Detection and species determination of malaria parasites by microscopy, antigen detection, polymerase chain reaction, and loop-mediated isothermal amplification assay in a tertiary care hospital

Background: Malaria is a major health issue in tropical and subtropical areas. Out of all subtypes, Plasmodium falciparum (Pf) is the most dangerous form accounting for high mortality and morbidity. It is transmitted by infected female anopheles mosquitoes and infected blood transfusions. Aims and Objectives: The aim of the study is to establish correct diagnosis by direct microscopy, Immunochromatographic test (ICT), and molecular studies. Materials and Methods: This prospective study was conducted in the PG Department of Microbiology, SCB Medical College, Cuttack. Thick blood smears were drawn and then stained with Leishman’s stain to visualize falciparum rings. DNA was extracted from infected blood samples by phenol chloroform method with some modification as described by Sambrook and Russel for molecular analysis. Results: In the present study, 150 cases of malaria were analyzed. The male: female ratio was 1.7:1 and age ranged from 0 to 56 years. The Plasmodium vivax positivity was compared with thin smear to 21 (84%) in ICT, 100% both polymerase chain reaction (PCR) and loop mediated isothermal amplification assay (LAMP) assays followed by the Pf positivity as 76 (92.7%) in ICT, 82 (100%) both PCR and LAMP assays, respectively. The results obtained were statistically significant with P < 0.001. The PCR and LAMP showed 100% response to specificity and positive predictive value. Conclusion: The present study established the role of molecular tests such as PCR and LAMP are highly specific for diagnosis of Plasmodium species whereas they are more or less similar in sensitivity as compared to other diagnostic methods such as ICT and microscopy.

An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

Establishment and evaluation of a LAMP-CRISPR-based nucleic acid detection method for Schistosoma japonicum / 中国热带医学

@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

Value of rifampicin-resistant real-time fluorescence quantitative nucleic acid amplification detection technology in the fast diagnosis of pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To evaluate the value and significance of rifampicin-resistant real-time fluorescence quantitative nucleic acid amplification detection technology (GeneXpert MTB/RIF) in the diagnosis of pulmonary tuberculosis. 　　Methods　The clinical data of 228 patients with suspected pulmonary tuberculosis, who admitted to Hebei Chest Hospital from January 2018 to December 2019, were analyzed retrospectively. The sputum was collected for GeneXpert MTB/RIF, sandwich cup liquid-based bacterial acid-fast staining smear microscopy (referred to as “sandwich cup method”) and Loop-Mediated isothermal amplification (referred to as “LAMP method”) and the results were statistically analyzed by SPSS 17.0 software. Results　Among the 228 patients with suspected cases, 200 cases were clinically diagnosed as pulmonary tuberculosis and 28 were non-tuberculosis. The positive detection rate of GeneXpert MTB/RIF (81.0%, 162/200) was significantly higher than that of sandwich cup method (62.5%, 125/200) and LAMP method (72.5%,145/200) (χ2=16.885, 4.049, P<0.05). Taking clinical diagnosis as gold standard, the sensitivity of GeneXpert MTB/RIF (80.00%,160/200) was significantly higher than that of sandwich cup method (60.00%, 120/200) and LAMP method (70.50%, 141/200) (χ2=19.048, 4.846, P<0.05). The diagnostic consistency of GeneXpert MTB/RIF (K=0.73) was higher than that of sandwich cup method (K=0.39) and LAMP method (K=0.56). Conclusions The GeneXpert MTB/RIF detection method is rapid and simple, and can diagnose pulmonary tuberculosis rapidly and simultaneously detect rifampicin resistance of Mycobacterium tuberculosis with high sensitivity. It has high clinical value for early diagnosis of pulmonary tuberculosis and guidance of treatment in general and specialized hospitals.

