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BACKGROUND: Diabetes mellitus (DM) increases the risk of TB disease and poor treatment outcomes such as delayed sputum culture conversion due to inadequate drug exposure. Therapeutic drug monitoring (TDM) has improved these outcomes in some settings.METHODS: To compare treatment outcomes in programs with routine TDM vs. programs that did not use TDM, we conducted a retrospective study among people with DM and TB at health departments in four US states.RESULTS: A total of 170 patients were enrolled (73 patients in the non-TDM group and 97 patients in the TDM group). Days to sputum culture conversion and total treatment duration were significantly shorter in the TDM group vs. the non-TDM group. In adjusted analyses, patients who underwent TDM were significantly more likely to achieve sputum culture conversion at 2 months (P = 0.007).CONCLUSION: TDM hastened microbiological cure from TB among people with DM and a high risk for poor treatment outcomes in the programmatic setting.

Objective To compare lesion conspicuity on synthetic screening mammography (SM) plus digital breast tomosynthesis (DBT) versus full field digital mammography (FFDM) plus DBT. Materials and methods Seven breast imagers each prospectively evaluated 107–228 screening mammograms (FFDM, DBT, and SM; total 1206 examinations) over 12 weeks in sets of 10–50 consecutive examinations. Interpretation sessions alternated as follows: SM + DBT, then FFDM, or FFDM + DBT, then SM. Lesion conspicuity on SM versus FFDM (equal/better versus less) was assessed using proportions with 95% confidence intervals. DBT-only findings were excluded. Results Overall 1082 of 1206 (89.7%) examinations were assessed BI-RADS 1/2, and 124 of 1206 (10.3%) assessed BI-RADS 0. There were 409 evaluated findings, including 134 masses, 119 calcifications, 72 asymmetries, 49 architectural distortion, and 35 focal asymmetries. SM conspicuity compared to FFDM conspicuity for lesions was rated 1) masses: 77 (57%) equal or more conspicuous, 57 (43%) less conspicuous; 2) asymmetries/focal asymmetries: 61 (57%) equal or more conspicuous, and 46 (43%) less conspicuous; 3) architectural distortion: 46 (94%) equal or more conspicuous, 3 (6%) less conspicuous; 4) calcifications: 115 (97%) equal or more conspicuous, 4 (3%) less conspicuous. SM had better conspicuity than FFDM for calcifications and architectural distortion and similar conspicuity for most masses and asymmetries. Conclusion Compared to FFDM, SM has better conspicuity for calcifications and architectural distortion and similar conspicuity for most masses and asymmetries.

Summary Background Since August, 2019, US public health officials have been investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). A spectrum of histological patterns consistent with acute to subacute lung injury has been seen in biopsies; however, autopsy findings have not been systematically characterised. We describe the pathological findings in autopsy and biopsy tissues submitted to the US Centers for Disease Control and Prevention (CDC) for the evaluation of suspected EVALI. Methods Between Aug 1, 2019, and Nov 30, 2019, we examined lung biopsy (n=10 individuals) and autopsy (n=13 individuals) tissue samples received by the CDC, submitted by 16 US states, from individuals with: a history of e-cigarette, or vaping, product use; respiratory, gastrointestinal, or constitutional symptoms; and either pulmonary infiltrates or opacities on chest imaging, or sudden death from an undetermined cause. We also reviewed medical records, evaluated histopathology, and performed infectious disease testing when indicated by histopathology and clinical history. Findings 21 cases met surveillance case definitions for EVALI, with a further two cases of clinically suspected EVALI evaluated. All ten lung biopsies showed histological evidence of acute to subacute lung injury, including diffuse alveolar damage or organising pneumonia. These patterns were also seen in nine of 13 (69%) autopsy cases, most frequently diffuse alveolar damage (eight autopsies), but also acute and organising fibrinous pneumonia (one autopsy). Additional pulmonary pathology not necessarily consistent with EVALI was seen in the remaining autopsies, including bronchopneumonia, bronchoaspiration, and chronic interstitial lung disease. Three of the five autopsy cases with no evidence of, or a plausible alternative cause for acute lung injury, had been classified as confirmed or probable EVALI according to surveillance case definitions. Interpretation Acute to subacute lung injury patterns were seen in all ten biopsies and most autopsy lung tissues from individuals with suspected EVALI. Acute to subacute lung injury can have numerous causes; however, if it is identified in an individual with a history of e-cigarette, or vaping, product use, and no alternative cause is apparent, a diagnosis of EVALI should be strongly considered. A review of autopsy tissue pathology in suspected EVALI deaths can also identify alternative diagnoses, which can enhance the specificity of public health surveillance efforts. Funding US Centers for Disease Control and Prevention.

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

To compare ultrasound visibility of selected biopsy markers in animal tissue models simulating axillary echotexture. Four breast biopsy markers were selected based on size, shape, and composition and compared to an institutional standard for testing in beef steak and pork loin phantoms. BD® UltraCor™ Twirl™; Hologic® Tumark® Professional series Q, Vision, and X; and BD® UltraClip™ Dual Trigger wing-shaped (institutional standard) biopsy markers were deployed at superficial (0–2.0 cm) and deep (2.1–4.0 cm) depths in the animal models. An animal model without a biopsy marker served as control. Four participating breast imagers blinded to marker shape and location assessed ultrasound visibility of each biopsy marker using a handheld 5–12 MHz linear array transducer with a 4-point grading system (0, not visible; 1, unsure if visible; 2, visible with difficulty; 3, definite visibility). Each breast imager was asked to select the three most easily visualized biopsy markers. Total visibility scores with the four-point grading system demonstrate highest score for the Twirl™ (48/48 points), followed by the Tumark® Q (42/48) and Tumark® Vision (41/48) biopsy markers. Overall individual accuracy scores across all biopsy marker types ranged from 83.3 to 95.8%. Visibility scores based on subjective radiologist assessment also demonstrate the highest vote for the Twirl™ (11), followed by the Tumark® Vision (7) and Tumark® Q (6) biopsy markers. The wing-shaped biopsy marker had the lowest visibility and voter score. The Twirl™ followed by the Tumark® Q and Vision biopsy markers demonstrates the highest visibility scores using a four-point grading system and by radiologist vote.

