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Testing of clinical plasma specimens by the COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV), COBAS AMPLICOR CMV MONITOR Test (CAM CMV), and a laboratory-developed assay using analyte-specific reagents (LC CMV) demonstrated substantial result bias for CAP/CTM CMV versus CAM CMV (r = 0.436) and CAP/CTM CMV versus LC CMV (r = 0.773).

We evaluated a commercial multiplex polymerase chain reaction (PCR) assay in a cross-sectional study among 81 adult and pediatric outpatients-40 cases with upper respiratory infection symptoms and 41 asymptomatic controls-from February to April 2008. Two specimens (throat swab and nasal swab) from each participant were tested using the EraGen MultiCode-PLx Respiratory Virus Panel that detects 17 viral targets. Throat swabs were also tested for Group A Streptococcus (GAS) by PCR. Respiratory viruses were detected in 22/40 (55%) cases and in 3/41 (7%) controls (P < 0.001). GAS was detected in 10 (25%) cases; GAS and respiratory virus co-infection was found in 4 (10%). Agreement between nasal and throat swabs for viral detection was 69% in cases and 95% in controls. Of 22 cases with a detectable virus, 12 (54%) were picked up by only 1 (throat or nasal) specimen, and the detection rate was increased by combining results of nasal and throat swab testing.

This article covers the laboratory diagnosis of infections that occur predominantly in the tropics. The discussion includes diagnosis of blood and tissue parasites, intestinal parasites, and tropical infections caused by fungi, bacteria, and mycobacteria. The laboratory performance of techniques for the identification of intestinal parasites and special requirements for the collection of specimens for virology testing are also discussed. Images demonstrating the characteristic features of selected tropical parasites and fungi are included for reference.

This chapter covers protozoa, helminths, and arthropods. 1. Protozoa are single-celled, microscopic eukaryotic organisms like amebae and Giardia. Helminths are parasitic worms including nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). Arthropods, like ticks and mites, are generally considered parasites. Specific organisms reviewed include Giardia lamblia, Cyclospora cayetanensis, Blastocystis hominis, Entamoeba histolytica, Plasmodium falciparum, Babesia microti, and Toxoplasma gondii. Diagnosis and treatment of different types of infection are also reviewed.

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.

Acute gestational hepatitis from herpes simplex virus (HSV) infection is a rare but potentially life-threatening condition. We present the first reported case of primary HSV type 2 hepatitis in a pregnant woman who was diagnosed by detection of HSV-2 viremia via real-time polymerase chain reaction. The patient was successfully treated with acyclovir and delivered a healthy infant.

Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause.We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis.On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris.We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.).

In June, 2008, a 47-year-old woman presented to us with a 1-day history of crackling sensations in her left ear. On otoscopic examination, a tick was seen on the left tympanic membrane (fi gure). An otorhinolaryngologist removed the tick with endoscopic-guided forceps. There was no bleeding, perforation, hearing loss, or other sequelae. The crackling sensations resolved immediately. The tick was identifi ed as Dermacentor variabilis, commonly known as the American dog tick. Our patient lived in a rural area of Minnesota, USA where the tick-borne diseases lyme and human anaplasmosis are endemic. She denied any known tick exposure, but reported that her dog often slept in her bedroom at night. Fortunately, Dermacentor variabilis is not a primary vector for Borrelia burgdorferi and Anaplasma phagocytophilum, which cause lyme disease and human analasmosis respectively. D variabilis is a known vector of rocky mountain spotted fever and tularaemia, neither of which are common in Minnesota.

We aimed to determine the clinical features, predisposing factors, and outcome of left-sided Pseudomonas aeruginosa endocarditis in persons with no history of injection drug use. We performed a retrospective review of patient medical records from Mayo Clinic (Rochester, MN; Scottsdale, AZ; and Jacksonville, FL) for all cases of left-sided P. aeruginosa endocarditis. We identified 4 cases. We present these cases, as well as a review of the English-language medical literature. Data gathered included the year the case was reported; the valve involved; treatment, including valve replacement surgery; and outcome, if known. Left-sided P. aeruginosa endocarditis in persons without injection drug use is a rare but serious infection, with a history of instrumentation as a common predisposing condition. Valvular surgery is indicated, when possible, for the best chance of survival, along with extended therapy with combination antibiotics for complete recovery.

