Diagnosing schistosomiasis-induced liver morbidity: implications for global control

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 Inob...

269KB Sizes 0 Downloads 49 Views

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;

cr

1. 2. 3. 4. 5. 6.

ip t

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†

us

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

__________________________________________________________________________________

an

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.

cr

Keywords. Schistosomiasis, morbidity, ultrasound, liver fibrosis, serum makers

us

Word count: Abstract = 197; Text = 3,664

an

42 43 44 45 46 47 48 49 50

ip t

37

INTRODUCTION

M

51

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

Ac ce

pt

ed

52

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

cr

ip t

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

an

us

cr

ip t

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

Ac ce

pt

105

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.

cr

us

an

M

ed

pt

Ac ce

135

ip t

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:

pt

ed

M

an

us

cr

ip t

140

157 158

Ac ce

156

ELF score = 2.278 + 0.851 ln(CHA) + 0.751 ln(CP3NP) + 0.394 ln(CTIMP-1)

159

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

163

6 Page 6 of 26

Ethical consent for the study was obtained from the ethical review boards of the Department

165

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

169

two divided doses, followed by another treatment of the same dose two weeks later. Stool-

170

negative individuals were treated with a single oral dose of 40mg/kg.

cr

us

171 172

ip t

164

Statistical Analysis

an

173

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.

181

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

184

PIIINP), both individually, as well as their combined quantitative results (ELF score) in

185

identifying subjects with high grade liver fibrosis (grade II-III). A significance level of 5%

186

was used for statistical inference.

Ac ce

pt

ed

M

174

187 188

RESULTS 7 Page 7 of 26

189 190

Study Descriptors

191

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).

us

cr

ip t

192

199

an

198

Prevalence, intensity and morbidity outcomes

M

200

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

Ac ce

pt

ed

201

8 Page 8 of 26

larger (P = 0.0001) than those without fibrosis (mean length = 87.7 mm; 95% CI 86.4–88.9

215

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).

ip t

214

219

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

231

1). The data suggests that a two year follow-up period may be too short of a time frame to

232

assess disease regression following treatment.

234

us

an

M

ed

pt

Ac ce

233

cr

220

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

237

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.

us

cr

ip t

239

247

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

M

ed

pt

Ac ce

259

an

248

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

Ac ce

pt

ed

M

an

us

cr

ip t

264

11 Page 11 of 26

289

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

ip t

293 294

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

Ac ce

pt

ed

M

an

us

cr

295

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

us

cr

ip t

Individuals with significant

an

323

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

Ac ce

pt

ed

M

324

337

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).

Ac ce

pt

ed

M

an

us

cr

ip t

338

360 361

CONCLUSIONS

362

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

cr

TIMP-1

above

8

Ac ce

pt

ed

M

an

us

high

ip t

363

15 Page 15 of 26

385

References

6. 7. 8. 9.

10.

11.

12. 13. 14.

ip t

cr

us

5.

an

4.

M

3.

ed

2.

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.

pt

1.

Ac ce

386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

16 Page 16 of 26

20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

ip t

cr

us

19.

an

18.

M

17.

ed

16.

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.

pt

15.

Ac ce

434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482

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.

ip t

483 484 485 486 487 488 489 490 491 492

Ac ce

pt

ed

M

an

us

cr

493 494

18 Page 18 of 26

494 N= 20,000 Eligible residents aged 5-65 years from 22 barangay

cr

ip t

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