Journal of Bacteriology & Parasitology

Development and Testing of Trial Samples for Specific Serodiagnosis of Fasciola gigantica via External Quality Assessment

Vu Quang Huy^1,2,3*, Tran Thien Trung^1,2, Ha Manh Tuan^1,2, Vu Tri Thanh^1,2, Le Van Chuong^1,2, Nguyen Lam Duc Vu², Huynh Thi Diem Phuc³, Bui Quang Sang³ and Van Hy Triet^{1,2 1}HCMC University of Medicine and Pharmacy, Vietnam
²HCMC University Medical Center, Vietnam
³Quality Control Center for Medical Laboratory under supervision of the Ministry of Health, HCMC University of Medicine and Pharmacy, Vietnam
^*Correspondence: Vu Quang Huy, HCMC University of Medicine and Pharmacy, Vietnam, Tel: +84 913586389, Email:

Received: 09-Dec-2018 Published: 24-Dec-2018, DOI: 10.35248/2155-9597.1000351

Introduction

External quality assessment (EQA) is one of the critical elements of a laboratory quality management system in accordance with ISO Guide 15189:2012 [1]. It is also a criterion for assessing the level of quality of a medical laboratory based on decision No. 2429/QD-BYT of the Vietnamese Ministry of Health. In addition, EQA provides objective evidence of reliable results for all customers using the services of the laboratory.

The specific serodiagnosis of anti-Fasciola gigantica antibodies for EQA was designed to improve the quality of screening and diagnostic tests for anti-Fasciola gigantica antibodies. It plays a key role in the control and evaluation of the quality of a laboratory via interlaboratory comparisons. EQA participation is vital for all medical laboratories [2]. At present, there is no study on production of trial samples for specific serodiagnosis of anti-Fasciola gigantica antibodies for EQA. Herein, we developed a procedure for producing standard samples that contain specific anti-Fasciola gigantica antibodies for use in EQA.

Materials and Methods

Sample collection

All samples were collected from patients who had clinical manifestations that met Fascioliasis -foodborne trematode infection criteria [3] comprising fever with unknown reason, right side abdominal pain, decreasing hemoglobin, hepatosplenomegaly and diagnostic imaging showed parenchymal heterogeneity characterised by multiple subcapsular and coalesced into tubular or tortuous structures.

Fifty serum samples that were expected to contain specific IgG anti- Fasciola gigantica antibodies were collected at the Institute of Malaria Parasitology and Entomology, Binh Dinh province, Vietnam.

From September to December 2017, collected serum samples that met the inclusion criteria were extracted into Eppendorf tubes and stored at -20°C. All the samples were transported to the Quality Control Center for a medical laboratory under supervision of the Ministry of Health, HCMC University of Medicine and Pharmacy, for the next steps in the research process.

Hemolytic serum samples or sera that turned dark after 48 hours of storage were excluded from this study. To ensure homogeneity of serum samples, all samples were kept at -20°C during transportation and storage. The samples were tested by both ELISA (Diagnostic Automation/Cortez Diagnostics, California, USA) and Western blotting (LDBIO Diagnostics, Lyon, France) to confirm the presence of specific IgG anti-Fasciola gigantica antibodies.

To prevent the cross reaction, samples were tested by ELISA technique and found negative result for other helminth antibodies including Taenia solium , Echinococcus sp. (Diagnostic Automation/ Cortez Diagnostics, California, USA) and Toxocara canis (Creative Diagnostics, New York USA) especially of trematode antibodies: Schistosoma mansoni (Diagnostic Automation/Cortez Diagnostics, California, USA), Paragonimus westermani and Clonorchis sinensis (Creative Diagnostics, New York USA). All samples were also tested and found negative for antibodies to HIV-1, -2, HCV, and HBV were selected for further analysis.

Negative trial samples for EQA were collected from researchers and tested negative for anti-HIV-1 and -2 antibodies, anti-HCV antibodies, HBs antigen, and anti-Fasciola gigantica antibodies. Then, the samples were frozen at −80°C at OD=0.062 ± 0.012.

