A rapid slide agglutination test for the diagnosis of neurocysticercosis in the rural health set up.

BACKGROUND: Simple and rapid latex-based diagnostic tests have been used for detecting specific antigens or antibodies in several diseases. AIMS: The aim of the present study was to standardize and evaluate the latex agglutination test (LAT) for the detection of Taenia solium metacestode antigen in the cerebrospinal fluid (CSF) and serum for the diagnosis of neurocysticercosis (NCC). SETTINGS AND
DESIGN: The study was conducted at Department of Microbiology, Jawaharlal Institute of Post graduate medical education and research after obtaining informed consent from the study subjects.
MATERIALS AND METHODS: In the present study, CSF and serum samples were collected from clinically suspected NCC, CT/MRI proven cases of NCC, non-cysticercal central nervous system infection control and from healthy control subjects. CSF was not collected from healthy controls. Polyclonal antisera raised in rabbits against porcine T. solium metacestode complete homogenate antigen, was used in the LAT to detect the antigen in the specimens. STATISTICAL ANALYSIS USED: The statistical analysis was carried out using Epi Info. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAT were calculated.
RESULTS: The LAT exhibited sensitivity of 64.7% and specificity of 85.7% with CSF samples and sensitivity of 52.08% and specificity of 96% with serum samples.
CONCLUSIONS: Results of the present study shows that the LAT can be employed as a moderately sensitive and specific test for the detection of T. solium metacestode antigen in the CSF and serum specimens for the diagnosis of NCC in poorly equipped laboratories.

INTRODUCTION

Cysticercosis, caused by Taenia solium metacestode (larval form) of the pork tape worm T. solium, is an important health problem in many countries of Asia, Africa, and Latin America with inadequate sanitary conditions.[1] The presence of cysticerci in the central nervous system(CNS), a condition known as neurocysticercosis (NCC), causes seizures and other neurological problems.

Several immunological tests for the serum and the cerebrospinal fluid (CSF) have been developed for antibody and antigen detection. Detection of the antibody in the serum or CSF have limitations of only indicating exposure to infection and not necessarily established viable infection.

Hence, now the focus remains on antigen detection in the serum and CSF which indicate recent or viable infection. For the first time,[2] demonstrated the presence of T. solium metacestode antigens in the CSF by the latex agglutination test (LAT) using polyclonal antibodies.

Thus, in the present study, an attempt was made to standardize and evaluate the inexpensive and simple LAT for detection of T. solium metacestode antigen in the serum and CSF specimens for the diagnosis of NCC in poorly equipped laboratories.

MATERIALS AND METHODS

Study groups

The study was carried out at the Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research, (JIPMER) India, where the patients and controls were categorized into different groups as described in the earlier studies.[34]

Group 1 (Clinically suspected cases of NCC)

Group 2 (CT/MRI proven cases of NCC)

Group 3 (Non-cysticercal CNS infection controls)

Group 4 (Healthy Controls)

Specimen collection

It was done after obtaining informed consent from all human adult participants and from parents or legal guardians of minors involved in the study.

Cerebrospinal fluid

1-2 ml of CSF was collected by lumbar puncture under aseptic precautions and stored at -20°C till use. CSF specimens were collected from 5 subjects (Group 1), 12 subjects (Group 2), and 14 subjects (Group 3). CSF was not collected from Group 4.

Serum

5 ml of venous blood was collected under aseptic conditions and allowed to clot. The serum was separated and preserved with 0.05 mol/l sodium azide and stored at -20°C till use. Serum specimens were collected from 25 subjects (Group 1), 23 subjects (Group 2), 25 subjects (Group 3), and 25 subjects (Group 4).

Preparation of T. solium metacestode antigen

The preparation of T. solium metacestode complete homogenate antigen was carried out according to the method described in the earlier studies.[5]

The pork tissue cysts were homogenized with PBS (pH 7.2) containing 0. 1 mM phenyl methyl sulphonyl fluoride (PMSF) under cooling condition. Then it was sonicated and centrifuged at 20,050×g for 30 min at 40°C. The supernatant was collected as the porcine T. solium metacestode complete homogenate antigen, and stored at -20°C.

Preparation of hyperimmune cysticercus antiserum

Hyperimmune cysticercus antiserum was raised in rabbits as per the procedure described in the earlier studies.[6]

Briefly, porcine T. solium metacestode complete homogenate antigen was emulsified with Freund's complete adjuvant and injected in an adult rabbit weighing (3-4 kg). After 6 weeks, it was reinjected with the same antigen emulsified with the incomplete adjuvant. After 10 days, blood samples were collected by rabbit ear vein bleeding and monitored for the antibodies to porcine T. solium metacestode complete homogenate antigen by the indirect hemagglutination test (IHA).

