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BACKGROUND:Vector-borne pathogens are a significant public health concern worldwide. Infections with these pathogens, some of which are emerging, are likely under-recognized due to the lack of widely-available laboratory tests. There is an urgent need for further advancement in diagnostic modalities to detect new and known vector-borne pathogens. We evaluated the utility of metagenomic shotgun sequencing (MGS) as a pathogen agnostic approach for detecting vector-borne pathogens from human blood samples. METHODS:Residual whole blood samples from patients with known infection with Babesia microti, Borrelia hermsii, Plasmodium falciparum, Mansonella perstans, Anaplasma phagocytophilum or Ehrlichia chaffeensis were studied. Samples underwent DNA extraction, removal of human DNA, whole genome amplification, and paired-end library preparation, followed by sequencing on Illumina HiSeq 2500. Bioinformatic analysis was performed using the Livermore Metagenomics Analysis Toolkit (LMAT), Metagenomic Phylogenetic Analysis (MetaPhlAn2), Genomic Origin Through Taxonomic CHAllenge (GOTTCHA) and Kraken 2. RESULTS:Eight samples were included in the study (2 samples each for P. falciparum and A. phagocytophilum). An average of 27.5 million read pairs was generated per sample (range, 18.3-38.8 million) prior to removal of human reads. At least one of the analytic tools was able to detect four of six organisms at the genus level, and the organism present in five of eight specimens at the species level. Mansonella and Ehrlichia species were not detected by any of the tools; however, mitochondrial cytochrome c oxidase subunit I amino acid sequence analysis suggested the presence of M. perstans genetic material. CONCLUSIONS:MGS is a promising tool with the potential to evolve as a non-hypothesis driven diagnostic test to detect vector-borne pathogens, including protozoa and helminths.

Blastomycosis, caused by dimorphic fungi from the genus Blastomyces, is an endemic disease of the midwestern United States that typically presents as pulmonary disease mimicking community acquired pneumonia. Extrapulmonary blastomycosis occurs in up to 50% of cases and most commonly involves the skin. Genitourinary blastomycosis is a rare entity most often seen in disseminated systemic blastomycosis, and rarely as the presenting manifestation of blastomycosis. Here we describe a case report of a 78-year-old Midwestern man incidentally found to have a large kidney mass and multiple pulmonary nodules. Renal biopsy revealed multiple sites of granulomatous inflammation and budding yeast consistent with Blastomyces species. We present the patient’s clinical history, treatment regimen, and clinical course.

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.

We detected the DNA of an Anaplasma bovis–like bacterium in blood specimens from 4 patients from the United States with suspected tickborne illnesses. Initial molecular characterization of this novel agent reveals identity to A. bovis–like bacteria detected in Dermacentor variabilis ticks collected from multiple US states.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020 and 2022 and used Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest calculated introduction into Minnesota was to Hennepin County from a domestic source around 22 January 2020; 6 weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota and, eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International Airport) totaling 45 (out of 107) over the 2-year period. Southern Minnesota counties were most common for domestic introductions, with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois, which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n = 772) over the 2-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases nor the major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have an impact on virus diversity across each individual county.IMPORTANCEWe analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission “sinks.” In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

Abstract The emergence of highly transmissible SARS-CoV-2 variants and vaccine breakthrough infections globally mandated the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 2.13 million SARS-CoV-2 genomes from 188 countries/territories (up to June 2021) and performed whole-genome viral sequencing from 102 COVID-19 patients, including 43 vaccine breakthrough infections. We identified 92 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a 3-month window. Deletions in the Spike protein N-terminal domain were highly enriched for these ‘surge-associated mutations’ (Odds Ratio = 14.19, 95% CI 6.15–32.75, p value = 3.41 × 10–10). Based on a longitudinal analysis of mutational prevalence globally, we found an expanding repertoire of Spike protein deletions proximal to an antigenic supersite in the N-terminal domain that may be one of the key contributors to the evolution of highly transmissible variants. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences from 102 patients and identified 107 unique mutations, including 78 substitutions and 29 deletions. In five patients, we identified distinct deletions between residues 85–90, which reside within a linear B cell epitope. Deletions in this region arose contemporaneously on a diverse background of variants across the globe since December 2020. Overall, our findings based on genomic-epidemiology and clinical surveillance suggest that the genomic deletion of dispensable antigenic regions in SARS-CoV-2 may contribute to the evasion of immune responses and the evolution of highly transmissible variants.