Pandemic (H1N1) 2009 influenza A virus originated due to triple reassortment of human, avian, and swine viruses (6). The pandemic epicenter was in Mexico, where the first human case was reported on 12 April 2009 (1). Thereafter, 16,813 pandemic H1N1 influenza A-related deaths have been reported to the World Health Organization (WHO) by 213 countries between April 2009 and March 2010 (8). From a laboratory perspective, detection and discrimination of pandemic H1N1 from seasonal and swine H1N1 is crucial for accurate diagnosis and disease surveillance. We previously described a laboratory-developed influenza A virus real-time reverse transcription-PCR (rRT-PCR) assay (Mayo FLU A) for simultaneous identification and subtype discrimination of influenza A virus RNA using melting temperature (Tm) analysis (3, 9). As discussed in the paper by Dhiman et al., loss of subtype discriminatory ability occurred in just 3 months of testing for the pandemic virus, due to multiple point mutations in the targeted 242-base region of the highly conserved matrix (M) gene as determined on a subset of 12 isolates (3). In the current paper, we have extended our analysis to a significantly larger data set to conduct mutational and amino acid analysis and characterize the genetic changes in the M gene of the pandemic H1N1 influenza A virus. Between 1 May 2009 and 31 December 2009, we detected 1,731 influenza A virus clinical isolates out of 9,614 isolates tested (18% positivity rate) using the Mayo FLU A assay; the majority were identified as the pandemic H1N1 subtype based on predefined Tm criteria (50.5°C to 53.2°C). However, between 14 August and 31 December 2009, 48 clinical samples were identified with Tms outside the validated range for pandemic H1N1 (48 of 1,579 isolates [3.0%] during this period), with an overall lower mean Tm of 46.71 ± 1.73°C. Only one specimen, from Ann Arbor, MI, had a higher Tm of 55.5°C, which is unexpected, given that mutations typically cause a decrease rather than an increase in Tm. The significance of this result is unknown, as we did not have additional specimens from that geographical area to confirm if this result was an actual trend or an outlier. All atypical Tm samples were confirmed as pandemic H1N1 using the CDC swine flu assay. We sequenced the 242-base oligonucleotide amplicon in 37 isolates to identify the mutations responsible for the atypical Tms. Mutational analysis revealed multiple point mutations in the 57-bp region detected by the probes compared to the original pandemic H1N1 gene sequence published by the WHO on 28 April 2009 (2) (Table ​(Table1).1). Six of these were silent mutations, resulting in nucleotide changes GAG→GAA, ATC→ATT, GCG→GCA, AGA→AGG, CTG→TTG, and AAC→AAT at amino acid positions 23, 24, 25, 27, 28, and 36, respectively, compared to the original pandemic H1N1 gene sequence. Of interest, two additional nonsynonymous mutations, CTG→ATG and ACA→ATA, resulted in downstream changes in amino acid sequence at positions Leu28Met and Thr37Ile, respectively (Fig. ​(Fig.1).1). We also observed geographic clustering among the mutations in these analyses (Table ​(Table11). FIG. 1. Amino acid sequence of the 57-bp discriminatory region of pandemic (H1N1) 2009 influenza A virus. TABLE 1. Sequence alignment in the discriminatory region of the amplified matrix gene from 37 clinical isolates with atypical Tms using the Mayo FLU A assay Since the emergence of pandemic H1N1, several studies have tried to characterize the mutational trends of this new virus (4, 5, 7). Pan et al. described the emergence of a signature residue at the position of nucleoprotein 100 (NP-100) (valine to isoleucine) that exhibited a dominant change in as many as 93% of isolates by the later phases of the pandemic. In addition, four nonsignature residues, at positions neuraminidase 91 (NA-91), NA-233, hemagglutinin 206 (HA-206), and nonstructural protein 1 (NS1) to NS123, were observed during a short period of time within the epidemic (5). All of these mutant residues were characterized in the viral functional domains, suggesting potent roles in the human adaption and virulence. Mutations were also observed in M1/M2 genes, but they were of low frequency with an unknown role. Potdar et al. reported several mutations throughout the viral genome, including a predominant D225G mutation in the H gene present in the receptor binding domain of Indian novel H1N1 isolates (7). Nelson et al. conducted a whole-genome phylogenetic analysis on 290 isolates of pandemic H1N1 influenza virus collected globally and identified seven major clades that have cocirculated worldwide since April 2009 (4). Several amino acid changes were also identified, predominantly in the HA, NA, and NP genes. However, no mutations were observed in the matrix gene in the latter two studies. In our study, we found eight mutations in a very short sequence span of only 57 bp of the influenza A virus matrix gene, of which two were nonsynonymous (Leu28Met and Thr37Ile). This is an unexpected finding, as the influenza A virus matrix gene is relatively conserved and is not thought to be under the same genetic selective pressures as other genes, such as HA and NA, that have very high mutability rates. The implications of these findings for viral adaptation and virulence have yet to be determined. However, from a diagnostic perspective, these mutations resulted in the loss of viral subtype discriminatory ability using Tm analysis within just 3 months of the pandemic and hindered laboratory diagnosis. Evidence for mutability in the highly conserved M gene of influenza virus calls for pandemic as well as routine in silico monitoring of primers and probes for optimal target coverage.