Diagnosis and treatment of culture negative endocarditis remains a challenge. This report describes a rare cause of endocarditis in humans, Bartonella vinsonii, identified through next generation sequencing of plasma microbial cell-free DNA with confirmation of cardiac valve tissue infection through immunohistochemical staining and polymerase chain reaction.

A successful breast core needle biopsy program provides numerous advantages for patients, radiologists, pathologists, surgeons, biomedical research, and the healthcare system. In order to obtain optimal results, it is essential that multiple healthcare providers and disciplines closely communicate and fully cooperate. The complexity of the process from the detection of a lesion, to biopsy, to ultimate outcome creates many opportunities for error. This chapter is a comprehensive guide for best practices for core needle biopsies. The chapter begins with an explanation of how imaging modalities (mammography, ultrasound, and MRI) detect breast abnormalities and how these findings correlate with pathologic lesions. Types of breast needle biopsies and the resulting specimens are then discussed in conjunction with information necessary to determine radiologic/pathologic correlation by both the radiologist and the pathologist. Recommendations for specimen handling from the removal by the radiologist to the creation of the glass slides by the histotechnologist are included. These topics are followed by optimal practices for identifying, excising, and examining a lesion previously sampled by needle biopsy. Throughout the chapter, commonly seen artifacts and possible pitfalls are discussed and illustrated. The chapter concludes with a review of rare cancer recurrences in needle tracks.

Background The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and replication in various tissues. Methods We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case patients (age range, 1 month to 84 years; 21 COVID-19 confirmed, 43 suspected COVID-19) by SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR). For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in situ hybridization (ISH), subgenomic RNA RT-PCR, and whole-genome sequencing. Results SARS-CoV-2 was identified by RT-PCR in 32 case patients (21 COVID-19 confirmed, 11 suspected). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR–positive case patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes, and macrophages of lungs; epithelial cells of airways; and endothelial cells and vessel walls of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. The D614G variant was detected in 9 RT-PCR–positive case patients. Conclusions We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes., By analyzing tissues from patients with fatal COVID-19, using in situ hybridization and RT-PCR, we identified cellular targets of SARS-CoV-2 tropism and replication in the lungs, airways, and vascular endothelium. These findings provide important insights into COVID-19 pathogenesis and severe outcomes.

We report a case of Rickettsia honei infection in a US tourist returning from India and the Himalayas. This case highlights a need for awareness of various Rickettsia species endemic to India and the importance for physicians to consider rickettsial diseases in returning travelers with eschar or rash-associated febrile illnesses.

Powassan virus (POWV) is a flavivirus of the tick-borne encephalitis serogroup that causes a rare and potentially life-threatening neuroinvasive disease. Viral transmission occurs during zoonotic spillover from mammals by the bite of an infected tick in endemic regions of North America. The number of reported POWV cases has recently increased in the United States. We report a fatal case of POWV meningoencephalomyelitis in Northern Wisconsin following a documented tick bite. Histologic examination of the brain demonstrated widespread intraparenchymal and perivascular lymphohistocytic infiltration, microglial nodule formation, and marked neuronal degeneration, most severely involving the substantia nigra, anterior horn of spinal cord and cerebellum. Although no viral inclusions were seen in routine light microscopy, electron microscopy identified multiple neurons containing cytoplasmic clusters of virus particles ∼50 nm in diameter. POWV infection was confirmed using immunohistochemical analysis and reverse transcription-polymerase chain reaction. This report demonstrates in detail regional central nervous system involvement and ultrastructural characteristics of Powassan viral particles by transmission electron microscopy, while highlighting the utility of evaluating fixed autopsy tissues in cases of unexplained meningoencephalomyelitis.

Diving remains the principal means of exploring and exploiting shallower underwater zones. Immersion and rapid change in pressure with depth cause most problems unique to diving. Gas density, partial pressures, and solubility vary proportionately with ambient pressure. At elevated partial pressure, nitrogen becomes narcotic, as can other inert gases, and contaminants barely detectable at the surface can become toxic. Hyperoxia irritates the lungs and the central nervous system, sometimes causing generalized seizures. A safe gas mixture at depth can become hypoxic as the partial pressure of oxygen decreases during the return to surface. Ventilation is compromised at depth and failure of CO2 elimination increasingly limits activity. Some divers are not distressed by elevated CO2, but this does not protect them from its toxic effects.