Se reportan seis casos de encefalitis amebiana admitidos en el Instituto Nacional de Enfermedades Neoplásicas entre los años 1994-2010 en Perú; estos casos ingresaron por sospecha clínica de tumor cerebral primario maligno y uno como sarcoma orbito-nasal. Todos los casos provenían de departamentos costeros; tres tenían menos de 24 años de edad y cuatro de sexo masculino. Los síntomas más frecuentes fueron cefalea y convulsiones. Tres casos presentaron más de una lesión cerebral. Se realizó biopsia por estereotaxia en tres pacientes y el diagnóstico anatomopatológico diferencial, en dos casos, fue glioma de alto y bajo grado. Se logró confirmar el diagnóstico mediante técnicas moleculares en muestras parafinadas en tres casos. Todos los pacientes fallecieron en menos de 15 días desde su ingreso al instituto. La encefalitis amebiana puede ser erradamente interpretada como una neoplasia cerebral, ocasionando retraso en el manejo del cuadro infeccioso.

Se reportan seis casos de encefalitis amebiana admitidos en el Instituto Nacional de Enfermedades Neoplásicas entre los años 1994-2010 en Perú; estos casos ingresaron por sospecha clínica de tumor cerebral primario maligno y uno como sarcoma orbito-nasal. Todos los casos provenían de departamentos costeros; tres tenían menos de 24 años de edad y cuatro de sexo masculino. Los síntomas más frecuentes fueron cefalea y convulsiones. Tres casos presentaron más de una lesión cerebral. Se realizó biopsia por estereotaxia en tres pacientes y el diagnóstico anatomopatológico diferencial, en dos casos, fue glioma de alto y bajo grado. Se logró confirmar el diagnóstico mediante técnicas moleculares en muestras parafinadas en tres casos. Todos los pacientes fallecieron en menos de 15 días desde su ingreso al instituto. La encefalitis amebiana puede ser erradamente interpretada como una neoplasia cerebral, ocasionando retraso en el manejo del cuadro infeccioso.

The published literature on cervicovaginal cytology includes fewer than ten reported cases of Cokeromyces recurvatus identified in Pap test samples. We report a unique case of an asymptomatic 27-year-old female with persistent gynecologic tract colonization by C. recurvatus in which distinctive fungal microorganisms were identified in three samples collected over three consecutive years.

# Background Schistosomiasis is a parasitic disease that affects the health of hundreds of millions of people worldwide. Affordable diagnostic methods are necessary for developing countries to monitor schistosomiasis rates and treat infected individuals, particularly in, Madagascar, a country with the world’s fifth highest rate of schistosomiasis. # Methods We established the prevalence rate of schistosomiasis among the local population served by a clinic in rural northwestern Madagascar. Additionally, we compared different low-cost methods for schistosomiasis detection, including empirical diagnostic methods in use before this study. 500 patients were recruited in this study, of whom 380 received three tests—urine microscopy, point-of-care circulating cathodic antigen (POC-CCA) testing, and urinalysis to screen for *Schistosoma haematobium* and *Schistosoma mansoni*. # Results By combining these modalities, we recorded a schistosomiasis prevalence rate of 64.47%, which was significantly higher than the 4.84% of patients who were diagnosed and treated through previous empirical methods. # Conclusions Our results indicate that screening tests are a necessary component of schistosomiasis control programs. The clinic continued to use urinalysis strip testing and urine microscopy to detect schistosomiasis after this study and decided to discontinue the use of POC-CCA *S. mansoni* urine testing because of cost. Increased awareness of schistosomiasis resulting from this screening program led to the installation of multiple infrastructure projects in local communities to improve access to potable water, demonstrating community benefits of schistosomiasis screening that extend beyond the identification of infected individuals.

Staphylococcus aureus, the most common cause of wound infection, produces several exotoxins, including superantigens (SAgs). SAgs are the potent activators of the immune system. Given this unique property, we hypothesized that SAgs produced by S. aureus in wounds would have local, as well as systemic immunologic effects. We tested our hypothesis using a novel staphylococcal skin wound infection model in transgenic mice expressing HLA-DR3. Skin wounds were left uninfected or colonized with S. aureus strains producing SAgs or an isogenic strain not producing any SAg. Animals with wounds challenged with SAg-producing S. aureus had increased morbidity and lower serum IL-17 levels compared to those challenged with the SAg non-producing S. aureus (p = 0.027 and p = 0.032, respectively). At Day 8 following microbial challenge, compared to mice with uninfected wounds, the proportion of Vβ8+CD4+ T cells was increased, while the proportion of Vβ8+CD8+ T cells was decreased only in the spleens of mice challenged with SAg-producing S. aureus (p < 0.001). No such changes were measured in mice challenged with SAg non-producing S. aureus. Lungs, livers and kidneys from mice challenged with SAg-producing, but not SAg non-producing, S. aureus showed inflammatory changes. Overall, SAg-mediated systemic immune activation in wounds harboring S. aureus may have clinical implications.

