Search

Initial microbiologic diagnosis of infective endocarditis (IE) relies on blood cultures and Bartonella and Coxiella burnetii serology. Small case series and one prospective study have preliminarily reported application of metagenomic sequencing on blood or plasma for IE diagnosis. Here, results of a prospective pilot study evaluating targeted metagenomic sequencing (tMGS) for blood-based early pathogen detection and identification in IE are reported. Subjects diagnosed with possible or definite IE at a single institution were prospectively enrolled with informed consent from October 2020 to July 2021. Blood was drawn and separated into whole blood and plasma. Both specimen types were subjected to nucleic acid extraction and PCR targeting the V1-V3 region of the 16S ribosomal RNA gene, followed by next-generation sequencing on an Illumina MiSeqTM platform. 35 subjects, 28 (80%) with definite and 7 (20%) with possible IE were enrolled, including 6 (17%) with blood culture-negative endocarditis (BCNE). Overall, 20 whole blood (59%) and 16 plasma (47%) samples tested positive (P = 0.47). When results of whole blood and plasma testing were combined, a positive tMGS result was found in 23 subjects (66%). tMGS identified a potential pathogen in 5 of 6 culture-negative IE cases. Although further study is needed, the results of this pilot study suggest that blood-based tMGS may provide pathogen identification in subjects with IE, including in culture-negative cases.

Rapid and accurate diagnostic testing is essential to bring the ongoing COVID-19 pandemic to an end. As the demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing continues to increase amid supply shortages, many laboratories have investigated the use of sources other than nasopharyngeal (NP) swabs. Saliva and midturbinate (MT) nasal swabs are attractive alternatives, as they allow for self-collection and are well accepted by patients. Saliva also requires limited consumables. We compared the performance of health care provider-collected NP swabs, patient-collected MT swabs, and patient-collected saliva specimens for SARS-CoV-2 detection using a laboratory-developed PCR assay that had received Emergency Use Authorization by the FDA. Of 281 total evaluable samples, 33 (11.7%) NP swabs, 33 (11.7%) MT swabs, and 32 (11.4%) saliva specimens were positive for SARS-CoV-2 following resolution of discordant results. Compared to NP swabs, saliva exhibited a sensitivity of 90.9% (30/33) and specificity of 99.2% (246/248), while patient-collected MT swabs exhibited a sensitivity of 93.9% (31/33) and specificity of 99.2% (246/248). When comparing to the consensus standard, the sensitivity was found to be 100% (31/31) for both NP and MT swabs and 96.8% (30/31) for saliva specimens, while specificity was the same in both NP swabs and saliva specimens (98.8% [247/250]) and 99.2% (248/250) for MT swabs. Pretreatment of saliva with proteinase K and heating for 15 min prior to extraction reduced the invalid rate from 26.7% (52/195) to 0% (0/195). These data show that midturbinate nasal swabs and saliva are suitable sources for self-collection in individuals who require routine monitoring for SARS-CoV-2 infection.

Clinical microbiology laboratories play a crucial role in patient care using traditional and innovative diagnostics. Challenges faced by laboratories include emerging pathogens, rapidly evolving technologies, health care-acquired infections, antibiotic-resistant organisms, and diverse patient populations. Despite these challenges, many clinical microbiology laboratories in the United States are not directed by doctoral level microbiology-trained individuals with sufficient time dedicated to laboratory leadership. The manuscript highlights the need for medical microbiology laboratory directors with appropriate training and qualifications.

The taxonomy of parasites of medical and public health importance is rapidly evolving. This minireview provides an update of taxonomic revisions and additions in the field of medical parasitology from January 2018 to May 2020. Several established human parasites have been reassigned to different genera over the past 2 years, while a number of novel parasites of humans have been identified. A comprehensive summary of these changes is provided here, and Taenia suihominis is proposed as a replacement name for Taenia asiaticus Eom et al., which is a homonym of Taenia asiatica von Linstow.