Influenza A viruses are well known for their genetic diversity. They are constantly evolving through point mutations in their hemagglutinin (H) and neuraminidase (N) genes and through genetic reassortment of their segmented RNA genome (2). The current pandemic/2009/novel H1N1 influenza A virus (called novel H1N1 herein) is a product of antigenic shift resulting from triple reassortment of human, avian, and swine viruses (7). Between 19 April 2009 and November 2009, 199 countries have reported nearly 6,000 novel influenza A H1N1-related deaths to the World Health Organization (WHO) (3). Several molecular tests have been developed for detection and discrimination of novel H1N1 and seasonal influenza A subtypes (1, 5, 6, 8). Notably, the Centers for Disease Control and Prevention (CDC) have developed two separate real-time reverse transcriptase PCR (rRT-PCR) TaqMan assays for detection and subtyping of the novel and seasonal influenza viruses. These assay target regions of the matrix (M) and H genes and require multiple PCRs for detection and subtyping (9). The Mayo Clinic uses a previously described influenza A rRT-PCR (Mayo FLU A) (10), which targets the M protein gene via specific primers and fluorescence resonance energy transfer (FRET) probes. Samples are extracted on the Roche MagNA Pure system and then amplified and detected using the Roche LightCycler 2.0. Reverse transcription and amplification take place in a one-step reaction, and subsequent melting temperature (Tm) analysis allows for confirmation of amplicon identity. Polymorphisms may also be detected through use of Tm analysis, since a single base mutation may cause a shift in the Tm. Early on in the novel H1N1 pandemic, we performed a pilot study to determine if discrimination between the three influenza A virus subtypes in current circulation (novel H1N1, seasonal H1N1, and seasonal H3N2) could be performed using Tm analysis. This would allow for simultaneous identification of influenza A RNA and subtype discrimination in a single rRT-PCR, without the need for a separate reflex assay. Included in the study were 104 novel H1N1, 37 seasonal H1N1, and 20 seasonal H3N2 isolates. The subtype of each isolate was confirmed by the CDC influenza A and swine flu panels and/or the xTAG respiratory viral panel (unsubtypeable samples positive for M protein but negative for seasonal H1 and H3 were assumed to be novel H1N1). This study showed that each major virus subtype had discrete, reproducible Tm ranges (50.5°C to 53.2°C, 47.1°C to 49.2°C, and 62.6°C to 66.8°C for novel H1N1, seasonal H1N1, and seasonal H3N2 isolates, respectively) (Fig. ​(Fig.11). FIG. 1. Melting curves and Tm data from confirmed clinical novel H1N1, seasonal H1N1, and seasonal H3N2 isolates in the original pilot study, showing the data distribution for each subtype. Tm is denoted by the vertical lines (top). The mean, median, and mode ... Between 1 May 2009 and 31 October 2009, we detected 1,414 influenza A clinical isolates out of 6,739 tested (20.98% positive), using the Mayo FLU A assay; the majority had a Tm between 50.5°C and 53.2°C and were identified as the novel H1N1 subtype. However, between 14 August and 31 October 2009, 19 clinical samples (19 patients) were identified with Tm values outside the validated range for novel H1N1 (19/1,261 [1.51%] isolates during this period); 18 were within or below the Tm range for seasonal H1N1 (mean ± 1 standard deviation [SD] = 47.0 ± 1.3°C), and one had a Tm between the validated range for novel H1N1 and that for seasonal H3N2 (55.5°C). All atypical Tm samples were identified as novel H1N1 by using the CDC swine flu assay (performed using the Roche LightCycler 480). To identify the mutations responsible for the atypical Tm values, sequencing of the 242-base oligonucleotide amplicon and alignment of the portions of the amplicon under the FRET probes were performed with 12 isolates. Analysis revealed multiple point mutations, compared to the original novel H1N1 gene sequence published by the WHO on 28 April 2009 (4) (Table ​(Table1).1). Of interest, some degree of geographic clustering was observed among the mutations in this small analysis. TABLE 1. Alignment of the sequences in the discriminatory region of the amplified M gene from 12 clinical isolates with atypical Tm values, using the Mayo FLU A assay Our results confirm that influenza A virus is constantly evolving and these mutations may have important implications in clinical diagnosis and possibly treatment, management, and vaccine development. In our case, we observed a loss of viral subtype discriminatory ability, using Tm analysis, within just 3 months of the pandemic. This was unexpected, given that the influenza A M gene is relatively conserved and is not thought to be under the same genetic selective pressures as the H and N genes. Fortunately, the longer length of the FRET probes, compared to that of alternative probe formats (e.g., TaqMan), and the ability to perform Tm analysis allows for continued detection of influenza A isolates with the Mayo FLU A assay, although subtyping cannot be performed. PCR-based systems that do not allow for detection of polymorphisms may lose sensitivity over time due to high mutability of the virus, and this may hinder laboratory diagnosis.