We report an imported case of myositis caused by a rare parasite, Haycocknema perplexum, in Australia in a 37-year-old man who had progressive facial, axial, and limb weakness, dysphagia, dysphonia, increased levels of creatine kinase and hepatic aminotransferases, and peripheral eosinophilia for 8 years. He was given extended, high-dose albendazole.

Purpose: To present a case of subretinal abscess associated with pneumonia and meningitis caused by Cryptococcus gattii in an immunocompetent host. Observations: A 37-year-old man presented with sub-acute painless unilateral vision loss and a white submacular elevation. Systemic evaluation revealed a lung lesion and cerebrospinal fluid evidence of Cryptococcus gattii infection. Conclusions and importance: While Crypococcus neoformans has been well described as a cause of chorioretinitis in immunocompetent and immunocompromised hosts, this report demonstrates that Cryptocuccus gattii is a related uncommon pathogen to be considered in similar presentations. Submacular surgical debridement may be challenging and OCT imaging may be helpful to detect full-thickness retinal necrosis.

Bed bugs are common urban pests. Unlike many other blood-feeding human ectoparasites, bed bugs are not known to be vectors of human infectious diseases, but clinical and epidemiological studies to directly interrogate this link have been limited. Here, we aimed to determine whether bed bugs were associated with infectious diseases in a set of infested patients presenting to emergency departments (ED) in the greater Cleveland, OH area. We performed a retrospective case-control study involving 332 ED patients with bed bugs and 4,952 control patients, seen from February 1, 2011, through February 1, 2017. Cases and controls were matched by age, sex, and the presenting ED. Additionally, data were adjusted for ≥20 sociodemographic variables, triage data, and comorbidities in multivariable regression analyses. Seventeen laboratory values, ten different ED and inpatient diagnoses, chest radiographs, infectious disease consults, and blood cultures were examined. The odds of bed bug infestation were significantly higher for patients that had positive blood cultures, had blood cultures growing coagulase-negative Staphylococcus, were diagnosed with pneumonia, were diagnosed with cellulitis, received an infectious disease consult, received a chest radiograph, and had higher percentages of eosinophils in the blood (P < .05 for all). Additional investigations are needed to determine whether bed bugs directly contribute to disease by transmitting causative agents, whether bed bug exposure contributes secondarily contributes to infections, or whether these associations are better explained by other environmental and social determinants of health.

The rapidly spreading COVID-19 pandemic demanded immediate organizational pivots in departments of laboratory medicine and pathology, including development and implementation of severe acute respiratory syndrome coronavirus 2 diagnostics in the face of unprecedented supply chain shortages. Laboratory medicine and pathology educational programs were affected in numerous ways. Here, we overview the effects of COVID-19 on the large, academic Department of Laboratory Medicine and Pathology educational practice at Mayo Clinic, highlighting lessons learned for the post-pandemic era and planning for the possibility of a future pandemic.

Infection is a rare cause of orbital apex syndrome (OAS) and most commonly occurs in immunocompromised hosts. We report a case of OAS in an elderly immunocompetent female due to invasive aspergillosis and Staphylococcus aureus co-infection. The patient required both surgical debridement and prolonged courses of antibiotic and antifungal therapy. Invasive fungal disease must be considered in cases of OAS, even in patients without classic risk factors.

Background: Cutaneous myiasis is a self-limited skin infestation by developing fly larvae, with three clinical subtypes: furuncular, migratory, and wound myiasis. Furuncular myiasis is endemic throughout much of Africa; however, few reports are from the Horn of Africa. Clinical presentation: An 85-year-old woman in Somaliland presented with a 12-day history of multiple painful and pruritic nodules on the temple, arm, chest, breast, flank, and legs. The posterior of a larva was visible within several lesions. One larva was extracted from an arm nodule and identified as Cordylobia anthropophaga (tumbu fly) by morphologic examination. The patient was instructed to occlude the other nodules with petroleum jelly and return in 3 days. Instead, she visited a traditional healer who extracted the remaining larvae. Conclusions: We present a case of furuncular cutaneous myiasis due to Cordylobia anthropophaga. Treatment options for this infestation include occlusion with petroleum jelly to cause larvae to exit, surgical extraction, and oral ivermectin. Occlusion may not be acceptable for some patients. Extraction may cause significant inflammatory response if the larva is damaged during the process. To our knowledge, this is the first published report of myiasis in Somaliland, although it is probably underreported. Myiasis is a common dermatosis associated with travel to endemic areas. Furuncular myiasis can easily be misdiagnosed as furunculosis or cellulitis. Dermatologists must be familiar with the clinical features and management of this dermatosis. Keywords: myiasis, cutaneous myiasis, furuncular myiasis, tumbu fly, fly larvae, Somaliland