The antiserum was purified as per the method described.[7] In this method, the serum-saline mixture was added dropwise to equal volume of cold saturated ammonium sulfate and then centrifuged in the cold. After discarding the supernatant, the precipitate was suspended in saline and the procedure was repeated until the supernatant was colorless. The final precipitate was suspended in 1 ml and dialyzed against PBS (pH 7. 2). Titre of the purified antiserum was then detected by the IHA test which was 1 in 1024.

T. solium metacestode antigen detection by the latex agglutination test

The T. solium metacestode antigen was detected in both the serum and cerebrospinal fluid samples by the latex agglutination test (LAT) as described below.

Latex agglutination test

Preparation of latex suspension

The polystyrene latex suspension of particle size 0.81 μm (SIGMA, St. Louis, MO, USA) was used in this test. 1 percent standardized polystyrene latex suspension was prepared by mixing 0.1 ml latex suspension with 9.9 ml glycine buffered saline (GBS) pH 8.4. This was stored at 4°C until used.

Sensitization of latex particles

One milliliter of 1% latex suspension was mixed with 1 ml of purified hyperimmune antisera raised against porcine T. solium metacestode complete homogenate antigen in rabbits. The mixture was incubated at 37°C for 2 h in the water bath. After incubation, antibody sensitized-latex particles were washed two times with GBS (pH 8. 4) and centrifuged at 3000×g for 5 minutes. Then the centrifuged pellet consisting of antibody-sensitized latex particle were emulsified with GBS (pH 8.4) with 1% BSA to make a suspension of 2%. The reagents were stored at 4°C until used. Latex particles coated with normal rabbit serum were used as control.

Test procedure

The test was performed on a clean glass slide divided with a glass marking pen into two halves. A drop of the test serum/CSF sample was placed on each half of the slide. An equal volume of the sensitized latex reagent was added to the specimen placed on one half and the same volume of control latex suspension was added to the specimen placed on the other half as control. The slide was then manually rotated for 2 min and inspected. Agglutination with the sensitized latex reagent and not with the control latex reagent was considered to be positive. Known positive and negative serum/CSF controls were included each time the tests were performed.

Statistical analysis of the immunoassay

The statistical analysis was carried out using Epi Info package supplied by the WHO. The CSF and serum samples were tested in a single blind manner. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAT were calculated according to the method described.[8]

RESULTS

In a positive LAT, the presence of T. solium metacestode antigen in the serum and CSF specimens were detected by the formation of visible clumps of latex particles and clearing of the suspension within 2 min of addition of the sensitized latex particles to the test serum/CSF samples. In a negative LAT, no visible clumping was observed.

Detection of T. solium metacestode antigen in the cerebrospinal fluid by the LAT

The results of the LAT with CSF specimens which were collected from the clinically suspected cases of NCC, CT/MRI proven cases of NCC, and non-cysticercal CNS infection control are summarized in Table 1. T. solium metacestode antigen was demonstrated by the LAT in the CSF in 4 of 5 (80%) patients of clinically suspected cases of NCC, in 7 of 12 (58.3%) CT/MRI proven cases of NCC and a false positive reaction in 2 of 14 (14.2%) non-cysticercal CNS infection controls. CSF was not collected from any healthy control, hence were not tested for the T. solium metacestode antigen.

Table 1

Evaluation of the LAT for antigen detection in the cerebrospinal fluid and serum for the diagnosis of neurocysticercosis

Detection of circulating T. solium metacestode antigen in the serum by the LAT

The results of the LAT with the serum specimens collected from all the four groups of NCC cases and controls are also summarized in Table 1. The antigen was demonstrated by the LAT in the serum specimens, in 14 of 25 (56%) clinically suspected cases of NCC, 11 of 23 (47. 8%) CT/MRI proven cases of NCC. A false positive reaction was observed with 1 of 25 (4%) non-cysticercal CNS infection controls and 1 of 25 (4%) healthy controls.

The LAT exhibited sensitivity of 64.7%, specificity of 85.7%, positive predictive value 84.6%, and negative predictive value of 61.1% with CSF samples, and sensitivity of 52.08%, specificity of 96%, positive predictive value of 92.59%, negative predictive value of 67.6 % with serum samples.