Development of fluorescent loop-mediated isothermal amplification for detection of sdaB of Streptococcus pyogenes / 中国热带医学

@#Abstract: Objective To establish a method for detecting sdaB virulence gene of Streptococcus pyogenes with loop mediated isothermal amplification (LAMP). Methods According to the conserved sequence of Streptococcus pyogenes sdaB gene published in GenBank (GenBank: 69901515), LAMP primers were designed with Primer Explorer V5.0 software. Main components of LAMP reaction system were optimized including of fluorescent dye, MgSO4, betaine, deoxyribonucleosidetriphosphate (dNTP), and Bst DNA polymerase, with the concentration of MgSO4 from 0 mmol/L to 12 mmol/L, betaine from 0 mol/L to 2.4 mol/L, dNTP from 0.2 µmol/L to 2 µmol/L, forward inner primer (FIP) and backward inner primer (BIP) from 0.2 µmol/L to 2 µmol/L respetively, forward outer primer (F3) and backward outer primer (B3) from 0.2 µmol/L to 0.4 µmol/L, Bst DNA polymerase from 0.16 U/µL to 0.96 U/µL, fluorescent dye from 0.2 µmol/L to 2 µmol/L. With the optimized system, the methodological specificity and the minimum detection limit were evaluated on the ABI7500 real-time fluorescent quantitative PCR analyzer, and 13 standard strains including Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus viridis, Enterococcus faecalis, Enterococcus faecium, Neisseria gonorrhoeae, Lactobacillus acidophilus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were detected. Finally, 103 clinical samples were tested. Results The optimized reaction system contained 25 µL reaction mixture, including 0.8 µL of 25 µmol/L fluorescent dye, 1 µL of 100 mmol/L MgSO4, 6 µL of 5 mol/L betaine, 1.4 µL of 25 mmol/L dNTP, 2 µL of 20 µmol/L FIP and BIP, 0.5 µL of 20 µmol/L F3 and B3, 1 µL of 8 U/µL Bst DNA polymerase, and 2 µL of template. After adding deionized water, the mixture was incubated at 63°C for 45 min to complete the reaction. The limit of detection (LOD) was 500 pg/µL. All 12 non-S. pyogenes strains tested were negative. Compared with the culture method, the clinical sensitivity and specificity were 100.0% (16/16) and 96.6% (84/87), respectively, for 103 clinical samples. Conclusions This LAMP assay is reliable for the detection of Streptococcus pyogenes in clinic and is suitable for field detection with good specificity and sensitivity, as well as simply operation.

Reverse-Transcription Loop-Mediated Isothermal Amplification and alternative protocols for lower cost, large-scale COVID-19 testing: lessons from an emerging economy / Amplificación isotérmica mediada por bucle de transcripción inversa y protocolos alternativos para pruebas COVID-19 a gran escala y de bajo costo: lecciones de una economía emergente

Abstract Introduction: Most successful cases of COVID-19 pandemic mitigation and handling have relied on extensive reverse-transcription quantitative polymerase chain reaction (RT-qPCR). However, many emerging economies have struggled with current molecular testing demands due to economic, technical and technological constraints. Objective: To define a potential diagnostic protocol to increase testing capacity in current and post-pandemic conditions. Methods: We reviewed the literature, patents and commercial applications, for alternatives. Results: We found a good potential in saliva samples, viral inactivation and quick RNA extraction by heating; the use of an isothermal technology such as loop mediated isothermal amplification (LAMP) and naked eye test-result visualization by in-tube colorimetry or turbidity. Conclusions: Saliva samples with quick RNA extraction by heating and colorimetric LAMP are promising options for countries with economic and infrastructure limitations.

Resumen Introducción: La mayoría de los casos exitosos de mitigación y manejo de la pandemia de COVID-19 se han dado mediante pruebas basadas en la reacción en cadena de la polimerasa cuantitativa (RT-qPCR por sus siglas en inglés). Sin embargo, muchas economías emergentes han tenido problemas con las demandas actuales de pruebas moleculares debido a limitaciones económicas, técnicas y tecnológicas. Objetivo: Definir un protocolo de diagnóstico potencial para aumentar la capacidad de prueba en las condiciones actuales y posteriores a la pandemia. Métodos: Revisamos la literatura, las patentes y las aplicaciones comerciales, en busca de alternativas. Resultados: Encontramos un buen potencial en muestras de saliva, inactivación viral y extracción rápida de ARN por calentamiento; el uso de una tecnología isotérmica como la amplificación isotérmica mediada por horquillas (LAMP, por sus siglas en inglés) y la visualización del resultado de la prueba a simple vista mediante colorimetría o turbidez en el tubo. Conclusiones: Las muestras de saliva con extracción rápida de ARN por calentamiento y LAMP colorimétrico son opciones prometedoras para países con limitaciones económicas y de infraestructura.