Cyclospora cayetanensis is an intestinal coccidian parasite transmitted to humans through the consumption of oocysts in fecally contaminated food and water. Infection is found worldwide and is highly endemic in tropical and subtropical regions with poor sanitation. Disease in developed countries is usually observed in travelers and in seasonal outbreaks associated with imported produce from endemic areas. Recently, summertime outbreaks in the United States have also been linked to locally grown produce. Cyclosporiasis causes a diarrheal illness which may be severe in infants, the elderly, and immunocompromised individuals. The increased adoption of highly sensitive molecular diagnostic tests, including commercially available multiplex panels for gastrointestinal pathogens, has facilitated the detection of infection and likely contributed to the increased reports of cases in developed countries. This manuscript reviews important aspects of the biology, epidemiology, and clinical manifestations of C. cayetanensis and provides an in-depth discussion of current laboratory diagnostic methods.

Granulomatous inflammation is a histologic pattern of tissue reaction which appears following cell injury. Granulomatous inflammation is caused by a variety of conditions including infection, autoimmune, toxic, allergic, drug, and neoplastic conditions. The tissue reaction pattern narrows the pathologic and clinical differential diagnosis and subsequent clinical management. Common reaction patterns include necrotizing granulomas, non necrotizing granulomas, suppurative granulomas, diffuse granulomatous inflammation, and foreign body giant cell reaction. Prototypical examples of necrotizing granulomas are seen with mycobacterial infections and non-necrotizing granulomas with sarcoidosis. However, broad differential diagnoses exist within each category. Using a pattern based algorithmic approach, identification of the etiology becomes apparent when taken with clinical context.The pulmonary system is one of the most commonly affected sites to encounter granulomatous inflammation. Infectious causes of granuloma are most prevalent with mycobacteria and dimorphic fungi leading the differential diagnoses. Unlike the lung, skin can be affected by several routes, including direct inoculation, endogenous sources, and hematogenous spread. This broad basis of involvement introduces a variety of infectious agents, which can present as necrotizing or non-necrotizing granulomatous inflammation. Non-infectious etiologies require a thorough clinicopathologic review to narrow the scope of the pathogenesis which include: foreign body reaction, autoimmune, neoplastic, and drug related etiologies. Granulomatous inflammation of the kidney, often referred to as granulomatous interstitial nephritis (GIN) is unlike organ systems such as the skin or lungs. The differential diagnosis of GIN is more frequently due to drugs and sarcoidosis as compared to infections (fungal and mycobacterial).Herein we discuss the pathogenesis and histologic patterns seen in a variety of organ systems and clinical conditions. Keywords: Foreign-body, Granulomatous inflammation, Granuloma, Mycobacterial, Sarcoidal, Tuberculous

Six cases of amoebic encephalitis admitted to the National Institute of Neoplastic Diseases between the years 1994-2010 in Peru are reported. These cases were admitted for clinical suspicion of malignant primary brain tumor and one orbital-nasal sarcoma. All cases came from coastal regions; three were less than 24 years of age and four were male. The most common symptoms were headache and seizures. Three cases had more than one brain lesion. Stereotactic biopsy was performed in three patients and the differential pathological diagnosis in two cases was glioma of high and low grade. It was possible to confirm the diagnosis using molecular techniques in paraffin-embedded samples in three cases. All patients died within 15 days of admission to the institution. Amoebic encephalitis may be erroneously interpreted as a cerebral neoplasm, causing delay in the management of the infection.

We present the unusual case of a 74 year-old female with a history of breast cancer who presented with acute painless orbital swelling and vertical diplopia. MRI revealed a focal enhancing mass within the superior rectus muscle. As the concern for metastatic disease was high, surgical biopsy was performed and revealed an unusual mimicker of metastatic disease, the parasitic infection dirofilariasis.