Clinical observation of Van Herick method for evaluating anterior chamber angle width / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore a more convenient and accurate method for evaluating the anterior chamber angle width based on the Van Herick method.METHODS:A total of 58 patients(69 eyes)with age-related cataract who visited our hospital between January and December 2021 were included. They were divided into the chamber angle width ≥1/2 corneal thickness(CT)group(44 eyes of 37 cases)and <1/2CT group(25 eyes of 21 cases)according to the Van Herick method. The central anterior chamber depths and the peripheral anterior chamber angle degrees were measured by ultrasound biomicroscopy.RESULTS: There were statistically significant differences in central anterior chamber depth between the two groups(2.64±0.27 mm vs. 2.23±0.29 mm, P<0.01), and the differences of chamber angle degrees of quadrants of superior, temporal, inferior and nasal compared between two groups were all statistically significant(P<0.01). The difference of chamber angle degrees of quadrants of superior and inferior in chamber angle width ≥1/2CT group was not statistically significant(P>0.05), while the differences of chamber angle degrees of other quadrants were all statistically significant(P<0.05). The differences of chamber angle degrees of quadrants of superior and nasal, temporal and the chamber angle degrees of quadrants of inferior and temporal were all statistically significant in chamber angle width <1/2CT group(P<0.05).CONCLUSION: In the overall evaluation of the anterior chamber angle, it would be more simple, fast and accurate when evaluating the temporal chamber angle width and inferior quadrant of chamber angle width by using the Van Herick method under silt lamp.

Outcomes of toric intraocular lens realignment surgery done using slit-lamp method and wavefront aberrometry method

Purpose: To compare the slit?lamp method and wavefront aberrometry method based on outcomes of toric realignment surgeries. Settings: Tertiary care ophthalmic hospital. Design: Retrospective study. Methods: This study included all eyes undergoing toric intraocular lens (TIOL) realignment surgery between January 2019 and December 2021 for which TIOL axis assessment by slit?lamp method and wavefront aberrometry method was available. Data were retrieved from electronic medical records, and we documented demographics, uncorrected visual acuity (UCVA), subjective refraction, and TIOL axis by slit?lamp and wavefront aberrometry methods on postoperative day 1 and day 14. In patients with misalignment, TIOL was realigned to the original position in group 1 (27 patients) and to an axis based on calculations provided by wavefront aberrometer in group 2 (25 patients). Post?realignment surgery, UCVA, subjective refraction, and TIOL axis by slit?lamp and wavefront aberrometry methods were assessed and analyzed. Results: We analyzed 52 eyes and found that the mean preoperative misalignment with the slit?lamp method (44.9° ±20.0°) and wavefront aberrometry (47.1° ±19.5°) was similar. The corresponding degrees of misalignment post?TIOL repositioning surgeries were 5.2° ±5.2° (slit?lamp method) and 4.7° ±5.1° (wavefront aberrometry) (P = 0.615). Both groups showed significant improvement in median log of minimum angle of resolution (logMAR) UCVA and reduction in median refractive cylinder. Conclusions: Slit?lamp method is as good as wavefront aberrometer method to assess TIOL axis. Toric realignment surgery is found to be safe, and realigning TIOL based on either slit?lamp method or wavefront aberrometer method equally improved UCVA and decreased residual refractive cylinder.

Proposal of a new slit-lamp shield for ophthalmic examination and assessment of its effectiveness using computational simulations / Avaliação da eficácia e proposta de um novo protetor de lâmpada de fenda durante exame oftálmico por meio de simulações computacionais

ABSTRACT Purpose: This study aimed to use computational models for simulating the movement of respiratory droplets when assessing the efficacy of standard slit-lamp shield versus a new shield designed for increased clinician comfort as well as adequate protection. Methods: Simulations were performed using the commercial software Star-CCM+. Respiratory droplets were assumed to be 100% water in volume fraction with particle diameter distribution represented by a geometric mean of 74.4 (±1.5 standard deviation) μm over a 4-min duration. The total mass of respiratory droplets expelled from patients' mouths and droplet accumulation on the manikin were measured under the following three conditions: with no slit-lamp shield, using the standard slit-lamp shield, and using our new proposed shield. Results: The total accumulated water droplet mass (kilogram) and percentage of expelled mass accumulated on the shield under the three aforementioned conditions were as follows: 5.84e-10 kg (28% of the total weight of particle emitted that settled on the manikin), 9.14e-13 kg (0.045%), and 3.19e-13 (0.015%), respectively. The standard shield could shield off 99.83% of the particles that would otherwise be deposited on the manikin, which is comparable to 99.95% for the proposed design. Conclusion: Slit-lamp shields are effective infection control tools against respiratory droplets. The proposed shield showed comparable effectiveness compared with conventional slit-lamp shields, but with potentially enhanced ergonomics for ophthalmologists during slit-lamp examinations.