African tick bite fever (ATBF), caused by infection with the bacterium Rickettsia africae and transmitted by Amblyomma hebraeum or Amblyomma variegatum ticks (Figure 1), is the most commonly diagnosed rickettsial disease among international travelers.1,2 Although the disease is primarily associated with travel to Africa, R. africae has been identified in A. variegatum ticks in other parts of the world, such as the Caribbean.3 Nearly 14% of all returned travelers from sub-Saharan Africa seeking health care are diagnosed with a spotted fever group rickettsiosis (SFGR), and of these infections, most are attributable to R. africae.1 Other tick-borne rickettsial pathogens endemic to the African continent include Rickettsia conorii, Rickettsia aeschlimannii, Rickettsia sibirica mongolitimonae, and Rickettsia monacensis.4 Among patients presenting to GeoSentinel clinics, SFGR is the most common individual diagnosis for ill travelers returning from South Africa and the third most frequently reported diagnosis for travelers returning from Botswana, Lesotho, Mozambique, Namibia, Swaziland, Zimbabwe, or Zambia.5 African tick-bite fever may exceed malaria as the most common vector-borne infection among short-term visitors to southern Africa.6,7 Unlike other well-known tick-borne rickettsioses, such as Rocky Mountain spotted fever (caused by Rickettsia rickettsii) and Mediterranean spotted fever (caused by R. conorii), ATBF is typically a self-limited disease and there are no known deaths attributable to infection with R. africae.2,4 Nonetheless, some patients who do not receive appropriate antibiotic therapy may remain febrile for 1–3 weeks, and in rare cases, the illness may be associated with more severe manifestations that include convalescent-phase asthenia, reactive arthritis, peripheral neuropathy, or myocarditis.2,8,9 Open in a separate window Figure 1. Principal tick vectors of Rickettsia africae. Amblyomma hebraeum female (top left) and male (bottom left). Amblyomma variegatum female (top right) and male (bottom right). All feeding stages (larvae, nymphs, and adults) of both species demonstrate highly aggressive host-seeking behavior and a host is often attacked simultaneously by multiple ticks. Both species are distributed across much of southern Africa, including Botswana, Mozambique, South Africa, Swaziland, and Zimbabwe. The distribution of A. variegatum is even broader and includes most countries of sub-Saharan Africa and eight islands of the Caribbean.2,3 Tick specimens were provided by Lorenza Beati, MD, PhD; images courtesy of James Gathany. This figure appears in color at www.ajtmh.org.

Tropheryma whipplei, the agent of Whipple disease, causes a rare bacterial disease that may be fatal if not treated. The classical form of the disease includes diarrhoea, weight loss, arthritis, endocarditis and neurological manifestations. Genotyping studies done in Europe, Africa and Asia showed high genetic diversity with no correlation between genotypes and clinical features, but contributed to a better understanding of the epidemiology of the disease. More than 70 genotypes have been described. No similar assessment of T. whipplei in the USA and the Caribbean has been performed. In this study, we describe genetic analysis of DNA from histopathological samples obtained from 30 patients from the Americas with Whipple disease and compare the genotypes with those previously identified. Complete genotypes were obtained from 18 patients (60%). Only 4 genotypes were previously described, and 14 were newly reported, confirming the diversity of T. whipplei strains.

Background Hand, foot, and mouth disease (HFMD), classically a childhood viral infection, has an atypical and severe clinical presentation in adults. Coxsackievirus A6 is a leading cause of atypical HFMD, but current diagnostic methods utilizing formalin-fixed, paraffin-embedded skin biopsy specimens often lack sensitivity and specificity. Methods Formalin-fixed, paraffin-embedded skin biopsies from seven case patients with clinical and histopathological suspicion of atypical HFMD were evaluated by coxsackievirus A6 (CVA6) immunohistochemistry, enterovirus-specific conventional reverse transcriptase-PCR with subsequent Sanger sequencing targeting the 5’UTR, and CVA6-specific real-time PCR targeting the VP1 gene. Results The CVA6-specific antibody demonstrated appropriate antigen distribution and staining intensity in keratinocytes in all cases. Conventional RT-PCR and sequencing also detected the presence of enterovirus, and CVA6-specific real-time RT-PCR analysis identified CVA6. Conclusion Applying these immunohistochemistry and molecular techniques to formalin-fixed, paraffin-embedded tissues, CVA6 was determined to be the causative infectious agent in seven cases of atypical hand, foot, and mouth disease.

The likely vector was Amblyomma triste, a Neotropical tick species only recently recognized in the United States., In the United States, all previously reported cases of Rickettsia parkeri rickettsiosis have been linked to transmission by the Gulf Coast tick (Amblyomma maculatum). Here we describe 1 confirmed and 1 probable case of R. parkeri rickettsiosis acquired in a mountainous region of southern Arizona, well beyond the recognized geographic range of A. maculatum ticks. The likely vector for these 2 infections was identified as the Amblyomma triste tick, a Neotropical species only recently recognized in the United States. Identification of R. parkeri rickettsiosis in southern Arizona demonstrates a need for local ecologic and epidemiologic assessments to better understand geographic distribution and define public health risk. Education and outreach aimed at persons recreating or working in this region of southern Arizona would improve awareness and promote prevention of tickborne rickettsioses.

Dielectric barrier discharge actuators are promising candidates for supersonic boundary-layer transition control because of their ability to concentrate energy transfer in receptive regions in the depth of the boundary layer. A detailed understanding of the plasma–flow coupling mechanisms and optimum scaling in compressible flows is necessary to develop efficient actuation schemes. In this work, the dielectric barrier discharge momentum transfer to an unstable laminar boundary layer over a flat plate under different Reynolds and freestream Mach numbers is studied. A new coupled plasma–compressible flow solver using a block-structured adaptive mesh refinement algorithm is presented, which was developed to model the force field generated by the actuator. The spectral components of the force are used as source terms for the study of the influence of flow conditions and length scales on linear receptivity.

