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.

Seasonal levels of the major American cockroach allergen per a 9 (arginine kinase) in Bangkok and their rele-vance for disease severity.

Monitoring the levels of cockroach (CR) allergen in the environment has medical relevance as a clear dose response relationship between CR allergen exposure, sensitization and hospitalization has been reported. In this study, a cross-sectional survey of the levels of a major American cockroach (Periplaneta americana) allergen, i.e. Per a 9 (arginine kinase) in dust samples collected in various seasons throughout the year 2007 from 76 houses of CR allergic Thai patients in the Bangkok metropolitan area were determined. A monoclonal antibody-polyclonal antibody (MAb-PAb) based-sandwich ELISA was used. The MAb was specific to Per a 9 and the PAb was raised in a rabbit against the crude extract of P. americana. The detection limit of the assay was 122 pg of the allergen or 0.024 μg per gram of fine dust powder. The concentrations of Per a 9 were found to be highest during the winter months and lowest in summer. The levels of this CR allergen had a direct correlation with disease exac-erbation; i.e. the majority of the CR allergic patients had their most severe clinical manifestations during winter. Moreover, the CR allergen levels were found to be higher in wood based-houses than in concrete houses.

Visceral leishmaniasis caused by Leishmania infantum in Thailand.

Thailand is considered as a non-endemic area for leishmaniasis. We report the first case of visceral leishmaniasis caused by Leishmania infantum in a Thai man living in Bangkok.

Sensitive detection of Pneumocystis jirovecii DNA in gastric wash using nested polymerase chain reaction.

One hundred and five samples of gastric washes were obtained from 52 pediatric patients. Eleven of the 105 samples (10%) gave positive results using immunofluorescence antibody test (IFA) for Pneumocystis jirovecii. Single-step polymerase chain reaction (PCR) produced 13% (14 samples), whereas detection by nested PCR was increased to 65 samples (62%). Moderate agreement (kappa = 0.5) was found between test results of IFA and single-step PCR, but no agreement was found between the results of IFA and nested PCR (kappa = 0.1).

Ganoderma lucidum: a cause of pseudoparasitosis.

We report a pseudoparasitosis case due to Ganoderma lucidum, (lingzhi or reishi mushroom); we believe this to be a first reported case in Thailand. A 49-year-old male patient with non-Hodgkins lymphoma presented with chronic watery diarrhea. He had a history of consumption of powdered lingzhi extract as a dietary supplement and herbal medicine. Stool examination demonstrated many spores of G. lucidum, which must be differentiated from intestinal helminth ova and coccidia. After discontinuation of mushroom spores ingestion, the diarrheal symptoms improved and fecal examination subsequently showed no Ganoderma spores. Many artifacts in the stool may be confused with parasites. Differentiation of parasites from artifacts depends on characterization of the size, shape, structure, and reactivity with common stains.

Determination of fecal occult blood in primary schoolchildren infected with Trichuris trichiura.

A correlation of Trichuris trichiura infection and fecal occult blood detection was conducted in 146 primary schoolchildren in Narathiwat Province, Thailand. The Kato-Katz thick smear method was used for determining egg counts and stated as eggs per gram of feces (epg). The number of T. trichiura eggs was categorized as class I (1-499 epg), class 11 (500-4,999 epg), and class III (> 5,000 epg), according to the relation between infection intensity and reduced hemoglobin concentration. Each fecal sample was processed to detect occult blood using a guaiac-based test (Hema-Screen, USA) and an immunochromatographic-based test (HEXAGON OBTI test, Germany). There were 50 schoolchildren without parasitic infection in the control group. Of 96 cases with T. trichiura infection, 85 and 11 children were classified in the class I and class II groups, respectively, but no subjects were in the class III group. Positive occult blood detection results in the control, class I, and class II groups using the guaiac and the immunochemical tests were 0, 3.5, and 9.1% (p=0.19), and 0, 2.4, and 36.4%, (p<0.0001) respectively. This study suggests that T. trichiura infection with an intensity of 500 epg or greater may be associated with intestinal bleeding.

A laboratory comparison of immunofluorescent assay and polymerase chain reaction (PCR) methods for detection of pneumocystis jirovecii in bronchoalveolar lavage.

