|Year : 2012 | Volume
| Issue : 2 | Page : 119-123
Evaluation of dried blood spots collected on filter paper for serodiagnosis of human hydatidosis by enzyme-linked immunosorbent assay
Naveen Kumar, Rakesh Sehgal, Kapil Goyal, Praveen Tripathi
Department of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Acceptance||25-Nov-2012|
|Date of Web Publication||28-Dec-2012|
Department of Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh -160 012
| Abstract|| |
Background and Objectives: Serological diagnosis of hydatidosis is usually performed by detecting the circulating antibodies in serum by ELISA. The present study was carried out to standardize and evaluate procedure of the ELISA using elute from dried blood spots (DBS) on filter paper and blood stored at different temperatures and at different durations for its further application under field conditions. Materials and Methods: Dried blood spots were collected from fifty study subjects and fifty control subjects and evaluated for the detection of IgG antibodies against hydatid. Samples were stored at room temperature and 4°C and tested by ELISA at 0, 15 and 30 days. Results: The present study shows that elute of DBS on filter paper can be stored at room temperature for a maximum of 30 days without a decrease in antibody titer as compared to serum samples tested by ELISA. Conclusions: The collection of blood sample on filter paper may serve useful purpose in resource limited countries for carrying out sero-epidemiological surveys at a cost effective level.
Keywords: Dried blood spots,enzyme-linked immunosorbent assay, hydatidosis
|How to cite this article:|
Kumar N, Sehgal R, Goyal K, Tripathi P. Evaluation of dried blood spots collected on filter paper for serodiagnosis of human hydatidosis by enzyme-linked immunosorbent assay. Trop Parasitol 2012;2:119-23
|How to cite this URL:|
Kumar N, Sehgal R, Goyal K, Tripathi P. Evaluation of dried blood spots collected on filter paper for serodiagnosis of human hydatidosis by enzyme-linked immunosorbent assay. Trop Parasitol [serial online] 2012 [cited 2022 Jan 20];2:119-23. Available from: https://www.tropicalparasitology.org/text.asp?2012/2/2/119/105177
| Introduction|| |
The detection of circulating antibodies against hydatid antigens is a major tool for the diagnosis of symptomatic hydatidosis patients and for seroepidemiological surveys, as well as in the active search for asymptomatic carriers. Intensive use of these immunological procedures within the context of adequate epidemiological surveillance and appropriate medical care has made it possible to correlate the findings of hydatid cysts with serological results in a high number of both symptomatic and asymptomatic patients. About 65% of hydatid cyst carriers have circulating antibodies at detectable levels and may therefore be diagnosed by immunological procedures. The most commonly used serological test for the diagnosis of hydatidosis is ELISA. The blood sample is collected by venipuncture, using disposable material to ensure asepsis by trained personnel. The serum sample required for ELISA is separated from clotted blood. The blood sample collection and serum separation also requires the container and equipment for centrifugation, refrigerator for storing the serum samples until they are tested or sent to reference laboratory from peripheral area. Since minute amounts of blood are required for the ELISA, elute of DBS collected on filter paper could be used to simplify blood sampling procedures and reduce costs. Conditions for preserving and recovering antibodies absorbed on filter paper have already been described and filter paper blood elutes have been-used in serological surveys or the diagnosis of several parasitic infections. ,,, However, data regarding the collection of blood sample on filter paper for carrying out serological investigations for hydatidosis is scanty in India. The purpose of the present study was to determine and standardize the best procedure for the ELISA using elute of DBS on filter paper, stored at different temperatures and to evaluate the efficiency and reliability of the ELISA-elute system for the detection of hydatid patients as compared with the ELISA-serum system currently being used in our laboratory. The present study shows that filter paper blood elutes can be stored at room temperature for a maximum of 30 days without any decrease in antibody titre as compared to serum samples tested by ELISA. The collection of blood sample on filter paper may serve useful purpose in resource limited countries for carrying out sero-epidemiological surveys at a cost effective level.
| Materials and Methods|| |
A total of 50 patients (Group A) clinically suggestive of hydatidosis and confirmed by ELISA for hydatidosis from serum samples were enrolled in the study. Study subjects were the patients of either sex, having hydatidosis presenting to OPD clinic (outdoor patient department) or admitted in wards of a tertiary care hospital of north India. The blood samples of confirmed cases of hydatidosis among OPD patients were collected on filter paper when they came to laboratory to collect their reports according to WHO memorandum. 
