Isolation of Acanthamoeba Genotype T4 from a Non-Contact Lens Wearer from the Philippines

We report the case of a 76-year old Filipino male who presented with pain, redness, and blurring of vision of the right eye. Corneal scraping was done and sent to the St. Luke’s Research and Biotechnology Group for detection and identification of the infectious agent. Morphological detection was performed by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA.S1 confirmed the morphological identification. Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present in the eye of keratitis patients

Lack of Efficacy of High-Dose Intravenous Immunoglobulin Treatment of Severe Thrombocytopenia in Patients with Secondary Dengue Virus Infection

Because most cases of secondary dengue virus infection are associated with an increased level of platelet-associated IgG, a high dose of intravenous immunoglobulin (IVIG) may have an effect on the development of severe thrombocytopenia in this disease. A randomized, controlled study was conducted with two treatment groups consisting of a treatment (IVIG) group (n = 15) and a non-treatment (non-IVIG) group (n = 16) to determine whether a high dose of IVIG is effective in hastening the recovery from thrombocytopenia in patients with secondary dengue virus infection. No significant difference was found in the baseline demographic data between the two groups. No adverse effect of IVIG was observed, but no effect in hastening the recovery of platelet counts was found in patients with secondary dengue infections. The lack of efficacy of IVIG suggests that platelet clearance by macrophages through Fc {gamma} receptors is not a primary mechanism in this disease

Increased phagocytosis of platelets from patients with secondary dengue virus infection by human macrophages.

The relationship between the percent phagocytosis of platelets by differentiated THP-1 cells was examined using flowcytometry and the peripheral platelet counts as well as platelet-associated IgG (PAIgG) in 36 patients with secondary dengue virus (DV) infections. The percent phagocytosis and the levels of PAIgG were significantly increased in these patients during the acute phase compared with the healthy volunteers. The increased percent phagocytosis and PAIgG found during the acute phase significantly decreased during the convalescent phase. An inverse correlation between platelet count and the percent phagocytosis (P = 0.011) and the levels of PAIgG (P = 0.041) was found among these patients during the acute phase. No correlation was found, however, between the percent phagocytosis and the levels of PAIgG. Our present data suggest that accelerated platelet phagocytosis occurs during the acute phase of secondary DV infections, and it is one of the mechanisms of thrombocytopenia in this disease

Comparison of clinical and environmental isolates of Acanthamoeba based on morphology, protease and gelatinase activity, and the cysteine proteinase gene

Acanthamoeba spp. are opportunistic pathogens that cause amebic keratitis and granulomatous amebic encephalitis in man. Recent attempts to correlate pathogenicity with species have been proven difficult due to inconsistencies in morphology-based classification. The objectives of this study were: (1) to compare clinical and environmental isolates based on morphology, protease and gelatinase activity, and the cysteine proteinase (CP) gene, and (2) to determine whether these features can be used to differentiate the isolates. Results show some degree of variation in trophozoite and cyst morphology. Zymography, demonstrated gross differences in banding patterns, and the protease activity of clinical isolates was greater than the environmental isolates (p-value < 0.01). Amplification of the CP gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length. In contrast, only one band, either the 755 bp or 440 bp band was amplified in the clinical isolates. The results confirmed the limitations of morphology in differentiating Acanthamoeba species, and suggest that zymography, protease activity, and detection of the CP gene are useful reference tests to distinguish pathogenic from non-pathogenic isolates

Serological studies on Philippine naegleria isolates

Mice immunized intravenously (IV) and intraperitoneally (IP) with NSRI and RITM Naegleria isolates produced agglutination titers of 1:256 and 1:643 respectively for both homologous and heterologous antisera. Intraperitoneal immunization with adjuvant produced the same titer as in IV route while no agglutinins were detected via the intranasal route of immunization. Slight difference in the degrees of agglutination between homologous and heterologous reactions suggest that the 2 amoebaisolates are closely related and possibly belong to the same species. Ouchterlony gel diffusion test using RITM and NSRI crude membrane antigens (CMA) produced 4 and 3 homologous precipitin lines respectively. Heterologous reaction produced 3 and 2 precipitin lines for RITM and NSRI CMA. Homologous and heterologous reaction using the soluble antigen (SA) of both isolates produced only one precipitin line suggesting that there are more antigenic determinants in the membrane than in the soluble substances in the cell

Preliminary screening for microsatellite instability and loss of heterozygosity in the deleted in colorectal cancer (dcc) gene among Filipino patients with colorectal adenocarcinoma

Objective. This study aimed to detect the presence of microsatellite instability (MSI) and loss of heterozygosity (LOH) of the Deleted in Colorectal Cancer (DCC) gene in normal and tumor tissues of Filipino colorectal cancer patients and examine its correlation with age, gender, tumor grade, tumor stage and site of lesion. Methods. Paired frozen normal and tumor tissues from thirty-nine (39) patients with colorectal adenocarcinoma were used in this study. Genomic DNA was extracted and amplified by polymerase chain reaction (PCR). Single strand conformation polymorphism - polyacrylamide gel electrophoresis (SSCP -PAGE) was used to determine MSI and restriction fragment length polymorphism (RFLP) was used to study LOH. Results. Based on our data, out of the 39 patients, 10 showed LOH of the DCC gene using the LOH markers VNTR, M2 and M3, while no MSI was detected in the samples using the MSI markers BAT25 and BAT26. Correlation with clinicopathological characteristics showed that there is significance for the site of lesion. The LOH has correlation with tumor samples from the colon but not with those from the rectum. Conclusion. Preliminary screening for MSI and LOH of the DCC gene shows that occurrence of colorectal cancer among Filipino patients can be correlated with LOH of the DCC gene but not with MSI. This may be the first study to examine the correlation or lack thereof of MSI and LOH of the DCC gene with colorectal cancer in a Filipino sample population