Development of a diagnostic single nucleotide polymorphism (SNP) panel for identifying geographic origins of Cochliomyia hominivorax, the New World screwworm.

The New World screwworm, Cochliomyia hominivorax, causes myiasis in livestock, humans, and other warm-blooded animals in much of South America and the Caribbean. It has been eradicated from North and Central America using the sterile insect technique and a biological barrier is currently maintained at the Panama - Colombian border. However, C. hominivorax is still a threat to eradicated areas as outbreaks can and do occur. In order to identify the origin of a fly involved in an outbreak scenario, diagnostic tools would be beneficial. Recently, the geographic population structure of this species was identified using single nucleotide polymorphisms (SNPs). Here we characterize the three major regional clusters: South America, the Inner Caribbean, and the Outer Caribbean. The objective of this study was to develop a SNP (single nucleotide polymorphism) panel to distinguish between these three clusters. A panel was developed using two unique SNPs per region for a total of six SNPs. This diagnostic SNP assay will allow for rapid source determination of flies from future incursions in order to intercept introductory pathways and aid in the control of New World screwworm.

(1,3)-ß-D-glucan is able to predict therapeutic failure of patients with candidemia and not only mortality.

BACKGROUND: Candidemia is a major cause of bloodstream infection in tertiary hospitals worldwide and fungal biomarkers may provide early diagnosis. OBJECTIVES: To evaluate the performance of (1-3)-ß-D-glucan (BDG) in the diagnosis of candidemia and its ability to predict therapeutic failure. PATIENTS AND METHODS: This was a prospective, multi-centre study conducted in 3 Brazilian hospitals. Clinical outcome was evaluated along 2 weeks of treatment, and therapeutic failure was defined as the occurrence of persistent candidemia, Candida deep-seated infection or death. Baseline BDG detection was performed with the Fungitell® assay (Associates of Cape Cod, Falmouth-USA). RESULTS: We enrolled a total of 71 patients with candidemia and a control group with 110 healthy volunteers. The sensitivity and specificity of BDG for diagnosing candidemia were as follows: 71.8% (95% confidence interval [95% CI] 59.7% - 81.5%) and 98.2% (95% CI 92.9% - 99.7%), respectively. The only predictor of therapeutic failure was a higher BDG value at diagnosis of candidemia; a value > 226 pg/mL predicted failure with sensitivity and specificity of 75% and 78%, respectively. CONCLUSIONS: A high baseline serum BDG value was associated with therapeutic failure.

A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.