Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Molecular neonatal screening for glucose-6-phosphate dehydrogenase deficiency in Guiyang region / 重庆医学

Objective To investigate the prevalence of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency and distribu‐tion of mutations in G6PD gene in Guiyang region .Methods A total of 515 DNA samples taken from newborn umbilical cord blood were collected ,15 mutations and one single nucleotide polymorphism in G6PD gene were detected by using of the Sequenom Mas‐sArray MALDI‐TOF‐MS system .Results Among the 515 samples ,10 were determined to have one of the G6PD gene mutations with a detection rate of 1 .94% ,5 mutation types were detected as follow :1388G>A accounted for 40 .0% (4/10 cases) ,1024C> T and 519C> T accounted for 20 .0% (2/10 cases) respectively ,1376G> T and 95A>G accounted for 10 .0% (1/10 cases) respec‐tively .The single nucleotide polymorphism allele frequency of 1311C>T was 12 .79% .Conclusion Guiyang is a region with higher prevalence of G6PD deficiency ,1388 G>A is the most common mutation of G6PD gene in this region .

Molecular analysis of oral squamous cell carcinoma: A tissue microarray study.

Background : An intriguing aspect of Oral Squamous Cell Carcinomas (OSCC) is its behavioral disparity. Among patients who present with the similar clinicopathological features, some have a better prognosis than others. Identification of molecular alterations responsible for this may contribute to a greater understanding of tumor behavior. Tissue microarray (TMA) approach is a high throughput technology that enables analysis of multiple molecular targets simultaneously without causing any morphological alteration to tissue specimens. Aim and Objective : To assess the tumor behavior based on the expression of p53, Bcl-2 and E-cadherin using TMA technology. Settings and Design : This was a case series analysis using tissue microarray technology. Materials and Methods : Formalin-fixed Paraffin-embedded (FFPE) tissue blocks of histological proven cases of OSCC (n = 30) were retrieved from the department archives. Tissue microarray blocks were constructed; 4 ΅m thick sections were cut and immunostained for p53, Bcl-2 and E-cadherin. Stastistical Analysis : Mean (SD) was used to summarize age, frequencies with percentages was used to summarize categorical variable and Chi-square test was used to find association between histopathology evaluation and expression of Bcl-2, p53, E-cadherin. Results and Conclusion : Bcl-2 was the most frequently expressed biomarker. The expression of Bcl-2 was inversely related to the degree of differentiation (P = 0.005). The follow-up data showed that 63.6% of the cases that were positive for both Bcl-2 and E-cadherin were disease-free following treatment. Tissue microarray technology is a promising way to analyse multiple biomarkers simultaneously. The molecular data obtained from TMA will enhance diagnosis, provide better prognostication and will improve cancer treatment for individual patients.

Development of a molecular screening test for hereditary hearing loss and genetic susceptibility to aminoglycoside toxicity for Chinese population / 北京大学学报(医学版)

Objective: To develop a molecular screening test for genetic defects on hearing loss related genes has significant impacts on early identification of hereditary hearing loss and genetic susceptibility to aminoglycoside ototoxicity. Early identification of pre-lingual hearing loss is very important for patient's language development, academic achievement, and social skill. Two common mutations, the 235delC in GJB2 gene and the mutation A1555G in mitochondrial DNA, are included in the newly developed screening panel for Chinese population. Methods: A molecular genetic assay, based on fluorescent labeled multiplex PCR and automatic DNA fragment analyzing techniques, was developed to detect both mutations simultaneously. Results: This assay was able to detect both mutations from patient's samples, and pooled DNA tests, as well as suitable to detect mutation from the DNA extracted from dried blood spot and buccal swab. Conclusion: This assay could be a useful tool for newborn screening and carrier screening for the hereditary hearing loss for the Chinese population.