intensity were 34% and 123 epg
300
respectively. Baseline ultrasonography revealed a high level of schistosomiasis-induced
301
morbidity among subjects with signs and symptoms suggestive of schistosomiasis. Eighty-
302
nine per cent of the patients had left lobe liver enlargement (≥ 70 mm) and 25% had grade II
303
or III liver fibrosis. Splenomegaly (≥ 100 mm) was seen in 13% of the population. Infection
304
intensity was significantly (P < 0.0002) correlated with grade III fibrosis. Hepatitis B virus,
305
alcoholism and malaria were excluded as possible causes. The results suggest that the
306
national control strategy for schistosomiasis (i.e., human treatment with 40 mg/kg PZQ) is
307
inadequate in the study area. Low drug coverage (<36%), low drug compliance (<40%), and
308
inability of PZQ to prevent re-infection given the zoonotic nature of the disease may have
309
contributed to the findings.22
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310 311
Two years after baseline chemotherapy (i.e., 40-60 mg/kg PZQ), as expected, a greater
312
reduction in both the prevalence and intensity of infection were noted. However, treatment
313
had less of an impact on lowering schistosomiasis-induced morbidity. Overall, hepatic
12 Page 12 of 26
314
fibrosis (grades I-III) regressed in only 24.3% of those who received a single treatment and in
315
only 19.3% of those who received two PZQ doses. This outcome was similar to that reported
316
for S. japonicum morbidity in China.9 The baseline grade II-III fibrosis prevalence was
317
25.2%, but remained unchanged two years post-treatment.
318
morbidity despite standard treatment will need a longer treatment course at a higher dose
319
(i.e., 60-80 mg/kg) to reverse fibrosis and improve clinical outcome. Approximately 10% of
320
residents in S. mansoni and S. japonicum endemic areas develop severe hepatosplenic
321
disease.23-25 These observations have been partly explained by genetic factors regulating
322
disease development.23-25
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Individuals with significant
an
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Liver fibrosis is a complex process involving production and deposition of insoluble
325
components that comprise the extracellular matrix (ECM).9 Quantitative and qualitative ECM
326
changes in liver fibrosis can be measured in blood or urine using indirect and direct
327
biomarkers.5 These biomarkers of hepatic matrix metabolism have also been drawn together
328
into multi-parametric scores to produce promising results.21,26 The Enhanced Liver Fibrosis
329
(ELF) test is an ECM marker set comprising an algorithm of three direct liver fibrosis
330
markers (HA, TIMP-1, and PIIINP). The ELF test was one of the first commercially available
331
serum fibrosis tests21, and its diagnostic potential has been explored in identifying and staging
332
liver fibrosis among patients with hepatitis C viral infection21,27, alcoholic liver disease26, and
333
non-alcoholic fatty liver disease (NAFLD).28 The test is said to be comparable with
334
FibroScan (transient elastography)29,30 and more accurate or reproducible than simple panels
335
(such as aspartate aminotransferase to platelet ratio and Fibrosis-4) in detecting advanced
336
fibrosis.31
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13 Page 13 of 26
Our study assessed four promising direct biomarkers (HA, PIIINP, TIMP-1, and sICAM-1)
339
based on their performance in previous studies.5,9,14,15 To the best of our knowledge, this is
340
the first time that an ELF score was deployed in the evaluation of liver fibrosis due to
341
schistosomiasis. The best correlation between serum marker level and ultrasonographic
342
fibrosis grading was demonstrated with TIMP-1. The TIMP-1 levels increased as the degree
343
of fibrosis increased both at baseline and at endpoint. The marker levels also decreased in all
344
fibrosis grade categories at endpoint (after treatment) suggesting the TIMP-1 serum levels
345
can accurately assess liver fibrotic disease due to schistosomiasis. Also, TIMP-1 serum levels
346
correlated positively with intensity of infection, suggesting that the marker may also respond
347
to inflammatory reactions to eggs deposited in the liver. HA and sICAM-1 also showed some
348
correlation but not as significant as shown for TIMP-1. Moreover, the ROC analysis revealed
349
that HA as an individual marker showed the highest sensitivity and specificity in discerning
350
higher fibrosis grades from the lower grades. The diagnostic performance of the ELF score
351
was equally as useful as that shown for HA alone. However, the AUC values generated using
352
both the ELF score and serum biomarkers individually indicated only moderate
353
discrimination between grades II-III and 0-I fibrosis (i.e., AUC’s 0.6-0.7).33 Moreover,
354
consistent with other studies, age should be considered as a factor when generating ELF
355
scores.21 An unadjusted ELF score may misclassify normal individuals younger than 18 years
356
of age as having moderate-to-severe schistosome-induced liver fibrosis. If the diagnostic
357
value of the ELF test can be improved as a reliable stand-alone test and suitable for field
358
application, then it could potentially deployed as a component of the diagnostic and treatment
359
algorithm for S. japonicum infected patients (Figure 5).