Procedure

Trial samples were divided into three batch then stocked in different condition comprising frozen samples were stored at -80°C, freezedried samples were stored at -20°C and one batch were stocked by both conditions. Each batch contained 100 tubes, which were subdivided into two lots, 50 tubes each.

The methods for testing the homogeneity and stability were in accordance with ISO Guide 35 and ISO 13528 [4]. Fourteen tubes from each lot were selected randomly. Optical density (OD) of each tube was measured by ELISA with the wavelength of 450 nm and was used to evaluate homogeneity. Then, stability was assessed after 2, 4, 8, 12 and 24 weeks, by taking three random tubes from each lot and determining OD by the ELISA technique as well as calculating the average of OD values at the corresponding time points.

Data analysis

The F-test analysis of variance (one-way ANOVA) was carried out to evaluate homogeneity of samples (F statistics<F distribution). The t test (independent-sample t test), sig (2-tailed) >0.05, at α=0.05, allowed us to evaluate stability of the samples. SPSS 20.0 software was used for data analysis.

Results

All collected samples were contained an anti-Fasciola gigantica antibody by ELISA. The specific IgG antibodies against Fasciola gigantica antigens were found to be positive for all three proteins, 8–9 kDa protein (P 8-9), 28 kDa (P 28), and 42 kDa (P 42), by the Western blot technique as shown in Figure 1. None of them were positive for other helminth as well as HIV, Hepatitis B and Hepatitis C.

Figure 1. Protein bands detected by western blotting that correspond to F. gigantica antigens.

Three batches of trial samples contain specific IgG antibodies to Fasciola gigantica were divided into three concentrations:

- The freeze-dried samples (Lot DK1) had OD=0.350 ± 0.037.

-The frozen samples (Lot DL1) had OD=0.850 ± 0.047.

-The freeze-dried and frozen samples (Lots DK3 and DL3) had OD=0.653 ± 0.046.

Strips number 1 and 4 contain the reaction with a positive control sample; strips number 2, 3, 5, and 6 show a reaction with positive collected samples; strip number 7 contains the reaction with a collected negative sample.

Evaluation of homogeneity

Table 1 shows the results of assessment of the homogeneity of samples DK1, DL1, and DK3–DL3 with F statistics<F distribution (F statistic was 0.367, 0.087, and 0.465 in comparison to F distribution at 3.231, 3.231, and 2.525, respectively), with sig value>0.05 (sig value was 0.695, 0.917, 0.761, respectively). We assumed that the H₀ hypothesis is accepted. It means that trial samples for EQA that were produced by both the freeze-drying and freezing method were homogeneous.

Table 1:Evaluation the homogeneity of the freeze-dried and frozen samples.

Evaluation of stability

The stability of the frozen samples 1

Table 2 shows that the t statistics at the time of evaluation were less than the t distribution value of 18 (n1+n2−2=16+4−2) degrees of freedom and sig (2-tailed) >0.05. It is likely that the frozen samples 1 (DL1) were stable during 24 weeks.

Table 2: Results of the OD value of DL1 during the study period.

The stability of the freeze-dried samples 1

Table 3 shows that the t statistics at the time of evaluation were less than the t distribution value of (n1+n2–2=20−2) degrees of freedom and sig (2-tailed) >0.05. It is likely that the freeze-dried samples 1 (DK1) were stable during 24 weeks.

Table 3: Results on the OD values of freeze-dried samples 1 during the study period.

Table 4 shows that the t statistics at the time of evaluation were less than the t distribution value of (n1+n2−2=16+4−2) degrees of freedom and sig (2-tailed) >0.05. It is likely that freeze-dried samples 3 (DK3) were stable during 24 weeks.

Table 4: Results on the OD value of Lot DK3 during the study period.

The stability of frozen samples 3

Table 5 shows that the t statistics at the time of evaluation were less than the t distribution value of (n1+n2-2=16+4-2) degrees of freedom and sig (2-tailed) >0.05. It is likely that frozen samples 1 (DL3) were stable during 24 weeks.

Table 5: Results on the OD value of DL3 during the study period.