RESUMO Introdução: Os oftalmologistas têm alto risco de contrair a doença do Coronavírus-19 devido à proximidade com os pacientes durante os exames com lâmpada de fenda. Usamos um modelo de computação para avaliar a eficácia das proteções para lâmpadas de fenda e propusemos uma nova proteção ergonomicamente projetada. Métodos: As simulações foram realizadas no software comercial Star-CCM +. Os aerossóis de gotículas foram considerados 100% de água em fração de volume com distribuição de diâmetro de partícula representada por uma média geométrica de 74,4 ± 1,5 (desvio padrão) μm ao longo de uma duração de quatro minutos. A massa total de gotículas de água acumulada no manequim e a massa expelida pela boca do paciente foram medidas em três condições diferentes: 1) Sem protetor de lâmpada de fenda, 2) com protetor padrão, 3) Com o novo protetor proposto. Resultados: A massa total acumulada das gotas de água (kg) e a porcentagem da massa expelida acumulada no escudo para cada uma das respectivas condições foram; 1) 5,84e-10 kg (28% do peso total da partícula emitida que assentou no manequim), 2) 9,14e-13 kg (0,045%), 3,19e-13 (0,015%). O escudo padrão foi capaz de proteger 99,83% das partículas que, de outra forma, teriam se depositado no manequim, o que é semelhante a 99,95% para o projeto proposto. Conclusão: Protetores com lâmpada de fenda são ferramentas eficazes de controle de infecção contra gotículas respiratórias. O protetor proposto mostrou eficácia comparável em comparação com os protetores de lâmpada de fenda convencionais, mas potencialmente oferece uma melhor ergonomia para oftalmologistas durante o exame de lâmpada de fenda.

A study establishing sensitivity and accuracy of smartphone photography in ophthalmologic community outreach programs: Review of a smart eye camera

Purpose: Diseases affecting the cornea are a major cause of corneal blindness globally. The pressing issue we are facing today is the lack of diagnostic devices in rural areas to diagnose these conditions. The aim of the study is to establish sensitivity and accuracy of smartphone photography using a smart eye camera (SEC) in ophthalmologic community outreach programs. Methods: In this pilot study, a prospective non?randomized comparative analysis of inter?observer variability of anterior segment imaging recorded using an SEC was performed. Consecutive 100 patients with corneal pathologies, who visited the cornea specialty outpatient clinic, were enrolled. They were examined with a conventional non?portable slit lamp by a cornea consultant, and the diagnoses were recorded. This was compared with the diagnoses made by two other consultants based on SEC videos of the anterior segment of the same 100 patients. The accuracy of SEC was accessed using sensitivity, specificity, PPV, and NPV. Kappa statistics was used to find the agreement between two consultants by using STATA 17.0 (Texas, USA). Results: There was agreement between the two consultants to diagnosing by using SEC. Above 90% agreements were found in all the diagnoses, which were statistically significant (P?value < 0.001). More than 90% sensitivity and a negative predictive value were found. Conclusion: SEC can be used successfully in the community outreach programs like field visits, eye camps, teleophthalmology, and community centers, where either a clinical setup is lacking or ophthalmologists are not available.

Universal slit-lamp–mounted mobile adapter with 20 D lens for fundus photography: “Litter to glitter”

Fundus photography is an arduous task as it involves using 90 D in one hand and a smartphone attached on an eyepiece of a slit-lamp biomicroscope in the other hand. Similarly, with a 20 D lens, the filming distance is adjusted by moving the lens or mobile forward or backward, which makes it difficult to adjust and focus the image in busy ophthalmology outpatient departments (OPDs). Moreover, fundus camera costs thousands of dollars. Authors describe a novel technique of performing fundus photography with a 20 D lens and a universal slit-lamp–mounted mobile adapter made from trash. By the use of this simple, yet frugal innovation, primary care physicians or ophthalmologists without a fundus camera can easily snap a fundus photo and subject it to digital analysis by retina specialists across the world. This will help in simultaneous ocular examination and fundus photos taken via mounted 20 D on a slit lamp itself and also reduce the need for unnecessary retina referrals to tertiary eye care centers.

