An ELISA kit with two detection modes for the diagnosis of lymphatic filariasis.

The aim of this study was to develop a low-cost antifilarial immunoglobulin (Ig) G4 detection kit for the diagnosis of lymphatic filariasis. The kit was designed to be used by minimally trained personnel without the constraints of expensive laboratory equipment. We provide a description of the development and validation of a single-serum-dilution based enzyme-linked immunosorbent assay (ELISA) kit with ready-to-use reagents for measuring antifilarial IgG4 antibodies. The kit was tested on residents in Brugia malayi-endemic areas in southern Thailand. Detection was performed by naked-eye observation of the resultant colour of the immunological reactivity. The coefficient of variation (CV) was used to assess the reproducibility of the results. Long-term stability was measured over a 6-month period. Sensitivity of the test kit was 97% when compared with microfilariae detection in thick blood smears. Specificity was 98.7% based on the sera of 57 patients living outside the endemic areas who were infected with other parasites and 100 parasite-free subjects. All positive CVs were < 10%. The test kit was remarkably stable over 6 months. Field validation was performed by the detection of antifilarial IgG4 in 4365 serum samples collected from residents of brugian filariasis-endemic areas and compared with outcome colours of the test samples by the naked eye. Subsequent ELISA evaluation of these results using an ELISA reader indicated high agreement by the kappa statistic. These results demonstrate that the test kit is efficient and useful for public health laboratories as an alternative tool for the diagnosis of lymphatic filarial infection.

A case report of Brugian filariasis outside an endemic area in Thailand.

A 2-year-old boy living outside the endemic area of lymphatic filariasis in Surat Thani Province, Thailand, developed a high fever. To investigate the cause of his presenting symptoms, blood was collected and microfilariae were detected and identified as Brugia malayi using thick blood smear staining. The sources of the infection were investigated. Microfilariae from two domestic cats residing in the boy's village were detected and identified as B. pahangi using a high-resolution melting real-time polymerase chain reaction analysis. The possible sources of this cryptic infection are discussed.

The impact of a national program to eliminate lymphatic filariasis in selected Myanmar immigrant communities in Bangkok and Ranong Province, Thailand.

Some immigrants from Myanmar to Thailand have brought Wuchereria bancrofti infections with them, causing a community health problem for Thai citizens. The seroprevalence of bancroftian filariasis was detected in 438 and 512 Myanmar immigrants residing in Bangkok and Ranong Provinces, respectively, along with 81 Thai citizens living in Bangkok. The immunochromatograpy card test was positive in 5 Myanmar immigrants living in Bangkok and 1 living in Ranong for a prevalence of 0.63%. Antifilarial IgG4 antibodies were found in 21 Myanmar immigrants living in Bangkok and 14 living in Ranong for a prevalence of 3.68%. None of the samples from Thai citizens were positive with either test. These prevalence rates are lower than those observed between 2001 and 2005. The Thai mass drug administration program to eliminate lymphatic filariasis among Myanmar immigrants appears to be a successful public health strategy.

A cohort study on anti-filarial IgG4 and its assessment in good and uncertain MDA-compliant subjects in brugian filariasis endemic areas in southern Thailand.

The prevalence of Brugia malayi was surveyed in three highly endemic provinces in southern Thailand as part of an ongoing evaluation of the effectiveness of two rounds of mass drug administration (MDA). Prior to MDA IV, and thereafter every 6 months up to 18 months, including MDA V at 12 months, nocturnal blood sampling was assayed for microfilaria (mf) and IgG4 antibodies in 200 subjects from Nakorn Srithammarat and Surathani provinces. From an additional 300 subjects from Narathiwatt province, daytime blood was also examined for IgG4 antibodies for 1 year. Subjects who tested positive, with reciprocal IgG4 titres above 100, were dichotomized in the 'good compliant' or 'uncertain compliant' groups. Stool samples were examined for intestinal parasites from all 500 subjects simultaneously. Only two subjects (1%) tested positive for mf, while 98 subjects (19.6% of 500) tested positive for IgG4. In general, the good drug-compliant subjects were less likely to exhibit the IgG4 response than subjects in the uncertain compliant group. Only six subjects (1.2%) exhibited lymphoedema while 68 subjects (13.6%) had a total of 105 parasitic infections, with female instances of protozoan infections exceeding male instances of helminth infections. It was concluded that the two MDA rounds were highly successful in not only in reducing mf to negligible levels but also in lowering antifilarial IgG4 titres in the good compliant subjects. The IgG4 assay is a sensitive and cost-effective surveillance tool for the early detection of brugian infections that is not contingent on nocturnal blood collections.

Scanning electron microscopy of Aedes lineatopennis (Diptera: Culicidae) eggs.