We previously developed a real-time polymerase chain reaction (PCR) assay for detection of the four Plasmodium species that infect humans. Recent studies have shown that natural transmission of the simian parasite Plasmodium knowlesi to humans occurs frequently in Southeast Asia. We have expanded our PCR assay to include detection of P. knowlesi.

Sarcocystis spp. are parasitic protists acquired when undercooked, cyst-laden meat is consumed. While both Sarcocystis hominis and S. cruzi encyst in beef, only S. hominis is pathogenic to humans. In this study, we used histological methods and novel molecular techniques to determine the regional prevalence and identity of Sarcocystis spp. in retail beef. Of 110 samples, 60 supported amplification of parasite rRNA by PCR. All 41 sequenced representatives were identified as S. cruzi. To compare detection methods, 48 samples were then examined in parallel by histology and PCR, and 16 and 26 samples, respectively, were positive. Five samples positive by initial histologic sections were not amplified by PCR. Fifteen PCR-positive samples did not contain sarcocysts on initial histologic section, but additional sections from these samples revealed sarcocysts in an additional 12 samples. When combined, histology with additional sections and PCR detected 31 positive specimens of the 48 total specimens. We found no evidence of human pathogen S. hominis and confirm that cattle pathogen S. cruzi is highly prevalent in this regional sample. PCR assays may increase the detection sensitivity of Sarcocystis spp. and contribute diagnostic precision.

Although parasites of the genus Sarcocystis have likely cycled between bovine herbivores and canine carnivores for tens of millions of years, humans may have profoundly influenced the ecology and evolution of those prevalent in domesticated dogs and cattle. To preliminarily assess the possibility of such anthropogenic effects, we surveyed genetic variation in conserved (18S small subunit) and variable (ITS-1) portions of ribosomal DNA from a large sample of Sarcocystis cruzi occurring in taurine beef cattle raised in the United States and Uruguay, and compared these data to available homologues, including those reported from zebu cattle, water buffalo, and bison. For additional context, we compared the apparent diversity of cattle parasites to that reported from congeneric parasites in other hosts. We find that the S. cruzi of taurine cattle, whether derived from the Americas or Asia, are devoid of variability in the sequenced portion (80%) of the small subunit rDNA. By contrast, geographically limited samples of related parasites in other hosts, including those of wildlife, are more variable. At the adjacent ITS-1 locus, allelic distribution patterns did not indicate any regional barriers to gene flow, suggesting that the parasite may have been introduced to the Americas via a common source such as domesticated dogs or cattle. Thus, human impact on this parasite's distribution and diversification would seem to have been great.