The blood samples of admitted patients were collected at their bed sites in their respective wards. Fifty controls were included in the study. The controls were 25 patients (Group B) suffering from cysticercosis, toxoplasmosis, malaria, amoebiasis and leishmaniasis (5 each) and 25 normal healthy subjects (Group C). Commercially available Whatman filter paper no. 1 (WHO Memorandum, 1974),  circular in shape having a diameter of 125 mm was taken for blood sample collection. Blood was collected as a spot on filter paper near its circumference by finger prick. After explaining the procedure to patient and taking aseptic precautions, blood was allowed to make a spot on filter paper by touching the filter paper at the site where prick was made. Six such spots were allowed to dry on different filter papers [Figure 1]. Both the filter papers were allowed to dry at room temperature for 45 min. After drying at room temperature, one filter paper was kept at 4°C by wrapping it in tin foil in a seal proof box. Second filter paper was kept at room temperature by wrapping it in tin foil in a seal proof box. Days were counted as day 0, from the time of collection of blood sample on filter paper. On day 0, blood spot was punched out with the help of 10 mm diameter punch [Figure 2]. The punched filter paper was kept in test tube containing PBST (700 μl) for 24 h to elute the dried blood spot collected on the filter paper. Next day test tube was gently vortexed for proper mixing. The eluted sample was used for hydatid serology by in-house ELISA. Similarly blood samples collected on filter paper were checked at both the temperatures at day 15 and 30 along with the serum sample for hydatid serology. The 25 negative control samples were taken from the healthy individuals of non endemic area who were negative for hydatid serology by ELISA. The antibody detection was done by micro ELISA technique by standard protocol  with slight modifications. The optimum concentration of antigen to be coated per well and optimum dilution of serum samples were found to be 2 μgm/well and 1:800 respectively as determined by checker board titration. ELISA was carried out according to the standard technique with certain modifications where ever required. The steps followed were as follows:
- One hundred micro liter of antigen diluted in coating buffer (2 μgm/well) was used for coating the well of micro titer plate and the plate was kept at 4°C overnight covered with tin foil and ELISA plate cover, so as to minimize evaporation.
- Next morning the antigen coated plate was washed thrice with washing buffer PBST (Phosphate buffer saline with 0.02% tween 20)
- Two percent bovine serum albumin (BSA, 100 μl) was added to each well to block the remaining unbound sites on the plate. Plate was incubated at 37°C for 1 h and washed thrice with washing buffer.
- Test and control sera was diluted in PBST as 1:800 and 1:1600 (optimum dilution) and 100 μl of dilution was added to appropriate labelled wells of micro titer plate and incubated at 37°C for 1 h.
- After washing with PBST 100 μl of enzyme labelled anti-human IgG horseradish peroxidise (HRP) conjugate (Sigma-Aldrich), diluted as 1:40,000 (optimum dilution was added to each well. The plate was again incubated at 37°C for 1 h.
- This was followed by washing thrice with PBST and 100 μl of ortho phenylene diamine (OPD) and H 2 O 2 was added in each well as substrate in dark. The plate was kept at room temperature for 15 min in dark.
- The reaction was stopped with 1M H 2 SO 4 and read in ELISA (Biotek, Model No. ELX800MS, US) reader at 490 nm.
The results of the test and control samples were recorded as optical densities (OD). The cut off OD for positivity was determined by adding 2 standard deviations (SD) to the mean of five negative samples. The results were compared between patients and control groups and standard formulae were used for calculating sensitivity and specificity. Criteria for true positives were based on clinical, surgical, radiological and serological (indirect ELISA positive from serum samples) findings for the diagnosis of human hydatidosis.
| Results|| |
Profile of subjects and controls
Among the 50 subject patients, 23 were adult males, 19 were adult females, 3 were male children, 5 were female children with males to female ratio of (M:F = 1.08:1). The age group of adult patients ranged from 18-83 years while of children ranged from 4-12 years. Among the healthy controls, 15 were males and 10 were females. All were adults and age group ranging from 26-50 years. Among the other parasitic disease controls, out of five cysticercosis patients 3 were adult females, 1 adult male and 1 male child. All the five amoebiasis patients were adult males. Out of 5 malaria patients, 4 were adult males and 1 was adult female. All toxoplasmosis patients were adult females. Among leishmaniasis patients 3 were adult males and 2 were adult females.
Majority (92%) of the patients had liver cyst with a history of pain in the abdomen as the main symptom; some had a feeling of fullness in the abdomen. Among the patients with lung cysts, pain and breathlessness were the main symptoms.
Location and number of the cysts
The anatomical location of hydatid cysts in 50 patients as reported by radiological techniques were as follows:
Out of 46 patients with hepatic cysts, 44 had single cysts and 2 had multiple cysts while remaining patients had single cyst.
Out of 50 patients (Group A), positive antibody response was observed in all the patients 50/50 (100%). Among all the healthy controls (n = 25) and parasitic disease controls (n = 25) a total of six samples (one and five among the healthy controls and parasitic disease controls respectively) were positive by indirect ELISA for hydatid IgG [Table 1]. Among all healthy controls (n = 25) one sample and among parasitic disease controls (n = 25) five samples were positive for hydatid IgG by indirect ELISA.
|Table 1: Results of IgG antibodies against hydatid detected by ELISA from serum and elute samples at day 0, 15, 30. (The results were similar for samples stored either at room temperature or 4°C)|
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All the subject patient (n = 50) and control patient samples Group B (n = 25) and Group C (n = 25) were collected on filter paper and stored at room temperature and 4°C for 30 days. The serum and elute samples were tested for IgG antibodies against hydatid on day 0, 15 and 30. The elute from all the DBS of subject patients were positive for hydatid IgG by indirect ELISA and 88% of elutes from control groups (Group B and C) were negative for hydatid IgG on day 0, 15 and 30. The results were similar for samples stored either at room temperature or 4°C. The sensitivity and specificity for ELISA from serum and elute of dried blood spots was 100% and 80% respectively.
