LAMP Technique Research Articles

Automatización para el registro de resultados de laboratorio al utilizar la técnica de amplificación isotérmica mediada por bucle (LAMP)

Digital image processing has a variety of applications in different areas, such as engineering, robotics, industry, agriculture, and health, to mention a few. When researchers use some colorimetric techniques, certain situations arise in which the support of computational tools is required to be able to accurately identify the color and tone of the results obtained, thus reducing the probability of making errors. The LAMP technique is a tool applied to nucleic acid amplification and is an example of a colorimetric procedure. The proposed solution presented in this research report is the development of a computer program that uses digital image processing to collect the information obtained from the use of the LAMP technique. Feature extraction techniques based on color and shape are used as part of the solution methodology; with the goal of identifying the elements that belong to the samples of interest. After carrying out a series of tests with different configurations, the automatic generation of files that store the description of results from the LAMP technique was achieved.

Simultaneous differential detection of H5, H7 and H9 subtypes of avian influenza viruses by a triplex fluorescence loop-mediated isothermal amplification assay.

H5, H7, and H9 are pivotal avian influenza virus (AIV) subtypes that cause substantial economic losses and pose potential threats to public health worldwide. In this study, a novel triplex fluorescence reverse transcription-loop-mediated isothermal amplification (TLAMP) assay was developed in which traditional LAMP techniques were combined with probes for detection. Through this innovative approach, H5, H7, and H9 subtypes of AIV can be simultaneously identified and differentiated, thereby offering crucial technical support for prevention and control efforts. Three primer sets and composite probes were designed based on conserved regions of the haemagglutinin gene for each subtype. The probes were labelled with distinct fluorophores at their 3' ends, which were detached to release the fluorescence signal during the amplification process. The detection results were interpreted based on the colour of the TLAMP products. Then, the reaction conditions were optimized, and three primer sets and probes were combined in the same reaction system, resulting in a TLAMP detection assay for the differential diagnosis of AIV subtypes. Sensitivity testing with in vitro-transcribed RNA revealed that the detection limit of the TLAMP assay was 205 copies per reaction for H5, 360 copies for H7, and 545 copies for H9. The TLAMP assay demonstrated excellent specificity, no cross-reactivity with related avian viruses, and 100% consistency with a previously published quantitative polymerase chain reaction (qPCR) assay. Therefore, due to its simplicity, rapidity, sensitivity, and specificity, this TLAMP assay is suitable for epidemiological investigations and is a valuable tool for detecting and distinguishing H5, H7, and H9 subtypes of AIV in clinical samples.

Transition probabilities and suitable coupling scheme for 6p→6s transition array of neutral xenon using hollow cathode discharge lamp technique

In the present work, analyses have been performed to determine spectroscopic parameters, including transition probabilities, and relative line strengths of discharge-produced xenon plasma. We present the transition probabilities of twenty-three emission lines originating from the 5p56p →5p56s transition array of neutral xenon covering the wavelength range from 450 to 1100 nm using a xenon-filled hollow cathode discharge lamp. The experimental absolute transition probabilities have been measured by combining the reported lifetimes of upper levels with the integrated spectral intensities of xenon transitions. The results agree well with the reported data in the NIST database, where available. The theoretical line strengths for all spectral transitions originating from the 5p56p→5p56s transition array have been calculated in the LS, LK, jj, and jck coupling schemes using the 3j, 6j, and 9j symbol relations. Furthermore, the normalized experimental line strengths have also been measured and compared with theoretical line strengths, which confirm that the jj and jck coupling scheme is more appropriate for the level designation of xenon.

Study on the applicability of novel molecular targets of Vibrio species by pan-genome mining in the field of nucleic acid detection

Vibrio spp. are ubiquitous in marine areas worldwide and can cause diseases in humans. Aqueous environments favor the transfer of genes between strains, resulting in the loss of strain-specific target genes. This study aimed to explore new molecular targets for the detection of Vibrio strains at the species level using pan-genome analysis for nucleic acid detection and differentiation of major pathogenic Vibrio in aquatic products. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: three for Vibrio parahaemolyticus, one for Vibrio alginolyticus, three for Vibrio vulnificus, three for Vibrio fluvialis. We constructed primers for the selected genes, and we verified their specificity using various Vibrio species and non-Vibrio strains. After systematic optimization, a novel molecular target-based 4-multiplex PCR (4-mPCR) method was developed, exhibiting a detection limit of 103 CFU/mL for pure cultures and 104 CFU/g for fish samples. The initial inoculum was approximately 1 Log10 CFU/g, and all samples were positive after 4 h of enrichment. Not only that, the feasibility of the mined new targets in the field of nucleic acid detection was further validated by the 4-mqPCR and LAMP techniques. These results suggest that novel targets mined by pan-genomic have good generalizability for use in a variety of nucleic acid detection techniques and can help improve the accuracy of nucleic acid-based detection techniques in the detection of pathogenic Vibrio species in aquatic samples, with potential applications for food safety investigations and disease diagnosis.