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.

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.

Advances in loop-mediated isothermal amplification in the diagnosis of tuberculosis / 中国热带医学

@#Abstract: The loop-mediated isothermal amplification (LAMP) technique is a technique for the specific and efficient amplification of target fragments at a constant temperature using two pairs of specially designed primers and a strand displacement activity DNA polymerase. LAMP technique is a simple, rapid, specific, sensitive and cost-effective nucleic acid amplification method, and therefore has a promising future in the field rapid detection of Mycobacterium tuberculosis and grassroots applications. In this review, the basic principles and characteristics of the LAMP technique, the main molecular markers for the diagnosis of tuberculosis, and the use of different molecular markers and various types of novel techniques in the diagnosis of pulmonary tuberculosis, extrapulmonary tuberculosis, and drug-resistant tuberculosis were described. The LAMP technique has been widely used in the diagnosis of tuberculosis with high sensitivity and specificity, but the technique still has some shortcomings. This paper reviews the progress of its application in tuberculosis in recent years and provides an outlook on its development, with a view to providing a rational research direction for rapid diagnosis of tuberculosis in a resource-limited environment.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Rapid detection of pneumocystis carinii infection in immunocompromised patients by loop mediated isothermal amplification technique / 中国小儿急救医学

Objective:To explore the value of detecting pneumocystis carini(PC)rapidly in immunocompromised patients by loop mediated isothermal amplification(LAMP).Methods:Respiratory tract specimens of immunocompromised children suspected of pneumocystis carinii pneumonia(PCP) at Shanghai Children′s Medical Center, School of Medicine, Shanghai Jiao Tong University were collected from May 2020 to May 2021.PCR and LAMP methods were used to detect PC.Firstly, LAMP primers of PC were synthetized according to the conserved region of PC gene, and the LAMP reaction system and reaction conditions were optimized to evaluate the sensitivity and specificity.Then, the results of pathogens were compared with those of PCR detection.Results:The established LAMP detection technology for PC had high specificity and super sensitivity.The detection results could be obtained within 1 hour.In 12 clinical samples, 10 cases were positive and 2 cases were negative, the coincidence rate of LAMP and PCR technique was 100%.Conclusion:LAMP can detect PC more rapidly and sensitively than PCR, and it can provide a good support for clinical rapid diagnosis of PCP.

Rapid detection of pneumocystis carinii infection in immunocompromised patients by loop mediated isothermal amplification technique / 中国小儿急救医学

Objective:To explore the value of detecting pneumocystis carini(PC)rapidly in immunocompromised patients by loop mediated isothermal amplification(LAMP).Methods:Respiratory tract specimens of immunocompromised children suspected of pneumocystis carinii pneumonia(PCP) at Shanghai Children′s Medical Center, School of Medicine, Shanghai Jiao Tong University were collected from May 2020 to May 2021.PCR and LAMP methods were used to detect PC.Firstly, LAMP primers of PC were synthetized according to the conserved region of PC gene, and the LAMP reaction system and reaction conditions were optimized to evaluate the sensitivity and specificity.Then, the results of pathogens were compared with those of PCR detection.Results:The established LAMP detection technology for PC had high specificity and super sensitivity.The detection results could be obtained within 1 hour.In 12 clinical samples, 10 cases were positive and 2 cases were negative, the coincidence rate of LAMP and PCR technique was 100%.Conclusion:LAMP can detect PC more rapidly and sensitively than PCR, and it can provide a good support for clinical rapid diagnosis of PCP.

Application of loop mediated isothermal amplification in etiological diagnosis of infectious diseases / 国际儿科学杂志

Infectious diseases are still the most important diseases in infants and young children.Due to young age and imperfect immune function, infectious diseases in children are characterized by "urgent" , "serious" and "complex pathogens" , which often lead to serious complications.Rapid and accurate diagnosis is particularly important for children.However, the existing pathogen detection methods are inefficiency, subject to experimental conditions and person, and impopularized in hospitals.The pathogen detection method based on loop mediated isothermal amplification presents high specificity, low-cost and simple operation and is suitable for promotion in primary medical units, which has great application value in the detection of pathogens of children.In addition, the combination of loop mediated isothermal with paper-based micro-gene chip, intelligent automation and digital system will make progress to clinical etiological diagnosis.

