Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection.

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

Antimicrobial resistance dashboard application for mapping environmental occurrence and resistant pathogens.

An antibiotic resistance (AR) Dashboard application is being developed regarding the occurrence of antibiotic resistance genes (ARG) and bacteria (ARB) in environmental and clinical settings. The application gathers and geospatially maps AR studies, reported occurrence and antibiograms, which can be downloaded for offline analysis. With the integration of multiple data sets, the database can be used on a regional or global scale to identify hot spots for ARGs and ARB; track and link spread and transmission, quantify environmental or human factors influencing presence and persistence of ARG harboring organisms; differentiate natural ARGs from those distributed via human or animal activity; cluster and compare ARGs connections in different environments and hosts; and identify genes that can be used as proxies to routinely monitor anthropogenic pollution. To initially populate and develop the AR Dashboard, a qPCR ARG array was tested with 30 surface waters, primary influent from three waste water treatment facilities, ten clinical isolates from a regional hospital and data from previously published studies including river, park soil and swine farm samples. Interested users are invited to download a beta version (available on iOS or Android), submit AR information using the application, and provide feedback on current and prospective functionalities.

Static self-directed sample dispensing into a series of reaction wells on a microfluidic card for parallel genetic detection of microbial pathogens.

A microfluidic card is described for simultaneous and rapid genetic detection of multiple microbial pathogens. The hydrophobic surface of native acrylic and a novel microfluidic mechanism termed "airlock" were used to dispense sample into a series of 64 reaction wells without the use of valves, external pumping peripherals, multiple layers, or vacuum assistance. This airlock mechanism was tested with dilutions of whole human blood, saliva, and urine, along with mock samples of varying viscosities and surface tensions. Samples spiked with genomic DNA (gDNA) or crude lysates from clinical bacterial isolates were tested with loop mediated isothermal amplification assays (LAMP) designed to target virulence and antibiotic resistance genes. Reactions were monitored in real time using the Gene-Z, which is a portable smartphone-driven system. Samples loaded correctly into the microfluidic card in 99.3% of instances. Amplification results confirmed no carryover of pre-dispensed primer between wells during sample loading, and no observable diffusion between adjacent wells during the 60 to 90 min isothermal reaction. Sensitivity was comparable between LAMP reactions tested within the microfluidic card and in conventional vials. Tests demonstrate that the airlock card works with various sample types, manufacturing techniques, and can potentially be used in many point-of-care diagnostics applications.

Spherules, hyphae, and air-crescent sign.

Coccidioidomycosis is endemic in the southwestern United States, resulting in 100,000 infections annually. The majority of these infections are asymptomatic or manifest as community-acquired pneumonia. In rare cases, patients can present with a mononuclear-cell predominant pyopneumothorax. The presence of spherules in tissue specimens is pathognomonic of this condition. A 72-year-old man born in Arizona with a heavy smoking history, presented with a 1-month history of weakness, night sweats, exertional dyspnea, and left pleuritic chest pain. The physical examination was remarkable for decreased breath sounds and dullness to percussion at the left lung base. His initial laboratory examination showed leukocytosis, eosinophilia, and elevated C-reactive protein. Computed tomography of the chest revealed a left lower lobe infiltrate, a cavity with air-crescent sign and hydropneumothorax. The pleural fluid was sampled and revealed an eosinophilic exudate with normal pH. Bacterial and fungal cultures of the pleural fluid were negative. Biopsy of the cavity wall showed chronic inflammation, fungal hyphae, and rare spherule-like structures. The surgical specimen culture grew Coccidioides immitis. Complement fixation for coccidioidomycosis performed on a serum sample was positive at a titer of 1:2 but a latex agglutinin test was negative. The patient was diagnosed with chronic fibrocavitary pneumonia with pyopneumothorax secondary to C. immitis infection and discharged on itraconazole for 1 year. Coccidioidomycosis can present in a variety of forms and should be part of the differential diagnosis in patients presenting with cavitation, air-crescent sign, eosinophilic pleural effusion, and hyphae and spherules on the tissue specimen. Chronic fibrocavitary pneumonia should be especially considered in patients who lived in endemic areas and have risk factors such as diabetes mellitus or pulmonary fibrosis related to smoking.

Decreased diversity of the fecal Microbiome in recurrent Clostridium difficile-associated diarrhea.

Antibiotic-associated diarrhea due to Clostridium difficile (CDAD) is thought to reflect colonization of a disrupted microbial community by the pathogen. We profiled the fecal microbiota of patients with CDAD (both initial and recurrent episodes) by culture-independent phylogenetic analysis of 16S rRNA-encoding gene sequences. Compared with those from control subjects and patients with an initial episode, the fecal communities in patients with recurrent CDAD were highly variable in bacterial composition and were characterized by markedly decreased diversity. Preservation and restoration of the microbial diversity could represent novel strategies for prevention and treatment of recurrent CDAD, which is often recalcitrant to existing therapies.

Genetic diversity of Campylobacter sp. isolates from retail chicken products and humans with gastroenteritis in Central Michigan.

Multilocus sequencing was used to compare Campylobacter sp. strains isolated from retail chicken products and humans with gastroenteritis in central Michigan. Sequence comparisons demonstrated overlapping diversity between chicken and human isolates. Campylobacter jejuni isolates from clinical sources had a greater diversity of flagellin alleles and a higher rate of quinolone resistance than isolates from retail chicken products.