Zika virus infection during pregnancy can cause congenital microcephaly and brain abnormalities (1), and detection of Zika virus RNA in clinical and tissue specimens can provide definitive laboratory evidence of recent Zika virus infection. Whereas duration of viremia is typically short, prolonged detection of Zika virus RNA in placental, fetal, and neonatal brain tissue has been reported and can provide key diagnostic information by confirming recent Zika virus infection (2). In accordance with recent guidance (3,4), CDC provides Zika virus testing of placental and fetal tissues in clinical situations where this information could add diagnostic value. This report describes the evaluation of formalin-fixed paraffin-embedded (FFPE) tissue specimens tested for Zika virus infection in 2016 and the contribution of this testing to the public health response. Among 546 live births with possible maternal Zika virus exposure, for which placental tissues were submitted by the 50 states and District of Columbia (DC), 60 (11%) were positive by Zika virus reverse transcription-polymerase chain reaction (RT-PCR). Among 81 pregnancy losses for which placental and/or fetal tissues were submitted, 18 (22%) were positive by Zika virus RT-PCR. Zika virus RT-PCR was positive on placental tissues from 38/363 (10%) live births with maternal serologic evidence of recent unspecified flavivirus infection and from 9/86 (10%) with negative maternal Zika virus immunoglobulin M (IgM) where possible maternal exposure occurred >12 weeks before serum collection. These results demonstrate that Zika virus RT-PCR testing of tissue specimens can provide a confirmed diagnosis of recent maternal Zika virus infection.

We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients’ isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection.

Background Pandemic 2009 H1N1 influenza A (pH1N1) virus has caused substantial morbidity and mortality globally and continues to circulate. Although pH1N1 viral antigens have been demonstrated in various human tissues by immunohistochemistry (IHC), cellular localization of pH1N1 RNA in these tissues has largely remained uninvestigated. Objectives To examine the distribution of pH1N1 RNA in tissues of fatal cases in order to understand the virus tissue tropism, replication and disease pathogenesis. Study design Formalin-fixed, paraffin embedded autopsy tissues from 21 patients with confirmed pH1N1 infection were analyzed by influenza A IHC and by in situ hybridization (ISH) using DIG-labeled sense (detects viral RNA) and antisense probes (detects positive-stranded mRNA and cRNA) targeting the nucleoprotein gene of pH1N1 virus. Results pH1N1 RNA was localized by ISH in 57% of cases while viral antigens were detected by IHC in 76%. However, in cases with a short duration of illness (1–3 days), more cases (69%) were positive by ISH than IHC (62%). Strong ISH staining was detected by antisense probes in the alveolar pneumocytes of the lungs, mucous glands and in lymph nodes. IHC staining of viral antigens was demonstrated in the lung pneumocytes and mucous glands, but no immunostaining was detected in any of the lymph nodes examined. Conclusions This study demonstrates cellular localization of positive-stranded pH1N1 RNA in the lungs, mucous glands and lymph nodes that suggests viral replication in these tissues. The novel ISH assay can be a useful adjunct for the detection of pH1N1 virus in tissues and for pathogenesis studies.

Context.—Paget disease is an uncommon skin manifestation of breast cancer, associated with either invasive carcinoma or ductal carcinoma in situ in the underlying breast. In very rare cases, tumor cells within the epidermis invade through the basement membrane of the skin into the dermis. Objectives.—To identify a series of cases of Paget disease with direct dermal invasion and to investigate the clinicopathologic features and outcome. Design.—Cases were identified during a 6-year period from the files of 2 hospitals. The clinical histories, imaging studies, and pathology reports were reviewed. Results.—Seven patients were identified, 5 with microinvasion ( Conclusions.—Patients with direct dermal invasion from Paget disease had a favorable outcome during the available follow-up period. This type of dermal involvement must be distinguished from locally advanced invasive carcinomas with skin invasion classified as T4b in the American Joint Cancer Commission staging system, as cancers with other types of skin invasion are associated with a poor prognosis.

Complex or radial sclerosing lesions (CSL/RSL) are uncommon diagnoses on core needle biopsy with a reported upgrade rate ranging between 0% and 23%. As a result, their management remains controversial. In this study, we sought to determine the rate of malignancy on excision for patients with pure CSL/RSL on core biopsy, and to evaluate future breast cancer risk when CSL/RSL is managed without excision. We retrospectively reviewed 118 cases of CSL/RSL diagnosed on image-guided breast biopsies between 2005 and 2014 at our institution. Of 98 analyzed patients, 34 (35%) underwent excision and 64 (65%) were observed. Demographic and clinical variables between excision and observation groups were compared. In excised specimens, factors associated with upgrade to malignancy were evaluated. The median age at diagnosis was 49 years (range, 27-88 years). In the excision group, 3/34 cases were associated with malignancy, an overall upgrade rate of 9%. All malignant cases had core needle biopsies interpreted as discordant and were BIRADS 4B or more on imaging. In the observation group, at a median follow-up of 2.2 years, 3/64 (5%) patients developed ipsilateral cancers, all of which were distant from the index CSL/RSL. In our series, we report a 9% malignancy rate on excision of BIRADS >4C lesions characterized as CSL/RSL on core biopsy. In patients with concordant biopsies and BIRADS 4A or lower lesions who underwent observation, we found a low rate of subsequent ipsilateral cancers. Further studies are needed to confirm that for CSL/RSL in concordant core biopsies and BIRADS 4A or lower, nonpalpable lesions, observation may be a reasonable alternative to excision.

