Saturday, October 23, 2021

Determination of Ganciclovir in Plasma of Newborns with Congenital CMV Infection

Determination of Ganciclovir in Plasma of Newborns with Congenital CMV Infection


The transmission of cytomegalovirus (CMV) to the fetus is a result of maternal viremia and transplacental infection often maternal CMV infections are subclinical. Mortality in symptomatic infants is about 10%. Surviving newborns can contract mental retardation and other neurologic deficits [1,2]. The treatment of congenitally cytomegalovirus infected neonates involves the use of ganciclovir or its prodrug valganciclovir (GCV). Unfortunately, their use is limited by neutropenia, carcinogenicity, gonadal dysgenesis risks, development of viral resistance and drug–drug interactions [3,4]. Several strategies have been suggested to prevent high plasma ganciclovir concentrations and toxicity. Dosage reduction is recommended in patients with renal damage. Temporary discontinuation of an interacting medication, adjustment of dosages and careful monitoring of the patient is suggested to prevent any potential adverse effects of such interactions [5].

Material and Method


Ganciclovir and Trifluoroacetic acid (TFA) were purchased from Sigma Chemical Co. (St. Louis, MO). and 1-heptanesulfonic acid. Distilled water and perchloric acid were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA).

Blood Samples Preparation

Blood samples (1.5 ml) were collected in gel separating tubes. The blood was then centrifuged for 5 min at 13000 rpm. 1 ml of the serum thus obtained were added with 250 l of TFA 20 % and the suspension was then centrifuged at 13000 rpm for 10 min. Aliquots of deproteinated serum were stored at-80°C until use.

GCV Extraction

100l of serum was added with 95 l of distilled water and 50l of perchloric acid 7 %.

Assay Application

8 newborns with congenital CMV infection were treated with 15 mg/kg of Valganciclovir (GCV prodrug) twice daily. Blood samples were collected before (TO) and after 2 hours (T1) of the morning administration.

Chromatographic Conditions

The chromatographic system consisted of an HPLC Jasco LCNet II/ADC (JASCO International Co., Ltd. Tokyo, Japan) with a 20 μL Rheodyne 8125 injector (Rheodyne, Ronhert Park, CA, USA), an UV Detector UV “Jasco MD-2010 plus” (JASCO International Co., Ltd. Tokyo, Japan) (σ254nm), and a reversed phase Jupiter C-18 5 σm (150x4.6 mm) column (Phenomenex, California, USA). Using Distilled water as mobile phase and 1.2 ml/min flow rate.


Recovery of GCV

Recovery of ganciclovir from the extraction process was calculated by using the next formula:

C1= analyte concentration measured after addition

C2= concentration of added standard

Results are reported in Table 1.


Table 1: Recovery.

GCV Stability

GCV concentrations were stable in standard solutions after one month of storage at 4°C, in plasma for at least 5 h at room temperature, in biological matrices when stored at 220°C for at least six months.

Method Validation

The method was validated, considering linearity, LLOD, LLOQ, precision and accuracy, according to European Medicines Agency guidelines Validation [6]. The method of constant addition was used for the construction of calibration curves Figure 1.


Figure 1: Chromatogram of blood samples collected after 2 hours of treatment with Valganciclovir.

Method Selectivity

The selectivity of the assay was defined by analysis of blank plasma, with and without internal standard. There are no interfering peaks present at the retention time for GCV peaks.

Method Linearity

The correlation coefficient of the linear regression was reported in Figure 2.


Figure 2: Linearity of the assay.

Method Sensitivity

The method detection limit was tested by repeated analysis of blank samples.

The detection limits were determined as the concentration giving a peak height three times the noise background

Lower limit of Detection (LLOD) = 3,3/ S = 0,021 μg /mL

Lower Limit of Quantitation (LLOQ) = 10 / S = 0,065 μg/mL

σ=DS of the y intercept of regression line

S=slope of regression line

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. LLOQ (0.065 μg/ ml), was >10

Method Accuracy

The accuracy of the method was tested by relative error calculation. The accuracy values in intra-day variation studies at low, medium and high concentrations of GCV in plasma were within acceptable limits (n=5) Table 2.


Table 2: Relative error & Relative standard deviation of regression line slope.

Acceptable limits:<15%

Method Precision

The precision of the method was expressed by the coefficient of variation of intra- and inter-day variations of the assay under the same operating conditions. The acceptance criterion was set at 15% Table 3.


Table 3: Intra and Inter-day variability and stability.

