Diagnostic Testing for Zika Virus: a Postoutbreak Update.

Since the emergence and dissemination of Zika virus (ZIKV) in late 2015, our understanding of the biology, transmission, clinical disease, and potential sequelae associated with infection has markedly expanded. Over the past 2 years, the number of diagnostic assays for ZIKV has increased from none in 2015 to 5 serological assays and 14 molecular assays in 2017, all with emergency use authorization granted through the U.S. Food and Drug Administration. Here we provide an update on ZIKV, addressing what we have collectively learned since the outbreak began, including a summary of currently available diagnostic assays for this virus.

Rapid identification of bacteria and Candida using PNA-FISH from blood and peritoneal fluid cultures: a retrospective clinical study.

BACKGROUND: Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens. METHODS: Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH. RESULTS: In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI -92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI -57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day. CONCLUSIONS: In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.

Utility and Recommendations for the Use of Multiplex Molecular Gastrointestinal Pathogen Panels.

BACKGROUND: Many molecular gastrointestinal pathogen panels (GIPs) are Food and Drug Administration (FDA) cleared but it is still unclear how to best utilize these new diagnostic tools. GIPs are highly sensitive and specific, simultaneously detect multiple pathogens in one reaction, and can shorten the overall time of diagnosis for infectious gastroenteritis but are also expensive with relatively poor insurance reimbursement. CONTENT: In this review, we take a comprehensive approach to discuss issues with utilization of GIPs from a physician perspective, and implementation from a laboratory perspective. The information presented is to assist physicians in deciding on appropriate use of GIPs in diagnostic algorithms for their patients, and to provide information to laboratories that may be considering the addition of these powerful diagnostic assays to their test menu. Some of the important topics discussed are inpatient vs outpatient use, the appropriate panel size and organisms to include, interpretation of results, laboratory validation, and reimbursement. SUMMARY: The information in this review provides clear guidance to both clinicians and laboratories in deciding the best use of GIPs for a specific patient population. While this technology provides many benefits over traditional methods, it can also complicate result interpretation and comes with a high cost, which necessitates the need for use recommendations.

Viral pathogen detection using multiplex gastrointestinal molecular panels: The pros and cons of viral target inclusion.

Diagnosis of gastrointestinal infections has been revolutionized by the development of in vitro diagnostic (IVD) multiplex molecular panels for the detection of viral nucleic acids. In addition to a high degree of accuracy, these panels are commercially available and relatively simple to perform in the clinical laboratory. However, use of these panels must be carefully considered owing to the laboratory costs of the test, limited reimbursement, and potential for overuse. In this review from the Pan American Society for Clinical Virology, we focus on the viral components of GI multiplex panels (GIPs), presenting a brief overview of pathogens included on most panels and a discussion of advantages and challenges of the inclusion of viral targets on GIPs that should be considered before implementation in the clinical laboratory.

Classification of fungal genera from microscopic images using artificial intelligence.

Microscopic image examination is fundamental to clinical microbiology and often used as the first step to diagnose fungal infections. In this study, we present classification of pathogenic fungi from microscopic images using deep convolutional neural networks (CNN). We trained well-known CNN architectures such as DenseNet, Inception ResNet, InceptionV3, Xception, ResNet50, VGG16, and VGG19 to identify fungal species, and compared their performances. We collected 1079 images of 89 fungi genera and split our data into training, validation, and test datasets by 7:1:2 ratio. The DenseNet CNN model provided the best performance among other CNN architectures with overall accuracy of 65.35% for top 1 prediction and 75.19% accuracy for top 3 predictions for classification of 89 genera. The performance is further improved (>80%) after excluding rare genera with low sample occurrence and applying data augmentation techniques. For some particular fungal genera, we obtained 100% prediction accuracy. In summary, we present a deep learning approach that shows promising results in prediction of filamentous fungi identification from culture, which could be used to enhance diagnostic accuracy and decrease turnaround time to identification.

Invasive Nocardiosis Versus Colonization at a Tertiary Care Center: Clinical and Radiological Characteristics.