Slit-lamp assisted TORic Marker (STORM): A novel corneal axis marker

Background: Toric Intraocular lenses (IOLs) are supposed to be aligned at a particular axis for spectacle?free vision for distance. The evolution of topographers and optical biometers has made it quite achievable for us to aim the target. However, the result sometimes remains unpredictable. A big aspect of this depends on the preop axis marking for toric IOL alignment. Errors in axis marking have been reduced recently with the array of different toric markers in the market, but still we see postoperative refractive surprises due to faulty marking. Purpose: In this video, we present a novel slit lamp–based toric marker innovation, STORM, which gives us a hands?free approach to a reliable and accurate axis marking on the cornea. The axis marker is a simple modification to our age?old marker, with the advantage of no touch and slit?lamp assistance, which will make it error free and easy to use. Synopsis: The present innovation answers the problem statement of stable, economical, and accurate marking solution. Many a times, hand?holding devices create inaccurate and stressed condition while marking the cornea before corneal surgery. Highlights: The invention can be used for marking of accurate and easy astigmatic axis of a toric IOL preoperatively, that is, before the surgery. If the appropriate device is used to mark the cornea, it would impact the outcome of surgery. This device also makes the patient and the surgeon comfortable to mark the cornea with accuracy and without hesitation

Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene

Background & objectives: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. Methods: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. Results: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. Interpretation & conclusion: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition

Inhibition of LAMP3 mediates the protective effect of vitamin D against hypoxia/reoxygenation in trophoblast cells

Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Emergency response of infectious snails detected after interruption of schistosomiasis transmission in Hannan District, Wuhan / 中国热带医学

@#Abstract: Objective　To analyze the emergency response and long-term intervention effects after the detection of infectious snails epidemic by loop-mediated isothermal amplification (LAMP) assays in Hannan District, Wuhan City, and to explore the application of LAMP in early surveillance and early-warning of schistosomiasis transmission. Methods Snails picked up by the risk monitoring system in Hannan District were examined by anatomical microscopy and LAMP technology to identify the schistosomiasis infection. Emergency response and intensive intervention were initiated in the environment where positive snails appeared, and the long-term effects were evaluated. Results In May 2018, the infectious snails were detected by LAMP technology in Hannan District, and the positive snails were located in Zhujiacha, Dongzhuang Village, Obstacles and weeds were removed and buried by machine in Zhujiacha. 12 700 m2 of snails were killed by drugs, and the mortality rate of snails was more than 80%; no new seropositive persons were found in the emergency examination within 500 m of the positive snail sites. 506 people were examined in Dong Zhuang Village at the end of the year, and 30 positive IHA cases were detected with a blood positive rate of 5.93%, no positive fecal test was found, and all positive blood test patients took preventive medication. The monitoring results of sentinel rats and wild feces were all negative. Health education was carried out, 7 warning signs were deployed and refreshed, and 500 publicity brochures were distributed. After nearly three years of intensified intervention and monitoring in the villages where the positive environment is located, and the density of snails on the stubborn snail has dropped from 0.094/frame to 0.027/frame, and the positive rate of blood test in Dongzhuang Village has steadily dropped from 5.93% to 3.74%. Conclusions The infected snails missed by microscopy were detected by LAMP in Hannan District, which created conditions for the rapid emergency treatment of environment and elimination of positive snail and improved the sensitivity of the surveillance and early warning system in transmission-interrupted areas.

Application and Modification of RT-LAMP for Rapid Detection of SARS-CoV-2 Viral Genome

@#COVID-19 outbreak caused by the newly discovered SARS-CoV-2 has become a major public health threat around the world and has create a tremendous effect on the global economy. Hence, there is a high demand for rapid and accurate diagnosis to contain the spread of the disease. The Reverse-Transcription Polymerase Chain Reaction (RTPCR), the current standard for diagnosis of COVID-19 however possesses certain drawbacks that limits its application to meet the high demand of the continually increasing COVID-19 cases. Conversely, Loop-Mediated Isothermal Amplification (LAMP) is another nucleic acid amplification method that shows a great potential as an alternative tool in rapid diagnosis of COVID-19 due to its simplicity and rapidity. This review summarized the recent published research articles related to the application and modification of RT-LAMP assay for the rapid detection of COVID-19 in comparison with other available diagnostic methods.

