Background and Objective: Schistosoma japonicum is the causative agent of schistosomiasis in the Philippines. Current diagnostics suffer from low sensitivity and accuracy, hence an accurate and reliable diagnosis of schistosomiasis is essential for its prevention and control. In this study, a PCR-based assay for the detection of Schistosoma japonicumfor patient stool and serum samples was developed. Methodology: Three candidate primer sets targeting mitochondrial genes COX3, NAD4, and NAD5 were assessed. COX3 primer pair was used for the rest of the study for sensitivity, specificity, and performance testing. Lastly, the assay using COX3 primer pair was compared to Kato-Katz and circumoval precipitin test (COPT). Results: COX3 and NAD5 primers showed suitability for the assay as sequencing analyses gave high similarities of 96-98% for S. japonicum, while NAD4 showed no similarity to any organisms. The PCR-assay was shown to have a detection limit of 4 ng/ul DNA and was specific only to S. japonicum. The assay detected seven out of ten S. japonicum-spiked stool samples and ten out of ten S. japonicum-spiked serum samples. Comparative performance testing with Kato-Katz and COPT showed high specificity of 100% for both samples, but low sensitivity for formalin-fixed stool samples and stored serum samples. Conclusion This study developed a sensitive and specific PCR-based assay to detect S. japonicum from human samples. Results suggest that this PCR assay could be useful for the detection of S. japonicum in fresh clinical samples and can be further improved as a reference to improve other diagnostic assays for schistosomiasis.
Schistosoma japonicum ; Schistosomiasis ; Polymerase Chain Reaction

Introduction. Several studies have been conducted on the use of Immunoglobulin Y (IgY) technology in the fields of diagnostics and therapeutics. IgY is the avian counterpart of the mammalian immunoglobulin G (IgG) which is exclusively transferred from the hen to the yolk thus conferring passive immunization to the growing embryo. However, despite the advantages it offers over the use of mammalian immunoglobulin, IgY technology has remained underutilized. Objective. The objective of this study is to produce an IgY with activity against synthetic peptide analogs of known immunogenic epitopes of the Hepatitis B Surface Antigen (HBsAg) - a molecular marker of Hepatitis B infection. Methods. Chickens were immunized with synthetic peptide analogs of previously reported immunogenic epitopes of the S and the pre-S1 regions of the Hepatitis B surface antigen (HBsAg). IgY specific for the synthetic peptides was isolated by delipidation and salt precipitation and was further purified by affinity chromatography. Purity and molecular weights of the whole IgY molecule and its subunits were assessed and determined by SDS-PAGE. Anti-peptide activity and specificity were determined by indirect ELISA. The study was approved by the Ethical Review Board (ERB) and Technical Review Board of the Research Implementation and Development Office (RIDO), University of the Philippines Manila. Results and Conclusion. The IgY that was purified in this study had an approximate molecular weight of 165 kilodaltons. The heavy and light chains are 60 and 28 kilodaltons, respectively. The affinity purified IgY demonstrated anti-peptide antibody activity against synthetic peptide analogs of known immunogenic epitopes of the HBsAg. Specific binding against a battery of synthetic peptides also revealed that the affinity purified IgY specifically binds to the known immunogenic epitope of the HBsAg.
HEPATITIS B SURFACE ANTIGENS ; HEPATITIS B ANTIGENS ; HEPATITIS B VIRUS ; IGY

Background. Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. Objective. To detect and characterize mutations in rpoB, katG and gyrA. Methods. Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. Results. Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. Conclusion. These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.
TUBERCULOSIS ; THERAPEUTICS ; THERAPY

BACKGROUND AND AIM: Current standard diagnostic tests for leptospirosis are time-consuming and laborious. The urine DOT-ELISA for leptospirosis is a non-invasive, rapid test utilizing monoclonal antibody to detect leptospiral antigens in the urine. We aimed to determine the clinical utility of urine monoclonal antibody-based DOT-ELISA in the rapid diagnosis of leptospirosis compared with the standard microscopic agglutination test (MAT) and urine and blood cultures.

METHODS: This cross-sectional study was done in a university tertiary hospital in Manila, Philippines. We obtained specimens from patients for blood and urine cultures for leptospirosis, MAT and urine ELISA upon admission and 7 days later.

RESULTS: We included 117 patients over a 15-month period. The median duration of illness before inclusion was 9 days (IQR 7-14) and 64 percent received antibiotics before testing. During the acute phase of illness, the sensitivity, specificity, negative and positive predictive values of the urine ELISA compared to culture and MAT were 27.3 percent (95 percent CI 13.2-48.2 percent), 97.7 percent (95 percent CI 80.0-98.3 percent), 66 percent (95 percent CI 51.7-77.8 percent) and 75 percent (95 percent CI 40.9-92.5 percent), respectively. For patients with symptoms more than 7 days of illness, the sensitivity increased to 40.9 percent (95 percent CI 23.3, 61.3 percent) and the specificity increased to 97.5 percent (95 percent CI 87.1, 99.9 percent), with negative and positive predictive values of 75 percent (95 percent CI 61.8, 84.8 percent) and 90 percent (95 percent CI 59.6, 99.5 percent), respectively. On sub-analysis of the effect of antibiotic use, the sensitivity rates differed significantly among patients with antibiotic use versus for those without antibiotic use, 48.4 percent vs. 15.4 percent, respectively (p = 0.001). The specificity rates did not vary with antibiotic use, 98 percent vs. 97 percent.

CONCLUSION: The DOT-ELISA urine antigen test is highly specific with fair accuracy of 73 percent. Its low sensitivity makes it a poor screening test but its high specificity makes it a useful confirmatory test in the diagnosis of leptospirosis. (Author)