DISCUSSION

Detection of circulating antigen in the cerebrospinal fluid have been described in a number of parasitic diseases such as toxoplasmosis,[9] gnathostomiasis,[10] malaria[11] and angiostrongyliasis.[12]

T. solium metacestode antigens are released into the CSF as a consequence of immune response or excreted/secreted (ES) by live cysticerci. Their detection indicates active infection. Cysticercus antigen has been detected in the CSF by various immunoassays. The ELISA has been used extensively for the detection of CSF cysticercus antigens for the diagnosis of NCC. Several authors have reported the use of standard ELISA, sandwich ELISA, dot-ELISA, or high pressure liquid chromatography ELISA (HPLC-ELISA) employing either monoclonal or polyclonal antibodies for demonstration of CSF antigen in the cases of NCC.[13] In addition to the ELISA, the LAT has also been used for the detection of the CSF antigen in NCC.[2]

The LAT is one of the simplest slide agglutination serological tests available in the diagnostic parasitology laboratory. The LAT was first described by Severin[14] for the diagnosis of meningococcal meningitis. Since then, the LAT has been used to detect antibodies in a variety of parasitic diseases such as visceral leishmaniasis,[15] toxoplasmosis,[16] cystic echinococcosis,[17] and neurocysticercosis.[18]

In our present study, the LAT using polystyrene latex particles coated with the polyclonal cysticercus antisera was able to detect the presence of cysticercus antigen in the CSF. This test has been employed earlier in our laboratory, to detect the presence of hydatid antigen in the serum and urine for the diagnosis of cystic echinococcosis for the first time.[19] Results of the present NCC study with the LAT had shown a sensitivity of 58. 3% and 80% for the diagnosis of CT/MRI proven cases and clinically suspected cases of NCC respectively [Table 1] using CSF samples. The LAT also showed a sensitivity of 64.7% and specificity of 85.7% for the cysticercal CSF antigen detection [Table 2] in the present sudy. Sensitivity of this test as indicated in the study compared well with that of ELISA using polyclonal sera raised against T. solium cysticerci and Taenia crassiceps cysticerci vesicular fluid to detect CSF antigen in patients with NCC.[20]

Table 2

Statistical analysis of the latex agglutination test in the diagnosis of neurocysticercosis

A ntigen detection in the serum provides a suitable alternative. Several assays have been developed to detect cysticercus antigen, but with variable results. ELISA-based assays have been applied to detect the parasite antigen in the serum samples.[2122] ELISA though moderately sensitive and specific, it is expensive and requires high technical expertise, constant availability of reagents, and difficult to adopt in poorly equipped laboratories. Hence, there is a need for simple, yet equally sensitive and specific test for use in such laboratories. The use of a reliable antigen-detection assay system such as the LAT employing serum samples, as observed in the present study, will help in the diagnosis of NCC cases. The serum antigen detection test will also supplement the results of antibody-based assays particularly in the situations where imaging methods are either not available or inconclusive. It is a recognized fact that antigen levels correlate to the number of viable cysticerci and also correlate with the imaging and clinical effectiveness of the antiparasitic therapy.[23] The serum antigen assay will also contribute to the understanding of the pathogenesis of NCC, particularly the process of death or degeneration of the cysticerci.

Results of the present NCC study with the LAT had shown a sensitivity of 47.8% and 56% for the diagnosis of CT/MRI proven cases and clinically suspected cases of NCC respectively [Table 1] using serum samples. The LAT showed a sensitivity of 52.08% and specificity of 96% for the cysticercal serum antigen detection [Table 2] in the diagnosis of NCC. Sensitivity of this LAT was less than the sensitivity of the ELISA as reported by various authors for the serum antigen.[2122]

Also in the present study, false positive reactions were observed with both the CSF and serum specimens from patients with tubercular meningitis [Table 1]. This may be due to cross-reactivity of antigenic fractions of Cysticercus cellulosae antigens with samples from patients with tuberculous meningitis. Similar observations have been reported by other workers with CSF samples from patients with tuberculous meningitis.[24]

In conclusion, the LAT for the detection of cysticercus antigen in the CSF and serum specimens for the diagnosis of NCC is a simple slide agglutination test, which can be performed even by a paramedical health personnel in a rural health centre using a microscopic glass slide. The test is rapid and results can be obtained in minutes and is economical. As it is much less expensive than ELISA or EITB, it can be afforded by the rural population where NCC is highly prevalent, Also the test does not require any special equipment or technically trained manpower. These above advantages make the LAT an ideal test for the rural setup