Reported cases of tick-borne diseases have steadily increased for more than a decade. In the United States, a majority of tick-borne infections are caused by bacteria. Clinical diagnosis may be challenging as tick-borne diseases can present with similar symptoms. Laboratory diagnosis has historically relied on serologic methods, which have limited utility during the acute phase of disease. Pathogen-specific molecular methods have improved early diagnosis, but can be expensive when bundled together and miss unexpected or novel pathogens. To address these shortcomings, we developed a 16S ribosomal RNA (rRNA) gene PCR with next-generation sequencing approach to detect tick-borne bacteria in whole blood. A workflow was optimized by comparing combinations of two extractions platforms and two primer sets, ultimately pursuing DNA extraction from blood with the MagNA Pure 96 and PCR amplification using dual-priming oligonucleotide primers specific to the V1-V3 region of the 16S rRNA gene. The amplified product underwent modified Illumina 16S metagenomics sequencing library preparation and sequencing on a MiSeq V2 Nano flow cell, with data analysis using Pathogenomix RipSeq NGS software. Results with the developed method were compared to those from a V1-V2 16S rRNA gene primer set described by the Centers for Disease Control and Prevention (CDC). The V1-V3 assay demonstrated equivalent performance to the CDC assay, with each method showing concordance with targeted PCR results in 31 of 32 samples, and detecting 22 of 23 expected organisms. These data demonstrate the potential for using a broad-range bacterial detection approach for diagnosis of tick-borne bacterial infection from blood.

We would like to thank Drs. Mathirus Mungthin, Saovanee Leelayoova, and Suradej Siripattanapippong for their letter in response to our manuscript “Medical Parasitology Taxonomy Update, January 2018 to May 2020” published in Journal of Clinical Microbiology (2021) Vol. 59, Issue 2, 12 e01308-20 (1).…

The taxonomy of parasites of medical and public health importance is rapidly evolving. This minireview provides an update of taxonomic revisions and additions in the field of medical parasitology from January 2018 to May 2020. Several established human parasites have been reassigned to different genera over the past 2 years, while a number of novel parasites of humans have been identified. A comprehensive summary of these changes is provided here, and Taenia suihominis is proposed as a replacement name for Taenia asiaticus Eom et al.

A 25-year-old Caucasian American woman without significant medical history presented to the travel clinic with a 1-week history of worsening diarrhea and bloating. She had recently returned from an 8-month trip to Ethiopia, where she worked as a Peace Corps volunteer. While in Ethiopia, she resided

Shotgun metagenomic sequencing can detect nucleic acids from bacteria, fungi, viruses, and/or parasites in clinical specimens; however, little data exist to guide its optimal application to clinical practice. We retrospectively reviewed results of shotgun metagenomic sequencing testing requested on cerebrospinal fluid samples submitted to an outside reference laboratory from December 2017 through December 2019. Of the 53 samples from Mayo Clinic patients, 47 were requested by neurologists, with infectious diseases consultation in 23 cases. The majority of patients presented with difficult-to-diagnose subacute or chronic conditions. Positive results were reported for 9 (17%) Mayo Clinic patient samples, with 6 interpreted as likely contamination. Potential pathogens reported included bunyavirus, human herpesvirus 7, and enterovirus D-68, ultimately impacting care in two cases. Twenty-seven additional samples were submitted from Mayo Clinic Laboratories reference clients, with positive results reported for three (11%): two with potential pathogens (West Nile virus and Toxoplasma gondii) and one with Streptococcus species with other bacteria below the reporting threshold (considered to represent contamination). Of 68 negative results, 10 included comments on decreased sensitivity due to high DNA background (n = 5), high RNA background (n = 1), insufficient RNA read depth (n = 3), or quality control (QC) failure with an external RNA control (n = 1). The overall positive-result rate was 15% (12/80), with 58% (7/12) of these interpreted as being inconsistent with the patient’s clinical presentation. Overall, potential pathogens were found in a low percentage of cases, and positive results were often of unclear clinical significance. Testing was commonly employed in cases of diagnostic uncertainty and when immunotherapy was being considered.

Volume 57, no. 2, e01067-18, 2019, [https://doi.org/10.1128/JCM.01067-18][1]. In our article, Neobalantidium was proposed as a revised taxon to accommodate Balantidium coli . Since the publication of the article, we were made aware of the rediscovery of the genus Balantioides , which was described

On behalf of the Microbiology Committee of the College of American Pathologists (CAP), we respond to the recent commentary by Kirby and colleagues on the topic of bringing in new antimicrobial susceptibility testing ([1][1]). The authors state that “there is a lack of clarity regarding