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360 361
CONCLUSIONS
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14 Page 14 of 26
We demonstrated a high level of schistosomiasis-induced morbidity among subjects with
364
signs and symptoms suggestive of the disease in a moderately endemic area of the Philippines
365
that has been under active community-based chemotherapy for over three decades. Hepatic
366
fibrosis, the main cause of hepato-splenic schistosomiasis, takes time to reverse with
367
chemotherapy. Individuals with hepato-splenic schistosomiasis should be identified and
368
treated as early as possible to prevent disease progression and possible death. Serum fibrosis
369
markers, particularly
370
may potentially guide program managers on where to direct limited health-care resources
371
for schistosomiasis control. More specifically, if made affordable for field application, the
372
ELF test may assist clinicians in assessing patients with heavy intensities of infection (>400
373
epg) and/or with physical signs of advanced disease (e.g. MCL > 3) in determining their risk
374
of severe liver fibrosis.
375 376 377
Acknowledgments: We thank the Australian National Health and Medical Research Council for providing financial support for our research.
378 379 380
Financial support: Our work is supported by the Australian National Health and Medical Research Council. Professor McManus is a Senior Principal Research Fellow with the Australian National Health and Medical Research Council.
381 382
Potential conflicts of interest: All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential.
383 384
Conflicts of Interest: Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
and
HA
levels,
and
ELF
scores
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TIMP-1
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8
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385
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Fagan KJ, Pretorius CJ, Horsfall LU, Irvine KM, Wilgen U, Choi K, et al. ELF score >/=9.8 indicates advanced hepatic fibrosis and is influenced by age, steatosis and histological activity. Liver international : official journal of the International Association for the Study of the Liver 2015;35:1673-81. Yoo EJ, Kim BK, Kim SU, Park JY, Kim do Y, Ahn SH, et al. Normal enhanced liver fibrosis (ELF) values in apparently healthy subjects undergoing a health check-up and in living liver donors in South Korea. Liver international : official journal of the International Association for the Study of the Liver 2013;33:706-13. Hosmer DW, Lemeshow S. Applied Logistic Regression. 2nd ed: John Wiley & Sons, Inc.; 2000.
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494 N= 20,000 Eligible residents aged 5-65 years from 22 barangay
cr
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n = 18,221 Completed individual medical questionnaire
n = 58 Individuals without head of hous e questionnaire
n = 10,435 (57%) Com plete stool data
n = 7,786 Incomplete stool data
an
us
n = 18,163 (99%) Indivduals with head of house ques tionnaire
ed
M
n = 6,074 (33%) Physical exam ination
pt
n = 736 (4%) Ultras ound examination
496 497 498 499 500
n = 171 incomplete stool data
n = 424 Ultrasound Follow-up + Stool Follow-up
n = 141 Incom plete stool data
Ac ce
495
n = 565 Ultrasound exam ination + Baseline stool exam ination
Figure 1. Study profile and compliance among 18,221 residents from 22 schistosomiasisendemic barangays in Palapag and Laoang, Northern Samar, the Philippines. All inhabitants aged 5–65 years were invited to participate in the questionnaire and provide 2 stool samples for parasitological examination. A subset of patients (n = 736) were selected for ultrasound investigations.
19 Page 19 of 26
ip t cr us Figure 2. Prevalence and intensity of infection in subjects with S. japonicum by age before (2012) and after (2015) two years of praziquantel treatment (i.e., 40-60 mg/kg; n = 473).