Method Applicability

Applicability of the method was confirmed by analyzing samples from 8 newborns with congenital CMV infection treated with 15 mg/kg of Valganciclovir (GCV prodrug) twice daily. GCV concentration in blood samples collected before (TO) and after 2 hours (T1) of the morning administration are reported in Table 4.


Table 4: Assay application.

The present HPLC method for the determination of GCV was useful for the therapeutic monitoring of antiviral medication.


Amperometry detection [7], fluorescence detection, ionpairing agents [8,9], and UV detection [10-13], are general used to determine GCV. The difficult to detect GCV in newborn plasma are small volume of plasma available and little GCV concentration. The new analytical method developed was validated according to European Medicines Agency guidelines Validation. For the validation of the extraction procedures, samples of blank serum were spiked with known amounts of GCV. The recovery was determined by computing the ratio of the amount extracted from spiked samples to the amount added, determined by the injection of spiked solution. Ganciclovir was completely recovered during the extraction. Good ability to obtain results directly proportional to the concentration of analyte (not more than some number close to 1), fine detection limit, high precision demonstrated by very good intra- and inter-day repeatability (mean recoveries were near 100% and %RSD values were low) make this method a useful tool for the therapeutic monitoring of GCV medication. Applicability of the method was confirmed by analyzing samples from 8 newborns with congenital CMV infection treated with 15 mg/kg of Valganciclovir (GCV prodrug) twice daily.


The analytical method developed and validated is rapid and controllable in quality simple. The method showed high resolution, high sensitivity, good linearity, precision, accuracy and sensitivity. The inter-day variability is very low, and samples are stable in the condition tested up to 7 days. The recovery of GCV gave rise to a satisfactory recovery, thus avoiding the use of internal standards. Comparing our method to other proposed in the literature, it determines the GCV in smaller plasma concentrations, and is less expensive as it use a smaller variety of solvents. Results obtained in this study demonstrate its usefulness for therapeutic monitoring.

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Facilitating Production of Rice Dreg Peptides via Lactic Acid Bacterial Fermentation on Anti-Hypertension Efficacy

Facilitating Production of Rice Dreg Peptides via Lactic Acid Bacterial Fermentation on Anti-Hypertension Efficacy


Rice (Oryza sativa L.) represents one of the leading food crops in the world. A global annual production was about 480 million metric tons (milled rice basis) in 2015 [1]. It is cultivated today in more than 100 countries except Antarctica. It is the staple food for over half the world’s population, mainly in Asian countries, where it provides a considerable proportion of the protein intake for millions of people [2-3]. The total food protein production of rice per hectare is second only to that of wheat, although the yield of utilisable protein is actually higher for rice than for wheat, due to the superior quality of rice proteins [3-4]. Rice is known to have nutritional, hypoallergenic and healthy properties, which are retained by Rice Dreg Peptides (RDP) derived from rice starch by-products that contains more than 50% protein content and is used as a protein source [5]. With the massive of rice starch syrups industry in China, RDP is available in large amounts and at minimal costs. RDP are currently used as animal feed with low economic benefits in China. In China, the development of efficient methods to recovering RDP for human consumption that could significantly increase economic and social benefits [6].

High blood pressure has now become a major global health concern. The disease affected approximately 73 million people in USA [7]. There will be about 1.56 billion people suffered with hypertension worldwide in 2025 [7-8]. At present, hundreds of peptides with antihypertensive activity have already been reported and novel peptides are also discovered every day [7]. These biofunctional peptides were derived from 35 major sources that include animal matrix as milk, egg, fish, pork, and chicken and plant as soybean etc. [7]. In general, plant-derived functional peptides are considered more safe and healthy compounds. They are low molecular weight, possess different biological activities, and are easily absorbed by the small intestine in human [9-10]. Usually, plant-derived functional peptides may show not only antioxidant properties but also a wide range of other physiologic and biological activities as anti-hypertension, anti-hypercholesterolemia, immunomodulation, and anti-microbial actions [5,9-10].

Angiotensin Converting Enzyme (ACE) increases blood pressure by converting the inactive angiotensin I (decapeptide) to the active angiotensin II (octapeptide). Several chemicals have been verified to act as anti-hypertensive drugs. These clinical anti-hypertensive drugs included as nifedipine, captopril, fosinopril, lisinopril etc., which act either by direct inhibition of ACE or block of the angiotensin II receptors. Although these chemicals are effective in the blood pressure control, however, there are many side effects found [11-12]. For the example, in the nifedipine intake, there are many side effects found as mild dizziness, flushing (warmth, redness, or tingly feeling), weakness, headache, mood changes, heartburn, nausea, tremors, muscle cramps, cough, wheezing, sore throat, stuffy nose, taste disturbance, skin rashes, kidney failure etc [7].

