Incomplete tissue product tracing during an investigation of a tissue-derived tuberculosis outbreak.

In the United States, there is currently no system to track donated human tissue products to individual recipients. This posed a challenge during an investigation of a nationwide tuberculosis outbreak that occurred when bone allograft contaminated with Mycobacterium tuberculosis (Lot A) was implanted into 113 patients in 18 US states, including 2 patients at 1 health care facility in Colorado. A third patient at the same facility developed spinal tuberculosis with an isolate genetically identical to the Lot A outbreak strain. However, health care records indicated this patient had received bone allograft from a different donor (Lot B). We investigated the source of this newly identified infection, including the possibilities of Lot B donor infection, product switch or contamination during manufacturing, product switch at the health care facility, person-to-person transmission, and laboratory error. The findings included gaps in tissue traceability at the health care facility, creating the possibility for a product switch at the point of care despite detailed tissue-tracking policies. Nationally, 6 (3.9%) of 155 Lot B units could not be traced to final disposition. This investigation highlights the critical need to improve tissue-tracking systems to ensure unbroken traceability, facilitating investigations of recipient adverse events and enabling timely public health responses to prevent morbidity and mortality.

Outbreak of Mycobacterium orygis in a Shipment of Cynomolgus Macaques Imported from Southeast Asia - United States, February-May 2023.

Nonhuman primates (NHP) can become infected with the same species of Mycobacteria that cause human tuberculosis. All NHP imported into the United States are quarantined and screened for tuberculosis; no confirmed cases of tuberculosis were diagnosed among NHP during CDC-mandated quarantine during 2013-2020. In February 2023, an outbreak of tuberculosis caused by Mycobacterium orygis was detected in a group of 540 cynomolgus macaques (Macaca fascicularis) imported to the United States from Southeast Asia for research purposes. Although the initial exposure to M. orygis is believed to have occurred before the macaques arrived in the United States, infected macaques were first detected during CDC-mandated quarantine. CDC collaborated with the importer and U.S. Department of Agriculture's National Veterinary Services Laboratories in the investigation and public health response. A total of 26 macaques received positive test results for M. orygis by culture, but rigorous occupational safety protocols implemented during transport and at the quarantine facility prevented cases among caretakers in the United States. Although the zoonotic disease risk to the general population remains low, this outbreak underscores the importance of CDC's regulatory oversight of NHP importation and adherence to established biosafety protocols to protect the health of the United States research animal population and the persons who interact with them.

Second Nationwide Tuberculosis Outbreak Caused by Bone Allografts Containing Live Cells - United States, 2023.

During July 7-11, 2023, CDC received reports of two patients in different states with a tuberculosis (TB) diagnosis following spinal surgical procedures that used bone allografts containing live cells from the same deceased donor. An outbreak associated with a similar product manufactured by the same tissue establishment (i.e., manufacturer) occurred in 2021. Because of concern that these cases represented a second outbreak, CDC and the Food and Drug Administration worked with the tissue establishment to determine that this product was obtained from a donor different from the one implicated in the 2021 outbreak and learned that the bone allograft product was distributed to 13 health care facilities in seven states. Notifications to all seven states occurred on July 12. As of December 20, 2023, five of 36 surgical bone allograft recipients received laboratory-confirmed TB disease diagnoses; two patients died of TB. Whole-genome sequencing demonstrated close genetic relatedness between positive Mycobacterium tuberculosis cultures from surgical recipients and unused product. Although the bone product had tested negative by nucleic acid amplification testing before distribution, M. tuberculosis culture of unused product was not performed until after the outbreak was recognized. The public health response prevented up to 53 additional surgical procedures using allografts from that donor; additional measures to protect patients from tissue-transmitted M. tuberculosis are urgently needed.

Pathogen Detection in Early Phases of Experimental Bovine Tuberculosis.