Comparative Analysis of PCR, Real-time PCR and LAMP Techniques in the Diagnosis of Trypanosoma vivax Infection in Naturally Infected Buffaloes and Cattle in the Brazilian Amazon

Comparative Analysis of PCR, Real-time PCR and LAMP Techniques in the Diagnosis of Trypanosoma vivax Infection in Naturally Infected Buffaloes and Cattle in the Brazilian Amazon

An experimental study on the visual identification of Fritillaria ussuriensis based on LAMP and nucleic acid colloidal gold technique

Fritillaria ussuriensis Maxim is one of the traditional Chinese valuable herbs, which is the dried bulb of Fritillaria, a plant of the lily family. The identification of authenticity about F. ussuriensis is still technically challenging. In this study, visual identification was performed by ring-mediated isothermal amplification and nucleic acid colloidal gold techniques. Firstly, multiple sequence comparative analysis was performed by DNAMAN to find the differential sites of F. ussuriensis and its mixed pseudo-products, and the specific identification primers of F. ussuriensis were designed. Genomic DNA was extracted by the modified CTAB method, and the reaction system and reaction conditions were optimized to construct LAMP for the visual detection of F. ussuriensis, meanwhile, the genuine product was cloned and the extracted plasmid was sequenced. The specificity and sensitivity were detected, and also verified by nucleic acid colloidal gold method, and 20 commercially available samples were tested. The extracted DNA met the requirements of the experiment, and the genuine F. ussuriensis PCR product titrated on a test strip showed two bands on the T and C lines, while the counterfeit and negative control showed only one band on the C line, which matched the LAMP results. The specificity was 100 %, and the sensitivity of LAMP assay was up to 0.01 ng μL−1, while that of colloidal gold assay was 0.1 ng μL−1, thus the LAMP assay had high sensitivity. 14 out of 20 commercially available samples of F. ussuriensis were qualified, and 6 were unqualified, and the results of the two methods of identification were consistent. In this study, the combined detection method of LAMP and colloidal gold for nucleic acid was established to be specific, rapid, precise and visualized, which can provide a new technical idea for the detection of F. ussuriensis.

Estandarización de una prueba isotérmica como una nueva herramienta diagnóstica en México para la detección molecular de Chlamydia abortus en pequeños rumiantes

Ovine Enzootic Abortion (OEA) is caused by Chlamydia abortus and has recently been recognized as endemic in Mexico. Due to the impact of OEA on intensive lamb production, it is necessary to establish a sanitary control plan based on an efficient diagnosis. The loop-mediated isothermal amplification technique (LAMP) is a molecular test that can specifically identify C. abortus. The aim of this study was to standardize the LAMP technique for the specific detection of C. abortus from ovine vaginal exudate. Primers were identified and modified from an electronic database. The effective conditions of the technique were determined using synthetic DNA and positive biological samples by PCR as references. The assay detection limit was 1 × 10-5 of genetic material, equivalent to 8.5 copies. The standardized LAMP test is proposed as a novel molecular tool for the rapid and specific diagnosis of C. abortus infection from ovine vaginal exudate samples in Mexico.

Quantitative classification of melasma with photoacoustic microscopy: a pilot study.