Objective: To describe the clinical and radiographic findings in a large cohort of patients with positive cultures for Nocardia emphasizing the differences between invasive disease and colonization. Patients and Methods: We conducted a single-center, retrospective cohort study of 133 patients with a positive Nocardia isolate between August 1, 1998, and November 30, 2018, and a computed tomography (CT) of the chest within 30 days before or after the bacteria isolation date. Results: Patients with colonization were older (71 vs 65 years; P=.004), frequently with chronic obstructive pulmonary disease (56.8% vs 16.9%; P<.001) and coronary artery disease (47.7% vs 27%, P=.021), and had Nocardia isolated exclusively from lung specimens (100% vs 83.1%; P=.003). On CT of the chest, they had frequent airway disease (84.1% vs 51.7%; P<.001). Patients with invasive nocardiosis had significantly (P<.05) more diabetes, chronic kidney disease, solid organ transplant, use of corticosteroids, antirejection drugs, and prophylactic sulfa. They had more fever (25.8% vs 2.3%; P<.001), cutaneous lesions (14.6% vs 0%; P=.005), fatigue (18% vs 0%; P=.001), pulmonary nodules (52.8% vs 27.3%; P=.006), and free-flowing pleural fluid (63.6% vs 29.4%; P=.024). The patterns of nodule distribution were different-diffuse for invasive nocardiosis and peribronchiolar for Nocardia colonization. Conclusion: The isolation of Nocardia in sputum from a patient with respiratory symptoms does not equal active infection. Only by combining clinical and chest CT findings, one could better differentiate between invasive nocardiosis and Nocardia colonization.

Use of Self-Collected Saliva Samples for the Detection of SARS-CoV-2.

OBJECTIVE: Using a US Food and Drug Administration (FDA) emergency use authorization (EUA) reverse transcription polymerase chain reaction (RT-PCR) method, we examined the analytic performance accuracy of saliva specimens as compared to nasopharyngeal (NP) specimens in symptomatic patients. Correlation between test results and symptoms was also evaluated. METHODS: Over a 5-week period in 2020, 89 matched saliva and nasopharyngeal swabs were collected from individuals exhibiting symptoms consistent with SARS-CoV-2. Specimens were tested with an FDA EUA-approved RT-PCR method, and performance characteristics were compared. RESULTS: The concordance rate between saliva and nasopharyngeal testing was 93.26%. The mean cycle threshold value of saliva when compared to the NP specimen was 3.56 cycles higher. As compared to NP swab, saliva testing demonstrates acceptable agreement but lower sensitivity. CONCLUSION: When compared to a reference method using NP swabs, the use of saliva testing proved to be a reliable method. Self-collected saliva testing for SARS-CoV-2 allows for a viable option when trained staff or collection materials are in short supply.

Precision across the analytical measuring range of a quantitative real-time PCR assay for cytomegalovirus detection among three clinical laboratories.

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.

The first case of Janibacter hoylei bacteremia in an adult.

The Janibacter species are Gram positive, coryneform bacteria that belong to the Actinobacteria phylum and have been linked to bacteremia in immunocompromised children. We present the first documented adult case of Janibacter hoylei bacteremia. The patient was a 52-year-old woman with a history of recurrent Clostridioides difficile infection, sinus tachycardia and high-risk AML who had been admitted one month prior to presentation for matched unrelated donor hematopoietic stem cell transplant with reduced intensity fludarabine-melphalan. Thirty days post-transplant, the infectious disease team was consulted because blood cultures grew Janibacter hoylei, from one of two blood cultures It took nine days to identify the species. She was treated with linezolid and imipenem. Janibacter are rarely implicated in human pathology, and therein, usually identified in the context of malignancy and relative immunosuppression. J. hoylei was only previously reported from the bloodstream of a previously healthy 8-week-old infant without underlying medical conditions. Antimicrobial susceptibility testing is challenging as only in vitro susceptibility testing of Janibacter terrae has been reported. Given these challenges, it is our hope to illustrate the clinical approach to diagnosis as well as subsequent recommendations for treatment in a particularly challenging case of bacteremia in an AML patient.

Invasive Nocardiosis in Transplant and Nontransplant Patients: 20-Year Experience in a Tertiary Care Center.

