Treatment related renal dysfunction after 3 monthly doses of 60 mg/kg of praziquantel for Schistosome haematobium infection

Background: Praziquantel (PZQ) is the standard treatment for Schistosomiasis in sub-Saharan Africa. However, there is evidence suggesting praziquantel treatment failure in Schistosome infections with associated potential renal impairment. The objective of this study was to determine the effect of three monthly doses of 60 mg/kg/day PZQ on schistosome egg count, liver and renal function during the treatment of urinary schistosomiasis in Ghana. Methods: A nested case-control study was designed from a cohort screened for schistosomiasis; 28 schistosomiasis positive cases by microscopy matched with 53 healthy controls by age and gender. The study population was urban dwellers from the Asokwa sub-metropolitan area, Kumasi in Ghana. Participants were within the age range of 6 to 30 years. We assessed S. haematobium egg counts in urine and its associated impact on liver and renal function at baseline, treatment and post-treatment phases. Results: Of the 28 cases and 53 controls, 78.6% and (81.1% were males respectively. Globulin levels before treatment was higher in cases [(36.92±1.46 vrs 27.88±1.49), p=0001] at pre-treatment but not at post-treatment [(35.35±1.28 vrs 37.26±1.77), p= 0.391]. Estimated cure rate was 42.9%, 46.4% and 96.4% after first, second and third dose respectively. Schistosome egg counts dropped significantly (p= 0.001) from before second dose to post-treatment. Similarly, levels of alanine aminotransferase (p=0.001), aspartate aminotransferase (p=0.028) and gamma glutamyl transferase (p=0.001) significantly declined towards post-treatment. Estimated glomerular filtration rate significantly improved from before second dose to post-treatment using both the Chronic Kidney Disease Epidemiology Program (p=0.001) and 4-variable Modification of Diet in Renal Disease (p=0.002) equations. Conclusion: Treatment of urinary

. This oral schistosomicidal agent is constituted of a racemate mixture, with activity both in vivo and in vitro [2,3]. Evidence from studies conducted on Schistosoma mansoni and Schistosomajaponicum although not very clear, indicate the mode of action of PZQ is the targeting of calcium channels and antigen exposure rendering the worm susceptible to elimination by antibodies [1,4].
After oral administration, PZQ is rapidly absorbed, metabolized and excreted by the kidney.
Metabolism of PZQ is primarily via the cytochrome P450 system leading to the production of toxic metabolic intermediates, which are potentially harmful to hepatocytes [5]. Plasma levels of PZQ are also reported to be affected by inducers and inhibitors of cytochrome P450 activity [6,7].
Several studies, predominantly in Asian populations where S.japonicum infections are endemic, state conflicting findings on hepatotoxicity associated with PZQ treatment against the helminth [8,9]. PZQ treatment is reported to be associated with elevated serum concentrations of liver aminotransferase [8]. However, in a large retrospective study from China, there was insignificant (less than 1%) prevalence of hepatotoxicity among populations treated for S.japonicum with PZQ [9].
Therapy for Schistosomiasis in sub-Saharan Africa has mainly been documented based on intestinal S. mansoni infections [1]. As a result, there is paucity of data on urinary S.haematobium and its associated drug metabolism effects on organs involved in metabolizing and excretion of PZQ. This leaves a gap in knowledge about the protective or destructive effect of metabolizing the drug in S.haematobium infection. It has further been shown that varied degrees of reduction in prevalence and infection rates of S.haematobium are reported with mostly single PZQ dosage of 40 mg/kg/day in both children and adults [1]. There are also indications of drug resistance to single doses of PZQ for treating schistosomiasis [10]. This heightens the need to probe the outcome of repeated PZQ treatment on urinary schistosome counts viz a vis its implication on liver and renal function.
The aim of this study was to assess the effect of PZQ on schistosome egg count, liver and renal function after 3 doses of 60mg/kg/day (PZQ60) in three months for treating urinary S.haematobium infection.