Pneumocystis jirovecii is one of the most common causative agents of Pneumocystis pneumonia in immunocompromised patients. Because of problems in the laboratory diagnosis, the majority of cases are diagnosed by medical response. In this study, an evaluation of the detection techniques for Pneumocystis jirovecii in bronchoalveolar lavage (BAL) was performed. Immunofluorescent assay (IFA), simple-polymerase chain reaction (simple PCR) and nested-polymerase chain reaction (nested PCR) methods were compared. This study was carried on sixty samples of BAL, from patients sent to the Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, were included in the study. Twenty-three of the samples (38.3%) were found to be positive by IFA. Twenty-five of them (41.6%) were found to be positive by PCR; and 56 of them (93.3%) were found to be positive by nested PCR. As a result, the simple and nested PCR, which are rapid and reliable diagnostic tests, can be used for the screening detection of this organism.

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.

Cerebral toxoplasmosis in AIDS patients: Clinical manifestations and laboratory diagnosis.

Objective : Patients with acquired immunodeficiency syndrome (AIDS) are susceptible to a variety of infections. Cerebral toxoplasmosis is one of the most common. The presumptive diagnosis is based on clinical manifestations, a positive Toxoplasma antibody test, characteristic neuroradiological abnormalities, and improvement after specific therapy. We evaluated not only the clinical manifestations and laboratory diagnosis but also its prevalence and the CD4 count in relationship to the development of cerebral toxoplasmosis in order to determine its place in the natural history of HIV infection. Methods : A retrospective cohort study was performed in 104 AIDS patients who were stratified into three levels: 35 with a definite diagnosis of cerebral toxoplasmosis and 41 with a probable diagnosis and 104 with a possible diagnosis. The criterion for diagnosing definite illness was a good response to specific treatment. Toxoplasma IgM were detected using solid-phase enzyme immunoassay and monoclonal captured ELISA methods respectively in all patients. 65 cases without a definitive diagnosis were further investigated with a CT scan of the brain. The patient data were followed up on day 7 and 14. Result : Toxoplasma IgG was present in 37 out of all case (35.6%) and Toxoplasma IgM was positive in only one case. The study showed that ELISA was a valuable method for diagnosis in addition of a CT scan of the brain. The ELISA detected antibodies in 24 out of 35 cases with a definite diagnosis (sensitivity 68.6%, 95% CI 5.7%-83.2%) and no antibody in 56 out of 69 cases with other diagnoses (specificty 81.2%, 95% CI 69.9%-89.6%). The CT scan showed positive features in 30 out of 33 cases with definite diagnosis (sensitivity 90.9%, 95% CI 75.7%-98.1%) but there were 4 (out of 32 cases) with a positive CT scan who were negative for the diagnosis, i.e., false positive, giving a specificity of 87.5%, 95% CI 71.0%-96.5%. The most common clinical presentations were headache and neurological deficit. Interestingly, neurological deficit was the only clinical manifestation that was associated with cerebral toxoplasmosis, when the group with a definite diagnosis was compared with the other diagnosis group (p-value 0.006). In HIV seropositive patients with previous Toxoplasma infection, cerebral toxoplasmosis was present in 24 out of 37 cases who were positive for Toxoplasma IgG (positive predictive value 64.9%), particularly in individuals with a CD4 count less than 100 cells/mm1. Conclusion : The two most common clinical manifestations were headache and neurological deficit. Detection of Toxoplasma gondii IgG and CT scan of the brain are valuable tools for the diagnosis of cerebral toxoplasmosis. Cerebral toxoplasmosis is mainly caused by reactivation, as Toxoplasma IgM antibodies were found in only one case.

Intestinal microsporidiosis in HIV-infected children with diarrhea.

Intestinal microsporidiosis is a common opportunistic disease associated with diarrhea in adult AIDS patients in Thailand; the data regarding this infection in children are scarce. The present study was designed to investigate the prevalence and clinical features of intestinal microsporidiosis in hospitalized HIV-infected and uninfected (free of HIV) children with diarrhea. Of the 95 HIV-infected children and 87 uninfected children, 24 (25.3%) and 13 (14.9%) respectively were diagnosed with intestinal microsporidiosis. Species identification of microsporidia spores, by transmission electron microscopy, demonstrated Enterocytozoon bieneusi in 5 cases. Cryptosporidium parvum was a common coinfective parasite; pneumonia was the most frequent concurrent disease found in children with intestinal microsporidiosis. Malnutrition was commoner in the HIV-infected group (79.2% vs 23.1%; p = 0.003). This study indicates that intestinal microsporidiosis is an important disease in both HIV-infected and uninfected Thai children with diarrhea.