Besides chemical drugs, diet and lifestyle also play a significant role in the prevention of hypertension. Lots of small molecular peptides with antihypertensive activity have been discovered and many of these peptides are part of proteins present in our daily food. They are not toxic in general and may represent a new therapeutic strategy for the prevention and treatment of hypertension [7]. Hence, RDP were obtained from the rice dreg fermentation with Pediococcus pentosauceus L11 in this study. This novel RDP with anti-hypertensive activity was verified via in vitro ACE inhibition assay and in vivo SHRs animal experiment. There is a substantial interest in discovering RDP with antihypertensive activity in this study.

Materials and Methodss

Food by Products, Chemicals and Reagents

Rice dreg (moisture: 80%) was provided from rice winery in Miaoli, Taiwan. Pediococcus pentosauceus L11 (L11) was isolated from soil in Miaoli, Taiwan. Angiotensin-I-converting enzyme (ACE) and Hipurry-L-histidyl-L-leucine (HHL) were ordered from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used were of analytical grades.

Fermentation of Rice Dreg

Rice dreg was adjusted to pH 7.0 using sodium hydroxide solution and fermented with 5% sucrose and 10% of L11 (final concentration of approximately 106 CFU/mL) and inoculated at 37oC for 48 h. Later, these fermented samples were collected at post-fermentation 0, 24, and 48 h, respectively. Then, the fermented rice dreg was centrifuged at 6,000 ×g for 30 min and removed insoluble materials. Collection of the supernatant was filtered (Advantec Grade No. 2 Qualitative Filter Paper) then stored at -30oC until analysis.

Measurement of RDP content

Measurement of the peptide content of the fermented rice dregs was followed as the method of Tsai and Wu [13] with some modification. Twenty-five milliliter of working solution was prepared by mixing with 12.5 mL of 100 mM borax, 1.25 mL of 20% (w/w) sodium dodecyl sulfate, 20 mg of o-phthaldialdehyde solution (dissolved in 0.5 mL of methanol) and 50 μL of β-mercaptoethanol and then adjusted the volume to 25 mL with deionized water. Twenty-five microliters of the fermented rice dregs was mixed with 2 mL of working solution and incubated for 2 min at ambient temperature then measured on light absorbance at 340 nm with Hybrid Multi-Mode Reader (Synergy™ H1, BioTek, Winooski, USA). The peptide content was quantified by L-leucine (Difco Laboratories, Sparks, MD, USA) as standard.

Determination of In Vitro ACE Inhibitory Activity

The ACE inhibitory activity was assayed by Cushman and Cheung [14] with some modification. Each 450 μL assay mixture contained the following components at the indicated final concentration: 100 mM borate buffer with 300 mM Sodium chloride 150 μL (pH 8.3); 15 mM HHL 150 mL; 150 μL of the fermented rice dregs and 8 mU ACE. The mixture was incubated at 37°C for 30 min. The reaction was stopped by adding 0.5 μL of 1 M HCl. The hippuric acid was extracted with ethyl acetate then removed ethyl acetate heat evaporation. The amount of hippuric acid was measured Hybrid Multi-Mode Reader (Synergy™ H1, BioTek, Winooski, USA) at 228 nm. The inhibition was calculated by the method of Tsai and Wu [13]. ACE inhibitory activity was calculated by using the following formula:

Inhibition (%) = Ac − As / Ac − Ab


Ac = Absorbance of control sample (HHL + buffer + ACE)

As = Absorbance of sample solution (HHL + sample + ACE)

Ab = Absorbance of blank solution (HHL + buffer)

Animal Care

All animal experiments were approved by the Institutional Animal Care and Utilization Committee (IACUC) of Agricultural Technology Research Institute (ATRI), Xiangshan, Hsinchu, Taiwan and animal care was performed in compliance with the guidelines of IACUC. WKY rats and SHRs were freely fed a standard laboratory diet and the sterile drinking water and kept on a 12-h light/ dark cycle at 24-27°C and 60-70% humidity using an automatic control system in the GLP Animal Laboratory, Animal Technology Laboratories, ATRI, Xiangshan, Hsinchu, Taiwan.

