Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Polymorphic and sex-limited phosphoglucomutase in Parastrongylus cantonensis (Nematoda: Angiostrongylidae)

Phosphoglucomutase was studied by polyacrylamide gel electrophoresis in the Thailand and Hawaii isolates of Parastrongylus cantonensis (also known as Angiostrongylus cantonensis). Two loci were present. The faster-moving locus (PGM-1) was polymorphic in the Hawaii isolate, represented by two alleles - the faster-moving, less common Pgm-1A and the slower-moving, more common Pgm-1B . It was monomorphic for the faster-moving allele Pgm-1A in the Thailand isolate. The slower-moving locus (PGM-2) was invariant, with a single band of enzyme activity, in the female worms of both the Thailand and Hawaii isolates. There was no detectable enzyme activity at this PGM-2 locus in the male worms of both isolates. The non-expression or 'null' PGM-2 phenotype in the male worms was presumed to be sex- limited. The present findings differ significantly in several aspects (polymorphic locus, proportion of polymorphic loci, heterozygosity, deviations from Hardy-Weinberg expectations, sex-limited expression) from the Japan isolate of P. cantonensis reported in the literature.

Blinded multi-laboratory evaluation of an in-house dot-blot ELISA kit for diagnosis of human parastrongyliasis.

An in-house membrane dot-blot enzyme-linked immunosorbent assay kit, for the detection of specific antibody to a 31 kDa protein of Parastrongylus cantonensis was evaluated in a blinded multi-laboratory study. The kit was provided to technologists, who were trained in its use, from nine regional laboratories of Thailand. With an identical set of 20 coded serum samples and reference positive and negative controls, the kit was found to have a diagnostic sensitivity of 100% and a diagnostic specificity of 100% in all the laboratories. There was no obvious variation in quality among five lots of the antigen-coated nitrocellulose strips evaluated. The shelf life of the kit was > or = 6 months when the test components were stored at 4 degrees C and 18 months at -20 degrees C. The enzyme-linked immunoassay dot technique is easy to perform and does not require sophisticated electrical equipment; the result is available within 3 hours. If appropriate technical training is included, the application of this dot-blot ELISA kit in clinical laboratories throughout Thailand should be possible.

Eosinophilic meningitis due to Angiostrongylus cantonensis.

We report a 23 year old female who presented with a history of headache. She was admitted to a hospital in Nakornrachasrima province. Eosinophilic meningitis was diagnosed. However, releasing pressure of cerebrospinal fluid (CSF) by lumbar puncture, supportive and symptomatic treatment were performed resulting in appropriate treatment. The patient was referred to Siriraj Hospital due to the persisted headache. Multidisciplinary investigation such as imaging modalities, cytology and serological test for specific antibodies were carried out. Antibody against an A. cantonensis-specific 31-kDa antigen was detected in the serum sample obtained from this patient. In conclusion, A. cantonensis is the possible causative agent of headache in this patient.

Neurocysticercosis : Clinical manifestations and assessments.

Objective : In patients who have symptoms of epilepsy and other neurological deficits, one of the most common parasitic infections is neurocysticercosis (NCC) caused by Cysticercus cellulosae, the larval form of Taenia solium. In order to evaluate the characteristics of this illness which define the diagnosis, we assessed clinical manifestations, a positive ELISA for cysticercosis and neurological imaging in patients suspected of having NCC at a teaching hospital. Methods : This retrospective cohort study assessed 44 patients, who had laboratory results and clinical manifestations compatible with a diagnosis of NCC, between November 1998 and November 2001. The criteria for diagnosing this illness modified from these of Del Brutto and colleagues\\\' criteria. All patients were investigated with serum ELISA, 18 with CSF ELISA, if there was no contraindication. T. solium vesicular cyst soluble antigen was used for the detection of cysticercosis (T. solium) antibodies by solid-phase enzyme immunoassay. 35 patients were further investigated by a computerized tomography (CT) scan of central nervous system (CNS), if the results of other investigations were inconclusive. Using these criteria, we stratified all patients into three groups: possible NCC (19), probable NCC (10), and definite NCC (15). All definite patients were further examined with ELISA for Angiostrongylus cantonensis and Western blot for Gnathostoma spinigerum in order to look for cross-reactivity of the ELISA for cysticercosis. Results : In this study, a prevalence of NCC (34.1%) in the group of patients in whom there was clinical suspicion of NCC makes us concerned that Thailand is still an endemic area for this disease. The three most common clinical presentations were seizures, neurological deficit, and signs of increased intracranial pressure. Serum ELISA showed a sensitivity of 33.3% and a specificity of 93.1%. CSF ELISA showed a sensitivity of 40.0% and a specificity of 100%. CT scan brain showed a high sensitivity (92.9%) with a modest specificity (52.4%). Cross-reaction with other parasitic proteins was found with A. cantonensis (6.7%), but not with G. spinigerum. Conclusion : NCC is still endemic in Thailand. In the approach to this illness, we conclud that the diagnosis of NCC should be based on the patient's history and physical signs in conbination with radiological and serological investigations. Moreover, in areas where A. cantonensis and G. spinigerum were also prevalent, case should be taken in the interpretation of a positive ELISA for cystercercosis because of cross-reactivity with antibodies to A. cantonensis which should be checked individually.

Study of urinary protein and complete blood counts in patients with pyuria.

Examination of 330 patients with pyuria, aged 18 months to 84 years, who attended the Outpatient Department of Siriraj Hospital during the period 1984 to 1988, disclosed proteinuria, and blood leukocytosis with neutrophilia in 70, 55 and 20 percent, respectively. Haemoglobin concentration and haematocrit value were decreased in 59 and 50 percent of the patients. Urine specimens containing over 30 white blood cells / high dry field yielded positive protein test. Nevertheless, there were no correlation between the number of white blood cells in urine and amount of urine protein, white blood cells and neutrophil in blood (correlation coefficient 0.37, 0.17 and 0.21, respectively); neither were correlation between the number of white blood cells in urine, haemoglobin and haematocrit (correlation coefficient – 0.14 and – 0.17, respectively).