The classification of melasma is critical for correct clinical diagnosis, treatment selection, and postoperative measures. However, preoperative quantitative determination of melasma type remains challenging using conventional Wood's lamp and optical dermoscopy techniques. Using photoacoustic microscopy (PAM) to simultaneously obtain the two diagnostic indicators of melanin and blood vessels for melasma classification and perform quantitative analysis to finally achieve accurate classification, rather than relying solely on physicians' experience. First, the patients were classified by experienced dermatologists with Wood's lamp and optical dermoscopy. Next, the patients were examined in vivo using the PAM imaging system. Further, the horizontal section images ( plane) of epidermal melanin and dermal vascular involvement were extracted from the 3D photoacoustic imaging results, which are important basis for PAM to quantitatively classify melasma. PAM can quantitatively reveal epidermal thickness and dermal vascular morphology in each case and obtain the quantitative diagnostic indicators of melanin and blood vessels. The mean vascular diameter in lesional skin () of epidermal M+V-type was much larger than that in non-lesional skin (), and the mean vascular density in lesional skin was more than three times that in non-lesional skin. Importantly, vascular diameter and density are important parameters for distinguishing M type from M+V type. PAM can obtain the data of epidermal thickness, pigment depth, subcutaneous vascular diameter, and vascular density, and realize the dual standard quantitative melasma classification by combining the parameters of melanin and blood vessels. In addition, PAM can provide new diagnostic information for uncertain melasma types and further refine the typing.

Low Prevalence of Schistosoma mekongi Infection and High Prevalence of Other Helminth Infections among Domestic Animals in Southern Lao People's Democratic Republic.

The prevalence of Schistosoma mekongi in humans in the Lao People's Democratic Republic (Lao PDR) has been relatively well monitored and has decreased due to effective interventions such as preventative chemotherapy with mass drug administration of praziquantel and community awareness programs. However, the prevalence among potential domestic reservoir animals remains broadly unclear, except for a few villages in the endemic area. Therefore, we conducted S. mekongi surveys for the domestic animals that had contact with Mekong River water. We conducted a cross-sectional study of the domestic animals in the seven sentinel villages in the Khong and Mounlapamok Districts of Champasak Province in southern Lao PDR in 2018 by random sampling with a statistically reliable sample size. Stool samples of the five predominant domestic animal species, cattle (n = 160), pig (n = 154), buffalo (n = 149), dog (n = 143), and goat (n = 85), were collected and examined using parasitological FECT method and the LAMP technique. The microscopic analysis did not detect any eggs of S. mekongi in the stool samples of any animal species. However, S. mekongi DNA was detected by the LAMP test in dog stool samples (0.7%; 1/143). On the other hand, the prevalence of other helminths was quite high and heterogeneous among animal species and sentinel sites by the microscopic analysis. These findings suggested that an intervention for S. mekongi infection should focus solely on human populations. However, periodic surveillance for S. mekongi infection among dogs should be conducted to monitor a possible resurgence of S. mekongi infection in the domestic animal population.

Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System.

The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays.

LAMP-Based Point-of-Care Nucleic Acid-Based Detection Method Can Be Useful for Quick Decision-Making for Diagnosis of Acute COVID-19 Emergency Cases in Hospital Settings

Real-time RT-PCR is used as a gold standard method for the diagnosis of COVID-19. Since real-time RT PCR is nucleic acid-based, it is a highly sensitive and specific test. However, this test takes 4–8 h to generate results and, in emergency settings, this delay may prove fatal for certain patients. The frequent surge in COVID cases increases patient load in emergency settings. Thus, a nucleic acid-based rapid POC test is required that can generate results quickly as well as being comparable to real-time RT-PCR. In this study, comparison of real-time RT-PCR was carried out using the rapid nucleic acid-based LAMP method. Nasopharyngeal swabs were taken in duplicate from patients visiting the kiosk and were analyzed for the presence of the SARS-CoV-2 virus by both real-time RT-PCR and LAMP techniques ID NOW(bbott). Out of 14 positive and 31 negative samples tested by real-time RT-PCR, 13 samples were identified as positive and 31 were observed as negative with the LAMP-based test. Hence, the sensitivity and specificity of this method were found to be 92.9% and 93.5%, respectively. Therefore, LAMP-based point-of-care testing has the potential to be used in hospital emergency settings for quick diagnosis of critically ill patients, and the information generated here will further draw the attention of policymakers toward such nucleic acid-based rapid tests.

Molecular and serological diagnosis of toxoplasmosis: a systematic review and meta-analysis.