Monitoring and analysis of kelch13 C580Y mutation in imported Plasmodium falciparum in Wuhan, China / 中国热带医学

@#Abstract: Objective To investigate the prevalence of C580Y mutation of kelch13 gene in the imported P. falciparum cases in Wuhan City, China, and to provide a reference basis for the prevention and treatment of imported falciparum malaria. Methods From 2009 to 2015, blood samples were collected from returnees who infected with P. falciparum in endemic areas of Africa and Southeast Asia in Wuhan City. The P. falciparum DNA was extracted from the blood samples, and kelch13 gene was amplified by loop-mediated isothermal amplification (LAMP), and the distribution of C580Y mutation was analyzed. Results C580Y mutation was detected in 16 of the 208 cases tested by LAMP. No mutations were detected in 69 cases of imported falciparum malaria from Africa during 2009-2012, while 13 cases of the C580Y mutation were detected in 114 cases from 2013 to 2015, with a mutation rate of 11.4%. The mutation rate in South Africa, West Africa, and Central Africa was 12.5%, 9.6%, and 19.0%, respectively, with no mutations detected in cases from North Africa and East Africa. Among the 25 cases of falciparum malaria from Southeast Asia between 2009 and 2013, three cases were positive for the C580Y mutation, all from Myanmar, with a mutation rate of 14.3% (3/21) in Myanmar and 12.0% (3/25) in Southeast Asia. There was no significant difference in the mutation rate between Africa and Southeast Asia after 2013 (P>0.05). Conclusions Our findings highlight the varying degrees of C580Y mutations of kelch13 gene in imported P. falciparum cases in Wuhan and suggest the need for enhanced monitoring and evaluation of related resistance genes.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

Assessment of optic disk by disk damage likelihood scale staging using slit-lamp biomicroscopy and optical coherence tomography in diagnosing primary open-angle glaucoma

Purpose: The current study was aimed at assessment of optic disk by disk damage likelihood scale (DDLS) staging using slit?lamp biomicroscopy and optical coherence tomography (OCT) in diagnosing primary open?angle glaucoma (POAG) patients. Methods: This was a cross?sectional observational study of 106 POAG patients, which was conducted from April 2017 to April 2018. All patients underwent slit?lamp fundoscopy with a +78 D lens and high?definition (HD)?OCT, and the vertical cup disk ratios (VCDRs) were recorded. Disk size and neuroretinal rim assessment were done, and the disk was then staged using the recent version, which stages the optic nerve head (ONH) from 1 to 10 as read from the DDLS nomogram table. DDLS scores >5 indicate glaucomatous damage. Pearson coefficient was used to correlate the DDLS staging by slit?lamp biomicroscopy with best?corrected visual acuity (BCVA), intraocular pressure (IOP), disk size, and VCDR and VCDR, mean deviation, and DDLS staging by HD?OCT. Results: The mean age of the patients was 59.54 ± 6.61 years. The male: female ratio was 2:1. The mean IOP was 16.04 ± 1.97 mmHg, and BCVA was 0.72 ± 0.13 LogMAR units. The mean VCDR on 78 D slit?lamp biomicroscopy was 0.76 ± 0.09 (standard deviation [SD]) (range 0.1–0.77), whereas on HD?OCT, the mean VCDR was 0.81 ± 0.09 (SD) (range 0.07–0.81). The mean deviation on visual field testing in decibels was ?14.43 ± 3.31 (SD). The correlation coefficient between DDLS staging by slit?lamp biomicroscopy and DDLS staging by HD?OCT parameters was r = 0.96. Conclusion: There is a positive correlation between the DDLS system of optic disk evaluation on slit?lamp biomicroscopy and most of the HD?OCT evaluation parameters

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Evidence of dengue virus in eviscerated specimens of panophthalmitis secondary to dengue fever: A possible cause-effect phenomenon