Methods
This was a nested-Case Control study conducted among children and adults of ages 6-30 years from the urban Asokwa District in Kumasi, Ghana (see plate 1). This was part of a larger study to assess plasmodium transmission in persons infected with Schistosomiasis (NCT02769013). Ethical approval was obtained from the Committee for Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Ghana. All participants were required to sign an informed consent and an assent form in case of minors. Cases were respondents diagnosed to have S.haematobium by routine microscopic examination of urine samples while controls were from similar populations age-sex matched having similar characteristics as the case population and living in the same area but without laboratory or clinical detection of urinary schistosomiasis infection.

Study area
Apromase, Deduako, Emena and Kokoben were the study communities in the urban Asokwa District with a population of 140,161 inhabitants in 36, 183 households [11]. These communities are located between latitude 6°30' and 7°00' North and longitude 1°30' and 2°00 West of Kumasi the capital city of the Ashanti Region of Ghana. The four communities have Saman (Kokoben and Apromase), Oda (Deduako) and Subin (Emena) as names of three rivers running through it.
Climatic conditions are tropical with temperatures varying from 20.2 °C to 37.1 °C. Rainfall pattern is seasonally bimodal with major rains extending from late April to August with a minor one from September to October [12]. The average annual rainfall for the area is 6.25mm with peaks of 214.3mm and 16.2mm in June and September respectively. The dry season (harmattan) is from November to March with humidity ranging between 53% and 93%.

Screening and enrolment
A census of the selected communities was conducted with the ages and number of inhabitants per building collected along with corresponding GPS coordinates using Personal Digital Assistants (PDAs).
Households within the buildings were selected and their members asked for written informed consent to be screened in the study. Twenty millilitres (20 ml) of urine sample was collected from consenting participant into well-labelled 30 ml urine containers. The urine samples were collected within the hours of 6:00 am and 12 noon. Subsequently, the samples were kept in cold boxes at temperature of 4-6 o C until transported to the laboratory at Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) about 15-20 minute drive from the study sites.
Asymptomatic schistosomiasis positive (SP) cases and schistosomiasis-negative (SN) controls based on screening results were invited to participate in the study.

Sampling procedures
A total of 1258 participants were screened for schistosomiasis out of which 104 were positive. All 104 schistosome positive participants ( Figure 1) were placed on PZQ60 treatment with 28 successfully completing the course.

Design of Experiment
Biochemical parameters and schistosome counts were analysed before and after treatment for both cases and controls ( Figure 2). In between pre-and post-treatment, the biochemical and schistosome counts were monitored before the 2 nd and 3 rd dosages of PZQ60 for cases.

Processing of Urine and S. haematobium quantification
Freshly voided urine was collected between 6:00am and 12:00pm in a sterile wide-mouthed screw-top plastic container (30 ml) and transported to the laboratory on ice at 4 o C to 8 o C. Urine processing and quantification were done as described by Cheesebrough [13]. Briefly, blunt-ended forceps were utilized to place a polycarbonate membrane filter of pore size of 12.0μm (Whatmann Nuclepore) on the filter-support of a filter-holder (Swinnex 25mm support chamber). The filter holder was reassembled and attached to a 10ml syringe filled with well-mixed urine which was filtered with the aid of the plunger. The filter was carefully removed and transferred with the face upwards to a clean glass-slide. A drop of physiological saline was added, covered with a cover-slip, and examined by two independent expert microscopists using the 10X objective (Carl Zeiss Microscope) with the condenser iris closed sufficiently to give good contrast. The entire filter was examined systematically for the presence of S. haematobium eggs. The number of the eggs counted per 10ml of urine was recorded and the average of the two counts was calculated. A slide was declared negative when no parasites were detected.

Blood sampling and processing
Blood samples were collected from the ante cubital vein with the aid of a tourniquet and the puncture site cleaned with 70% alcohol prep. Blood drawn into separator gel tubes (5 ml) were centrifuged at 1780 x g for 10 minutes at 4 o C to obtain the serum which were subsequently stored at -80 o C.

Biochemical Analysis
Assays for the liver function; albumin, globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) and renal function (urea and creatinine), were conducted using a chemistry analyzer (HumaStar 200, Human, Germany). Aliquots of the serum were dispensed into cuvettes after thawing and placed at pre-programmed positions in the auto-analyzer and analysis done in batches. Renal sufficiency was calculated based on the 4variable Modification of Diet in Renal Disease (4v-MDRD) and the Chronic Kidney Disease Epidemiology Program (CKDEPI) equations [14].

