Identification of connective tissue disease autoantibodies and a novel autoantibody anti-annexin A11 in patients with "idiopathic" interstitial lung disease.

BACKGROUND: Autoantibodies are a hallmark feature of Connective Tissue Diseases (CTD). Their presence in patients with idiopathic interstitial lung disease (ILD) may suggest covert CTD. We aimed to determine the prevalence of CTD autoantibodies in patients diagnosed with idiopathic ILD. METHODS: 499 patient sera were analysed: 251 idiopathic pulmonary fibrosis (IPF), 206 idiopathic non-specific interstitial pneumonia (iNSIP) and 42 cryptogenic organising pneumonia (COP). Autoantibody status was determined by immunoprecipitation. RESULTS: 2.4% of IPF sera had a CTD-autoantibody compared to 10.2% of iNSIP and 7.3% of COP. 45% of autoantibodies were anti-synthetases. A novel autoantibody targeting an unknown 56 kDa protein was found in seven IPF patients (2.8%) and two NSIP (1%) patients. This was characterised as anti-annexin A11. CONCLUSION: Specific guidance on autoantibody testing and interpretation in patients with ILD could improve diagnostic accuracy. Further work is required to determine the clinical significance of anti-annexin A11.

A novel syphilis Treponema pallidum lipoprotein peptide antigen diagnostic assay using red cell kodecytes in routine blood centre column agglutination testing platforms.

BACKGROUND AND OBJECTIVES: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. MATERIALS AND METHODS: Candidate Kode Technology function-spacer-lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non-reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non-reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. RESULTS: From three FSL-peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA-kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non-reactive samples the agreement rate for TmpA-kodecytes was 98.8%. CONCLUSION: TmpA-kodecytes are viable for use as cost-effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse-algorithm testing into low-volume laboratories, by utilizing existing transfusion laboratory infrastructure.

Bacterial Leaf Spot Susceptibility Screening of Chili Pepper Cultivars Using qPCR Determination of Xanthomonas euvesicatoria pv. euvesicatoria Titers.

Bacterial leaf spot is a serious disease of chili pepper (Capsicum spp.) caused by Xanthomonas euvesicatoria pv. euvesicatoria. Conventional resistance screening is time and resource intensive. It was considered that a quick and simple determination of cultivar susceptibility could be achieved through estimating bacterial titers of inoculated plants. A SYBR quantitative polymerase chain reaction (qPCR)-based assay was compared with conventional PCR, then used to detect and enumerate pathogen titers in serial dilutions and DNA extracted from infected plant leaves. The qPCR detection limit was approximately 1 CFU µl-1, 10 times more sensitive than conventional PCR. A linear correlation (R2 = 0.994) was obtained from the standard curve comparing plate-truthed serial dilutions of the pathogen with the qPCR cycle threshold. Six strains were used to inoculate cultivars Hugo and Warlock. One strain, X. euvesicatoria pv. euvesicatoria BRIP62403, was consistently the most virulent based on visual symptoms and pathogen titers in planta inferred by qPCR performed on DNA extracted from infected leaves 2 and 6 weeks postinoculation. Visual observations 6 weeks after inoculation were highly correlated (R2 = 0.8254) to pathogen titers. The qPCR method was used to categorize 20 chili pepper cultivars 2 weeks after inoculation. A high positive correlation (R2 = 0.6826) was observed between visual scoring and pathogen titers from 20 chili pepper cultivars, facilitating categorization of susceptible, intermediate, and resistant cultivars. The qPCR approach developed here facilitates susceptibility screening of chili pepper cultivars at an early stage of selection and could be readily adapted to a range of other pathosystems.

LAMP Assay to Detect Elsinoë necatrix, an Important Eucalyptus Shoot and Leaf Pathogen.

Eucalyptus scab and shoot malformation caused by Elsinoë necatrix is an emerging disease and a serious threat to the global commercial forestry industry. The disease was first discovered in North Sumatra, Indonesia, and now requires a simple and effective method for early pathogen detection. In this study, a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for E. necatrix. A unique region in a secondary metabolite gene cluster was used as a target for the assay. To test robustness of the assay, LAMP was verified in 15 strains of E. necatrix. A specificity test against 23 closely related Elsinoë species and three fungal species commonly isolated on Eucalyptus showed that the LAMP assay exclusively identified E. necatrix isolates. The assay had a high level of sensitivity, able to detect 0.01 ng (approximately 400 target copies) of pure E. necatrix DNA. Furthermore, using a simple DNA extraction method, it was possible to use this assay to detect E. necatrix in infected Eucalyptus leaves.