M
502 503 504
an
501
505
pt
508
Ac ce
507
ed
506
20 Page 20 of 26
508
513 514 515 516
pt
511 512
Figure 3. Baseline ROC curves (n = 177) for serum concentrations of ELF, HA, PIIINP, and TIMP-1 for differentiating grade II-III liver fibrosis from grade 0-I fibrosis.
Ac ce
510
ed
M
an
us
cr
ip t
509
517 518 519
21 Page 21 of 26
ip t cr us an 525
ed
M 524
Figure 4. Two year post-treatment follow-up ROC curves (n = 180) for serum concentrations of ELF, HA, PIIINP, and TIMP-1 for differentiating grade II-III liver fibrosis from grade 0-I fibrosis.
pt
521 522 523
Ac ce
520
22 Page 22 of 26
525
Patient with heavy intensity of infection ( > 400 epg) and/or liver enlargment (MCL .> 3 cm)
Grade II/III liver fibrosis unlikely Refer to local treament and follow-up
Grade II/III liver fibrosis likely Refer for ultrasound as sessment
an M
529 530
Ultras ound positive
Refer to Gastroenterologist
Ac ce
527 528
pt
ed
Ultras ound negative
526
cr
ELF score > 8
us
ELF Score < 8
ip t
Referral for ELF Test
Figure 5: Algorithm for the recommended diagnosis and management of severe hepatic fibrosis (grade II-III) due to gastrointestinal schistosomiasis infection.
531 532
23 Page 23 of 26
532
Table 1. Dynamics of schistosomiasis liver parenchyma grading before and after praziquantel treatment in a cohort of 424 subjects from Northern Samar, Philippines from 2012 – 2015.
Parenchyma Grading 2012
Parenchyma Grading 2015
Total (N)
Total (%)
Progressed (%)
Regressed (%)
Grade 1
Grade 2
Grade 3
Normal
156
54
3
0
213
50.24
Grade 1
40
40
17
7
104
24.53
Grade 2
8
17
33
22
80
18.87
27.5
31.3
Grade 3
2
1
14
10
27
6.37
0.0
63.0
Total (n)
206
112
67
39
424
103
82
535
0.0
23.1
38.5
cr
26.8
100
an
536
ip t
Normal
us
533 534
537
Ac ce
pt
ed
M
538
24 Page 24 of 26
538 539 540 541 542
Table 2. Area under the curve (AUC), and diagnostic sensitivities and specificities for ELF score cut-off values and individual serum fibrotic markers at baseline and two years after treatment using receiver operating analyses to distinguish between severe fibrosis (grade IIIII) from low grade fibrosis (grade 0-I).
543
0.68 0.69 0.56 0.65
<0.001 <0.001 0.515 <0.001
64 66 61 63
0.62 0.63 0.57 0.62
0.004 0.043 0.475 0.001
62 73 58 73
9.2 30.7 10.2 253.6
62 53 57 51
8.9 26.3 9.4 183.7
Ac ce
pt
ed
M
544
71 72 51 68
Cut-off
ip t
Sensitivity Specificity
cr
P
us
AUC
an
Fibrosis Marker Baseline (n=177) ELF Hyaluronic Acid PIINP TIMP1 Follow-up (n=180) ELF Hyaluronic Acid PIINP TIMP1
25 Page 25 of 26
544
Table 3. Mean fibrosis marker value by intensity of infection and fibrosis grade.
545
Intensity of Infection
n
ELF
HA
PIINP
TIMP
Negative
68
8.8
31.6
12.1
199.8
Mild
10
8.9
23
14.7
252.5
Moderate
2
8.5
21.4
8.2
278.1
Heavy
0
cr
Grade 1 0
Mild
7
8.8
23.2
13.7
295.3
Moderate
4
9
27.3
13.6
290.1
Heavy
1
9.4
12
27.6
594.5
Negative
14
9.3
41.1
Mild
17
9.1
46.5
Moderate
4
9.4
Heavy
1
7.7
Mild
546 547
an
M 10.3
274.6
10
241.1
5.6
10.5
277.1
pt
8
294.6
59.2
85.5
21.4
302.8
10
Ac ce
Negative
12.3
ed
Grade 2
us
Negative
Grade 3
ip t
Grade 0
10
9.6
64.6
11.2
279.5
Moderate
3
10.2
76.2
14.2
370.4
Heavy
2
8.7
24.1
10.3
457.8
548
26 Page 26 of 26