Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

Real-time qPCR coupled with high-resolution melting curve analysis for the detection of the internal transcribed spacer 1 of Angiostrongylus costaricensis.

Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Criteria for second generation comparator tests in validation of novel HPV DNA tests for use in cervical cancer screening.

While HC2 and GP5+/6+ PCR-EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second-generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second-generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC-group I), and show consistent non-inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first-generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta-analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High-Risk HPV Test (Abbott), (2) Cobas-4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Viral diarrheas - newer advances in diagnosis and management.

PURPOSE OF REVIEW: Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas. RECENT FINDINGS: Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures. SUMMARY: Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease.

Establishment of a Rapid Detection Method for Highly Virulent Klebsiella Pneumoniae Based on Multienzyme Isothermal Rapid Amplification Technology.

BACKGROUND: This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection. METHODS: For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated. RESULTS: In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%. CONCLUSIONS: The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.

Establishment of LAMP-CRISPR/Cas12a for rapid detection of Escherichia coli O157:H7 and one-pot detection.

Escherichia coli O157:H7 is a pathogenic serotype of Escherichia coli. Consumption of food contaminated with E. coli O157:H7 could cause a range of diseases. Therefore, it is of great importance to establish rapid and accurate detection methods for E. coli O157:H7 in food. In this study, based on LAMP and combined with the CRISPR/cas12a system, a sensitive and specific rapid detection method for E. coli O157:H7 was established, and One-Pot detection method was also constructed. The sensitivity of this method could stably reach 9.2 × 10° CFU/mL in pure culture, and the whole reaction can be completed within 1 h. In milk, E. coli O157:H7 with an initial contamination of 7.4 × 10° CFU/mL only needed to be cultured for 3 h to be detected. The test results can be judged by the fluorescence curve or by visual observation under a UV lamp, eliminating instrument limitations and One-Pot detection can effectively prevent the problem of false positives. In a word, the LAMP-CRISPR/cas12a system is a highly sensitive and convenient method for detecting E. coli O157:H7.

ERA-CRISPR/Cas12a system: a rapid, highly sensitive and specific assay for Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses a significant threat to global public health and imposes a considerable burden on the economy. However, existing laboratory diagnostic methods for M. tuberculosis are time-consuming and have limited sensitivity levels. Methods: The CRISPR/Cas system, commonly known as the "gene scissors", demonstrates remarkable specificity and efficient signal amplification capabilities. Enzymatic recombinase amplification (ERA) was utilized to rapidly amplify trace DNA fragments at a consistent temperature without relying on thermal cyclers. By integrating of CRISPR/Cas12a with ERA, we successfully developed an ERA-CRISPR/Cas12a detection system that enables rapid identification of M. tuberculosis. Results: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/µL and 90 copies/µL, respectively. Simultaneously, the detection system exhibited no cross-reactivity with various of respiratory pathogens and non-tuberculosis mycobacteria, demonstrating a specificity of 100%. The positive concordance rate between the ERA-CRISPR/Cas12a fluorescence system and commercial qPCR was 100% in 60 clinical samples. Meanwhile, the lateral flow system showed a positive concordance rate of 93.8% when compared to commercial qPCR. Both methods demonstrated a negative concordance rate of 100%, and the test results can be obtained in 50 min at the earliest. Discussion: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.

Synergy of Xpert (MTB/RIF) and Line probe assay for detection of rifampicin resistant strains of Mycobacterium tuberculosis.