Statistical analysis
Data were entered into excel and analyzed with Stata V.12 (StataCorp, USA). Continuous variables were reported as median with interquartile ranges and categorical variables as proportions. The Wilcoxon signed rank test was used to assess differences in continuous variables between groups with statistical significance set at p< 0.05.  (Table 1). Furthermore, the 4v-MDRD renal equation also showed no difference in levels between schistosome positive and negative groups at pre-treatment (p= 0.776).

Effect of treatment with 3 doses of PZQ60 on cure rate
Estimated cure rate was 42.9% a month after administering the first dose of PZQ60 (Table 2). This increased to 46.4% before administering the third dose and finally to 96.4% post-treatment.  (Table 3). Moreover, creatinine and urea levels did not statistically vary between cases and controls. No significant difference (p= 0.753) was recorded using CKDEPI equation at post-treatment (Table 3) between the two groups.

Comparing Schistosome count and biochemical parameters between cases and controls after treatment
Likewise, 4v-MDRD reported no significant difference post-treatment (p=0.866), between cases and controls.

Discussion
Schistosome eggs were high before administration of the second dose of 60mg/kg praziquantel and almost completely eliminated post-treatment with 96.4% cure rate. The estimated glomerular filtration rate dropped significantly after the first dose of praziquantel but resolved after the third dose. Moreover, levels of liver enzymes increased after the first dose and returned to pre-treatment levels in schistosome positive cases post-treatment.
Several studies assessing the efficacy of praziquantel treatment on S. haematobium has been based on single doses of 40mg/kg with cure rates ranging from 39.8% to 88.9% in mainly children below 17 years [15][16][17]  With respect to organ damage, much investigation have tried to analyze the efficacy of varying concentrations of PZQ on organ injury [25,26]. However, little is known of its effect to the liver and kidney as a result of repeated and higher doses for treating S. haematobium among sub-Saharan subjects. It is well established that metabolizing of PZQ analogs by cytochrome P450 can lead to highly oxidative intermediates [27] which overwhelm the body's defense resulting in varied levels of organ damage. This study found a significant increase in liver enzymes after the first dose, which steadily dropped to pre-treatment levels. The initial upsurge in liver enzymes could be explained as due to highly oxidative intermediates of PZQ than can be quenched by the antioxidant activity of superoxide dismutase, glutathione and glutathione S-transferase. Consequently, hepatic cells are disrupted leading to the release of compartmentalized liver enzymes. However, the steady return after two more doses of PZQ to pre-treatment levels could be proof of the body system's response of mopping up toxic metabolites by inducing higher expression of cells that synthesis these proteins involved in the body's redox systems.
Similarly, the significant drop in estimated glomerular filtration rate for both the CKDEPI and 4v-MDRD equation after the first 60mg/kg dose shows the body's initial intolerance to oxidative species from the metabolism of the drug which improved even after monthly repeated doses. It is plausible that sustained higher thresholds of these induced protective antioxidants persist after its initial deficit compared with the highly oxidative drug intermediates associated with organ damage. This suggests possible protective effect of monthly repeated doses of PZQ on the renal filtration with a corresponding decline in schistosome egg count.
It is possible other medications which could potentially have synergistic effect on secretion of liver enzymes but not looked at in this study may have confounded these findings. Moreover, the reported levels of liver enzymes and estimated glomerular filtration rates could have been influenced by viral infections and wasting of muscles respectively.

Conclusion
This study suggests that treatment of urinary Schistosomahematobium infections with a repeated high monthly dose of 60mg/kg of praziquantel for 3 months is an effective and safe alternative. It provides an option to consider for S. haematobium infection in endemic areas as the possibility for drug resistance persists.

Ethical approval and consent to participate
Ethical approval was obtained from the Committee for Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Ghana (CHRPE/AP/227/17). All participants were required to sign an informed consent and an assent form in case of minors.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests     Table 3. Schistosome count and biochemical parameters after treatment   Effect of three repeated monthly doses of praziquantel 60mg/kg on renal function and S.