Results on production of the trial serum samples containing an anti- Fasciola gigantica antibody for EQA: The procedure for production of samples for specific serodiagnosis of anti-Fasciola gigantica antibodies via an external quality assessment scheme have been developed as shown in Figure 2.

Figure 2. The workflow of preparation of trial samples containing an anti-Fasciola gigantica antibody used in EQ.

Discussion

Determination of quality of the trial samples containing an anti-Fasciola antibody used in EQA

The trial samples: The purpose of creating the trial samples with different concentrations of antibodies is to enhance the efficiency of quality assessment of participating laboratories. In comparison with other studies such as “External quality assessment scheme for parasite serology; a review of the scheme design and performance” by Collier [2], and “The United Kingdom National External Quality Assessment Service for parasitology: toxoplasma serology scheme” by Manser [5], in the EQA scheme of serological testing for Toxoplasma sp. in England, there is a bias at low concentrations. This observation means that our EQA sample production method yielding three different concentrations is in compliance with the trend of the EQA scheme of developed countries.

Homogeneity and stability of trial samples: In our study, trial samples produced by freeze-drying and by the freezing method had good homogeneity and stability. Nonetheless, within the limited period of our study, we could evaluate the stability of the resultant trial samples for only 24 weeks in contrast to other studies worldwide. For example, in a study by Michaut [6], serum samples stored frozen at -80°C were stable for 3.5 years, whereas in the study by Dard [7], the stability of IgG antibodies against Toxoplasma gondii antigens in frozen serum samples stored at -20°C lasted for 6 years. There was no change in the levels of the antibody after 6 years of storage and between the mean values before analysis (before freezing of samples) and after freezing.

The process of production of trial serum samples containing anti-Fasciola gigantica antibodies used in EQA

The process includes six steps as follows:

Step 1: We collected materials from the original serum sample with IgG antibodies against F. gigantica antigens had the clinical manifestations were mainly fever with unknown reason, right side abdominal pain, decreasing hemoglobin, hepatosplenomegaly and clearly diagnostic imaging.

Step 2: We selected the samples that were tested negative results for anti-HIV-1 & -2 antibodies, anti-HCV antibodies, HBsAg and negative screened immunoreactivity to other helminth antigens, especially of trematode antigens. This design suggests that our process of preparation of trial samples satisfies the requirements of a laboratory biosafety manual [8].

Step 3: We rechecked the IgG antibodies to Fasciola gigantica antigens from the original serum to eliminate false positive samples and confirm the true positive samples for production of trial samples.

Step 4: We detected protein bands by western blotting by means of the Fasciola gigantica antibodies including P 8-9, P 28, and P 42 kDa as specific antigens to produce the trial EQA samples.

Step 5: We generated the positive samples containing different antibody concentrations by means of the negative serum for dilution at a ratio of 1/5 and 1/10.

Step 6: We evaluated quality of the trial EQA samples in terms of homogeneity and stability: the two criteria in accordance with ISO 35: 2006 and ISO 13528: 2015.

Conclusion

1. The specific IgG antibodies against Fasciola gigantica antigens in collected serum samples were found to be reactive with all three protein bands (P 8-9, P 28, and P 42 kDa) in the western blot analysis.

2. The produced trial EQA samples of anti-Fasciola gigantica antibodies had good quality: homogeneity and stability during 24 weeks.

3. The process of preparation of the trial EQA samples of anti- Fasciola gigantica antibodies can be applied to produce serological EQA samples for parasitosis.

Acknowledgments

We would like to thank the Quality Control Center for the Medical Laboratory under supervision of the Ministry of Health – University of Medicine and Pharmacy at Ho Chi Minh City for funding and support of facilities. We owe special thanks to the Institute of Malaria, Parasitology, and Entomology, Binh Dinh province, for kindly providing serum samples. Furthermore, we kindly appreciate Do Duc Minh PhD - University of Medicine and Pharmacy at Ho Chi Minh City for proof reading the article.

REFERENCES

1. International Organization for Standardization (2012) Medical laboratories — Requirements for quality and competence. ISO 15189: 18-36.
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