Intestinal parasitic infections in HIV and non-HIV infected patients with chronic diarrhea in Thailand.

Chronic diarrhea is a common problem in AIDS patients, and enteric parasites are re-cognized as important causes. This study determined the prevalence of parasitic infections in HIV infected patients with chronic diarrhea. Ninety-one patients with AIDS who presented with chronic diarrhea and 103 patients who were HIV negative were enrolled in the study. Detection of stool parasites was made by microscopy of simple smear, formalin-ether concentration method, modified acid-fast and modified trichrome staining techniques. Species identification of microsporidia spores was made by transmission electron microscopy (TEM). Cases in which serial fecal examinations were negative were subjected to gastroduodenoscopy and/or colonoscopy. Parasitic infections were found in 51 (56%) AIDS patients with chronic diarrhea and in 18 (17%) non-HIV cases (p < 0.001). Microsporidia and Cryptosporidium parvum were the most common parasites found in HIV infected patients and were also significantly more prevalent than in non-HIV infected cases [29% vs 0% for microsporidia, (p < 0.001), and 25% vs 1% for C. parvum, (p < 0.001)]. Species identification of microsporidia spores in 26 patients using TEM revealed Enterocytozoon bieneusi in 19 cases. This study indicated that microsporidia and C. parvum are important pathogenic causes of chronic diarrhea in AIDS patients in Thailand.

The rapid detection of microsporidia in stool using a new trichrome-methylene blue stain: A preliminary report.

The increasing number of HIV-infected patients with intestinal microsporidiosis promt the physician to look for appropriate diagnostic techniques to identify microsporidia. The Weber’s modified trichrome stain has been established as a standard method for detection of microsporidia spores in fecal apecimens. This staninng is practical for routinely diagnosis. However, the original method requires approximately two hours to be completed. The new rapid trichrome-methylene blue technique was therefore developed to reduce the processing time from 120 to 15 minutes. Twenty-two formalinized stool samples known to be positive for microsporidia by transmission electron microscopy and 22 negative control specimens were stained by both original and new modified methods. With these two techniques, the parasitic morphology showed the same characteristic staining pattern, but the trichrome-methylene blue method provided a greater background contrast. The sensitivity and specificity of this modified technique were similar to the Weber’s method and the specificity was equal. This new less time-consuming procedure will be helpful in clinical management.

Myiasis of the maxillary sinus caused by cochliomyia macellaria larvae and review of literature.

A case of myiasis of the maxillary sinus caused by Cochliomyia macellaria was reported in a seventy-four-year old Thai woman. She had a history of enucleation of her left eye because of endophthalmitis due to chronic maxillary sinusitis. She presented with painful facial swelling and purulent nasal discharge and was admitted. The patient was treated successfully with parenteral antibiotics to control associated bacterial infection, and Caldwell-Luc operation with removal of the maggots and antrostomy. Chronic sinusitis was considered to be a contributing factor to the myiasis of the paranasal sinus in the patient. Previously reported cases in Thailand are reviewed.

The comparative effectiveness of metronidazole, furazolidone and cotrimoxazole in blastocystis hominis infectiont.

A study of the treatment of symptomatic Blastocystis hominis infection was carried out in order to compare the efficacy of the use of metronidazole, furazolidone and cotrimoxazole. Fifty-eight subjects were enrolled in this and were placed in three groups at random. The cure rates of the patients who received metronidazole on days, 7, 14 and 30 after treatment were 68.4, 68.4 and 73.7 percent respectively. The cure rates of the furazolidone treatment group on days, 7, 14 and 30 after treatment were 45.0, 70.0 and 85.0 percent respectively. The cure rates for cotrimoxazole on days, 7, 14 and 30 after treatment were 57.9, 84.2 and 78.9 percent respectively. There were no statistically significant differences in the efficacy of these drugs.