Detection of Blood Pressure of RDP-Administrated SHRse

6-8 weeks old WKY rats (n = 10) and SHRs (n = 35) were raised in GLP Animal Laboratory, ATRI. The RDP via L11 fermentation was dissolved in physiological saline at two doses of 50 and 200 mg/kg body weight (BW). According to blood-pressure level, 35 SHRs were randomized into 4 groups [negative control group: 5 rats; positive control group (20 mg/kg/day nifedipine administration): 10 rats; RDPlow group (50 mg/kg BW RDP): 10 rats; RDPhigh group (200 mg/ kg BW RDP): 10 rats]. On the other hand, 10 WKY rats were blank normal control group. RDP was administrated to SHRs by p.o. using disposable feeding needles (FN-9921, 20G × 1.5”; Kent Scientific, San Diego, CA, USA) once per day for 2 months. Twenty mg/kg/day nifedipine was administrated for SHRs as the same method of RDP.

The same volume and frequency of physiological saline was administrated to WKY rats by p.o. using disposable feeding needles. The blood pressure values were measured for each group once a week. The efficacy of RDP on the blood pressure was compared with each group. Following p.o. of sample, the blood pressure of rats was measured by a tail-cuff method (Model MK-2000ST; Muromachi Kikai, Tokyo, Japan) without warming rats in a chamber maintained at 38°C for 5 min. Five times at a time and take the average for the detection of blood pressure.

Statistical Analysis

Values of ACE inhibitor activity and production concentration of RDP are reported as mean ± Standard Deviation (S.D.). Values of blood pressure, body weight, and liver and kidney function indexes are represented as mean ± Standard Error of the Mean (SEM). Statistical evaluation was performed using Student’s t-test, one-way analysis of variance (ANOVA), and SAS 8.0 software. Differences between groups were considered statistically significant at *p < 0.05.


The Most Ideal Fermentation Conditions of Rice Dregs

The fermentation conditions as fermentation time (h), pH value, and bacterial concentrations (CFU/mL) were screened. The fermentation time involved 0, 5, 24 and 48 h ; pH value included 7.00, 6.99, 5.13, and 5.17; bacterial concentrations involved 1.0 × 106, 3.4 × 106, 3.9 × 109, and 3.6 × 109 (CFU/mL). The most ideal fermentation condition is set at fermentation time (48 h), pH value (5.17), and bacterial concentration (3.6 × 109 CFU/mL). According to the most ideal fermentation conditions, the concentrations of RDP (mg/mL) under three fermentation time (0, 24, and 48 h) were respectively calculated as 24.14 ± 0.09 (fermentation at 0 h), 31.79 ± 0.42 (fermentation at 24 h), and 30.62 ± 0.26 (fermentation at 48 h) (Table 2).


Table 1: Fermentation conditions of the rice dregs.

Effect of RDP on ACE Inhibitory Activity In Vitro

The ACE inhibitory activity (%) and IC5 (half maximal inhibitory concentration, mg/mL) of RDP was presented that 79.34 ± 4.61% and 15.21 mg/mL at 0 h-fermentation, 84.78 ± 7.72% and 18.75 mg/mL at 24 h-fermentation, 96.70 ± 5.85% and 15.83 mg/ mL at 48 h-fermentation (Table 2). RDP production concentration and ACE inhibitory activity were dependently fermentation time. Zero h, 24 h, and 48 h-fermentation compared with each other were significantly different (p < 0.05) on RDP production concentration and ACE inhibitory activity (Table 2).


Table 2: Effects of RDP on ACE inhibitory activity at various fermentation times.

Abbreviation: angiotensin I-converting enzyme (ACE); half maximal inhibitory concentration (IC5); rice dreg peptides (RDP); standard deviation (SD). Data were presented as mean ± SD. Different latters (a, b, and c) was presented significant difference intergroups.

Effect of RDP on BW, Anti-hypertension, and Liver and Kidney Functions In Vivo

The once per day- p.o. experiments with RDP in 6-8 weeks old SHRs. Thirty five SHRs were randomized into 4 groups: negative control group, positive control group (nifedipine administration), RDPlow group (50 mg/kg RDP), RDPhigh group (200 mg/kg RDP). Ten WKY rats were blank normal control group. RDG was administrated by p.o. through gavage per day for 2 months. The BW and blood pressure values were measured for each group once a week. The BW of all rats were increase continuously and were showed non-significantly different (p > 0.05) between 5 groups each other (Table 3). The blood pressure of SHRs before and after experiment in four groups were respectively 181.20 ± 5.51 mmHg; 192.20 ± 3.92 mmHg (negative control group), 170.50 ± 3.49 mmHg; 129.44 ± 3.80 mmHg (positive control group), 171.40 ± 5.29 mmHg; 167.00 ± 0.92 mmHg (RDPlow group), and 169.70 ± 6.69 mmHg; 159.70 ± 1.01 mmHg (RDPhigh group). The efficacy of anti-hypertension of nifedipine is significantly higher than RDP (p < 0.001).


