Identification of Mycobacterium leprae and Mycobacterium lepromatosis Cases with Broad-Range Molecular Assays at a Single Large Reference Laboratory

American Journal of Tropical Medicine and Hygiene · 2026-04-21

Definitively diagnosing Hansen's disease (HD) is challenging, but molecular methods can provide species identification of the causative mycobacteria (Mycobacterium leprae and Mycobacterium lepromatosis). Most studies have investigated targeted assays, and the performance of broad-range molecular approaches remains unknown. This study evaluated a multilocus (hsp65, 16S ribosomal RNA, and rpoB) broad-range polymerase chain reaction (PCR) assay in detecting HD-associated mycobacteria at a single large reference laboratory in the United States from 2009 to 2024. We identified 148 positive samples from 137 patients submitted from 26 states. Mycobacterium lepromatosis was identified in 8 of 137 patients (5.8%), and M. leprae was identified in the rest. Although most samples were skin biopsies (∼90%), 10 samples (7.9%) represented atypical locations, including bone marrow and liver. Half of all samples were identified by only a single target (n = 72/146 samples, 49.3%), and rpoB was the most likely to be positive (n = 123/146 samples, 84.2%). Overall, broad-range PCR successfully identified HD-associated mycobacteria, including M. lepromatosis, from skin and atypical anatomic sites, and it should be considered more frequently in the evaluation of suspected cases.

<i>Coxiella burnetii</i> Infections Identified by Molecular Methods, United States, 2006–2023

Emerging infectious diseases · 2025-03-18 · 1 citations

We identified 34 patients with Coxiella burnetii infection using PCR; 31 (86%) cases were diagnosed from cardiac specimens. Nearly half (15/31, 48%) of those cases were not reported to any channel of national disease surveillance, indicating substantial underreporting for diseases identified using molecular methods at noncommercial laboratories.

P-2226. Clinical Validation and Performance of Molecular Syphilis Diagnosis by Loop-Mediated Isothermal Amplification

Open Forum Infectious Diseases · 2025-01-29

Abstract Background Syphilis cases continue to increase rapidly in the US. Molecular detection of the pathogen, Treponema pallidum (TP) may speed turnaround time (TAT) and resolve ambiguous results from current diagnostics methods, which require stepwise serologic testing. Here we describe validation and clinical performance of a loop-mediated isothermal amplification (LAMP) assay for TP. Methods Candidate LAMP primers targeting the 23S rRNA locus of TP were evaluated and the best performing set was used for clinical validation per Clinical Laboratory Standards Institute guidelines. We determined the sensitivity, specificity, precision, accuracy, and reproducibility of the TP-LAMP assay in fresh and formalin-fixed tissue, body fluids, and whole blood using contrived specimens spiked with intact treponemes and residual clinical specimens. For whole blood, automated nucleic acid extraction was performed from 1mL ETDA-anticoagulated sample. Results The 95% limit of detection was determined to be 7-10 genomes/reaction by testing remnant DNA eluates spiked with TP gDNA. Assay specificity was evaluated by testing synthetic DNA targets representing non-cultivable targets and residual clinical samples containing DNA from 114 clinically relevant microbes, including non-TP spirochetes, bacteria, fungi, and viruses. TP-LAMP detected 100% of whole blood samples spiked with 120-125 organisms/mL; 88% of samples with 85 organisms/mL; and 80% of samples spiked with 60 organisms/mL. From 09/2022 - 07/2023 TP-LAMP had 110 results in non-blood samples and the mean TAT was 4.7 days. Orthogonal diagnostic results were available for a subset of 34 samples. In this group the assay was 100% sensitive and specific with 7 positive tests, comprising 2 cases of primary and 5 cases of secondary syphilis. Three tissue specimens with negative TP-LAMP results had false-positive immunohistochemical TP stains due to cross-reactivity with commensal treponemes. Conclusion This assay represents a novel, clinically available method for sensitive and specific syphilis diagnosis. Additionally, the assay can resolve ambiguous findings in tissue. Applications in blood may aid in diagnosis of early or latent disease and resolution of unclear serologic test results. Disclosures Stephen J. Salipante, M.D. Ph.D., Anavasi: Grant/Research Support Joshua Lieberman, MD, PhD, Anavasi: Grant/Research Support

The Brief Case: A case of tinea corporis caused by drug-resistant <i>Trichophyton indotineae</i> identified by broad-range fungal DNA sequencing

Journal of Clinical Microbiology · 2024-08-14 · 7 citations

Descriptive and molecular epidemiology of leishmaniasis diagnosed from clinical samples in the United States, 2021-2022