Purpose: To report a retrospective series of three cases of infectious panophthalmitis post?dengue fever with ex vivo confirmation of dengue virus ribonucleic acid (RNA) in the tissues of the eye. Methods: Four eyes of three patients, who were diagnosed with panophthalmitis following dengue fever and who underwent evisceration, were included. All demographic and clinical data were recorded. The eviscerated samples were subjected to direct microscopy, culture for bacteria, fungi, and parasites, and molecular virology (dengue virus [DENV] NS1?specific reverse transcription loop?mediated isothermal amplification (RT?LAMP) assay). Results: The time from the development of dengue fever to the occurrence of ocular symptoms was 4.33 ± 1.15 (median 5) days. DENV NS1 RNA, suggestive of the presence of the dengue virus, was confirmed in all evisceration specimens (uveal tissue, cornea). All the patients recovered completely from dengue fever and on follow?up had healthy eviscerated sockets. Conclusion: Demonstration of the DENV RNA in the eviscerated specimens of panophthalmitis following dengue fever implicates the DENV in the pathophysiology of the ocular infection.

Development of a RT-LAMP assay for detection of SARS-CoV-2

Background & objectives: The pandemic of SARS-COV-2 began in Wuhan, China in December 2019 and has caused more than 101 million cases worldwide. Diagnostic technologies possessing sensitivity and specificity equivalent to real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays are needed to ramp up testing capacity in most countries. Newer platforms need to be technically less demanding, require minimum equipment and reduce turn-around time for reporting results. The objective of this study was to exploit loop-mediated isothermal amplification (LAMP) for the detection of SARS-CoV-2 and evaluate its performance by comparison with rRT-PCR. Methods: Reverse-transcription LAMP (RT-LAMP) assay primers were designed to detect envelop (E) and nucleocapsid (N) genes of SARS-CoV-2. Positive control RNA was prepared by in vitro transcription of E and N genes clones. RT-LAMP amplification reactions were incubated at 65°C for 30 min. Results were recorded visually. RT-LAMP results were evaluated by comparing the results obtained with a commercial rRT-PCR kit. Results: The RT-LAMP assay for E and N genes was carried out in separate tubes. RT-LAMP detected about 40 copies of SARS-CoV-2 RNA per reaction. A total of 253 throat swabs were tested using the RT-LAMP assay. The overall diagnostic sensitivity and specificity of the LAMP assay were 98.46 and 100 per cent, respectively, as compared to the rRT-PCR. Interpretation & conclusions: SARS-CoV-2 RT-LAMP assay was designed, standardized and evaluated. The assay showed diagnostic sensitivity and specificity equivalent to rRT-PCR assays. The assay will be useful to increase testing capacity for the detection of SARS-CoV-2 in the country.

Accuracy of Toric Intraocular Lens Axis Alignment Using Manual Slit Lamp Method and CALLISTO Eye Image-Guided System

Aims: Image-guided systems are the gold standard for determining toric intraocular lens (IOL) axis alignment. However, their high cost prevents widespread use of these systems. As an alternative, a simpler and affordable method could be performed manually using a slit-lamp biomicroscope. This study aims to compare the accuracy of manual toric IOL axis marking using a slit-lamp compared to the CALLISTO eye image-guided system.Study Design: Prospective comparativeMethods: In this prospective study, toric IOL axis alignment of 42 eyes with cataract and coexisting corneal astigmatism were evaluated using manual slitlamp method and CALLISTO eye image-guided method. Preoperative and postoperative uncorrected visual acuity, best corrected visual acuity, amount of spherical and astigmatic refractive errors, and postoperative IOL axis alignment were evaluated. Intraclass correlation of the manual method was calculated and the difference of IOL axis alignment to the image-guided method was compared.Results: Toric IOL implantation reduced the amount of astigmatic refractive error from -1.63 � 0.65 D to -0.50 � 0.19 D in the image-guided group and from -1.93 � -0.90 D to -0.87 � 0.26 D in the manual slitlamp group. As many as 90.5% of eyes in the image-guided group and 81.0% of eyes in the manual slitlamp group reached the target induced astigmatism (p=0.38). Manual axis marking showed intraclass correlation of 99.3%. However, when the manual method was compared to the image-guided method a mean difference in axis alignment of 10.98o (95% confidence interval: 9.32o - 12.63o) was observed.Conclusions: Alignment of toric IOL axis using the manual method demonstrated a consistent result; yet producing a considerable difference to the result of the image-guided method.

Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.