Bovine tuberculosis is caused by Mycobacterium bovis, a member of the M. tuberculosis complex of mycobacterial species that cause tuberculosis in humans and animals. Diagnosis of bovine tuberculosis has relied on examinations of cell-mediated immune responses to M. bovis proteins using tuberculin skin testing and/or interferon gamma release assays. Even when using these methods, disease detection during the earliest phases of infection has been difficult, allowing a window for cattle-to-cattle transmission to occur within a herd. Alternative means of diagnosis could include methods to detect M. bovis or M. bovis DNA in bodily fluids such as nasal secretions, saliva, or blood. During the first 8 weeks after experimental aerosol infection of 18 calves, M. bovis DNA was detected in nasal swabs from a small number of calves 5, 6, and 8 weeks after infection and in samples of saliva at 1, 7, and 8 weeks after infection. However, at no time could culturable M. bovis be recovered from nasal swabs or saliva. M. bovis DNA was not found in blood samples collected weekly and examined by real-time PCR. Interferon gamma release assays demonstrated successful infection of all calves, while examination of humoral responses using a commercial ELISA identified a low number of infected animals at weeks 4-8 after infection. Examination of disease severity through gross lesion scoring did not correlate with shedding in nasal secretions or saliva, and calves with positive antibody ELISA results did not have more severe disease than other calves.

Diagnostic Evaluation of the IS1081-Targeted Real-Time PCR for Detection of Mycobacterium bovis DNA in Bovine Milk Samples.

The ability of Mycobacterium bovis (M. bovis) to survive in bovine milk has emerged as a serious public health concern. The first objective of this study was to evaluate the diagnostic utility of IS1081-targeted real-time PCR for the detection of M. bovis DNA in different fractions of bovine milk. In a model study, bovine milk samples were spiked with serially diluted M. bovis BCG to investigate the detection limit of M. bovis DNA in whole milk and milk fractions (cream, pellet, and pellet + cream combined) using IS1081 real-time PCR. The assay was then used to detect M. bovis DNA in whole milk and milk fractions from naturally infected animals. The results showed that the IS1081 real-time PCR was more sensitive when detecting M. bovis DNA in the cream layer alone and cream + pellet combined compared to whole milk or the pellet alone. While PCR-based diagnostic assays for the detection of M. bovis in milk samples provide a quicker diagnostic tool for bovine tuberculosis, safe processing, and handling of M. bovis-infected milk samples remain a challenge and pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to rapidly inactivate infected specimens while preserving nucleic acid for subsequent Molecular analysis. Therefore, the secondary objective of this study was to evaluate the ability of MTM to inactivate M. bovis BCG in spiked milk samples as well as its ability to preserve BCG DNA for the PCR assay. The results showed that MTM can successfully inactivate BCG alone or in spiked milk samples while preserving DNA for the PCR assay. The CT values of M. bovis BCG alone and spiked milk samples aliquoted in MTM and without MTM were similar at various dilutions. Taken together, our results indicate that using DNA extracted from the milk cream fraction alone or combined milk cream and pellet improved the recovery rate of M. bovis DNA in bovine milk samples. MTM has the potential to provide a safe and rapid sample processing tool for M. bovis inactivation in milk samples and preserve DNA for molecular diagnostics.

Whole genome sequencing of Mycobacterium bovis directly from clinical tissue samples without culture.

Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) for Mycobacterium bovis (M. bovis) genomes in clinical reference laboratories. To date, the M. bovis genome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequence M. bovis DNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially diluted M. bovis BCG DNA at the following concentration range: 0.1 ng/µl to 0.1 pg/µl (10-1 to 10-4). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS of M. bovis DNA extracted from tissue samples from naturally (n = 6) and experimentally (n = 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10-1 (mean Ct value: 20.3) and 10-2 (mean Ct value: 23.4), respectively. The M. bovis genome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping and M. bovis group assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing of M. bovis DNA from tissue samples has the potential to provide accurate sequencing of M. bovis genomes significantly faster than WGS from cultures in research and diagnostic settings.

National Prevalence of Caprine Prion Protein Genetic Variability at Codons 146, 211, and 222 in Goat Herds in the United States.

