Retrospective seroepidemiological study of chikungunya infection in South Asia, Southeast Asia and the Pacific region.

Chikungunya virus (CHIKV) and Ross River virus (RRV) of the genus Alphavirus, family Togaviridae are mainly transmitted by Aedes mosquitoes and the symptoms they cause in patients are similar to dengue. A chikungunya (CHIK) outbreak re-emerged in several Asian countries during 2005-2006. This study aimed to clarify the prevalence of CHIKV infection in suspected dengue patients in six countries in South Asia and Southeast Asia. Seven hundred forty-eight serum samples were from dengue-suspected patients in South Asia and Southeast Asia, and 52 were from patients in Fiji. The samples were analysed by CHIKV IgM capture ELISA, CHIKV IgG indirect ELISA and focus reduction neutralization test against CHIKV or RRV. CHIK-confirmed cases in South Asia, particularly Myanmar and Sri Lanka, were 4·6%, and 6·1%, respectively; and in Southeast Asia, particularly Indonesia, the Philippines and Vietnam, were 27·4%, 26·8% and 25·0%, respectively. It suggests that CHIK was widely spread in these five countries in Asia. In Fiji, no CHIK cases were confirmed; however, RRV-confirmed cases represented 53·6% of suspected dengue cases. It suggests that RRV is being maintained or occasionally entering from neighbouring countries and should be considered when determining a causative agent for dengue-like illness in Fiji.

Use of IgM-capture ELISA for confirmation of Japanese encephalitis infections in the Philippines.

In 2001, the Research and Biotechnology Division (RBD) of St Luke's Medical Center, in collaboration with the Institute of Tropical Medicine of Nagasaki University in Japan, initiated a long-term study of Japanese encephalitis in the Philippines. Laboratory confirmation of acute cases of Japanese. encephalitis was done by IgM-capture ELISA, which detects anti-JEV immunoglobulin M in cerebrospinal fluid (CSF) samples. In the period 2002-2004, a total of 614 CSF samples were submitted to RBD, and of these, 11.7% were positive for anti-JEV IgM: 17 in 2002, 18 in 2003, 32 in 2004, and 5 in 2005. Positive cases came from patients aged 2-77 years. In the 72 positive cases where gender was identified, 44 were male and 28 female. Possible co-infections with dengue virus were also detected by separate testing for anti-dengue IgM by ELISA in 17 CSF samples positive for JE.

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections.

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.

Distribution of three arbovirus antibodies among monkeys (Macaca fascicularis) in the Philippines.

Serum samples from 54 monkeys were collected from healthy individuals in a monkey farm in Luzon island, Philippines, in 1999, and examined by IgM-capture ELISA and indirect IgG ELISA for the presence of dengue (DEN), Japanese encephalitis (JE) and chikungunya (CHIK) viruses. The positive rates for IgM ELISA were 3.7, 35.2 and 14.8% against DEN, JE and CHIK, respectively. Higher positive rates were obtained when indirect IgG ELISA was used: 100% against flaviviruses (JE or DEN) and 59.3% against CHIK virus. The results indicate a high prevalence of flavivirus infections such as JE and DEN, and a lesser prevalence of CHIK virus infections, among monkeys in the Philippines. These findings suggest possible sylvatic transmission cycles of these viruses.

IgM-capture ELISA of serum samples collected from Filipino dengue patients.

Viral antigens for 4 dengue serotypes were produced in C6/36 Aedes albopictus cells. These were used as assay antigens for IgM-capture ELISA to detect IgM antibodies in sera of dengue patients from 3 hospitals in Metro Manila, Philippines. A total of 378 serum samples came from National Children's Hospital (NCH), San Lazaro Hospital (SLH), and St Luke's Medical Center (SLMC), from January to November 1995. Three hundred and four (304) out of 378 serum samples, or 80.42% showed positive IgM ELISA titer against at least one of the 4 assay antigens. Dengue type 4 (D4) antigen detected antibodies in 61.90% (234/378) of these serum samples, whereas type 1 (D1), type 3 (D3), and type 2 (D2) had detection rates of 60.05% (227/378), 50.79% (192/378) and 49.47% (187/378) respectively. Although the results show that both D1 and D4 are the most effective antigens in identifying dengue infections for this batch of samples, the use of a cocktail of antigens is still recommended. The results of this study are the basis for the IgM-capture ELISA protocol presently applied for the laboratory confirmation of dengue cases in the Philippines.

Cytopathogenicity of Acanthamoeba isolates on rat glial C6 cell line.

