Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2024 Update by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM).

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by experts in both adult and pediatric laboratory and clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including arboviral Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also addressed. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

Psychodid flies and their implicated role in human myiasis and pseudomyiasis.

Several psychodid flies are commonly associated with human-inhabited environments and have been increasingly implicated in cases of human myiasis. However, the basic biology of psychodid larvae is not well-suited for survival in the human intestinal or urogenital tract, making true, prolonged myiasis unlikely. In this review, we performed a systematic literature review of published cases of purported myiasis caused by psychodid flies, their identification, associated clinical findings, and treatment. We also discuss the anatomy and lifecycle of psychodid flies in relation to their purported ability to use human tissue as a nutritive source and survive in the human alimentary or urogenital tracts. Based on the range of non-specific and varied reported clinical manifestations, lack of observed collections, life cycle patterns of psychodid flies, the mechanics of their mouthparts, and breathing requirements, we conclude that most cases likely represent incidental findings, or in rare cases possibly pseudomyiasis, rather than true myiasis, and provide recommendations for clinical evaluation and reporting so that disease misclassification and unnecessary therapy do not occur.

Anaplasma bovis-Like Infections in Humans, United States, 2015-2017.

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

Medical Parasitology Taxonomy Update, June 2020-June 2022.

The taxonomy of medically important parasites continues to evolve. This minireview provides an update of additions and updates in the field of human parasitology from June 2020 through June 2022. A list of previously reported nomenclatural changes that have not been broadly adapted by the medical community is also included.

16S rRNA Gene PCR/Sequencing of Heart Valves for Diagnosis of Infective Endocarditis in Routine Clinical Practice.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively (P = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively (P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice.

The Landscape of Parasitic Infections in the United States.

The landscape of parasitic infections in the United States has shifted dramatically over the past century. Although infections such as malaria have been successfully eliminated, others remain endemic and pose a significant public health risk. Numerous parasitic infections are also imported each year. This article focuses on endemic parasitic infections that may be commonly seen in anatomical pathology preparations and discusses their biology, diagnostic histopathological features, and epidemiology.

Imported Haycocknema perplexum Infection, United States1.

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

Activity of Omadacycline in Rat Methicillin-Resistant Staphylococcus aureus Osteomyelitis.

Omadacycline, vancomycin, and rifampin, as well as rifampin combination therapies, were evaluated in an experimental rat model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. All treatment groups had less MRSA recovered than saline-treated animals. The emergence of rifampin resistance was observed in 3 of 16 animals with rifampin monotherapy and none with rifampin combination therapy. After treatment, the median tibial bacterial loads were 6.04, 0.1, 4.81, and 5.24 log10 CFU/g for saline-, rifampin-, vancomycin-, and omadacycline-treated animals, respectively. Omadacycline or vancomycin administered with rifampin yielded no detectable MRSA. Omadacycline administered with rifampin deserves evaluation in humans as a potential treatment for osteomyelitis.

Pathogen Detection in Infective Endocarditis Using Targeted Metagenomics on Whole Blood and Plasma: a Prospective Pilot Study.

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.

Transmission electron microscopy on a case of Cyclospora cayetanensis infection from an immune-competent case confirms and extends prior detailed descriptions of its notably small endogenous stage.

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite's life cycle remain unknown. Humans are the only known hosts, existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. In histological sections, its stages are less than 10 µm, making definitive identification difficult. Here, confirmation of cyclosporiasis in a duodenal biopsy specimen from an 80-year-old man without any recognized immunodeficiency patient is reported. Asexual forms (schizonts) and sexual forms (gamonts) were located within enterocytes, including immature and mature schizonts, an immature male gamont and a female gamont. Merozoites were small (<5 µm × 1 µm) and contained two rhoptries, subterminal nucleus and numerous micronemes and amylopectin granules. These parasite stages were like those recently reported in the gallbladder of an immunocompromised patient, suggesting that the general life-cycle stages are not altered by immunosuppression.

Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice.

BACKGROUND: Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear. METHODS: We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019. RESULTS: A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2-31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens. CONCLUSION: 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

The Need for Dedicated Microbiology Leadership in the Clinical Microbiology Laboratory.

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.

Medical Parasitology Taxonomy Update, January 2018 to May 2020.

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.

Detection of Tick-Borne Bacteria from Whole Blood Using 16S Ribosomal RNA Gene PCR Followed by Next-Generation Sequencing.

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 may miss unexpected or novel pathogens. To address these shortcomings, we developed a 16S rRNA gene PCR with a next-generation sequencing (NGS) approach to detect tick-borne bacteria in whole blood. A workflow was optimized by comparing combinations of two extraction 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.

Evaluation of Self-Collected Midturbinate Nasal Swabs and Saliva for Detection of SARS-CoV-2 RNA.

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.

Novel Applications of Metagenomics for Detection of Tickborne Pathogens.