Scrapie is a neurodegenerative disease that impacts sheep and goats, characterized by gradual and progressive changes in neurological function. Recent research shows that the scrapie incubation period is significantly influenced by specific variations in amino acids within the prion protein gene (PRNP). The objective of this study was to estimate the national prevalence of caprine PRNP genetic variability at codons 146, 211, and 222 in goat populations across the United States. A total of 3052 blood, ear tissue, and brain tissue samples were collected from goats from 50 states. The participating states were categorized into four Veterinary Service (VS) district regions. The samples underwent DNA extraction, and the PRNP variants corresponding to codons 146, 211, and 222 were amplified and sequenced. The analysis of PRNP variants, when compared to the PRNP reference sequence, revealed seven alleles in twelve genotypes. The homozygous 146NN, 211RR, and 222QQ alleles, which have been linked to an increased risk of scrapie, were found to be the most prevalent among all the goats. The heterozygous 222QK, 211RQ, 146SD, 146ND, and 146NS alleles and the homozygous 222KK, 146SS, and 146DD alleles, known to be associated with reduced scrapie susceptibility and a prolonged incubation period after experimental challenge, were found in 1.098% (222QK), 2.33% (211RQ), 0.58% (146SD), 3.13% (146ND), 20.68% (146NS), 0.005% (222KK), 3.31% (146SS), and 0.67% (146DD) of goats, respectively. The 222QK allele was found most frequently in goats tested from the east (VS District 1, 1.59%) and southwest (VS District 4, 1.08%) regions, whereas the 211RQ allele was found most often in goats tested from the Midwest (VS District 2, 8.03%) and east (VS District 1, 6.53%) regions. The 146NS allele was found most frequently in goats tested from the northwest (VS District 3, 29.02%) and southwest (VS District 4, 20.69%) regions. Our results showed that the prevalence of less susceptible genotypes at PRNP codon 146 may be sufficient to use genetic susceptibility testing in some herds. This may reduce the number of goats removed as part of a herd clean-up plan and may promote the selective breeding goats for less susceptible alleles in high-risk herds at the national level.

Vaccination of White-Tailed Deer with Mycobacterium bovis Bacillus Calmette-Guérin (BCG): Effect of Mycobacterium avium ssp. paratuberculosis Infection.

In many parts of the world, bovine tuberculosis eradication efforts are hampered by wildlife reservoirs of Mycobacterium bovis, which serve as a constant source of M. bovis for nearby cattle. The human tuberculosis vaccine, M. bovis BCG has been investigated for use in several wildlife species, including deer. In the US, white-tailed deer in Michigan have been the source of infection for over 82 cattle herds since M. bovis was discovered in free-ranging deer in 1995. The efficacy of BCG may be influenced by many factors, including prior exposure or infection with non-tuberculous mycobacteria, that is, species other than members of the M. tuberculosis complex. M. avium subspecies paratuberculosis (Map) infection is not uncommon in ruminants such as deer. Using natural exposure to Map and experimental infection with M. bovis, we demonstrate that Map infection increased BCG vaccine efficacy as measured by lesion severity scores.

Complete genome sequence of Candidatus Mycobacterium wuenschmannii, a nontuberculous mycobacterium isolated from a captive population of Amazon milk frogs.

A slow growing species of nontuberculous mycobacteria (NTM) was isolated from the liver of an Amazon milk frog. The complete genome of this isolate comprises 5,102,433 bp, exhibiting 66.86% GC content, 4,940 protein-coding sequences, 52 predicted RNA genes, and 39 repeat regions.

Nationwide tuberculosis outbreak in the USA linked to a bone graft product: an outbreak report.

BACKGROUND: Mycobacterium tuberculosis transmission through solid organ transplantation has been well described, but transmission through transplanted tissues is rare. We investigated a tuberculosis outbreak in the USA linked to a bone graft product containing live cells derived from a single deceased donor. METHODS: In this outbreak report, we describe the management and severity of the outbreak and identify opportunities to improve tissue transplant safety in the USA. During early June, 2021, the US Centers for Disease Control and Prevention (CDC) worked with state and local health departments and health-care facilities to locate and sequester unused units from the recalled lot and notify, evaluate, and treat all identified product recipients. Investigators from CDC and the US Food and Drug Administration (FDA) reviewed donor screening and tissue processing. Unused product units from the recalled and other donor lots were tested for the presence of M tuberculosis using real-time PCR (rt PCR) assays and culture. M tuberculosis isolates from unused product and recipients were compared using phylogenetic analysis. FINDINGS: The tissue donor (a man aged 80 years) had unrecognised risk factors, symptoms, and signs consistent with tuberculosis. Bone was procured from the deceased donor and processed into 154 units of bone allograft product containing live cells, which were distributed to 37 hospitals and ambulatory surgical centres in 20 US states between March 1 and April 2, 2021. From March 3 to June 1, 2021, 136 (88%) units were implanted into 113 recipients aged 24-87 years in 18 states (some individuals received multiple units). The remaining 18 units (12%) were located and sequestered. 87 (77%) of 113 identified product recipients had microbiological or imaging evidence of tuberculosis disease. Eight product recipients died 8-99 days after product implantation (three deaths were attributed to tuberculosis after recognition of the outbreak). All 105 living recipients started treatment for tuberculosis disease at a median of 69 days (IQR 56-81) after product implantation. M tuberculosis was detected in all eight sequestered unused units tested from the recalled donor lot, but not in lots from other donors. M tuberculosis isolates from unused product and recipients were more than 99·99% genetically identical. INTERPRETATION: Donor-derived transmission of M tuberculosis via bone allograft resulted in substantial morbidity and mortality. All prospective tissue and organ donors should be routinely assessed for tuberculosis risk factors and clinical findings. When these are present, laboratory testing for M tuberculosis should be strongly considered. FUNDING: None.

