Detection of active schistosome infection by cell-free circulating DNA of Schistosoma japonicum in highly endemic areas in Sorsogon Province, the Philippines.

The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the host's body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection.

Detection of Plasmodium knowlesi DNA in the urine and faeces of a Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection.

BACKGROUND: Diagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis. METHODS: Urine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene. RESULTS: Urinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine. CONCLUSIONS: The cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts.

Genetic structure inferred from mitochondrial 12S ribosomal RNA sequence of Oncomelania quadrasi, the intermediate snail host of Schistosoma japonicum in the Philippines.

Species and subspecies of the Oncomelania hupensis species complex are recognized as intermediate hosts of Schistosoma japonicum. Of these species and subspecies, O. quadrasi is distributed throughout the Philippines. This study used 12S ribosomal RNA sequences to explore the genetic structure of O. quadrasi populations in the Philippines. Three subspecies, O. h. hupensis, O. h. formosana, and O. h. chiui of this group were also examined. The phylogenetic tree and haplotypes network showed that O. quadrasi separated from the subspecies. Ten O. quadrasi haplotypes (Oq1-Oq10) clustered in relation to their geographic origin. Genetic differentiation (FST) and estimated gene flow (Nm) among populations showed significant differences, ranging from 0.556-1.000 to 0.00-0.74, respectively. Genetic differences among groups (FCT = 0.466), populations within a group (FSC = 0.727), and populations (FST = 0.854) were observed. These results indicate that the O. quadrasi populations in the Philippines have a substructure associated with their geographic origin.

Use of cell-free circulating schistosome DNA in serum, urine, semen, and saliva to monitor a case of refractory imported schistosomiasis hematobia.

This case of imported refractory schistosomiasis has highlighted the usefulness of cell-free parasite DNA as a diagnostic marker to assess active schistosome infection. In contrast to the rapid disappearance of ova in urine, parasite DNA remained persistent in several other specimen types even after the fourth treatment with praziquantel. This result was consistent with the presence of morphologically intact ova in bladder biopsy samples and with the corresponding symptoms.

Identification and differentiation of human schistosomes by polymerase chain reaction.

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.

High susceptibility of Neotricula aperta gamma-strain from Krakor and Sdau in Cambodia to Schistosoma mekongi from Khong Island in Laos.

Neotricula aperta gamma-strain snails collected from Krakor and Sdau in Cambodia were found to have the same or higher susceptibility to Schistosoma mekongi as N. aperta originally isolated from Khong in Laos. Infection rates of N. aperta gamma-strain snails exposed to 3 miracidia at week 8 were: Khong gamma-strain, 22.6%; Krakor gamma-strain, 33.3%; and Sdau gamma-strain, 67.4%. At week 10, the Sdau gamma-strain showed the highest infection rate of 83.3%. We thus found significantly high susceptibility of the Sdau gamma-strain to S. mekongi originally isolated from Khong. However, in another experiment, susceptibility of the Sdau gamma-strain was rather comparable to that of Khong and Krakor gamma-strain. We also found no significant differences in infection and survival rates between the Khong and Krakor gamma-strain when the snails were exposed to 3 or 6 miracidia. This is the first report to confirm the high susceptibility in the laboratory of N. aperta gamma-strain snails from endemic areas in Cambodia to S. mekongi originally isolated from Laos. The high susceptibility of N. aperta gamma-strain snails to S. mekongi in distant areas may be an important factor in the endemic transmission of human schistosomiasis.