During 2012-2014, five cases of Rickettsia parkeri rickettsiosis were identified by a single urgent care practice in Georgia, located approximately 40 miles southwest of Atlanta. Symptom onset occurred during June-October, and all patients had a known tick bite. Patients ranged in age from 27 to 72 years (median = 53 years), and all were male. The most commonly reported initial signs were erythema (n = 3) and swelling (n = 2) at the site of the bite. Two patients reported fever and a third patient reported a rash and lymphadenopathy without fever. Other symptoms included myalgia (n = 3), chills (n = 3), fatigue (n = 2), arthralgia (n = 2), and headache (n = 2). Eschar biopsy specimens were collected from each patient using a 4-mm or 5-mm punch and placed in 10% neutral buffered formalin or sterile saline. These specimens were tested by immunohistochemical (IHC) stains, quantitative polymerase chain reaction (qPCR) assays, or cell culture isolation to determine if there was evidence of infection with a Rickettsia species (1). IHC evidence of spotted fever group rickettsiae was found in the eschar biopsy specimens in all five cases. In four cases, the biopsy specimens were also positive for R. parkeri by qPCR. The fifth case (specimen positive only by IHC testing) was considered a probable R. parkeri case based on clinical signs and symptoms. R. parkeri was grown in cell culture from one specimen from which isolation was attempted. All patients were treated with oral doxycycline (100 mg twice daily) for a minimum of 10 days, and all recovered.

Rickettsia rickettsii, a bacterial tickborne pathogen that causes Rocky Mountain spotted fever (RMSF), stains poorly or not at all with conventional tissue Gram techniques, and contemporary visualization of the pathogen in formalin-fixed, paraffin-embedded tissues has relied almost entirely on immunohistochemical staining methods that are generally limited to specialized research laboratories or national reference centers. To our knowledge, previously described argyrophil-based histochemical techniques have not successfully detected rickettsiae in formalin-fixed, paraffin-embedded tissues. To investigate the ability of standard silver impregnation techniques to demonstrate the occurrence and distribution of R. rickettsii in tissues of patients with RMSF confirmed by molecular and immunohistochemical methods, three widely recognized and commercially available silver impregnation methods (Warthin-Starry, Steiner, and Dieterle's) were applied to various tissues obtained at autopsy from 10 patients with fatal RMSF. R. rickettsii bacteria were demonstrated in one or more tissues of all patients, using each of the argyrophil-based methods, and appeared as small, dark brown-to-black lanceolate rods, often in pairs and occasionally surrounded by a faint halo. Rickettsiae were identified most consistently in small arteries and arterioles of liver, kidney, and leptomeninges, and were localized predominantly to the cytoplasm of endothelial cells and less often within the internal elastic lamella and smooth muscle of the media. This validation of argyrophilic techniques to detect R. rickettsii demonstrates the utility of inexpensive core histochemical methods in the diagnosis of infectious agents in pathology specimens and may have utility in certain resource-limited settings where RMSF is endemic.

Background. Influenza B virus infection causes rates of hospitalization and influenza-associated pneumonia similar to seasonal influenza A virus infection and accounts for a substantial percentage of all influenza-related hospitalizations and deaths among those aged ,18 years; however, the pathogenesis of fatal influenza B virus infection is poorly described. Methods. Tissue samples obtained at autopsy from 45 case patients with fatal influenza B virus infection were evaluated by light microscopy and immunohistochemical assays for influenza B virus, various bacterial pathogens, and complement components C4d and C9, to identify the cellular tropism of influenza B virus, characterize concomitant bacterial pneumonia, and describe the spectrum of cardiopulmonary injury. Results. Viral antigens were localized to ciliated respiratory epithelium and cells of submucosal glands and ducts. Concomitant bacterial pneumonia, caused predominantly by Staphylococcus aureus, was identified in 38% of case patients and occurred with significantly greater frequency in those aged .18 years. Pathologic evidence of myocardial injury was identified in 69% of case patients for whom cardiac tissue samples were available for examination, predominantly in case patients aged ,18 years. Conclusions. Our findings suggest that bacterial pneumonia and cardiac injury contribute to fatal outcomes after infection with influenza B virus and that the frequency of these manifestations may be age related.

It is unknown why some apparently healthy persons become severely ill after influenza infection while others infected by the same strain remain asymptomatic or become only mildly ill. The presence of neutralizing antibody to a specific influenza strain is protective, and certain chronic medical conditions increase the risk for severe outcomes of influenza infections, but the risk factors for influenza-associated deaths among previously healthy persons remain largely unknown (1). Infectious disease mortality risk has a heritable component; children of parents who died of an infectious disease are ≈6× more likely to die of an infectious cause compared with the general population (2). A recent large family study that used genealogic databases found an elevated risk for influenza death among relatives of persons who died of influenza (3). By comparing the influenza mortality rate for relatives of persons who died of influenza with the influenza mortality rate for relatives of spouses of persons who died, the authors showed that the increased risk was not explained by shared exposure to influenza virus and thus may have a genetic component. However, to our knowledge, no published studies have examined the association between specific host genetic variants and severe influenza disease outcomes. To address the paucity of research on host genomics and influenza, the Centers for Disease Control (CDC) convened a meeting of experts in 2007 to solicit opinions on how to explore the role of host genomics in public health activities for influenza conducted by the agency. A study of host genomic factors related to severe influenza outcomes in children was recommended as an activity that CDC was well positioned to pursue. This article reports the findings of the study implemented in response to that recommendation. We conducted a hypothesis-generating pilot study to examine if host genetic variants were associated with fatal influenza virus infection by comparing prevalence of selected host genetic variants among children and young adults who died of influenza with population-based prevalence estimates. We focused on 8 single-nucleotide polymorphisms (SNPs) in 2 candidate genes important in the innate immune response to influenza infection and for which national prevalence estimates were available: the gene for tumor necrosis factor superfamily, member 2 (official symbol TNF) and the mannose-binding lectin gene (official symbol MBL2).