Large-scale survey of prion protein genetic variability in scrapie disease-free goats from the United States.

Scrapie is a slowly progressive neurodegenerative disease of small ruminants caused by an accumulation of an abnormal isoform of prion protein in the central nervous system. Polymorphisms of the prion protein gene (PRNP) strongly modulate scrapie resistance and incubation period in goats. The aim of this study was to identify PRNP genetic variability in goats across the United States. Blood from a total of 6,029 apparent scrapie disease-free goats from 654 operations and 19 breeds were analyzed. Sequencing of PRNP revealed 26 genotypes with different rates based on eight codons. The GG127, RR154, and QQ222 genotypes were predominant and showed a remarkably high rate across all goats. The QK222 and NS146 genotypes, known to be protective against scrapie, were found in 0.6% [with 95% CI = (0.3, 1.2)] and 22.0% [95% CI = (19.1, 25.2)] of goats, respectively. The QK222 genotype was found in 23.1% of Oberhasli goats tested, with 95%CI = (3.9, 68.7)] and 22.0% of Toggenburg goats tested with 95%CI = (9.7, 42.5)], while NS146 was found in 65.5% of Savannah goats tested, with 95%CI = (30.8, 89.9), 36.7% of Boer goats tested, with 95%CI = (33.1, 40.4), 36.3% of Nubian goats tested, with 95%CI = (27.0, 46.7)], and 35.6% of LaMancha goats tested, with 95%CI = (22.8, 50.8%). The MM142 and IM142 genotypes were found more frequently in goats on dairy operations, while the HR143, NS146, and ND146 genotypes were found more frequently in goats on meat operations. Goats in the east region had a higher percentage of goats with RH154, RQ211, and QK222 genotypes than goats in the west region. The results of this study showed high genetic variability of PRNP among the U.S. goat population, with differences by location and breed, and may serve as a rationale for development of goat breeding programs at the national level to mitigate the risk of scrapie.

Sarcoidosis.

An active duty male presents to your clinic with concerns of an increasing number of enlarging papules on his neck. How would you describe the morphology of these lesions? What questions should be included in your history? What would you include in your examination? What would you include in your differential diagnosis? What labs and/or tests would you order? This report discusses cutaneous sarcoidosis and its diagnosis and treatment.

Allergic contact dermatitis in Operation Iraqi Freedom: use of the T.R.U.E. Test in the combat environment.

BACKGROUND: Refractory dermatitis can frequently cause loss of personnel and poses an economic drain on the military due to the evacuation of civilians and soldiers out of theater. OBJECTIVE: Proof-of-concept retrospective analysis to review the utility of the T.R.U.E. Test in the combat environment. METHODS: Thirty T.R.U.E. Tests were performed by the dermatology clinic in Baghdad, Iraq, between January 15, 2008, and July 15, 2008. Thirty active-duty and civilian contractors referred to the dermatology clinic for dermatitis were tested and four others were clinically rechallenged for suspected bacitracin contact allergy. RESULTS: Of the 30 patients tested, 14 (46.7%) had a positive test reaction to at least one antigen. In these positive tests, nickel, neomycin, and thimersol each comprised 17% followed by neomycin, thimerosal, budesonide, epoxy resin, potassium dichromate, p-phenylenediamine, formaldehyde, quaternium-15, fragrance, and balsam of Peru. All four clinical rechallenges for bacitracin allergy were also positive. CONCLUSIONS: The use of the T.R.U.E. Test in the combat desert environment is an efficient, easy, and clinically relevant method of testing for allergic contact dermatitis. Continued study of the prevalence of allergic contact dermatitis at Ibn Sina Hospital, Baghdad, Iraq, is recommended.