| Discussion|| |
Human echinococcosis is a cosmopolitan zoonoses caused by larval stages of tape worms belonging to genus Echinococcus. Cystic echinococcosis (CE) continues to be a significant disease burden in humans and live stock in India and has been reported from all parts of the country. Diagnosis mainly depends upon radiology, immunological and molecular techniques. Radiological techniques are either too costly or radiological picture may mimic other pathologies. Therefore, the serodiagnosis is always required to confirm clinical or radiological diagnosis. The routine laboratory diagnosis of CE is heavily reliant on serological test, which is based on detecting circulating hydatid antibodies. Thus, detection of circulating antibodies against hydatid antigens is a major tool for the diagnosis of symptomatic hydatidosis patients and for seroepidemiological surveys, as well as in the active search for asymptomatic carriers within the framework of control programs. Intensive use of these immunological procedures in such programs within the context of adequate epidemiological surveillance and appropriate medical care has made it possible to correlate the findings of hydatid cysts with serological results in a high number; of both symptomatic and asymptomatic patients; about 65% of hydatid cyst carriers have circulating antibodies at detectable levels and may therefore be diagnosed by immunological procedures. ,,
The entire serological test requires the serum sample as a pre-requisite and collecting blood sample by venipuncture itself requires technical expertise and equipments for collecting blood, separating serum and refrigerator for storing the serum samples. Collection of blood samples on filter paper is a good alternative as only small amount of serum is required for the ELISA. Conditions for preserving and recovering antibodies absorbed on filter paper have already been described ,, and filter paper blood elutes have been used in serological survey, for the diagnosis of several infections. ,
In the present study results obtained by indirect in-house ELISA using filter paper elutes were similar to those observed with serum samples. The elutes were prepared after keeping the filter papers at room temperature and 4°C for a maximum of 30 days to evaluate the level of antibodies for serodiagnosis of human hydatidosis. The OD values of elutes from filter paper were similar when compared with the OD values of serum samples. There was no decrease in the antibody level after 30 days of storage. The sensitivity of detection of IgG antibody against hydatid from elutes of blood samples dried on filter paper was 100% as compared to results of serum samples. There were 6 false positive reactions both in serum and elute samples giving a specificity of 88%. Among all the healthy controls (n = 25) only one sample was found to be positive for hydatid IgG serology and among the parasitic disease controls five samples were found to be false positive due to cross reaction of the circulating antibodies. The results are similar to the previous published studies in which similar levels of antibodies were detected in elutes from filter paper as compared to serum samples. Colorti  et al., have shown that blood samples dried on filter paper at room temperature for approximately 7 days under conditions similar to those of surface transportation does not impair their immunodiagnostic capacity. However, there are studies which show a decrease in the antibody activity of the elutes of blood samples dried on filter paper and kept for 120 days at -20°C. ,, In the present study, the filter paper was stored for a maximum of 30 days and further studies of longer duration are required to check the level of antibodies in filter paper elutes. The results of the present study show that filter paper blood elutes stored at room temperature were as good as stored at 4°C. Thus, results show that even storing at room temperature would be a viable alternative, especially in rural areas where these may not be available. Furthermore, filter paper samples could easily be transported/mailed to tertiary labs for tests without any fear of compromising the results.
Though other biological samples such as saliva and urine samples have also been studied for detecting the specific antibody for the diagnosis of cystic echinococcosis with a sensitivity of 56% and 84% respectively and specificity of 76% in both the samples.  Though, the collection of saliva and urine sample is much easier and non invasive in nature as compared to blood samples but transportation of such samples is as difficult as transporting serum samples in vials. It is also recommended that the urine samples should be analyzed immediately. Another aspect is that even if the urine samples are preserved at 4°C then in the process of freezing and defrosting, there occurs a decrease in antibody titre. Thus, collection of dried blood spots have an added advantage over other methods of sample collection where storage and transportation is difficult. The drawing of blood samples by finger pricking and their collection on filter paper will lower the cost and will increase the number of individuals accessible for sampling and consequently, extend the coverage. This technique would be very beneficial in peripheral areas where there is no laboratory set up is available. Mass population surveys can be conducted easily by the health care workers in remote areas and samples can be transported just by ordinary mail to specialised laboratories for specific tests.
| Summary and Conclusion|| |
Thus the present study indicates that for the diagnosis of human hydatidosis, blood sample collected on filter paper can be used as an alternative to the serum samples for the detection of anti hydatid antibodies, particularly in remote rural areas where serum collection by venipuncture may be difficult. These filter papers with dried blood spots can be easily transported to the reference laboratory for specific diagnostic test.
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[Figure 1], [Figure 2]
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