OBJECTIVE: To present the clinical characteristics and outcome of transplant and nontransplant patients with invasive nocardiosis. PATIENTS AND METHODS: We conducted a retrospective chart review of 110 patients 18 years and older diagnosed with culture-proven invasive nocardiosis (defined as the presence of clinical signs and/or radiographic abnormalities) between August 1, 1998, and November 30, 2018. Information on demographic, clinical, radiographic, and microbiological characteristics as well as mortality was collected. RESULTS: One hundred ten individuals with invasive nocardiosis were identified, of whom 54 (49%) were transplant and 56 nontransplant (51%) patients. Most transplant patients were kidney and lung recipients. The overall mean age was 64.9 years, and transplant patients had a higher prevalence of diabetes and chronic kidney disease. A substantial proportion of nontransplant patients were receiving corticosteroids (39%), immunosuppressive medications (16%), and chemotherapy (9%) and had chronic obstructive pulmonary disease (20%), rheumatologic conditions (18%), and malignant neoplasia (18%). A higher proportion of transplant patients (28%) than nontransplant patients (4%) received trimethoprim-sulfamethoxazole prophylaxis. In both groups, the lung was the most common site of infection. Seventy percent of all Nocardia species isolated were present in almost equal proportion: N brasiliensis (16%), N farcinica (16%), N nova (15%), N cyriacigeorgia (13%), and N asteroides (11%). More than 90% of isolates were susceptible to trimethoprim-sulfamethoxazole, linezolid, and amikacin. There was no significant difference in mortality between the 2 groups at 1, 6, and 12 months after the initial diagnosis. CONCLUSION: The frequency of invasive Nocardia infection was similar in transplant and nontransplant patients and mortality at 1, 6, and 12 months was similar in both groups. Trimethoprim-sulfamethoxazole prophylaxis failed to prevent Nocardia infection.

Performance Accuracy of a Laboratory-Developed Real-Time RT-PCR Method for Detection of SARS-CoV-2 in Self-Collected Saliva Specimens.

OBJECTIVE: The ongoing COVID-19 pandemic caused by SARS-CoV-2 has challenged diagnostic laboratories to re-examine traditional methods for collecting specimens and sample types used in molecular testing. Our goal was to demonstrate that saliva can be used for detecting SARS-CoV-2 and correlates well with established molecular methods using nasopharyngeal (NP) swabs. METHODS: We examined use of a saliva collection device in conjunction with a laboratory-developed real-time reverse transcription-polymerase chain reaction (LDPCR) method for detecting SARS-CoV-2 in a symptomatic population and compared results with 2 US Food and Drug Administration (FDA)-approved methods (emergency use authorization [EUA]) that use specimens from NP swabs. RESULTS: The sensitivity of LDPCR compared with the reference methods was 75.0% (21/28); specificity, 98.1% (104/106). When cycle threshold values were compared between paired specimens using the LDPCR and a EUA reverse transcription PCR method, both targeting the open-reading frame gene, the mean value for saliva was 4.66 cycles higher than for NP specimens. CONCLUSION: Use of self-collected saliva in conjunction with an LDPCR for SARS-CoV-2 compared favorably with 2 FDA EUA methods using NP swabs. The use of an alternative sample type and assay method will aid in expanding the availability of testing during the ongoing COVID-19 pandemic.

Reduction in Health Care Facility-Onset Clostridioides difficile Infection: A Quality Improvement Initiative.

OBJECTIVE: To reduce health care facility-onset (HCFO) Clostridioides difficile infection (CDI) incidence by improving diagnostic stewardship and reducing the inappropriate testing of C difficile assays. PATIENTS AND METHODS: A multidisciplinary team conducted a quality improvement initiative from January 1, 2020, through March 31, 2021. Clostridioides difficile infection and inappropriate testing were identified via electronic health records using predefined criteria related to stool quantity/caliber, confounding medications, and laboratory data. An intervention bundle was designed including (1) provider education, (2) implementation of an appropriate testing algorithm, (3) expert review of C difficile orders, and (4) batch testing of assays to facilitate review and cancellation if inappropriate. RESULTS: Compared with a baseline period from January to September 2020, implementation of our intervention bundle from December 2020 to March 2021 resulted in an 83.6% reduction in inappropriate orders tested and a 41.7% reduction in HCFO CDI incidence. CONCLUSION: A novel prevention bundle improved C difficile diagnostic stewardship and HCFO CDI incidence by reducing testing of inappropriate orders. Such initiatives targeting HCFO CDI may positively affect patient safety and hospital reimbursement.

Candida auris: An Emerging Yeast Pathogen Posing Distinct Challenges for Laboratory Diagnostics, Treatment, and Infection Prevention.