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 filamentous bacterium actinomyces can cause serious gynaecological tract infections, including pelvic inflammatory disease (PID) and tubo-ovarian abscess. Thus, definitive diagnosis of actinomycotic granules (AMGs) in gynaecological specimens is clinically important. Non-infectious pseudoactinomycotic radiate granules (PAMRAGs) can mimic the microscopic appearance of AMGs. PAMRAGs may be more common than actinomycotic infections in specimens from patients using intrauterine devices and may be seen in patients with PID. Although the composition and aetiology of PAMRAGs is unclear and variable, a panel of histochemical stains can aid in diagnosis. On haematoxylin and eosin (H&E) stained sections, AMGs show as distinct granules with basophilic peripheral radiating filaments and a dense central eosinophilic core, whereas H&E stained sections of PAMRAGs feature refractile granules with irregular club-like peripheral projections and no central dense core. The filaments of AMGs are Gram positive on Brown and Brenn (B&B) stain and are highlighted with Gomori methenamine silver stain (GMS). They stain negatively with a modified acid fast bacillus (AFB) stain, aiding in the distinction of actinomyces from nocardia. PAMRAGs show negative or non-specific staining with B&B, GMS, and AFB stains. Therefore, knowledge of these staining properties and the distinguishing characteristics of PAMRAGs and AMGs enables recognition of this important diagnostic pitfall.

Context.—Cutting injuries pose an infrequent but serious threat to anatomic pathology personnel. Although cut-resistant gloves may reduce this danger, it is imperative to recognize specific behaviors that increase the chance of an injury.Objective.—To examine the incidence of cutting injuries in an academic pathology department and the mechanisms by which such injuries occurred.Design.—Hospital Report of Event forms completed for laboratory incidents of cutting injury from March 1998 to September 2003 were evaluated. Further information regarding the incidents was obtained, when possible, by interviews with those personnel involved.Setting.—A university-based pathology laboratory was the setting for this study. On average, 505 autopsies and 29 000 surgical specimens were processed each year during the 5.5-year time period.Participants.—Pathology attending physicians, residents, dieners, and pathologists' assistants who performed autopsies and surgical specimen examinations.Results.—Eight scalpel injuries occurred during the study period. No needle-stick injuries were reported. Searching for lymph nodes and cutting firm tissue each accounted for 3 of the injuries. Only 2 of the 8 individuals were in compliance with the departmental policy regarding protective glove wear. Hospital Report of Event forms alone failed to elicit sufficient detail regarding the mechanism of injury.Conclusions.—A laboratory-based form may be necessary to supplement the hospital form, so as to obtain full details of each injury. This information may then be disseminated to all who handle blades, with the goal of preventing future cutting injuries.

Establishing an accurate primary invasive breast cancer size is crucial for patient management. Although ultrasonographic measurement is reported to correlate reliably with the gold standard pathology measurement, few authors have examined the influence of histologic subtype on ultrasound measurement. The common subtypes of invasive breast carcinoma, ductal and lobular, have different growth patterns, which may influence the ability of ultrasound to predict pathologic size. For this analysis, ultrasound and pathology reports were retrospectively reviewed for 204 women with 210 invasive breast cancers, including 129 ductal, 41 lobular, and 40 mixed pattern ductal and lobular carcinomas. For each tumor, the largest pathology and ultrasound dimensions were compared using Pearson's correlations, linear regression, paired t-tests and Wilcoxon signed ranks tests, stratified by histologic subtype. The Hodges-Lehmann approach was used to obtain 95% confidence intervals (CI) for median difference of the sizes. Ultrasonography consistently underestimated pathologic tumor size; the overall median difference was 3.5 mm (CI: 2.5-4.0 mm) and for subtypes: 2.5 mm (CI: 1.5-3.5 mm) for ductal pattern; 3.0 mm (CI: 1.5-4.5 mm) for mixed pattern; and in contrast, 7.5 mm (CI: 5.0-13.5 mm) for lobular pattern tumors. Significant correlations of similar magnitude, were observed between size measurements for ductal, lobular, and mixed subtypes (r=0.816, 0.811 and 0.672, respectively; all P

We report an unusual case of extraintestinal infection with adult Enterobius vermicularis worms in the nares and ocular orbit of a 14-year-old girl in Illinois. Only one other similar case has been reported in the English-language literature.