Table 3: Change of body weight in rats during RDP administration.

Abbreviation: Wistar Kyoto (WKY); Spontaneously Hypertensive Rats (SHR); Standard Error of the Mean (SEM); Rice Dreg Peptides (RDP); Low Concentration of RDP (RDPlow); High Concentration of RDP (RDPhigh). Data were presented as mean ± SEM.

The mean blood pressure in two RDP groups was significant lower than negative control group (p < 0.01-p < 0.001) (Figure 1). Moreover, 4 indexes (GOT, GPT, BUN, and CRE) of the liver and kidney functions of RDP-administrated SHRs were located at the normal levels (Table 4).


Table 4: Effects of RDP on liver and kidney function indexes of WKY rats and SHR.

Abbreviation: Wistar Kyoto (WKY); Spontaneously Hypertensive Rats (SHR); Standard Error of the Mean (SEM); Glutamic Oxaloacetic Transaminase (GOT); Glutamic-Pyruvic Transaminase (GPT); Blood Urea Nitrogen (BUN); Creatinine (CRE); Rice Dreg Peptides (RDP); Low Concentration of RDP (RDPlow); High Concentration of RDP (RDPhigh). Data were presented as mean ± SEM.


Figure 1: Efficacy of RDP on the anti-hypertension in vivo. Thirty-five SHRs were randomized into 4 groups [negative control group: 5 rats; positive control group (20 mg/kg/day nifedipine): 10 rats; RDPlow group (50 mg/kg RDP): 10 rats; RDPhigh group (200 mg/kg RDP): 10 rats]. Ten WKY rats were blank normal control group.


Functional peptides can be isolated from the natural food or their by-products. These natural food sources include mainly animal matrix as milk, egg, meat, fish, etc. and plants as cereal, wheat, rice, garlic, spinach, grapes, etc. [15-23]. These functional peptides have also been reported from various biological processes such as enzymatic hydrolysis, fermentation, and chemical synthesis [7,24-26]. At present, lots of chemical drugs have been designed to act on anti-hypertension. Although these drugs are very effective in controlling blood pressure, there are a lot of side effects associated with them. Hence, search of a novel anti-hypertensive compound with no side effects is need. Rice is a major and fundamental cereal source of energy and protein in the world. It is consumed as a staple food for about half of the worldʼs population in over 100 countries. Rice protein has unique nutritional and hypoallergenic properties compared to other plant-sourced protein [27-28] and is suitable as a hypoallergenic protein source to replace milk and soy infant formulas [6,28-29]. Rice manufacturing industry produces large volumes of co- and by-products, which are generally undervalued and under-utilised. Although rice-sourced by-products were cheap. However, it can be renewable to producing the abundant antioxidant and bioactive compounds/peptides [5]. Currently, ricesourced by-products are mostly used as animal feed. These proteins of rice-sourced by-products were as a valuable source for the recovery of antioxidant peptides following whole cell or proteolytic enzyme treatments [5].

Many ACE inhibitory peptides have been characterized from food proteins such as gelatin, maize, fish, eggs, pea, and whey protein [30]. In this study, the source of RDP was rice dregs via lactic acid bacterial fermentation. This RDP is a valuable source for anti-hypertension according to the data of in vitro ACE inhibitory activity and in vivo animal experiment with SHRs. We have successfully R&D to evaluate the rice sourced by-products became the renewable bio-matrix and produced the anti-ACE and anti-hypertensive peptides (RDP). RDP with ACE inhibitory activity was mainly produced by many factories such as monosodium glutamate factory, glucose factory, fermentation industry etc. Commonly, rice dregs are severed as animal feed and they can cause environmental pollution problem. Moreover, rice dregs contain at least 65% protein content, which is higher than soybean protein content.

Therefore, rice dregs are good materials for producing the anti- hypertensive peptides [30]. In this study, ACE inhibitory peptides sourced from rice dregs has some effect on blood pressure regulation in vivo with SHRs. Additionally, Increase BW and normal liver and kidney functions of RDP-administrated SHRs were found in this study. We have found a safer, effective, and economical ACE inhibitor (RDP) for the remedy of hypertension. So, rice dregs-derived ACE inhibitory peptides via the lactic acid bacterial fermentation are safer and more efficient substitution for human health.