The recent influenza pandemic, caused by a novel H1N1 influenza A virus, as well as the seasonal influenza outbreaks caused by varieties of influenza A and B viruses, are responsible for hundreds of thousands of deaths worldwide. Few studies have evaluated the utility of real-time reverse transcription-PCR to detect influenza virus RNA from formalin-fixed, paraffin-embedded tissues obtained at autopsy. In this work, respiratory autopsy tissues from 442 suspect influenza cases were tested by real-time reverse transcription-PCR for seasonal influenza A and B and 2009 pandemic influenza A (H1N1) viruses and the results were compared to those obtained by immunohistochemistry. In total, 222 cases were positive by real-time reverse transcription-PCR, and of 218 real-time, reverse transcription-PCR-positive cases also tested by immunohistochemistry, only 107 were positive. Although formalin-fixed, paraffin-embedded tissues can be used for diagnosis, frozen tissues offer the best chance to make a postmortem diagnosis of influenza because these tissues possess nucleic acids that are less degraded and, as a consequence, provide longer sequence information than that obtained from fixed tissues. We also determined that testing of all available respiratory tissues is critical for optimal detection of influenza virus in postmortem tissues.

The purpose of our study was to determine which patient-related, target lesion-related, or procedure-related variables impact the duration of MRI-guided core needle breast biopsy.Between July 11, 2006, and September 26, 2007, data were collected for 75 single-target MRI-guided 9-gauge vacuum-assisted core needle biopsy procedures using a grid-guidance technique and performed at a single institution. The following variables were studied: MRI suite occupation time, number of operators, patient age and breast size, target morphology and location, approach to target, equipment used, number of image acquisitions and times the patient was moved in and out of the closed magnet, and occurrence of complications. Statistical analysis was performed using the Student's t test, analysis of variance, and Pearson's correlation, with p values0.05 considered significant.The mean duration was 57.9 minutes (SD, 17.2 minutes; range, 30-109 minutes). None of the patient- or target-related variables significantly impacted the duration, although lesions located in the anterior third of the breast showed a trend to prolong the procedure (p = 0.059). The time to complete a procedure was reduced when the operating radiologist was assisted by a breast imaging fellow-in-training (p = 0.01). Increasing numbers of image acquisitions and times the patient was moved in and out of the magnet significantly lengthened the procedure duration (p = 0.0001 for both). No major complications occurred. Biopsies yielded 16% (12/75) malignant and 84% (63/75) benign diagnoses.Variables that minimized procedure duration were number of image acquisitions, number of patient insertions or removals from the magnet, and assistance of a breast imaging fellow-in-training. No patient-related or target-related variables impacted procedure time.

The O-antigen-encoding region in the genomes of 14 isolates of Coxiella burnetii was examined by PCR. Five phase I isolates (Nine Mile clone 7, KAV, Ohio, Henzerling RSA 343, Q173) were analyzed and no deletions were detected. Two other isolates of unknown phase (Scottish, WAV) were examined, but no deletions were detected. In contrast, RSA 514 and three phase II isolates (Nine Mile phase II clone 4, Nine Mile phase II clone 1, Nine Mile Baca) contained large deletions, and the latter two were further characterized by DNA sequencing. Three other phase II isolates (Henzerling RSA 331, M44, Australian QD) contained no apparent deletions. Reactivity to phase I- and phase II-specific antibodies by immunofluorescence assay was used to further characterize isolates. Selected ORFs in Australian QD and M44 DNA were sequenced to detect mutations, and no significant changes were found. Australian QD RNA was examined by reverse transcriptase-PCR specific to the four ORFs hypothesized to encode the O-antigen sugar virenose, which this isolate has been shown to lack, as well as one that is predicted to encode part of the O-antigen ABC transporter. Each of these five genes was found to be expressed.

Massive placental perivillous fibrinoid deposition in the placenta is thought to be an immune-related condition associated with poor perinatal outcomes, including growth restriction and intrauterine fetal demise, with a high risk of recurrence. Rare cases have been associated with Coxsackievirus infection. We present such a case and review the literature.

Chemical−mechanical planarization (CMP) is a process of oxidizing and chelating the copper overburden present in an interconnect device while mechanically polishing the surface of the wafer. Because the use of condensed CO2 as the solvent for CMP would be environmentally and technically advantageous, several substituted bis(acetylacetonate)ethylenediimine (R4BAE, where R = CH3 or CF3) and lithium or sodium dialkyldithiocarbamate (M+(R2DTC-), where M+ = Li+ or Na+ and R = ethyl, n-propyl, n-butyl, or 1,1,1-trifluoroethyl) ligands were used with t-butylperacetate (t-BuPA, as oxidant) for the oxidative dissolution of copper(0) in supercritical (sc) CO2 at 40 °C and 170−210 bar or in hexanes at 40 °C and atmospheric pressure. The reaction products from the copper etching were determined to be Cu(R4BAE) or Cu(R2DTC)2, respectively. The R2DTC- ligands had higher etch rates than the R4BAE ligands with comparable substituents, and the lithium dialkyldithiocarbamate salts gave higher copper etching rates than thei...