Washington (Wash) C. Winn Jr., (Figure) died suddenly and unexpectedly on July 3, 2011. A remarkable Renaissance man; a warm, humane person; and outstanding academic physician and scientist, Dr. Winn is remembered vividly as a contributor to understanding of emerging infectious diseases; a contemporary, efficient diagnostic clinical microbiologist; and a treasured educator. Born in Richmond, Virginia, on April 2, 1941, he was a true scholar, graduating magna cum laude with honors in English from Yale University in 1963. All who have been a co-author with, been edited by, or shared an educational venue with him have experienced a captivating creativity, mastery of expression, and improvement in one’s own communication through his skills with the language. Figure Washington C. Winn, Jr. Dr. Winn earned his doctor of medicine degree from the University of Virginia (1967) and deepened his experience as a physician during an internship in medicine at Tufts–New England Medical Center where he received the Medical Intern Award. Subsequently, he served as a resident in pathology at Washington University in St. Louis (1968–1970). Dr. Winn also earned a master’s degree in business administration at from the University of Vermont in 1993. A critical period in his life (1970–1973) occurred when he was a medical officer at the Center for Disease Control in Atlanta. He served in the Arbovirus Reference Unit and the Viral Pathology Branch, Division of Viral and Rickettsial Diseases, where he attributed the mentoring influence of Frederick A. Murphy on the subsequent direction of his professional career. His research on rhabdoviruses and arenaviruses led to his first 7 scholarly publications, including contributions to our knowledge of the pathology of Lassa fever, an archetypal emerging infectious disease. His colleagues of that era remember him as “a warm and wonderful guy and a true Virginia gentleman” (Thomas Monath, pers. comm., 2011), “who lived a life of real grace in everything he did or thought” (Karl Johnson, pers. comm., 2011). After 4 years as assistant professor of pathology at the University of Virginia (1973–1977), Dr. Winn moved to the University of Vermont where he served as director of the clinical microbiology laboratory for 34 years. An outbreak of Legionnaires’ disease in the hospital led to investigations of this disease, which was emerging at the time. As principal investigator of a National Institutes of Health grant, he contributed to our knowledge of the pathology of Legionella pneumoniae and the basic pathogenesis of legionellosis and to methods for the clinical microbiologic diagnosis of the disease, with 29 peer-reviewed publications on these topics. Dr. Winn then made a conscious positively motivated decision to focus his career on clinical microbiology, with resulting tremendous benefits to the field nationally and internationally and to patients in his institution. His clinical microbiology laboratory at Fletcher Allen Health Care, the clinical program of the University of Vermont, was a model of success. Through the decades, he assembled an exceptional technical staff. He had honed laboratory policies and optimized procedural details to such a degree that problems were essentially nonexistent. He kept the laboratory up to date with the latest technologies. In 2006, his goal was to incorporate real-time PCR into the laboratory—something that was not available in any clinical or anatomic pathology laboratory at the institution. Within just 5 years, he transitioned an essentially nonmolecular test menu to one with a full spectrum of qualitative and quantitative real-time PCRs that encompassed locally developed and Food and Drug Administration–approved assays. His national contributions to clinical microbiology included membership on the Microbiology Test Committee of the American Board of Pathology and service as chairman, vice chairman, and member and advisor of the Microbiology Resource Committee of the College of American Pathologists. In these roles, he is remembered not only as a knowledgeable scientist and diagnostician but also as a person of character and genuine concern for those with whom he worked. He was always willing to share what he knew in an unselfish way that was endearing to everyone who learned from him. Another great contribution to the field of clinical microbiology was Dr. Winn’s involvement in the influential textbook Color Atlas and Textbook of Diagnostic Microbiology, in which he joined Elmer Koneman as co-author of the third edition (1988) (1). He wrote chapters on virology, antimicrobial drug susceptibility testing, and new technologies in the diagnosis of infectious diseases in the next 3 editions and added a section on ectoparasites to the parasitology chapter in the fifth edition. He became chief editor of the sixth edition (2006) (2), which in the opinion of Dr. Koneman was the crown jewel in the life of the book (E. Koneman, pers. comm.). Memories of Dr. Winn are indelible and include his extraordinary wealth of knowledge and passion for opera, particularly when the scenes and music were linked to medical practice, such as the final arias sung by Violetta (La Traviata) and Mimi (La Boheme) before dying of tuberculosis. He will be remembered for his joy in playing the banjo and fiddle, love of good wines and diverse cuisines, learning the German language and Egyptian hieroglyphics, expressing staunch Republican opinions in a sea of liberal colleagues, and cultivating bonsai and ancient varieties of roses. His wisdom, humor, insight, honesty, and kindness will be missed tremendously. Dr. Winn was also a member of the Emerging Infectious Diseases review panel.