Tick-borne diseases, due to a diversity of bacterial pathogens, represent a significant and increasing public health threat throughout the Northern Hemisphere. A high-throughput 16S V1-V2 rRNA gene-based metagenomics assay was developed and evaluated using13,000 residual samples from patients suspected of having tick-borne illness and1,000 controls. Taxonomic predictions for tick-borne bacteria were exceptionally accurate, as independently validated by secondary testing. Overall, 881 specimens were positive for bacterial tick-borne agents. Twelve tick-borne bacterial species were detected, including two novel pathogens, representing a 100% increase in the number of tick-borne bacteria identified compared to what was possible by initial PCR testing. In three blood specimens, two tick-borne bacteria were simultaneously detected. Seven bacteria, not known to be tick transmitted, were also confirmed to be unique to samples from persons suspected of having tick-borne illness. These results indicate that 16S V1-V2 metagenomics can greatly simplify diagnosis and accelerate the discovery of bacterial tick-borne pathogens.

Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository., Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository. Testing was performed to confirm the presence of tick-borne pathogens by real-time PCR, and a subset of samples was tested for Borrelia burgdorferi by culture. Serology was performed on all samples using the CDC’s standard two-tiered testing algorithm (STTTA) for LD. LD diagnosis was supported by laboratory testing in 82 cases, including positive results by use of the STTTA, PCR, or culture or positive results by two enzyme-linked immunosorbent assays for cases presenting with EM lesion sizes of >5 cm. The remaining 216 cases had negative laboratory testing results. For the controls, 43 were positive by at least one of the tiers and 6 were positive by use of the STTTA. The results obtained with this collection highlight and reinforce the known limitations of serologic testing in early LD, with only 29% of individuals presenting with EM lesion sizes of >5 cm yielding a positive result using the STTTA. Aliquots of whole blood, serum, and urine from clinically characterized patients with and without LD are available to investigators in academia and industry for evaluation or development of novel diagnostic assays for LD, to continue to improve upon currently available methods.

Parasite taxonomy continues to change as molecular and morphologic studies enhance our understanding of parasite relatedness. This minireview builds on the information provided in the last taxonomy update in this journal to summarize new and revised clinically relevant human parasite taxonomic changes that have occurred in 2016 and 2017.

There is substantial evidence that stool culture and parasitological examinations are of minimal to no value after 3 days of hospitalization. We implemented and studied the impact of a clinical decision support tool (CDST) to decrease the number of unnecessary stool cultures (STCUL), ova/parasite (O&P) examinations, and Giardia/Cryptosporidium enzyme immunoassay screens (GC-EIA) performed for patients hospitalized >3 days. We studied the frequency of stool studies ordered before or on day 3 and after day 3 of hospitalization (i.e., categorical orders/total number of orders) before and after this intervention and denoted the numbers and types of microorganisms detected within those time frames. This intervention, which corresponded to a custom-programmed hard-stop alert tool in the Epic hospital information system, allowed providers to override the intervention by calling the laboratory, if testing was deemed medically necessary. Comparative statistics were employed to determine significance, and cost savings were estimated based on our internal costs. Before the intervention, 129/670 (19.25%) O&P examinations, 47/204 (23.04%) GC-EIA, and 249/1,229 (20.26%) STCUL were ordered after 3 days of hospitalization. After the intervention, 46/521 (8.83%) O&P examinations, 27/157 (17.20%) GC-EIA, and 106/1,028 (10.31%) STCUL were ordered after 3 days of hospitalization. The proportions of reductions in the number of tests performed after 3 days and the associated P values were 54.1% for O&P examinations (P < 0.0001), 22.58% for GC-EIA (P = 0.2807), and 49.1% for STCUL (P < 0.0001). This was estimated to have resulted in $8,108.84 of cost savings. The electronic CDST resulted in a substantial reduction in the number of evaluations of stool cultures and the number of parasitological examinations for patients hospitalized for more than 3 days and in a cost savings while retaining the ability of the clinician to obtain these tests if clinically indicated.

In the United States, laboratories frequently offer multiple different assays for testing of cerebrospinal fluid (CSF) samples to provide laboratory support for the diagnosis of central nervous system Lyme disease (CNSLD). Often included among these diagnostic tests are the same enzyme immunoassays and immunoblots that are routinely used to detect the presence of antibodies to Borrelia burgdorferi in serum. However, performing these assays on CSF alone may yield positive results simply from passive diffusion of serum antibodies into the CSF. In addition, such tests are only U.S. Food and Drug Administration cleared and well validated for testing serum, not CSF. When performed using CSF, positive results from these assays do not establish the presence of intrathecal antibody production to B. burgdorferi and therefore should not be offered. The preferred test to detect intrathecal production of antibodies to B. burgdorferi is the antibody index assay, which corrects for passive diffusion of serum antibodies into CSF and requires testing of paired serum and CSF collected at approximately the same time. However, this assay also has limitations and should only be used to establish a diagnosis of CNSLD in conjunction with patient exposure history, clinical presentation, and other laboratory findings.