Our study mainly focused on the ACE inhibitory activity and anti-hypertension efficacy of RDP. The present study indicated the RDP had significant antihypertensive efficacy and no side effects were found in vitro and/or in vivo. Pediococcus pentosauceus L11-fermentated products (RDP) from rice dregs might serve as alternative sources of dietary health care products with good nutritional quality, safety, and protection against hypertension. Our data might contribute to further research into food derived antihypertensive compounds, meanwhile it also provides some reference for the clinical drug use of RDP in traditional Chinese medicine.

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Diagnosis of Faecal Schistosomiasis in the Context of its Epidemiology in Gabon: Molecular Tools as Alternative to Conventional Methods

Diagnosis of Faecal Schistosomiasis in the Context of its Epidemiology in Gabon: Molecular Tools as Alternative to Conventional Methods


A Brief Overview of the Epidemiology of Faecal Schistosomiasis in Central Africa

In Central Africa, faecal schistosomiasis is mainly caused by Schistosoma mansoni. Its wide distribution is associated with the presence of Biomphalaria, the intermediate snail host living in the fresh waters of Africa [1]. In Cameroon, S. mansoni was detected since 1959 in populations residing around the Olezoa fishponds in Yaounde, with an infestation rate of 64% [2]. It was also described in Nkolmébanga near Saa bordering the Sanaga in southern Cameroon [3] and Minkana in the Lekie Division [4]. In 1981, the Kosa and Dougué foci in the north of the Mandara mountains were highlighted [5]. Several urban and peri-urban areas are listed, such as Melen, which covers part of the border areas of the campus of the University of Yaoundé I [6,7]. Recent data from 63 of the 179 health districts of Cameroon indicate that infection with S. mansoni is the most frequent with an overall prevalence of 5.53% [8]. In Congo, studies carried out in Brazzaville in 1963 and 1976 revealed that the infection rates were 22.1% in Konilon, 19% in Niari and less than 5% in Lekoumon, Bouenza, Pool, Plateau, Cuvette, Likouala and Sangha [9]. In the Central African Republic, reports indicated the existence of populations infected with S. mansoni in almost the entire territory. However, only the south-western region, comprising of the Sangha departments, can be considered as a low-risk area with an infection prevalence of less than 0.5% [10]. In Chad, the distribution of intestinal schistosomiasis is mainly associated with the Logone and Chari basins. Outside the valleys of the south-west, this parasite is present only in Abeche in the mountains of Ouaddaï. In addition to this major species, S. guneensis in the region of the Gulf of Guinea and S. intercalatum in the Kinshasa region, Democratic Republic of Congo, are also encountered at very low levels. These last two faecal schistosomes are transmitted by intermediate snail hosts of the genus Bulinus: Bulinus forskalii for S. guineensis and B. globosus for S. intercalatum.

Epidemiology of Faecal Schistosomiasis in Gabon

In Gabon, faecal schistosomiasis is only caused by S. guneensis and was the first reported case of bilharziasis in the country [11]. To the best of our knowledge, there exist no published data on the local transmission of S. mansoni in Gabon despite its presence in other neighboring countries of Central Africa. Some cases were reported [12] but there was no evidence of the presence of this species in Gabon [13]. In 1995, a case of S. mansoni was diagnosed among patients diagnosed at the department of parasitologymycology of the Faculty of Medicine and Health Sciences of Libreville (FMSS). A case was diagnosed in a 14-year-old boy of Gabonese nationality who had always lived in Gabon. In 2009, a clinical case involved a girl complaining of abdominal pain (FMSS data). Coprological examinations (MIF concentration technique) showed the presence of S. mansoni eggs recognized through its characteristic lateral spur (unpublished data). Based on its public health importance, the distribution of faecal schistosomiasis in Gabon needs to be elucidated. A survey, carried out at Plaine Orety in Libreville, indicated the presence of 4 cases of S. mansoni from a total of 495 people analyzed [14]. Recently, a study reported that the northern and eastern health regions of Gabon were represented low transmission areas of faecal schistosomiasis [15].

