Detection of circulating cell-free DNA to diagnose Schistosoma japonicum infection.

Schistosomiasis occurs in 240 million people worldwide and is a major public health concern. Thus, early diagnosis and monitoring of schistosomiasis progression are needed to treat patients. Cell-free DNA (cfDNA) is present as fragments of parasite-derived DNA in host body fluids. Detection of this cfDNA in host blood may be a promising diagnostic marker of schistosomiasis. Therefore, in this study, we investigated the potential of internal transcribed spacer 2 (ITS2), a molecular taxonomy and barcoding marker, in diagnosing schistosomiasis using infected rabbit and mice sera. A 192 bp fragment of ITS2 was detected in the serum-isolated DNA from the infected host on different days after infection. We also determined the sensitivity of detecting ITS2 in mice with varying numbers of cercaria: cfDNA was present even in mice with low abundance of the parasite. Overall, our results show that cfDNA may be a potential tool for the early diagnosis and therapeutic evaluation of S. japonicum infection.

Evaluation of quantitative real-time PCR and Platelia galactomannan assays for the diagnosis of disseminated Talaromyces marneffei infection.

Talaromyces (Penicillium) marneffei is an emerging pathogen that causes significant morbidity and mortality in immunocompromised patients in endemic regions such as southeast Asia. The diagnosis of disseminated T. marneffei infection remains challenging in clinical practice. In the study, a well-validated real-time quantitative polymerase chain reaction (qPCR) target region of ITS1-5.8S-ITS2 and a Platelia galactomannan (GM) assay were compared for their diagnostic performance using serum samples from patients with or without human immunodeficiency virus (HIV). The results showed that this novel qPCR method is highly sensitive and specific for T. marneffei DNA detection in serum samples, and the limit of detection and species-specificity of qPCR were five copies of DNA and 100%, respectively. For detection in serum samples from 36 talaromycosis patients, the sensitivity of qPCR was 86.11% (31/36), including 20/20 (100%) patients with fungemia and 11/16 (68.75%) patients without fungemia. For the GM assay, the sensitivity was 80.56% (29/36) when the GM optical density cutoff index was ≥0.5, including 19/20 (95%) patients with fungemia and 10/16 (62.5%) patients without fungemia. These results indicate that the novel qPCR and GM assays can be used as a valuable tool in the diagnosis of T. marneffei infection. Serum samples are convenient hematological specimens for T. marneffei DNA quantification. Combining the GM assay and qPCR is more scientific and appropriate for diagnosing T. marneffei infection in endemic areas.

Molecular diagnosis of Eimeria stiedae in hepatic tissue of experimentally infected rabbits.

The early detection of Eimeria stiedae in the hepatic tissue of experimentally infected rabbits was investigated using molecular assay. Forty 6-week-old male New Zealand rabbits were divided into two groups. Group A (30 animals) was infected with 2.5 × 10(4) sporulated oocysts of E. stiedae per animal on Day 0 and Group B (10 animals) was used as the uninfected controls. Three animals from Group A and one from Group B were sacrificed at 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27 days post infection (PI). Gross and microscopic post-mortem findings were recorded. Polymerase chain reaction (PCR) of the E. stiedae internal transcribed spacer 1 genomic region was conducted on blood, liver tissue, and feces from the Group A experimentally infected animals. Macroscopically, the liver showed irregular yellowish white nodules pathognomonic to E. stiedae infection beginning on Day 15 PI. Hepatomegaly and ascites were obvious from Day 21-24 PI. The presence of different E. stiedae schizonts and gametocytes in the histopathological sections of the biliary epithelium were evident on Day 15 PI. The E. stiedae PCR was first positive in liver tissues on Day 12 and in fecal samples on Day 18 PI, but the blood samples were negative. In conclusion, the PCR can be used for early diagnosis and control of E. stiedae schizonts before shedding of the oocysts in feces.

Development of PCR primers and a DNA macroarray for the simultaneous detection of major Staphylococcus species using groESL gene.

Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10° target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10° target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.

Use of DNA sequencing analysis to confirm fungemia due to Trichosporon dermatis in a pediatric patient.

This is the first reported case of human disease caused by Tricosporon dermatis, an organism recently transferred to the genus Trichosporon from Cryptococcus and now confirmed to be a human pathogen.