INTRODUCTION: Early diagnosis and successful treatment of drug-resistant tuberculosis (TB) demands rapid, precise, and consistent diagnostic methods to minimise the development of resistance. Therefore, this comparative study was designed to evaluate the diagnostic performance of Xpert (MTB/RIF) and Line probe assay (LPA) for detecting drug-resistant TB. METHODOLOGY: This study comprised 389 (279 pulmonary and 110 extrapulmonary) samples from patients suspected of having TB. All samples were subjected to Xpert (MTB/RIF), LPA, solid culture, and drug-susceptibility testing. Out of 320 samples, only 180 culture (gold standard) positive were included in the final evaluation. The diagnostic characteristics for methods used were determined by calculating diagnostic sensitivity, specificity, and predictive values. The agreement between all methods was determined by calculating the kappa coefficient. RESULTS: The sensitivity and specificity for Xpert (MTB/RIF) for detecting TB were 88.5% and 96.4%, respectively, against the solid culture. On the other hand, LPA showed sensitivity and specificity at 94.3% and 100%, respectively. Xpert (MTB/RIF) showed moderate agreement (kappa 0.65, p < 0.01) - (73.3% sensitivity; 97.6% specificity) for the detection of rifampicin resistance. However, LPA achieved better diagnostic accuracy (kappa 0.80, p < 0.01) - (84.6% sensitivity; 98.4% specificity) against drug-resistant TB. CONCLUSIONS: Xpert (MTB/RIF) and LPA have outstanding diagnostic sensitivity and specificity against RIF resistance with a shorter turnaround time, which could result in a substantial therapeutic outcome. Our findings showed LPA superiority over Xpert (MTB/RIF) for drug resistance. However, due to operational challenges, the requirement of technical expertise and infrastructure issues, LPA cannot be used as point-of-care testing in resource-limited countries.

An at-home and electro-free COVID-19 rapid test based on colorimetric RT-LAMP.

INTRODUCTION: In the fight against virus-caused pandemics like COVID-19, diagnostic tests based on RT-qPCR are essential, but they are sometimes limited by their dependence on expensive, specialized equipment and skilled personnel. Consequently, an alternative nucleic acid detection technique that overcomes these restrictions, called loop-mediated isothermal amplification following reverse transcription (RT-LAMP), has been broadly investigated. Nevertheless, the developed RT-LAMP assays for SARS-CoV-2 detection still require laboratory devices and electrical power, limiting their widespread use as rapid home tests. This work developed a flexible RT-LAMP assay that gets beyond the drawbacks of the available isothermal LAMP-based SARS-CoV-2 detection, establishing a simple and effective at-home diagnostic tool for COVID-19. METHODOLOGY: A multiplex direct RT-LAMP assay, modified from the previously developed test was applied to simultaneously identify the two genes of SARS-CoV-2. We used a colorimetric readout, lyophilized reagents, and benchmarked an electro-free and micropipette-free method that enables sensitive and specific detection of SARS-CoV-2 in home settings. RESULTS: Forty-one nasopharyngeal swab samples were tested using the developed home-testing RT-LAMP (HT-LAMP) assay, showing 100% agreement with the RT-qPCR results. CONCLUSIONS: This is the first electrically independent RT-LAMP assay successfully developed for SARS-CoV-2 detection in a home setting. Our HT-LAMP assay is thus an important development for diagnosing COVID-19 or any other infectious pandemic on a population scale.

An alternative colorimetric RT-LAMP assay for the rapid detection of SARS-CoV-2: development and validation in Thailand.

INTRODUCTION: COVID-19, an emerging infectious disease caused by SARS-CoV-2, continues to be a global public health threat. The development of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) can extend the availability of simple, reliable molecular tests for the rapid detection of COVID-19. METHODOLOGY: The RT-LAMP assay was developed using a new primer set targeting a portion of SARS-CoV-2 orf8. The method was validated at 63 ºC for 60 minutes with naked-eye visualization of the color change. The clinical performance was compared to a real-time reverse transcription-polymerase chain reaction (rtRT-PCR) using 273 RNA samples extracted from nasopharyngeal swab specimens. RESULTS: The developed RT-LAMP was specific to SARS-CoV-2 with a limit of detection at 15 RNA copies per reaction. The assay demonstrated diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 90.48% (95% CI: 86.36-93.68%), 87.00% (95% CI: 81.53-91.33%), 100% (95% CI: 95.07-100%), 100% (95% CI: not available), and 73.74% (95% CI: 66.22-80.07%), respectively, compared to the rtRT-PCR. The greatest sensitivity of 98.03% (95% CI: 94.34-99.59%) was demonstrated in samples with the cycle threshold (Ct) values < 30 cycles. CONCLUSIONS: The RT-LAMP method in this study showed good performance. The assay can increase the scope of laboratory testing for rapidly detecting SARS-CoV-2 in Thailand. Due to a decrease in COVID-19 cases, its application is beneficial when commercial alternatives are unavailable.