Toxoplasmosis is an infection of vast worldwide distribution whose etiologic agent is Toxoplasma gondii. This disease can cause problems ranging from mild symptoms to serious conditions, such as encephalitis, miscarriage and blindness. Therefore, it is of utmost importance to perform a diagnosis with reproducible techniques in order to obtain a good prognosis. The aim of this review was to analyze the efficiency of toxoplasmosis diagnostic techniques based on sensitivity and specificity results. Five research platforms in English language were used (Eric, Elsevier, Google Scholar, PubMed and SciELO), which contained data on the diagnosis of toxoplasmosis. The search and selection were performed for studies published prior to June 2021. The search resulted in the inclusion of 13 articles published from 2005 to 2020. The data revealed the use of different samples in the standardization of techniques such as serum, total blood, colostrum and amniotic fluid. The flow cytometry, lateral flow immunoassay and qPCR techniques showed 100% sensitivity, whereas the ELISA, western blotting, qPCR and RE-LAMP techniques achieved 100% specificity. Significantly, the qPCR and LAMP techniques were more accurate when the likelihood ratio was assessed. The meta-analysis identified that ISAGA and western blotting have low sensitivity values and LIASON, ELFA and ELISA, using a silica bioconjugate, also have low specificity values. It was noted that a wide range of methods have high values of sensitivity and specificity. Therefore, the choice of the method will be based on the conditions and its financial viability.

Diagnostic techniques for infectious bovine rhinotracheitis: a review

Infectious bovine rhinotracheitis (IBR) is a group of the bovine respiratory disease multifaceted pathogens and a key disease of cattle, leading to significant economic losses to the dairy industry globally. The causative agent of IBR is Bovine herpesvirus type-1 (BoHV-1), which is a member of the genus Varicellovirus in the Alphaherpesvirinae subfamily, which belongs to the family Herpesviridae, order Herpesvirales. BoHV-1 can be categorized into three subtypes (BoHV-1.1, BoHV-1.2a, and BoHV-1.2b) that belong to one single viral species, which is a serologically indistinguishable strain. Therefore, a more optimal method for the rapid diagnosis of BoHV-1 infection is highly needed. Hence, the objective of this paper is to review the appropriate diagnostic techniques for the IBR virus in infected cattle. In this review, various rapid and confirmatory diagnostic methods used for the diagnosis of BoHV-1 infection were briefly described. BoHV-1 can be routinely detected by virus neutralization tests and enzyme-linked immunosorbent assays (indirect or blocking ELISA). IBRgE-ELISA is the most specific serological test for BoHV-1 and is recommended for marker vaccine to differentiate wild infection from vaccination schemes. Furthermore, virus isolation from tissue or swab samples by cell culture and DNA detection with LAMP, PCR, and real-time PCR techniques are all used to detect infected cattle. Direct sequencing of the entire genome using the Sanger sequencing method recently allowed for the differentiation of BoHV-1 subspecies and the distinction of the BoHV-1 field strain from vaccine strains based on single nucleotide polymorphisms. As the gold standard diagnosis for IBR is virus isolation in cell culture, commonly followed by BoHV-1 gene sequencing, it is also recommended.

Diagnostic Techniques for Infectious Bovine Rhinotracheitis: A Review

Infectious Bovine Rhinotracheitis (IBR) is a group of the bovine respiratory disease multifaceted pathogens and a key disease of cattle, leading to significant economic losses to the dairy industry globally. The causative agent of IBR is Bovine herpesvirus type-1 (BoHV-1), which is a member of the genus Varicellovirus in the Alphaherpesvirinae subfamily, which belongs to the family Herpesviridae, order Herpesvirales. BoHV-1 can be categorized into three subtypes (BoHV-1.1, BoHV-1.2a, and BoHV-1.2b) that belong to one single viral species, which is a serologically indistinguishable strain. Therefore, a more optimal method for the rapid diagnosis of BoHV-1 infection is highly needed. Hence, the objective of this paper is to review the appropriate diagnostic techniques for the IBR virus in infected cattle. In this review, various rapid and confirmatory diagnostic methods used for the diagnosis of BoHV-1 infection were briefly described. BoHV-1 can be routinely detected by virus neutralization tests and enzyme-linked immunosorbent assays (indirect or blocking ELISA). IBRgEELISA is the most specific serological test for BoHV-1 and is recommended for marker vaccine to differentiate wild infection from vaccination schemes. Furthermore, virus isolation from tissue or swab samples by cell culture and DNA detection with LAMP, PCR, and real-time PCR techniques are all used to detect infected cattle. Direct sequencing of the entire genome using the Sanger sequencing method recently allowed for the differentiation of BoHV-1 subspecies and the distinction of the BoHV-1 field strain from vaccine strains based on single nucleotide polymorphisms. As the gold standard diagnosis for IBR is virus isolation in cell culture, commonly followed by BoHV-1 gene sequencing, it is also recommended.

Host range and virulence diversity of Pectobacterium carotovorum subsp. brasiliense strain RDKLR infecting radish in India, and development of a LAMP-based diagnostics.