The pathogenicity of Acanthamoeba isolates from keratitis patients (the Hamburg isolate from Germany, H-1 and a Philippine isolate, IB-1-7) as well as an environmental isolate, W4 was assayed in vitro using rat glial C6 cell line. Results indicate that both live amebae and cell-free supenatants from H-1 and IB-1-7 clones produced cytopathic effects (CPE) on rat glial C6 cells in a dose-and-time-dependent fashion. A dose of 10(5) cells/ml induced death and moderate areas of destruction of individual cells after 48 hours of incubation. Results of both free zone capillary electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis suggest the release of amebic products to the culture medium that could at least partially explain the observed cytopathogenicity after 48 hours. Furthermore, results of SDS-PAGE indicate differences between the secretions of the isolates, with bands produced by the two ocular isolates that were not seen with the environmental isolates. That the secretions can produce a cytopathic effect (CPE) has been shown by the cytotoxicity assays using protein concentrations of the secretory products. Protein concentration of 0.30 microg/microl of culture supenatants from H-1 and IB-1-7 clones produced similar effects on the cell monolayers after 2 hours of incubation. This concentration caused the highest % cell death as measured by both trypan blue exclusion (TBE) and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) assays. In contrast, using W4 clone, corresponding concentrations of both trophozoites and culture supernatant did not cause significant cell death and cellular disintegration.

Degradation of vitellogenins by 170 kDa trypsin-like protease in the plasma of the tilapia, Oreochromis niloticus.

Proteolytic degradation of plasma vitellogenins during purification procedure has been noted in several teleost fishes. We have characterized here a trypsin-like serine protease in the plasma of the tilapia, Oreochromis niloticus, which degrades vitellogenins. The molecular mass of the protease was estimated as 230 kDa by gel filtration and as 170 kDa both by nondenaturing and by SDS-polyacrylamide gel electrophoresis. The protease efficiently hydrolyzed the synthetic peptide substrates for trypsin-like proteases but not the substrates for chymotrypsin-like proteases nor aminopeptidases. Hydrolysis of the peptide substrates was strongly inhibited by leupeptin, aprotinin and N-tosyl-L-lysine chloromethyl ketone and to certain extent by chymostatin, 3,4-dichloroisocoumarin, phenylmethanesulfonyl fluoride, and soybean trypsin inhibitor. Leupeptin and aprotinin also inhibited the degradation of a vitellogenin in the plasma. Although the physiological functions of the 170 kDa protease in vivo have not been elucidated, the results on exzymatic properties of this protease will be useful for the isolation and characterization of vitellogenin not only in tilapia but also in other organisms.

Vitellogenins of Oreochromis niloticus: identification, isolation, and biochemical and immunochemical characterization.

Two native forms of vitellogenin (EIP1 and EIP2) were identified in the plasma of Oreochromis niloticus. They were present in females and were estrogen-inducible in males. Both were phosphoglycolipoproteins and both immunoreacted with the antiserum raised against egg proteins. Two prominent bands (EIpp1 and EIpp2; corresponding to 185 and 120 kDa, respectively), observed on sodium dodecyl sulfate-polyacrylamide gels, were induced by estradiol treatment of males and immunoreacted with the antiserum against egg proteins. EIP1 and EIP2 were isolated by precipitation with Mg2+ ions and ethylenediamine tetraacetic acid, preparative polyacrylamide gel electrophoresis, and electroelution. During purification the fractions containing EIP1 and EIP2 also retained EIpp2 and EIpp1, respectively. Immunoblot analyses using affinity-purified antibodies against EIP1, EIP2, EIpp1, and EIpp2 confirmed that EIP1 and EIP2 were at least composed of EIpp2 and EIpp1, respectively. These results suggest that at least two immunochemically different proteins are induced and are secreted into the blood to serve as vitellogenin and to provide a source of nutrient for the developing embryo of Oreochromis niloticus.

Acanthamoeba sp. from the Philippines: electron microscopy studies on naturally occurring bacterial symbionts.

The isolation of two plasmid-like circular DNAs, measuring 52 and 42 kbp, from an Acanthamoeba sp. from the Philippines has led to the demonstration of a bacterial endosymbiont occurring in this free-living amoeba. The 52-kbp band hybridized with a short sequence of cytochrome b gene and was identified as the mitochondrial DNA, whereas the 42-kbp band was identified as plasmid DNA of the bacterial symbionts on the basis of electron microscopy. The endosymbionts are gram-negative, rod-shaped bacteria measuring approximately 1.3 x 0.43 microns and numbering about eight to ten cells per section. They are randomly distributed in both cysts and trophozoites and are surrounded neither by a phagolysosomal membrane nor by a clear or electron-translucent region. The endosymbiont membrane appears to have a close association with ribosomes, which are seen to be more concentrated within the vicinity of the symbionts than elsewhere within the cytoplasm. Attempts to grow the symbionts and the amoebae separately have failed.

The pathogenicity of a Philippine isolate of Naegleria sp. in mice: effects of dose levels and routes of infection.

The pathogenicity of a Philippine isolate of Naegleria sp. was evaluated using 3-4 week-old mice as experimental animals. Results showed that only the massive doses of 10(6) and 10(7) amebae/mouse inoculated intranasally could successfully establish ameba infection in the brain and cause death after 2-6 days. The effect of the ameba on the mortality rate of inoculated mice was dose-dependent. The amebae were recovered in the brain when inoculated through intracerebral and intranasal routes and in the lungs, liver, and intestines when administered through intranasal and oral routes. By intraperitoneal inoculation, recovery of amebae was positive in all major organs except in the heart. Intravenous inoculation resulted to positive recovery in the lungs, spleen, liver, and heart. Infectivity of the ameba isolate in major organs was route-dependent.