A DAMP-Based Assay for Rapid and Affordable Diagnosis of Bacterial Meningitis Agents: Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae.

The rapid and accurate diagnosis of meningitis is critical for preventing severe complications and fatalities. This study addresses the need for accessible diagnostics in the absence of specialized equipment by developing a novel diagnostic assay. The assay utilizes dual-priming isothermal amplification (DAMP) with unique internal primers to significantly reduce non-specificity. For fluorescence detection, the dye was selected among Brilliant Green, Thioflavin T, and dsGreen. Brilliant Green is preferred for this assay due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay was developed for the detection of the primary causative agents of meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae), and tested on clinical samples. The developed method demonstrated high specificity, no false positives, sensitivity comparable to that of loop-mediated isothermal amplification (LAMP), and a high S/B ratio. This versatile assay can be utilized as a standalone test or an integrated assay into point-of-care systems for rapid and reliable pathogen detection.

Comparative Bioinformatic Analysis Reveals Conserved Regions in SARS-CoV-2 Genome for RAPID Pandemic Response.

In the face of the SARS-CoV-2 pandemic, characterized by the virus's rapid mutation rates, developing timely and targeted therapeutic and diagnostic interventions presents a significant challenge. This study utilizes bioinformatic analyses to pinpoint conserved genomic regions within SARS-CoV-2, offering a strategic advantage in the fight against this and future pathogens. Our approach has enabled the creation of a diagnostic assay that is not only rapid, reliable, and cost-effective but also possesses a remarkable capacity to detect a wide array of current and prospective variants with unmatched precision. The significance of our findings lies in the demonstration that focusing on these conserved genomic sequences can significantly enhance our preparedness for and response to emerging infectious diseases. By providing a blueprint for the development of versatile diagnostic tools and therapeutics, this research paves the way for a more effective global pandemic response strategy.

Multicenter performance evaluation of the Alinity m CMV assay for quantifying cytomegalovirus DNA in plasma samples.

Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r ≥0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy.

Multi-site performance evaluation of the Alinity m Molecular assay for quantifying Epstein-Barr virus DNA in plasma samples.

Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy.

Comparison of Seegene AnyPlexTM II STI-7e with standard-of-care diagnostic methods for the detection of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

The AnyPlexTM II STI-7e panel assay (Seegene) detects seven sexually transmitted organisms (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, M. hominis, Ureaplasma urealyticum, U. parvum, and Trichomonas vaginalis). This study compared the performance of AnyPlexTM II STI-7e with standard-of-care diagnostic methods. Samples (cervical or vaginal swabs, or urine) from 1330 women were tested on standard-of-care assays; 83/1318 (6.3%) tested positive for M. genitalium (ResistancePlus® MG), 99/1317 (7.5%) positive for C. trachomatis and 11/1316 (0.8%) positive for N. gonorrhoeae (Hologic® Aptima Combo 2®), and 6/689 (0.9%) positive for T. vaginalis (wet mount microscopy). AnyPlexTM II STI-7e had good agreement for the detection of M. genitalium [Cohen's kappa of 0.80, 95% confidence intervals (CI) 0.74-0.87] and C. trachomatis (kappa of 0.87, 95% CI 0.82-0.92), with positive and negative % agreement >96% for both infections. There was lower agreement for the detection of N. gonorrhoeae (kappa of 0.37, 95%CI 0.19-0.55) and T. vaginalis (kappa of 0.521, 95%CI 0.25-0.80). In summary, the test performed well in this comparison for M. genitalium and C. trachomatis detection, but results were less conclusive for N. gonorrhoeae and T. vaginalis due to low prevalence in the population.

Utilization of a New Hundred-Genomes Pipeline to Design a Rapid Duplex LAMP Detection Assay for Xanthomonas euvesicatoria and X. vesicatoria in Tomato.

Xanthomonas euvesicatoria and X. vesicatoria are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. Therefore, this work aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of X. euvesicatoria and X. vesicatoria by loop-mediated isothermal amplification. In total, 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg µl-1 for both pathogens with an assay duration of 40 min on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with X. euvesicatoria and X. vesicatoria pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.

Development of a Molecular Tool for Identification of a New Neopestalotiopsis sp. Associated with Disease Outbreaks on Strawberry.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a ß-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.

Current Developments in Diagnostic Assays for Laboratory Confirmation and Investigation of Botulism.