Answer: a dicotyledonous plant seed. A differential identification of the object initially included both a small intestinal fluke and a cross-section of a nematode, among other possibilities. However, close inspection of the object did not reveal internal structures, such as reproductive and

A 13-year-old male presented to the Riley Hospital for Children at Indiana University Health emergency department with a 2-day history of constipation and a 1-day history of rectal bleeding. He had also passed several stools containing blood following administration of a laxative. At the time of evaluation, the patient was afebrile and denied anti-inflammatory drug ingestion, illicit substance abuse, epistaxis, hematemesis, travel outside Indiana, and consumption of raw or undercooked meat. Digital rectal examination revealed an ulcer in the posterior wall and a hemorrhoid in the anterior wall, while the remainder of the physical examination was unremarkable. A complete blood count and coagulation studies indicated mild anemia and an elevated neutrophil count, but other parameter values were within normal limits. A radiograph of the child's abdomen was also performed, which showed only a moderate amount of gas and stool retention. The patient's medical history was significant for developmental delay and Nissen fundoplication for esophageal reflux. Based on these findings, the patient was admitted for further evaluation by the gastrointestinal service. A colonoscopy performed the following morning confirmed the presence of a raised, erythematous, and friable rectal ulcer, while the rest of the colon and terminal ileum appeared normal. Biopsy specimens of the colon, terminal ileum, and rectal ulcer obtained during the colonoscopy did not reveal an obvious cause of the rectal ulcer, and the intestinal mucosa was unremarkable. However, one hematoxylin-and-eosin-stained section of the colon biopsy specimen revealed an ovoid object containing abundant eosinophilic granules measuring 1.0 by 0.75 mm in the greatest dimension (Fig. 1) in the lumen. The pathologist queried as to the presence of an intestinal parasite. FIG 1 Hematoxylin-and-eosin-stained colon biopsy specimen of the ovoid object (left; ×100 total magnification). The higher magnification demonstrates the cellular structure and outer wall (right; ×600 total magnification). (For answer and discussion, see page 3138 in this issue [doi:10.1128/JCM.00700-13].)

Membranoproliferative glomerulonephritis (MPGN) is a pattern of injury characterized by mesangial and endocapillary proliferation, double contours along the capillary walls, and lobular accentuation of the capillary tufts.1 Based on pathophysiology, MPGN is classified into MPGN mediated by immune complexes and Igs and complement-mediated MPGN.1, 2 Immune complex/Ig–mediated MPGN most often results from an underlying infection, autoimmune disease, or monoclonal gammopathy.3, 4, 5 Complement-mediated MPGN includes C3 glomerulopathy, which encompasses C3 glomerulonephritis and dense deposit disease.6, 7 C3 glomerulopathy is characterized by dominant staining for C3 and negative or minimal staining for Igs.8 The staining for C3 is at least 2 orders greater in magnitude than Ig staining. C3 glomerulopathy results from dysregulation of the alternative pathway of complement with accumulation of complement factors of the alternative and terminal pathways of complement.9, 10 Infections are an important cause of MPGN that are usually associated with an immune complex–mediated MPGN. Immune complex–mediated MPGN due to infections is characterized by the presence of Ig, usually IgG or IgM, along with complement factors of the classical and terminal pathways. In this report, we present an unusual form of MPGN characterized by negative Ig but by large deposits of complement factors of the classical and terminal pathways in a patient with leishmaniasis and HIV infection. Furthermore, Leishmania species amastigotes were detected within macrophages in the interstitium. This finding of both complement-mediated MPGN and interstitial inflammation associated with leishmaniasis is extremely unusual and should be kept in mind as an uncommon cause of renal disease in the immunocompromised host.