A process employing patient- or parent-collected pharyngeal swabs for group A Streptococcus (GAS) testing would expedite diagnosis and treatment, reduce patient exposure to the health care setting, and decrease health care costs. Our aim was to determine the concordance between patient- or parent-collected (self-collected) and health care worker (HCW)-collected pharyngeal swabs for detection of GAS by PCR. From 9 October 2012 to 21 March 2013, patients presenting with a sore throat meeting criteria for GAS testing and not meeting criteria for severe disease were offered the opportunity to collect their own pharyngeal swab. The HCW also collected a swab. Paired swabs were tested by GAS real-time PCR, allowing semiquantitative comparisons between positive results. Of the 402 participants, 206 had a swab collected by the patient and 196 a swab collected by the parent. The percent positivity results were 33.3% for HCW-collected swabs and 34.3% for self-collected swabs ( P = 0.41). The overall concordance between the two collection strategies was 94.0% (95% confidence interval [CI], 91.3 to 96.0). Twenty-four of the paired swabs had discordant results, with 10 and 14 positives detected only with the HCW- and self-collected swabs, respectively ( P = 0.41). The person collecting the swab in the self-collected arm, the order of collection, and prior swab collection training did not influence results. Among the 124 specimens that were positive by both collection methods, the amount of GAS DNA was higher in the self-collected versus the HCW-collected swabs ( P = 0.008). Self-collected pharyngeal swabs provide a reliable alternative to HCW collection for detection of GAS and offer a strategy for improved health care delivery.

We thank Hanscheid and Grobusch for their comments. While the focus of our report is the correct selection and use of specific laboratory tests for malaria diagnosis when the disease is suspected, we acknowledge that failure to include malaria in the differential diagnosis, particularly in settings

Microsporidia are highly specialized obligate intracellular organisms that are closely related to fungi. Although traditionally associated with diarrheal illness in patients with AIDS, extraintestinal infections involving various organs have been reported with increasing frequency in the past decade, particularly in immunocompromised hosts. Diagnosis is usually accomplished by light microscopic identification of spores in body fluids and tissues, using a variety of stains. Transmission electron microscopy, immunofluorescence assays, or molecular methods are necessary for identification to the genus and species level. Early diagnosis is essential for preventing the significant associated morbidity and mortality of extraintestinal microsporidiosis.

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Anisakidosis is a parasitic infection caused by anisakid nematodes in the genera Anisakis and Pseudoterranova . Infection is not uncommon in the United States due to increased raw seafood consumption. We report the first known case of parametrial anisakidosis in a 42-year-old woman and review existing literature.

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee ( n = 272) or hip ( n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture ( P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.

Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/μl for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10 −1 to 10 3 50% tissue culture infective dose (TCID 50 )/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs.

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia , Apophysomyces , Cunninghamella , Mucor , Rhizopus , and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/μl, and a specificity panel consisting of other filamentous fungi, yeasts ( Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.

We report an unusual case of a foreign body removed from the urinary bladder of a 63-year-old male which mimicked a parasitic worm. The foreign body was identified as an artificial fishing worm by morphological comparison to a similar commercially produced product and by infrared spectrum analysis.

This study measured the precision of a quantitative laboratory-developed real-time PCR test for cytomegalovirus performed at three different clinical laboratories that use the same methodology. The overall standard deviation (adjusted for analyte level) was 0.18 log 10 copies/ml, and there was no significant relationship between standard deviation and analytical measuring range.

A 65-year-old female with a history of multiple tick bites presented with fever and pancytopenia. Intracytoplasmic rickettsial morulae were detected on peripheral smear and bone marrow biopsy specimens, and PCR amplified Ehrlichia ewingii DNA from both specimens. To our knowledge, this is the first report of E. ewingii infection of human bone marrow.

A 65-year-old male with known hypertension and hypercholesterolemia sought medical attention because of a 3-month history of skin swelling on his upper back. Histopathology and molecular techniques were employed and identified an organism in the Onchocerca genus. This represents a very uncommon example of cutaneous infection by a zoonotic Onchocerca species.

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed ( Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

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.

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.

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].)