A simple color absorption analysis of colorimetric loop-mediated isothermal amplification for detection of Raillietina spp. in clinical samples using a 3D-printed tube holder coupled with a smartphone camera and notebook screen.

A simple method has been developed for semi-quantitative analysis of the colorimetric output of loop-mediated isothermal amplification (LAMP) using a 3D-printed tube holder with a smartphone and notebook for the detection of Raillietina, which is the cause of Raillietiniasis affecting free-range chicken farming. In this method, a light is directed from a notebook screen to the LAMP products in the tube holder and the color absorption of the LAMP products is measured by using the appropriate smartphone application. It was found that the malachite green dye-coupled LAMP (MaG-LAMP) assay showed the highest sensitivity and specificity for detecting Raillietina without any cross-reaction with other related parasites and hosts. The limit of detection was 10 fg/µL of DNA. A total of 60 fecal samples were infectively confirmed by microscopic examination and the results of microscopy compared with those of MaG-LAMP and triplex PCR assays. Microscopy and MaG-LAMP based on the color absorption demonstrated high agreement in Raillietina detection with kappa = 1. Rapid, simple, cost-effective, and easy interpretation of colorimetric LAMP assays and their high sensitivity make them superior to PCR and morphological investigation, demonstrating the feasibility of this assay in point-of-care screening to support farm management and solve chicken health problems. Our study presents is an alternative diagnostic method using semi-quantitative analysis of colorimetric LAMP based on the differing solution color absorptions between positive and negative reactions for infectious disease diagnosis.

Comparative analysis of metagenomic and targeted next-generation sequencing for pathogens diagnosis in bronchoalveolar lavage fluid specimens.

Background: Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. Methods: In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. Results: The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Conclusions: Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Molecular Diagnosis of Human Cutaneous Leishmaniasis and Identification of the Causative Leishmania Species in Iran: A Narrative Review

Cutaneous leishmaniasis (CL) is a common form of leishmaniasis in underdeveloped countries. Although CL tends to be self-limiting, it can cause significant scars and may progress to more severe manifestations. Additionally, Leishmania species vary in susceptibility to the available treatments. The selection of treatment and clinical outcome of CL depend on the accurate determination of the Leishmania species. This mini-review aims to provide an overview of the molecular diagnosis techniques such as PCR-based assays, nucleic acid sequence-based amplification, and loop-mediated isothermal amplification utilized in the identification of Leishmania species in Iran.

Diagnostic performance of metagenomic sequencing in patients with suspected infection: a large-scale retrospective study.

Background: Metagenomic next-generation sequencing (mNGS) has been widely reported to identify pathogens in infectious diseases (IDs). In this work, we intended to investigate the diagnostic value and clinical acceptance of paired-samples mNGS as compared to the culture method. Methods: A total of 361 patients with suspected infection were retrospectively included. With reference to the clinical diagnosis, we compared the diagnostic performance and clinical acceptance in pathogen detection between mNGS and culture tests. Moreover, the pathogen concordance of paired blood and respiratory tract (RT) samples in mNGS assay was investigated. Results: Among 511 samples, 62.04% were shown to be pathogen positive by mNGS, and that for clinical diagnosis was 51.86% (265/511). When compared to culture assay (n = 428), mNGS had a significantly higher positivity rate (51.87% vs. 33.18%). With reference to the clinical diagnosis, the sensitivity of mNGS outperformed that of culture (89.08% vs. 56.72%). Importantly, mNGS exhibited a clinically accepted rate significantly superior to that of culture. In addition, the mNGS result from 53 paired blood and RT samples showed that most pairs were pathogen positive by both blood and RT, with pathogens largely being partially matched. Conclusion: Through this large-scale study, we further illustrated that mNGS had a clinically accepted rate and sensitivity superior to those of the traditional culture method in diagnosing infections. Moreover, blood and paired RT samples mostly shared partial-matched positive pathogens, especially for pathogens with abundant read numbers in RT, indicating that both blood and RT mNGS can aid the identification of pathogens for respiratory system infection.

Development and Validation of the MAST ISOPLEX®VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay.

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.