This work aimed at determining the pathogenicity, molecular characterization, host range and rapid detection of Pectobacterium carotovorum subsp. brasiliense (Pcb) causing soft rot disease in radish. The four isolated isolates were inoculated to radish, typical soft rot symptoms were observed and Koch's postulates were proved. The most virulent strain RDKLR was morphologically and biochemically distinct. Pcb showed a positive potato soft rot test and elicited hypersensitivity response on Nicotiana tobaccum. The genes Pel2 and pmrA were used for subspecies characterization of Pcb. It has a wide host range and infection was observed on slices of carrot, tomato, radish, potato, cauliflower, cabbage, chilli, knol-khol, bell pepper and cucumber. Infectivity was also seen in seedlings under glasshouse conditions. Pcb produced cell wall degrading enzymes in semi-quantification assay and is a strong biofilm producer. The LAMP technique was standardized to help rapid detection and take prophylactic measures to manage the disease. This work reports Pcb as a new soft rot causing organism of radish in India. Pcb is highly virulent with a broad host range. The LAMP technique helps in rapid detection. Pcb-induced soft rot causes significant yield loss, decreased market value, damage in transit, storage and the market. Disease characterization and early identification aid in disease management and prevention in the field.

Influence of ancestry in racial disparities of squamous cell lung cancer.

e20554 Background: Significant progress has been made in the screening, diagnosis, and treatment of squamous cell carcinomas of the lung (LUSC). Despite the advancements, African American populations continue to experience worse disease burden and poorer overall survival, even after controlling for patient and tumor characteristics. While the interplay of various social determinants of health is well known to contribute to racial disparities in cancers, the molecular basis of racial disparity is less understood. Given the diversity of the US population that comprise of genetically admixed groups, understanding the role of ancestry and its influence on biology of cancers that drive incidence and prognosis is paramount to reducing racial disparities. Therefore, in this study, we aimed to investigate the influence of African (AFR) ancestry in genetic mutations of LUSC. Methods: We utilized The Cancer Genome Ancestry Atlas (TCGAA) to obtain data on the genetic ancestry and ancestral composition of 504 LUSC patients. The dataset contained 459 self-reported whites, 32 blacks, and 11 Asians. The percentage of each patient’s ancestral composition (European, AFR, Asian, and Native American) was revealed via the LAMP technique. The dominant ancestry was defined as those with genetic composition of ≥50% from one of four ancestral backgrounds. Genetic alteration profiles and clinical data of patients were obtained from LUSC TCGA PanCancer Atlas through GDC data portal. Of those with AFR dominant ancestry, we analyzed the differences in the gene mutation frequency based upon the proportion of AFR ancestry. Results: Ancestry analysis uncovered 30 AFR ancestry dominant cases with a mean AFR composition of 78.4%. We found 14 subjects with > 80% AFR ancestry and 16 subjects with 50-80% AFR ancestry. Two patients from the > 80% were excluded from the mutational analyses due to absence of genetic profiles in the data portal. TP53 was the most frequently mutated gene with a rate of 91.7% and 87.5% in > 80% and 50-80% AFR groups, respectively. Evaluation of the classic genetic mutations that drive disease pathology in LUSC (EGFR, ALK, PIK3CA, NRAS, KRAS) revealed 25% of > 80% AFR ancestry subjects carried mutations in at least one of these genes compared to 6.3% in the 50-80% AFR ancestry subjects. Also, a trend towards worse overall survival (OS) was seen in those with greater proportion of AFR ancestry (median OS of 27.2 months in > 80% group vs 43.9 months in 50-80% group). Conclusions: Despite the limited sample size, our study highlights substantial heterogeneity in tumor biology even within those of AFR ancestry and that genetic ancestry may impact tumor and host biology that contribute to disparities. Moving forward, engagement of the African American community into clinical trials and genomic studies are needed to explore ancestral influence on tumorigenesis, and to facilitate discovery of new actionable targets to help eradicate racial disparities in lung cancer.

