Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

Study on the Efficacy of Sentinel Lymph Node Identification by Radionuclide Method (Tc 99) Versus Combined Method (Radionuclide and Vital Stain) in the Staging of Breast Carcinoma

Introduction: Sentinel node biopsy is the gold standard for axillary assessment of patients with breast cancer without axillary metastases on clinical and radiological examination. Internationally accepted biopsy methods currently use a radioactive tracer (Te) or different variations of vital stain, or the combination of the two. Due to the high cost of technical and organizational difficulty related to the radioactive material, as well as the disadvantages of using the vital stain method, great effort is being made to find alternative solutions. The aim of this study was to determine the effectiveness of the exclusive use of vital stain versus the radioactive isotope, and the need to use the combined method. A second goal was the comparative analysis of the radioactive method and intraoperative assessment of suspicious (non-sentinel) lymph nodes. Materials and methods: This article is based on a prospective nonrandomized study conducted on 69 patients with early breast cancer in whom the combined method was used (injection of radionuclide and methylene blue vital stain). The comparatively monitored parameters were the following: the total and mean number of excised sentinel lymph nodes, the number of metastatic ganglia revealed by the 2 methods, and the risk of understaging in case only one technique was used. Results: We excised 153 sentinel nodes identified by the radioisotope method. Of these only 56 were stained with methylene blue (p <0.0001). We could also identify a significantly higher number of metastatic nodes with the aid of the radioactive method (p = 0.0049). Most importantly, a significant number of patients (57.14%) who would have been declared node-negative using vital staining could only be properly staged using the radionuclide or the combined method. On microscopic examination of 35 non-sentinel lymph nodes, we found 3 lymph nodes with metastases, and in 1 case the metastases were found only in the non-sentinel lymph node. Conclusions: Given the risk of understaging, exclusive use of the vital stain method is not recommended, especially under the ASGO Z 00011 Protocol, since the more accurate determination of the number of metastatic sentinel lymph nodes in a patient influences the decision whether to perform lymphadenectomy or not. Using the combined method confers benefits only during the learning curve, in our database we found no stained nodes which were not radioactive. It is very important that the intraoperative stage uses the radioactive method and the intraoperative assessment of suspicious lymph nodes, because 35 non-sentinel lymph nodes were identified in our study, 3 of which had metastases, while in 1 case the metastases were in the non-sentinel lymph node.

A Quick and Simple Method to Evaluate Anisogramma anomala Ascospore Viability

Viability of ascospores of Anisogramma anomala, cause of eastern filbert blight on European hazelnut, was assessed using the vital stain trypan blue (working solution of 0.05% in lactoglycerol). Viable ascospores only had faint blue staining around their cell walls while non-viable ascospores absorbed the stain and turned dark blue. The number of viable (non-stained) ascospores as determined by trypan blue was similar to the proportion of ascospores germinating on culture media. Viability of field collected ascospores from rainwater spore traps ranged from 41 to 68%. Disease incidence of hazelnut seedlings was more closely related to differences in ascospore abundance than to differences in ascospore viability. Accepted for publication 3 January 2013. Published 9 May 2013.