These results raised the question of a possible underestimation of faecal schistosomiasis due to the techniques and protocols used for its diagnosis. Although Kato-Katz is a widely used and recommended method for the diagnosis of faecal schistosomiasis, its efficacy is very low in areas with very low endemicity. Due to its low sensitivity, it was necessary to move towards molecular diagnosis which is a credible alternative to the Kato-Katz method [16,17]. The present mini review aimed at assessing the prospective of some molecular protocols versus conventional methods for the diagnosis of faecal schistosomiasis in the epidemiological context in Gabon. Indeed, an accurate diagnosis of helminthiasis is the starting point for better treatment of patients and effective control. The lessons learned can improve on patient management through the use of effective diagnostic tools in epidemiological surveys.

Conventional Methods

The methiolate-iodine-formaldehyde concentration (MIF-C) and Kato-Katz methods have been extensively used in surveys in Gabon [14,15,18]. These techniques are widely used and recommended by the WHO for the diagnosis of schistosomiasis in endemic areas. The Kato-Katz for instance is based on the principle of searching for eggs of schistosomes under a microscope using a very small amount of stool (maximum 50 mg). The advantages of using this method include simplicity, accessibility and its low cost. The critical analysis of these techniques indicates that despite the advantages listed, it is highly specific and advisable only in areas with high endemicity and not in areas with low endemicity. In low endemic areas, the intensity of infection (number of egg per gram of stool) is generally low and the conventional techniques (Katokatz and MIF-C) underestimates the level of infection, because of the very small amount (50 mg) of stool that it analyzes and despite the recommendations that strongly encourage the use of replicate analysis. Therefore, molecular tools can be an alternative to overcome these shortcomings

Molecular methods

Protocol According to Verweij et al. (2001)

The principle of this protocol is to amplify and demonstrate using PCR the presence of DNA of Schistosoma in a stool sample. Stool samples should be stored at -70°C prior to genomic DNA extraction and PCR. The protocol is based on the extraction of schistosome DNA from a stool solution in 0.5 g/ml of PBS buffer. The extraction is carried out using a Qiagen extraction kit (QIAamp Tissue Kit after treatment with 4% PVPP (polyvinylpolypyrolidone) (Sigma, Steinheim, Germany) and a mixture of Sodium dodecyl Sulphate-Proteinase K [19]. It can be more specific than Kato- Katz because PCR amplifications can be carried out with a very small amount of DNA, but it is much more expensive and more difficult to use because it can only be performed by very competent individuals in the domain of molecular biology who are acquainted to the reagents and equipment. It also has the disadvantage of not giving information on the intensity of the parasite.

Protocol According to Pontes et al. (2003)

It is based on the same principle of PCR amplification of schistosome DNA in a stool sample defined above. Stool samples should be frozen prior to usage. The protocol for DNA extraction uses 0.5 g of stool which is mixed with 1 ml of distil water [19]. DNA extraction is not carried out using a Qiagen kit but following the ROSE protocol [20]. This protocol is less expensive and easier than the extraction protocol with the Qiagen kit. It can make better diagnosis than the Kato-Katz because it uses small amount of sample and significant genomic DNA copies can be gotten from very small amount of such stool samples. The disadvantages with this technique are the lack of equipment as well as competent individuals who can manipulate such equipment. Also, in an area of low endemicity, it is likely that the chances of having the eggs and consequently the DNA are not optimized from only 0.25g of stool. This method does not give information on the intensity of the parasite.

Protocol According to Ten Hove et al. (2008)

It uses the same principle and the same steps defined in the protocol of Verweij et al. [19]. Here, the stool samples collected from subjects need to be processed by making a dilution of 0.25g/ ml using 70% ethanol within a 24hrs time frame and the PCR used is a real-time PCR [16]. The advantages, criticisms and disadvantages highlighted in the protocol of Verweij et al. [19] are also valid here. Unlike Verweij et al. [19], the PCR technique used here gives an idea of the amount of DNA and hence the intensity of infection.

The Hatching Technique

The principle of the hatching method consists of filtering a stool sample with saline solution (0.09%) in order to recover the eggs and hatch them to obtain miracidia through a sieve with fine mesh size. The stool can be stored in the refrigerator for 24hrs. Briefly, a mouse parasitized by S. mansoni and conserved dead for three days at 4°C results in a liver with eggs that hatch and release miracidia (unpublished data). The protocol of the hatching technique uses the entire available stool sample, at least 10 g [21]. Any parasitologist can perform this method. This protocol is ideal for field use in endemic settings. The only disadvantage with this technique is that it is long and laborious as it takes 2 to 3hrs on average just to filter a stool sample until hatching of the eggs and release of miracidia. This duration is necessary because it is required to give a real image i.e. twice the parasitic status of the sample of the stool analyzed and it equally allows for the identification of the eggs after filtration as well as in the identification of miracidia. In addition, eggs can be misplaced in the stool solution obtained after filtration, and the possibility of allowing them to hatch to give miracidia, which is more easily repairable, is an additional option for an optimal diagnosis. In this case, the confirmation of the species can be carried out using an additional biomolecular analysis on the miracidia obtained. In areas of low endemicity, this is the most credible way to have an accurate picture of the epidemiological situation. However, it is necessary to carry out a pilot study with the objective of evaluating the cost/ time and ease of execution of the results in order to validate on the need to invest in such a long protocol.