Microbiology Spectrum · 2024-09-09 · 5 citations

ABSTRACT Leishmaniasis is a rare disease in the United States, with an estimated annual incidence of dozens of cases occurring primarily in travelers, migrants, and military personnel. True disease incidence is unknown, since leishmaniasis is not a nationally notifiable condition. Here, we describe the results of molecular leishmaniasis over a 1-year interval (September 2021 to August 2022) when our laboratory served as the primary national reference laboratory for molecular diagnosis of civilian leishmaniasis. We tested 218 specimens submitted from 36 states yielding 94 of the 186 (50.5%) positive cases with species or species complex-level identification and 18 novel mini-exon alleles. Most species belonged to subgenus Viannia (75.6%) and associated with cutaneous or mucocutaneous disease. Cases were associated with recent travel (18.1%), travel timing unspecified (7.4%), migration (7.4%), remote travel (2.1%), military (1.1%), or unknown history (63.8%). These data illustrate the clinical utility of molecular testing for leishmaniasis and provide unique insight into disease epidemiology. IMPORTANCE Leishmaniasis is a disfiguring, neglected parasitic infection endemic to the Southern United States and the Americas. Despite significant populations at risk—travelers, military and foreign service members, and migrating persons—the epidemiology of the disease in the United States is poorly understood. Moreover, few clinical laboratories in the United States can test for the disease. Here, we present results from 1 year of testing for this disease at a major reference laboratory. These findings are particularly relevant because they coincide with a temporary “pause” on all clinical testing at the CDC. Our findings suggest at least several hundred cases occur each year in the United States. In particular, mucosal leishmaniasis may be more common than previously reported. We also highlight greater genetic diversity in Leishmania species endemic to the Americas than has been previously sampled, with implications for diagnostic specificity.

Evaluation of Fourier transform-infrared spectroscopy (FT-IR) as a control measure for nosocomial outbreak investigations

Journal of Clinical Microbiology · 2023-10-03 · 26 citations

ABSTRACT Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii ( n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli ( n = 16), two of Pseudomonas aeruginosa ( n = 2 and n = 5), two of Serratia marcescens ( n = 9 and n = 7), five of Staphylococcus aureus ( n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia ( n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive ( r = 0.43, P ≤ 0.0001), Gram-negative ( r = 0.1554, P = 0.0639), and all organisms combined ( r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa , and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.

<i>Bartonella</i> spp. Infections Identified by Molecular Methods, United States

Emerging infectious diseases · 2023 · 39 citations

• Biology
• Virology
• Microbiology

Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.

<i>In Silico</i> Evaluation of Variant Calling Methods for Bacterial Whole-Genome Sequencing Assays

Journal of Clinical Microbiology · 2023-07-10 · 8 citations

workflow to introduce single nucleotide polymorphisms (SNP) and indels into bacterial reference genomes, and computationally generate sequencing reads based on the mutated genomes. We then applied the method to Mycobacterium tuberculosis H37Rv, Staphylococcus aureus NCTC 8325, and Klebsiella pneumoniae HS11286, and used the synthetic reads as truth sets for evaluating several popular variant callers. Insertions proved especially challenging for most variant callers to correctly identify, relative to deletions and single nucleotide polymorphisms. With adequate read depth, however, variant callers that use high quality soft-clipped reads and base mismatches to perform local realignment consistently had the highest precision and recall in identifying insertions and deletions ranging from1 to 50 bp. The remaining variant callers had lower recall values associated with identification of insertions greater than 20 bp.

Pulmonary Co-Infections Detected Premortem Underestimate Postmortem Findings in a COVID-19 Autopsy Case Series

Pathogens · 2023-07-12 · 4 citations

Bacterial and fungal co-infections are reported complications of coronavirus disease 2019 (COVID-19) in critically ill patients but may go unrecognized premortem due to diagnostic limitations. We compared the premortem with the postmortem detection of pulmonary co-infections in 55 fatal COVID-19 cases from March 2020 to March 2021. The concordance in the premortem versus the postmortem diagnoses and the pathogen identification were evaluated. Premortem pulmonary co-infections were extracted from medical charts while applying standard diagnostic definitions. Postmortem co-infection was defined by compatible lung histopathology with or without the detection of an organism in tissue by bacterial or fungal staining, or polymerase chain reaction (PCR) with broad-range bacterial and fungal primers. Pulmonary co-infection was detected premortem in significantly fewer cases (15/55, 27%) than were detected postmortem (36/55, 65%; p &lt; 0.0001). Among cases in which co-infection was detected postmortem by histopathology, an organism was identified in 27/36 (75%) of cases. Pseudomonas, Enterobacterales, and Staphylococcus aureus were the most frequently identified bacteria both premortem and postmortem. Invasive pulmonary fungal infection was detected in five cases postmortem, but in no cases premortem. According to the univariate analyses, the patients with undiagnosed pulmonary co-infection had significantly shorter hospital (p = 0.0012) and intensive care unit (p = 0.0006) stays and significantly fewer extra-pulmonary infections (p = 0.0021). Bacterial and fungal pulmonary co-infection are under-recognized complications in critically ill patients with COVID-19.

Windows into leishmaniasis epidemiology in the United States: September 2021 through August 2022

medRxiv · 2023-05-28 · 4 citations

Abstract Leishmaniasis is a rare disease in the United States, with an estimated annual incidence of dozens of cases occurring primarily in travelers, migrants, and military personnel. True disease incidence is unknown, since leishmaniasis is not a nationally notifiable condition. Here, we describe the results of molecular leishmaniasis over a 1-year interval (September 2021 to August 2022) when our laboratory served as the primary national reference laboratory for molecular diagnosis of civilian leishmaniasis. We tested 218 specimens submitted from 36 states yielding 94/186 (50.5%) positive cases with species or species complex-level identification and 18 novel mini-exon alleles. Most species belonged to subgenus Viannia (75.6%) and associated with cutaneous or mucocutaneous disease. Cases were associated with recent travel (18.1%), travel timing unspecified (7.4%), migration (7.4%), remote travel (2.1%), military (1.1%), or unknown history (63.8%). These data illustrate the clinical utility of molecular testing for leishmaniasis and provide unique insight into disease epidemiology.