Performance of loop-mediated isothermal amplification (LAMP) assay for detection of Schistosoma japonicum infection in Oncomelania snails in schistosomiasis transmission-interrupted regions / 中国血吸虫病防治杂志

Objective To compare the effectiveness of loop-mediated isothermal amplification (LAMP) assay and microscopic examinations for detection of Schistosoma japonicum infections in Oncomelania hupensis in transmission-interrupted regions, so as to provide insights into the optimization of snail surveillance tools in these regions. Methods Four hilly schistosomiasis-endemic villages where transmission interruption was achieved were selected in Heqing County of Yunnan Province as the study villages, including Xinzhuang and Gule villages in hilly regions and Lianyi and Yitou villages in dam regions. Snail survey was performed by means of systematic sampling combined with environmental sampling in July 2018. All captured snails were identified for S. japonicum infections using microscopy. In addition, 10 to 20 snails were randomly sampled from each snail habitat following microscopy, numbered according to environments and subjected to LAMP assay. The positive rate of settings with S. japonicum-infected snails was compared among villages. Results A total of 7 949 living snails were captured from 83 snail habitats in 4 villages, and no S. japonicum infection was detected in snails. There were 226 mixed samples containing 1 786 snails subjected to LAMP assay, and positive LAMP assay was found in 3 mixed samples from 3 snail habitats in 2 dam villages. The positive rates of settings with S. japonicum-infected snails were comparable between Lianyi Village (one setting) and Yitou Village (2 set tings) (5.89% vs. 14.29%, P = 0.344). However, the overall positive rate of settings with S. japonicum-infected snails was significantly higher in dam villages (9.67%, 3/31) than in hilly villages (0) (P = 0.048). Conclusions LAMP assay is more sensitive to detect S. japonicum infections in O. hupensis than conventional microcopy method, which may serve as a supplementary method for detection of S. japonicum infections in O. hupensis in high-risk snail habitats in hilly transmission-interrupted regions.

Rapid screening of Salmonella by loop-mediated isothermal amplification combined with lateral flow dipstick / 上海预防医学

Objective:To establish a specific and sensitive method using loop-mediated isothermal amplification for rapid screening of Salmonella. Methods:The invA gene sequence of Salmonella was downloaded from GenBank. After homology comparison with DNAMAN software， amplification primers were designed in the conserved region， and a LAMP-LFD detection method was established. The reaction system was optimized， and the specificity and sensitivity of the method were verified. Results:The sensitivity of this method to detect Salmonella DNA was up to 1.0×101 copies/μL. The positive rate of anal swabs was the same as that of fluorescent PCR. Meanwhile， LAMP-LFD was easy to operate and did not need expensive instruments. The detection result could be obtained within 30 minutes. Conclusion:The LAMP-LFD method established in this study is rapid， simple， sensitive and specific， which is suitable for rapid screening of Salmonella.

Real-time PCR detection method for Fusarium solani, pathogen of Panax notoginseng root rot / 中草药

Objective: In order to establish an accurate and rapid real-time fluorescence quantitative PCR method for the detection of Fusarium solani, the pathogenic fungus of Panax notoginseng root rot. Methods: Based on aminoadipate reductase Lys2 gene of F. solani, specific primers Fs-QF and Fs-QR were designed. The recombinant plasmid standard was prepared and the SYBR Green I fluorescence real-time quantitative PCR method and real-time fluorescent 1oop-mediated isothermal amplication (LAMP) system for detecting F. solani was established. Fifteen samples including P. notoginseng plants with typical symptoms of root rot and black spot as well as soil of P. notoginseng planting area were collected. The total DNA of these samples were extracted as templates, and then detected by the real-time fluorescence quantitative PCR method and 1oop-mediated isothermal amplification established in this study. Results: The real-time fluorescent quantitative PCR method and 1oop-mediated isothermal amplification technique established in this study had high specificity. P. notoginseng plants infected with F. solani can be detected quickly. In addition, the method has high sensitivity and the concentration of detection template can be as low as 0.2 pg/μL. Conclusion: The method established in this study can be used to reveal the dynamic changes of F. solani concentration in the complex P. notoginseng planting soil and diseased plants. Furthermore, it can provide technical supports for the soil treatment, early diagnosis and dynamic monitoring of root rot, and rapid molecular detection of P. notoginseng seeds and seedlings.