Detection of botulinum neurotoxin or isolation of the toxin-producing organism is required for the laboratory confirmation of botulism in clinical specimens. In an effort to reduce animal testing required by the gold standard method of botulinum neurotoxin detection, the mouse bioassay, many technologies have been developed to detect and characterize the causative agent of botulism. Recent advancements in these technologies have led to improvements in technical performance of diagnostic assays; however, many emerging assays have not been validated for the detection of all serotypes in complex clinical and environmental matrices. Improvements to culture protocols, endopeptidase-based assays, and a variety of immunological and molecular methods have provided laboratories with a variety of testing options to evaluate and incorporate into their testing algorithms. While significant advances have been made to improve these assays, additional work is necessary to evaluate these methods in various clinical matrices and to establish standardized criteria for data analysis and interpretation.

ImprintSeq, a novel tool to interrogate DNA methylation at human imprinted regions and diagnose multilocus imprinting disturbance.

PURPOSE: Disruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research. METHODS: We report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations. RESULTS: Across a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified. CONCLUSION: ImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.

Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica.

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.

Circulating Pregenomic Hepatitis B Virus RNA Is Primarily Full-length in Chronic Hepatitis B Patients Undergoing Nucleos(t)ide Analogue Therapy.

Hepatitis B virus RNA is detectable in the serum of infected patients; however, the RNA species has been questioned. We tested 1827 specimens using a quantitative dual-target quantitative polymerase chain reaction assay and determined that full-length pregenomic RNA is the primary source. These results clarify the major identity of circulating HBV RNA species.

Next-Generation Sequencing (NGS) in COVID-19: A Tool for SARS-CoV-2 Diagnosis, Monitoring New Strains and Phylodynamic Modeling in Molecular Epidemiology.

This review discusses the current testing methodologies for COVID-19 diagnosis and explores next-generation sequencing (NGS) technology for the detection of SARS-CoV-2 and monitoring phylogenetic evolution in the current COVID-19 pandemic. The review addresses the development, fundamentals, assay quality control and bioinformatics processing of the NGS data. This article provides a comprehensive review of the obstacles and opportunities facing the application of NGS technologies for the diagnosis, surveillance, and study of SARS-CoV-2 and other infectious diseases. Further, we have contemplated the opportunities and challenges inherent in the adoption of NGS technology as a diagnostic test with real-world examples of its utility in the fight against COVID-19.

(1,3)-ß-D-glucan is able to predict therapeutic failure of patients with candidemia and not only mortality.

BACKGROUND: Candidemia is a major cause of bloodstream infection in tertiary hospitals worldwide and fungal biomarkers may provide early diagnosis. OBJECTIVES: To evaluate the performance of (1-3)-ß-D-glucan (BDG) in the diagnosis of candidemia and its ability to predict therapeutic failure. PATIENTS AND METHODS: This was a prospective, multi-centre study conducted in 3 Brazilian hospitals. Clinical outcome was evaluated along 2 weeks of treatment, and therapeutic failure was defined as the occurrence of persistent candidemia, Candida deep-seated infection or death. Baseline BDG detection was performed with the Fungitell® assay (Associates of Cape Cod, Falmouth-USA). RESULTS: We enrolled a total of 71 patients with candidemia and a control group with 110 healthy volunteers. The sensitivity and specificity of BDG for diagnosing candidemia were as follows: 71.8% (95% confidence interval [95% CI] 59.7% - 81.5%) and 98.2% (95% CI 92.9% - 99.7%), respectively. The only predictor of therapeutic failure was a higher BDG value at diagnosis of candidemia; a value > 226 pg/mL predicted failure with sensitivity and specificity of 75% and 78%, respectively. CONCLUSIONS: A high baseline serum BDG value was associated with therapeutic failure.

A Diagnostic TaqMan Real-Time PCR Assay for In Planta Detection and Quantification of Colletotrichum theobromicola, Causal Agent of Boxwood Dieback.

Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult to distinguish it from other closely related species based on morphology. Moreover, molecular identification of C. theobromicola requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Therefore, a diagnostic TaqMan real-time PCR assay was developed for early and accurate detection and quantification of C. theobromicola in boxwood. The study involved the design of species-specific primers and a TaqMan probe to differentiate C. theobromicola from other closely related Colletotrichum species. The primers and probe discriminate between C. theobromicola and other species in the C. gloeosporioides species complex and can detect C. theobromicola at very low concentrations, illustrating the high specificity and sensitivity of the assay. This TaqMan real-time PCR assay accurately and rapidly distinguishes boxwood dieback from other diseases with similar symptomatology, including Macrophoma blight, Phytophthora root rot, and Volutella blight, as well as some disorders produced by abiotic agents.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.