A novel rapid LAMP test for identification of cutaneous leishmaniasis: An evaluation and comparative analysis of three molecular methods

Leishmaniasis is a neglected and widespread parasitic disease that can lead to serious health problems. The conventional method in diagnostic health clinics is direct smear preparation of the lesion and staining with standard Giemsa to visualize the amastigote stage and by culturing the organism in an NNN (Novy-MacNeal-Nicolle) to observe the promastigote form of the parasite. In the case of urban-type leishmaniasis, microscopic diagnosis is sometimes not possible due to the reduction of amastigotes in patients' wounds. Because most endemic areas are located in regions that do not have access to laboratories equipped with molecular tools, access to a rapid test to diagnose the disease is essential. In this study, for the first time for DNA extraction, the scalpel used for sampling was washed and extracted by boiling method. Also, the LAMP technique in this study was modified so that the test can be performed in 10 min and the results can be recognized by color. We used four microscopic methods, conventional PCR, real-time PCR, and LAMP, to diagnose urban-type leishmaniasis and compared the results of these methods with each other. The sensitivity and specificity of LAMP were higher than other techniques used. Therefore, it allows rapid diagnosis for timely treatment of the disease to control the primary reservoir host more quickly in ACL as humans are the principal source of infection. This test is performed at a high-speed and is cost-effective. For its convenience, this test is highly recommended to be used in endemic areas.

Rotational extraction of incarcerated iris (REII): a slit lamp technique to reduce incarcerated iris after nonpenetrating deep sclerectomy for glaucoma.

Iris incarceration is a complication of glaucomafiltering surgery that often requires surgery. We describe a technique for reduction of incarcerated iris at the slit lamp, dubbed rotational extraction of incarcerated iris (REII). A retrospective analysis of visual function and intraocular pressure (IOP) was done in patients treated with REII after nonpenetrating deep sclerectomy. We retrospectively evaluated a cohort of patients who received REII for iris incarceration after nonpenetrating deep sclerectomy for glaucoma. IOP (applanation) and visual acuity (VA) were measured day-of, and 1, 3, 6, and 12months post-REII. Adverse events were recorded. Kaplan-Meier survival analysis was done with definitions of IOP control at 15, 18, and 21mmHg. Forty-one eyes of 41 patients were treated with REII. Median time to iris incarceration from glaucoma surgery was 50days (range 1-1906). Mean pre-REII IOP ± SD was 33.7 ± 14.1mmHg, which reduced to 11.5 ± 6.1mmHgday-of. LogMAR VA was 0.72 ± 0.8 log units at baseline and was unchanged at 12months (P = 0.53). Survival analysis demonstrated varying efficacy depending on the definition of success. 79.0 to 92.2% of eyes achieved IOP control immediately after REII, 39.5 to 71.1% at 1month, 26.3 to 52.6% at 3months, 21.1 to 44.3% at 6months, and 10.5 to 38.0% at 12months. Nearly half (47.4%) of eyes required a tube shunt by 12months. REII may be a safe, minimally invasive slit lamp procedure that can reduce incarcerated iris and delay more invasive intervention for 3-6months in most eyes.

Impact assessment of different DNA extraction methods for non-invasive molecular diagnosis of tegumentary leishmaniasis

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method – commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL.

Magnetic Multiplex Loop Mediated Isothermal Amplification (MM-LAMP) technique for simultaneous detection of dengue and chikungunya virus

Dengue and chikungunya viruses are arthropod borne virus spread through common vector instigating infection in human. There has been an increased recognition that more attention needs to be paid to similar sympotoms caused by both of the virus as they spread in the same region at same time. It warrants need of cost effective, user friendly and rapid multiplex diagnostic technique which could simultaneously diagnose and identify between two virus diseases in resource poor setting. A magnetic multiplex loop mediated isothermal amplification (MM-LAMP) technique was developed by coupling multiplex LAMP with magnetic particle-based naked eye visualization to overcome the shortcoming of simultaneous detection of both diseases. In recent years this technology has emerged as a particularly attractive candidate as amplification reaction process completes within 45 min. The first step involves multiplexing biotin and digoxigenin coated dengue and chikungunya primers respectively in LAMP reaction followed by precipitation of the amplified DNA with polyethylene glycol (PEG) buffer and finally clumping with streptavidin and anti-digoxigenin coated magnetic particle for virus discrimination and naked eye visualization. The DNA detection limit of MM LAMP visualization was 51.65 ng/μl which is comparable to the electrophoresis base UV light visualization. The results showed potential superiority over standard methods polymerase chain reaction (PCR). This current advancement empowers multiplex LAMP utility in resource limited setting without using any of the florescent dyes, turbidimeter, or the sophisticated quantitative PCR machine etc which restrict multiplex LAMP technique to laboratorial use only. We have proposed a novel method without such limitations. This technique has potential as a point of care technique for simultaneous detection of two diseases.