Recent Advances in the Diagnosis and Management of Pneumocystis Pneumonia / 결핵

In human immunodeficiency virus (HIV)-infected patients, Pneumocystis jirovecii pneumonia (PCP) is a wellk-nown opportunistic infection and its management has been established. However, PCP is an emerging threat to immunocompromised patients without HIV infection, such as those receiving novel immunosuppressive therapeutics for malignancy, organ transplantation, or connective tissue diseases. Clinical manifestations of PCP are quite different between patients with and without HIV infections. In patients without HIV infection, PCP rapidly progresses, is difficult to diagnose correctly, and causes severe respiratory failure with a poor prognosis. High-resolution computed tomography findings are different between PCP patients with HIV infection and those without. These differences in clinical and radiological features are due to severe or dysregulated inflammatory responses that are evoked by a relatively small number of Pneumocystis organisms in patients without HIV infection. In recent years, the usefulness of polymerase chain reaction and serum β-D-glucan assay for rapid and non-invasive diagnosis of PCP has been revealed. Although corticosteroid adjunctive to anti-Pneumocystis agents has been shown to be beneficial in some populations, the optimal dose and duration remain to be determined. Recent investigations revealed that Pneumocystis colonization is prevalent and that asymptomatic carriers are at risk for developing PCP and can serve as the reservoir for the spread of Pneumocystis by airborne transmission. These findings suggest the need for chemoprophylaxis in immunocompromised patients as well as infection control measures, although the indications remain controversial. Because a variety of novel immunosuppressive therapeutics have been emerging in medical practice, further innovations in the diagnosis and treatment of PCP are needed.

Loop-mediated Isothermal Amplification LAMP, Simple Alternative Technique of Molecular Diagnosis Process in Medicals Analysis: A Review

The Medical diagnostic process requires more various methods to cover all sorts of clinical demands. Those methods depend on the types of samples and the requested analysis. The Medical diagnostic is considered as a process until making a clinical decision about a physiopathology or infection through medicals analysis. Nucleic acid amplification methods continue to play a role in these medicals analysis processes, but, many gold conventional standard Methods are time-consumers, less sensitive, technically challenging. It is that reason why recently, after the development of the PCR technique; the real-time PCR became a frequently requested tool to diagnose the virus, bacteria, fungal and parasites infections in different pathologies, especially in developed countries. However, its availability remains a big problem in resource-limited countries like Africa continent. It is because of the issue due to cost, technology, human resource constraints and the remote areas with limited access to laboratory facilities which stays also a major problem. Nucleic acids amplification methods remain to be expensive, not accessible for all laboratories. In addition to this, they are easily inhibited and contaminated by the other biochemical reagents time-consumer, for this reason, other methods of nucleic acid amplification under isothermal conditions have emerged each with its particularities. Despite their successes, molecular biology should have a method that combines many of the advantages and accessible everywhere. So, in 2000, the LAMP method was developed and offered many advantages such as speed, specificity, high sensitivity and the cost of very low equipment. In this review, we present the different studies to confirm the advantages of LAMP that we consider as an alternative to PCR in molecular diagnostics with a wide range of use on clinical samples and in scientific research and also, the method to save the Africans patients from the high cost and time consuming of other amplification methods.

Evaluation of loop-mediated isothermal amplification assay for detection and typing of human papilloma virus 16 and 18 from endocervical samples

Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.

Institutional outbreak of varicella in a child welfare institute in Chandigarh, North India

Introduction: Varicella outbreaks are known to occur in developing nations as vaccine coverage is still low. Material and Methods: In the present study, an institutional outbreak from Chandigarh, India, is reported wherein the utility of non-invasive samples such as saliva and urine was studied for the molecular diagnosis of varicella by conventional polymerase chain reaction (PCR), real-time PCR and real-time loop-mediated isothermal amplification (real-time LAMP). Results: The results of the present study showed that saliva and urine samples can be used for outbreak investigation of varicella compared to varicella-zoster virus DNA in vesicular swab samples with reasonable sensitivity. Conclusion: Thus, molecular techniques may be useful in the early identification of the outbreak and timely isolation, and the treatment of cases can further prevent its spread.