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Carios capensis, a tick of seabirds, infests the nests of brown pelicans, Pelecanus occidentalis, and other ground nesting birds along the coast of South Carolina. This tick is associated with pelican nest abandonment and could pose a threat to humans visiting pelican rookeries if visitors are exposed to ticks harboring infectious agents. We collected ticks from a pelican rookery on Deveaux Bank, South Carolina and screened 64 individual ticks, six pools of larvae, and an egg mass for DNA from Bartonella, Borrelia, Coxiella, and Rickettsia by polymerase chain reaction amplification and sequencing. Ticks harbored DNA from "Borrelia lonestari", a novel Coxiella sp., and three species of Rickettsia, including Rickettsia felis and two undescribed Rickettsia spp. DNA from the Coxiella and two undescribed Rickettsia were detected in unfed larvae that emerged in the laboratory, which implies these agents are transmitted vertically by female ticks. We partially characterize the novel Coxiella by molecular means.

A versatile "top-down" method for the fabrication of particles, Particle Replication In Nonwetting Templates (PRINT), is described which affords absolute control over particle size, shape, and composition. This technique is versatile and general enough to fabricate particles with a variety of chemical structures, yet delicate enough to be compatible with sophisticated biological agents. Using PRINT, we have fabricated monodisperse particles of poly(ethylene glycol diacrylate), triacrylate resin, poly(lactic acid), and poly(pyrrole). Monodisperse particle populations, ranging from sub-200 nm nanoparticles to complex micron-scale objects, have been fabricated and harvested. PRINT uses low-surface energy, chemically resistant fluoropolymers as molding materials, which eliminates the formation of a residual interconnecting film between molded objects. Until now, the presence of this film has largely prevented particle fabrication using soft lithography. Importantly, we have demonstrated that PRINT affords the simple, straightforward encapsulation of a variety of important bioactive agents, including proteins, DNA, and small-molecule therapeutics, which indicates that PRINT can be used to fabricate next-generation particulate drug-delivery agents.

Phase-variable lipoproteins are commonly found in Mycoplasma species. Mycoplasma pulmonis contains a family of extensively studied phase- and size-variable lipoproteins encoded by the vsa locus. The Vsa surface proteins vary at a high frequency, the in vivo significance of which has yet to be determined. We investigated the role of Vsa phase variation in respect to tissue tropism and avoidance of the immune system in the mouse host. Mycoplasmas were cultured 3, 14, and 21 days postinoculation from the nose, lung, trachea, liver, and spleen of experimentally infected C57BL/6 (wild-type), C57BL/6-RAG-1 −/− (RAG −/− ), and C57BL/6-inducible nitric oxide synthase (iNOS) −/− (iNOS −/− ) mice. In wild-type and iNOS −/− mice, a large number of Vsa variants were seen by 21 days postinoculation. In contrast, little Vsa variation occurred in all tissues of RAG −/− mice. Analysis of isolates from 14 days postinoculation revealed less variation of the Vsa proteins in iNOS −/− mice than in the wild type. Western blot analysis of isolates from each strain of mouse demonstrated that Vsa phase variation occurred independently of size variation, indicating no obvious selection pressure for size variants. Additionally, these experiments provided no evidence that mycoplasmas producing particular Vsa proteins adhered only to specific tissues. The data strongly indicate that Vsa phase variation is a mechanism for avoidance of the immune system with no obvious contribution to tissue tropism.

ObjectiveThe goal of this study is to determine the impact of large core needle biopsy of suspicious breast lesions on the rate of malignancies found at surgical biopsy after wire localization.MethodsThe results of surgical biopsy after wire localization of non-palpable suspicious breast abn

Site-specific DNA invertible elements often control the production of bacterial surface proteins that are subject to phase variation (ON/OFF switching). Inversion of the DNA element occurs as a result of the reciprocal exchange of DNA catalysed by a specialized enzyme (recombinase) that acts at specific sites. By continually switching the orientation of the invertible element in the chromosome, and consequently the production of the variable protein(s), the cell population remains continually responsive to environmental change such as immunological challenge. In addition to phase-variable surface proteins, Mycoplasma pulmonis has a family of phase-variable restriction-modification enzymes. We report here that a single recombinase in M. pulmonis, HvsR, catalyses independent DNA inversions at non-homologous loci, causing variations in surface lipoproteins and in the DNA recognition sequence specificity of restriction enzymes. Thus, HvsR is a site-specific DNA recombinase with dual substrate specificity.