The High-Resolution Melting (HRM) Analysis

The HRM analysis is a technique that detects very small sequence variations from a nucleotide and is based on real-time PCR. It is based on the denaturation of the two strands of the DNA sequence by heating the latter following a temperature gradient. The denaturation temperature, repairable with the loss of the signal of the interacting agent (Syber green) active only on the double strand, is very specific and varies between two different sequences even of a single nucleotide. Today, it is a method of choice because it is rapid, specific, sensitive and effective, and allows us to answer specific questions concerning the interaction between some schistosome species in endemic settings [21].

Pitfalls of the Diagnosis of Faecal Schistosomiasis in Gabon

Diagnosis is based on the amount of stool sample to be analyzed and on the endemicity of the country. All parasitological techniques for the diagnosis of schistosomes are aimed at identifying the parasite (its eggs, its miracidia, and its DNA). It is therefore important to maximize the chances of detecting it. The first way to maximize these chances relies on the amount of stool to be examined. The major concern here is that the screening is carried out in areas with low parasitic load. The critical analysis of the different diagnostic techniques taught us some important lessons. If the conventional methods as the Kato-Katz underestimates the infection prevalence in a zone of low endemicity, it is because the parasites present in the stool examined (i.e. maximum 50 mg of stool) is weak. The quantity of stool examined is multiplied by 10 in proportions indicated by Pontes et al. [16], i.e. 0.5 g and by 200 in the proportions indicated for hatching i.e. about 10 g. It is therefore very useful to adapt the diagnostic technique to the field context, because the results highly depend on the amount of stool analyzed and the endemicity of the study area. The results obtained in Libreville [14] and in the northern and eastern health regions of Gabon [15] using the Kato-Katz method indicated that Gabon had a low prevalence and low intensity for S. mansoni. The review of the critical analysis of parasitological techniques used for the diagnosis of bilharziasis reveals that those that are widely used, notably Kato-Katz, were less sensitive in areas of low endemicity, like in the case of Gabon [15].

The hatching technique is a credible alternative to the latter, since it maximizes the chances of an optimal diagnosis by multiplying them by a factor of 200 compared to the Kato-Katz. Its guarantee is thanks to its approach and its principle of double observation of the parasite i.e. first on eggs and a second on miracidia. This technique does not only offer the possibility of diagnosis but also the possibility of studying the transmission dynamics on a small scale of the individuals or the possibility of extracting the strain necessary for analysis in different aspects in the laboratory. Thus, the hatching technique can make it possible to approach in a more integrated way the studies on schistosomiasis in Gabon. In Gabon, several species of Schistosoma prevail notably S. haematobium, S. guneensis and probable hybrids [22] and S. mansoni [14,15]. The obvious concern will be to distinguish the species and to show their hybrids. However, this can be carried out using the HRM technique. Indeed, it is known that the use of simple morphology or morphometry of the eggs as well as their localization is not enough to differentiate certain Schistosoma spp. [18]. The possibility of using the HRM gives some hope for the clarification of the possible interactions between the different species present in Gabon, insofar as the results will instruct decision-makers to effectively control this parasite. The use of the HRM technique to answer multiple questions about the epidemiology of schistosomiasis in Gabon may be possible through our collaboration with the laboratory of Perpignan whose expertise on this topic is well known.

In the context of low endemicity like ours, we strongly recommend the hatching method which guarantees a better diagnosis and offers the possibility of responding to many questions, in combination with the HRM in the context of the epidemiology of schistosomiasis in Gabon. The only inconvenience of these techniques is that they are very expensive and difficult to perform on a large scale or under field conditions [23]. DNA extraction is a key procedure, but it can be a methodological bottleneck in molecular diagnostic assays since preservation and initial quantity of stool to be examined directly affects the yield and quality of genomic DNA and thus the PCR results. It is often the most expensive part of DNA-based diagnosis, particularly when using commercially available extraction kits.

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