The eggs of Aedes lineatopennis (Ludlow) are described by means of a scanning electron microscopy. The Ae. lineatopennis egg has a rather boat-shaped, fragmented micropylar collar, and membrane-like wall enclosing many tubercles of exochorionic sculpture.

Toxoplasma infection in healthy persons and in patients with HIV or ocular disease.

Toxoplasma infection is associated with acquired immune deficiency syndrome (AIDS), pregnancy, chorioretinitis, etc. Since the number of AIDS patients is increasing rapidly in Thailand and there are few reports about T. gondii immune status in this country. Toxoplasma-specific IgG and IgM antibodies (Ab) were determined in healthy persons and patients with different symptoms who were suspected of toxoplasmosis. Specific IgG Ab were detected in 3.2% of healthy persons, 12.5% of patients with ocular disease and in 42.5% in HIV positive patients. Only 3.1% of patients with ocular disease were positive for specific IgM Ab. No specific IgM Ab were found in the other samples studied.

Immunoblotting and enzyme linked-immunosorbent assay for diagnosis of toxoplasma infection in HIV Thai patients.

Toxoplasma gondii has been recognized as an important cause of morbidity and mortality among immunocompromised persons. The diagnosis of T. gondii infection is most often based on serological tests results. Serological diagnosis can be limited in AIDS patients because of depressed antibody responses. Fifty serum samples were used in this study to investigate serological evidence of toxoplasmosis in HIV positive Thai patients by Platelia kit, the commercial enzyme-linked immunosorbent assay (ELISA) in which the membrane protein p-30 is the predominant antigen and immunoblot technique (IB). Sera of HIV positive Thai patients with Toxoplasma infection recognized the same antigenic component, the 32 kDa antigenic band, as is recognized by Toxoplasma positive sera from immunocompetent patients and it may represent a specific marker for diagnosis of Toxoplasma infection in HIV positive Thai patients.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in mosquitos.

The application of ethanol-extracted Gloriosa superba for metaphase chromosome preparation in adult and 4th larva Aedes aegypti revealed that 0.5-8% ethanol-extracted Gl. superba solution could be used instead of 1% colchicine in Hanks' balanced salt solution. For adult mosquitos, the metaphase rates and average number of metaphase chromosomes per positive mosquito after intrathoracic inoculation with 1-2% ethanol-extracted Gl. superba solution were 100% and 11.8 (2-16) -12.6 (3-28) in females, and 80-90% and 16.5 (1-52) - 29.89 (1-72) in males, whereas the inoculation with 1% colchicine solution yielded 80% and 50% metaphase rates, and 18.25 (1-40) and 16.5 (2-53) average number of metaphase chromosomes per positive mosquito in females and males, respectively. For 4th stage larvae, the metaphase rates and average number of metaphase chromosomes per positive mosquito after incubation with 0.5-8% ethanol-extracted Gl. superba solution were 90-100% and 14.42 (1-65) - 64 (19-137), while incubation with 1% colchicine solution yield 100% metaphase rate and 10.9 (7-15) average number of metaphase chromosomes per positive mosquito.

Aedes albolateralis, a potential vector of nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus.

The susceptibility of Aedes albolateralis to nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus was investigated by using artificial membrane feeding and intrathoracic inoculation techniques, respectively. The results indicated that Ae. albolateralis was susceptible to nocturnally subperiodic W. bancrofti (susceptibility rate = 9.43%) and dengue type 2 virus (susceptibility rate = 100%), suggesting the potential vector of the two pathogens.

Indirect immunoperoxidase staining of Crithidia luciliae for detecting anti-dsDNA: comparison with other serodiagnostic tests.

The indirect immunoperoxidase (IIP) method was modified and improved for detecting anti-dsDNA. Using fetal calf serum to block the non-specific antibodies and staining with chromatic substrate solution at pH 6.2, the performance time is substantially decreased and the patterns of positive and negative staining are easily distinguished. IIP was compared with indirect hemagglutination (IHA), latex agglutination (LA) and the standard method indirect immunofluorescent technique (IFT). The sensitivities and specificities of the three methods were studied in 507 sera from various sources. Using IFT as the gold standard, sensitivities of IIP, IHA, LA were 98.18%, 88.18% and 50.91% respectively, and the corresponding specificities were 99.5%, 98.24% and 100%. There was a good correlation between titers of IFT and IIP (Spearman correlation coefficient = 0.77, p < 0.001) with no significant difference between the titer of both tests (Wilcoxon Matched-Pairs Signed-Rank test, p = 0.143). These results indicate that the modified IIP method is both highly sensitive and specific and only needs a light microscope to perform it.

Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis.

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.