CONTEXT.­: Candida auris is an emerging yeast species that was first described in 2009. This ascomycetous yeast is notable for resistance to azole antifungal agents, for environmental persistence, and for its ability to contaminate health care environments, resulting in patient colonization and nosocomial infections. OBJECTIVE.­: To review the state of current knowledge addressing challenges in the accurate identification of C auris in the diagnostic microbiology laboratory, including application of phenotypic, proteomic, and genomic methodologies; characteristics that may predispose the human host to acquiring C auris; transmission; clinical presentations; treatment modalities; environmental decontamination; and infection prevention in health care settings. DATA SOURCES.­: The PubMed search engine was used to access peer-reviewed literature published from 2009 to 2019. CONCLUSIONS.­: The rapid emergence of C auris has presented unique challenges for the areas of laboratory diagnostics and infection prevention and in options for antifungal treatment, which are limited. The current lack of established antifungal susceptibility test breakpoints complicates therapeutic decision making. Enhanced awareness of this pathogen is essential to monitor outbreaks and to reduce the risk of spread within health care environments.

Real-time PCR method for detection of zygomycetes.

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/mul, 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.

Adult Intestinal Botulism: A Rare Presentation in an Immunocompromised Patient With Short Bowel Syndrome.

The cholinergic heat-labile neurotoxin produced by Clostridium species is primarily responsible for the clinical manifestations of botulism. The classic phenotypic presentation of botulism consists of subacute descending flaccid paralysis with intact sensory function. Traditionally, it is classified into 3 main forms (foodborne, wound-related, and infantile) on the basis of primary site of toxin entry into the human nervous system. Toxemia is the common pathophysiology in all forms of botulism. Adult intestinal toxemia botulism is an extremely rare form of the disease with pathogenesis similar to that of infant-type botulism. Symptomatic adults usually have an anatomic abnormality in the gastrointestinal tract leading to changes in normal gut flora. The current case is an addition to the growing literature on this unusual clinical variant of botulism.

Human Gongylonema pulchrum Infection: Esophageal Symptoms and Need for Prolonged Albendazole Therapy.

AbstractWe describe a case of human infection with Gongylonema pulchrum acquired in southeast Georgia. The patient presented with intermittent yet persistent nausea and vomiting for months. This case describes the need for extraction of worms on two occasions each followed by courses of albendazole treatment. Gongylonema pulchrum infections with high worm burden may relapse after extraction of the worm and a 3-day short course of albendazole therapy. Longer courses of albendazole may be indicated in selected circumstances.

Disseminated Mycobacterium interjectum Infection with Bacteremia, Hepatic and Pulmonary Involvement Associated with a Long-Term Catheter Infection.

We present a 49-year-old female with one year of intermittent fevers, chills, night sweats, and significant weight loss. Liver and lung biopsy showed evidence of a granulomatous process. Blood and liver biopsy cultures yielded growth of presumed Mycobacterium interjectum, thought to be related to a disseminated long-term central venous catheter infection. She successfully received one year of combined antimicrobial therapy after catheter removal without recurrence of disease. M. interjectum has been previously described as a cause of lymphadenitis in healthy children and associated with pulmonary disease in adults, although other localized infections have been reported. This is the first case described of a disseminated M. interjectum infection with bacteremia, hepatic and pulmonary involvement associated with a long-term catheter infection.

Morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review.

Previous studies have demonstrated high morbidity and mortality for adult patients with respiratory syncytial virus (RSV) infection. We performed a retrospective, multicenter, two-year chart review of all patients (n = 334) testing positive for RSV by the ProFlu + (®) Influenza A/B and RSV assay (Hologic, Bedford, MA). We analyzed indicators of morbidity and mortality in children <6 years old, immunocompetent and immunosuppressed adults, and transplant patients. Significant morbidity and mortality was observed among hematopoietic stem cell transplant patients (7.3%, 60-day mortality), solid organ transplant patients (13.3%, 60-day mortality), and COPD patients (12.8%, 60-day mortality). Of the patients positive for RSV, 144 (43.1%) of 334 received antibacterials or antifungals following diagnosis. Of these patients, a bacterial or fungal pathogen was not recovered from 60% of cases. Despite advances in RSV treatment, certain populations appear to be inadequately treated, while others appear to be inappropriately treated with unnecessary antimicrobials.