To the Editor: The influenza A pandemic (H1N1) 2009 virus rapidly created a global pandemic among humans and also appears to have strong infectivity for a broad range of animal species (1–3). The virus has been found repeatedly in swine and has been detected in a dog, cats, turkeys, and domestic ferrets and in nondomestic animals, including skunks, cheetahs, and giant anteaters (2–4). In some cases, animal-to-animal transmission may have occurred, raising concern about the development of new wildlife reservoirs (2). In 2009, the first recognized occurrence of pandemic (H1N1) 2009 in southern California in April was followed by a surge of cases during October through November (4). During this time, respiratory illness developed in a 12-year-old male American badger (Taxidea taxus taxus), a 19-year-old female Bornean binturong (Arctictis binturong penicillatus), and a 7-year-old black-footed ferret (Mustela nigripes) housed in a San Diego zoological garden. The 3 affected animals had clinical signs that included lethargy, inappetance, dyspnea, nasal discharge, and coughing. The severity of disease in the badger and binturong necessitated euthanasia; the ferret recovered with antibiotic and fluid therapy. Postmortem examination revealed bronchopneumonia with diffuse alveolar damage in the badger and interstitial pneumonia with diffuse alveolar damage in the binturong. Bacterial cultures and Gram stains of affected lung samples were negative. Molecular analyses for several groups of viruses, including Herpesviridae, Paramyxoviridae, Adenoviridae, and all influenza A viruses, were performed on frozen lung samples from the badger and binturong and on frozen conjunctival and pharyngeal swabs from the ferret. Results of PCRs specific for segments of influenza A nucleoprotein, matrix protein, hemagglutinin, and neuraminidase genes were positive in samples from all 3 animals, and DNA sequencing of amplicons identified the viruses as pandemic (H1N1) 2009. Influenza A virus was not detected in samples from the ferret after it recovered. Results of PCRs for all other viruses were negative. Immunohistochemical evaluation of lung samples from the badger for antigens of influenza A virus (5) showed rare staining in bronchiolar epithelial cells (Figure). Figure Lung section from an American badger showing immunohistochemical staining (red chromogen) specific for the pandemic (H1N1) 2009 virus within the nucleus and cytoplasm of bronchiolar epithelial cells and concurrent inflammatory cell infiltrates; hematoxylin ... Respiratory disease in all 3 affected animals seemed to be caused by pandemic (H1N1) 2009 virus. The badger and binturong were generally healthy, no other pathogens were detected, and pulmonary lesions were consistent with influenza pneumonia. In these animals, pandemic (H1N1) 2009 infection was especially aggressive, resulting in irreversible disease. Reports of pandemic (H1N1) 2009 virus in skunks and anteaters also describe severe disease in those species (2,3). In contrast, the infected black-footed ferret in our study had relatively mild clinical illness, consisting only of lethargy. This finding was surprising given recent experimental studies that reported the current pandemic (H1N1) 2009 virus was more pathogenic in domestic ferrets (Mustela putorius furo) than typical seasonal influenza viruses (6). However, several factors could have resulted in the low level of disease in this animal, such as prior immunity to influenza viruses or a low exposure dose. It is also possible black-footed ferrets are innately more resistant to influenza infection than domestic ferrets. The origin of infection in these cases was not determined but was most likely an infected human. All animals had some level of contact with caretakers or veterinarians and were housed separately from other wildlife species. None of the potential human sources of virus had clinical signs before the animals became ill; however, influenza infections in humans can often be mild (7). Wild animals, such as opossums and skunks, that occasionally enter the zoological garden, represent another possible source. Good hygiene and husbandry practices used within the enclosures of the badger, binturong, and ferret failed to prevent infection, which suggests pandemic (H1N1) 2009 is efficiently transmitted to these species. Descriptions of infection in giant anteaters and cheetahs kept under similar conditions also support high transmissibility of influenza A viruses to animals, as do ongoing findings for swine (3,4,8). Although ferrets are known to be susceptible to influenza A virus, to our knowledge, influenza in badgers and binturongs has not been reported. Badgers and binturong have been housed in zoological gardens for decades without incidence of influenza. Increased surveillance for influenza by the scientific community during the pandemic may have resulted in the novel recognition of infection in these species. Alternatively, the current pandemic (H1N1) 2009 virus may have a broader host range and stronger virulence than viruses in the past. Pandemic (H1N1) 2009 was first detected in humans in March 2009 and reached pandemic levels by June of that year, rapidly establishing a rich pool for the development of genetic variants. Naturally acquired disease has now been described in 10 animal species, and experimental infection has been reported in an additional 2 animals (mice and cynomolgus macaques) (9). The ubiquity of pandemic (H1N1) 2009 and its ability to infect a diverse range of hosts is worrisome for the health of wildlife and for the possibility of creating additional reservoirs that could alter the evolution of subtype H1N1 viruses by applying varied selection pressures and establishing new ways of generating unique reassortant strains.

Amblyomma maculatum (the Gulf Coast tick), an aggressive, human-biting, Nearctic and Neotropical tick, is the principal vector of Rickettsia parkeri in the United States. This pathogenic spotted fever group Rickettsia species has been identified in 8-52% of questing adult Gulf Coast ticks in the southeastern United States. To our knowledge, R. parkeri has not been reported previously from adult specimens of A. maculatum collected in Kansas or Oklahoma. A total of 216 adult A. maculatum ticks were collected from 18 counties in Kansas and Oklahoma during 2011-2014 and evaluated by molecular methods for evidence of infection with R. parkeri. No infections with this agent were identified; however, 47% of 94 ticks collected from Kansas and 73% of 122 ticks from Oklahoma were infected with "Candidatus Rickettsia andeanae" a spotted fever group Rickettsia species of undetermined pathogenicity. These preliminary data suggest that "Ca. R. andeanae" is well-adapted to survival in populations of A. maculatum in Kansas and Oklahoma, and that its ubiquity in Gulf Coast ticks in these states may effectively exclude R. parkeri from their shared arthropod host, which could diminish markedly or preclude entirely the occurrence of R. parkeri rickettsiosis in this region of the United States.