BACKGROUND: Tick populations have expanded in many parts of the globe, bringing with them an enhanced appreciation and discovery of novel tickborne pathogens, as well an increased in reported human cases of tickborne disease. Targeted and unbiased (shotgun) clinical metagenomic sequencing tests are increasingly used for detection of known and emerging infectious agents and have recently been employed for detection of tickborne pathogens. CONTENT: This review describes the types of metagenomic sequencing assays used for detection of emerging tickborne pathogens and reviews the recent literature on this topic. Important diagnostic and interpretative challenges are also covered. SUMMARY: Metagenomic analysis has emerged as a powerful tool for detection, discovery, characterization, and classification of tickborne pathogens. Shotgun metagenomics is especially promising because it allows for detection of all tickborne bacteria, viruses, and parasites in a single specimen. Despite the potential advantages, there are several important challenges, including high cost, complexity of testing and interpretation, and slow turnaround time. No doubt, these challenges will diminish with increased use and advances in the field.

Bed bugs are associated with anemia.

INTRODUCTION: Bed bugs are hematophagous insects that can be problematic in some urban emergency departments. The objective was to determine if red blood cell (RBC) and coagulation indices of bed bug-infested emergency department (ED) patients differed from those of noninfested control patients. METHODS: A chart review from a single health system was performed for ED patients between February 1, 2011, and February 1, 2017. Bed bug-infested patients were matched to noninfested control patients on the basis of age, sex, and the presenting ED. Variables were analyzed with the t-test and Pearson χ2 test and were modeled with multivariable logistic regression. RESULTS: The study had 332 bed bug-infested patients and 4952 controls. Infested patients had lower hemoglobin (11.7 g/dL vs 12.8 g/dL), hematocrit (35.0% vs 37.9%), RBC counts (4.1 × 109/L vs 4.4 × 109/L), mean corpuscular volume (86.0 vs 87.5 fL/cell), and mean corpuscular hemoglobin concentrations (33.2 vs 33.7 g/dL) and higher RBC distribution width-coefficient of variation (RDW-CV) (15.2% vs 14.2%) than noninfested patients (all P ≤ .003). Infested patients were more likely to be anemic (59.5% vs 36.9%) and to have severe anemia (4.4% vs 0.7%) (P < .001 for both). Blood transfusions were more common in those with bed bugs (5.1%) than those without bed bugs (2.3%) (P < .001). CONCLUSION: Bed bug infestated patients in the ED are associated with anemia.

Retrospective Review of Clinical Utility of Shotgun Metagenomic Sequencing Testing of Cerebrospinal Fluid from a U.S. Tertiary Care Medical Center.

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.

The Lyme Disease Biobank: Characterization of 550 Patient and Control Samples from the East Coast and Upper Midwest of the United States.

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.

Interpretable multimodal deep learning for real-time pan-tissue pan-disease pathology search on social media.

Pathologists are responsible for rapidly providing a diagnosis on critical health issues. Challenging cases benefit from additional opinions of pathologist colleagues. In addition to on-site colleagues, there is an active worldwide community of pathologists on social media for complementary opinions. Such access to pathologists worldwide has the capacity to improve diagnostic accuracy and generate broader consensus on next steps in patient care. From Twitter we curate 13,626 images from 6,351 tweets from 25 pathologists from 13 countries. We supplement the Twitter data with 113,161 images from 1,074,484 PubMed articles. We develop machine learning and deep learning models to (i) accurately identify histopathology stains, (ii) discriminate between tissues, and (iii) differentiate disease states. Area Under Receiver Operating Characteristic (AUROC) is 0.805-0.996 for these tasks. We repurpose the disease classifier to search for similar disease states given an image and clinical covariates. We report precision@k = 1 = 0.7618 ± 0.0018 (chance 0.397 ± 0.004, mean ±stdev ). The classifiers find that texture and tissue are important clinico-visual features of disease. Deep features trained only on natural images (e.g., cats and dogs) substantially improved search performance, while pathology-specific deep features and cell nuclei features further improved search to a lesser extent. We implement a social media bot (@pathobot on Twitter) to use the trained classifiers to aid pathologists in obtaining real-time feedback on challenging cases. If a social media post containing pathology text and images mentions the bot, the bot generates quantitative predictions of disease state (normal/artifact/infection/injury/nontumor, preneoplastic/benign/low-grade-malignant-potential, or malignant) and lists similar cases across social media and PubMed. Our project has become a globally distributed expert system that facilitates pathological diagnosis and brings expertise to underserved regions or hospitals with less expertise in a particular disease. This is the first pan-tissue pan-disease (i.e., from infection to malignancy) method for prediction and search on social media, and the first pathology study prospectively tested in public on social media. We will share data through http://pathobotology.org . We expect our project to cultivate a more connected world of physicians and improve patient care worldwide.