Performance Evaluation of Fast Microfluidic Thermal Lysis of Bacteria for Diagnostic Sample Preparation.

Development of new diagnostic platforms that incorporate lab-on-a-chip technologies for portable assays is driving the need for rapid, simple, low cost methods to prepare samples for downstream processing or detection. An important component of the sample preparation process is cell lysis. In this work, a simple microfluidic thermal lysis device is used to quickly release intracellular nucleic acids and proteins without the need for additional reagents or beads used in traditional chemical or mechanical methods (e.g., chaotropic salts or bead beating). On-chip lysis is demonstrated in a multi-turn serpentine microchannel with external temperature control via an attached resistive heater. Lysis was confirmed for Escherichia coli by fluorescent viability assay, release of ATP measured with bioluminescent assay, release of DNA measured by fluorometry and qPCR, as well as bacterial culture. Results comparable to standard lysis techniques were achievable at temperatures greater than 65 °C and heating durations between 1 and 60 s.

Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH).

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Infectious disease immunohistochemistry in placentas from HIV-positive and HIV-negative patients.

Studies comparing placental pathology between human immunodeficiency virus (HIV)-positive and HIV-negative patients have shown conflicting results. In addition, few studies have evaluated the infectious etiology of placental inflammation in HIV-positive patients. We examined a cohort of placentas from 73 HIV-positive and 41 HIV-negative patients to gain a better understanding of the spectrum of placental inflammatory lesions. Bacterial and viral immunohistochemistry (IHC) was run on a subset of placentas (12 HIV-positive and 7 HIV-negative) with the greatest amount of inflammation. Although few histologic differences were seen between the HIV-positive and HIV-negative groups, chorioamnionitis was of a higher stage in the HIV-positive placentas. An infectious agent was found by IHC in 3 of 7 HIV-negative patients (2 Neisseria spp. and 1 group B Streptococcus ). One HIV-positive placenta showed gram-positive cocci on fetal membranes; organisms were not detected by IHC. In 2 patients, the etiologic agent was not suspected prior to IHC. This study identified that acute inflammation is less common in placentas from HIV-positive patients, compared with HIV-negative patients. However, when severe inflammation is present, infectious organisms may be identified by IHC, providing a more specific diagnosis and offering a beneficial impact in maternal and fetal management.

Usefulness of immunohistochemical diagnosis of streptococcus pneumoniae in formalin-fixed, paraffin-embedded specimens compared with culture and gram stain techniques.

Streptococcus pneumoniae is the most frequent cause of pneumonia and meningitis. Because S pneumoniae can colonize the upper respiratory tract and antibiotic treatment may inhibit growth, culture-based diagnosis can be problematic. An immunohistochemical assay using a polyclonal antibody against pneumococci was used to test formalin-fixed, paraffin-embedded tissue samples from 46 patients for whom bacterial culture results were available. Samples from 26 patients demonstrated pneumococcal antigens in areas of pneumonia, meningitis, or osteomyelitis or within circulating inflammatory cells. Various specimens from 18 patients grew S pneumoniae, whereas 8 had cultures that grew mixed bacteria or had no growth but were polymerase chain reaction-positive for the S pneumoniae Ity A gene. Pneumococcal antigens were not present in 20 cases (7 grew Streptococcus pyogenes; 9, Staphylococcus aureus; and 4, Haemophilus influenzae). Compared with culture, the immunohistochemical assay showed 100% sensitivity and 71% specificity. Immunohistochemical analysis has the diagnostic advantage of correlating host inflammatory reaction with presence of pneumococci.

Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor.

Animal models for severe acute respiratory syndrome (SARS) coronavirus infection of humans are needed to elucidate SARS pathogenesis and develop vaccines and antivirals. We developed transgenic mice expressing human angiotensin-converting enzyme 2, a functional receptor for the virus, under the regulation of a global promoter. A transgenic lineage, designated AC70, was among the best characterized against SARS coronavirus infection, showing weight loss and other clinical manifestations before reaching 100% mortality within 8 days after intranasal infection. High virus titers were detected in the lungs and brains of transgene-positive (Tg+) mice on days 1 and 3 after infection. Inflammatory mediators were also detected in these tissues, coinciding with high levels of virus replication. Lower virus titers were also detected in other tissues, including blood. In contrast, infected transgene-negative (Tg-) mice survived without showing any clinical illness. Pathologic examination suggests that the extensive involvement of the central nervous system likely contributed to the death of Tg+ mice, even though viral pneumonia was present. Preliminary studies with mice of a second lineage, AC63, in which the transgene expression was considerably less abundant than that in the AC70 line, revealed that virus replication was largely restricted to the lungs but not the brain. Importantly, despite significant weight loss, infected Tg+ AC63 mice eventually recovered from the illness without any mortality. The severity of the disease that developed in these transgenic mice--AC70 in particular--makes these mouse models valuable not only for evaluating the efficacy of antivirals and vaccines, but also for studying SARS coronavirus pathogenesis.

Histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season.

BACKGROUND: The Centers for Disease Control and Prevention enhanced national surveillance for influenza-associated deaths among children because of early reports of pediatric deaths during the 2003-2004 influenza season. METHODS: We studied lung and upper airway specimens from 47 case patients who died who had at least 1 positive result for influenza virus tests using hematoxylin and eosin, special stains for bacteria and fungi, and immunohistochemical (IHC) assays for influenza A and B viruses and other potential viral and bacterial respiratory pathogens. RESULTS: Nineteen (40%) of the 47 patients were

Transmission of lymphocytic choriomeningitis virus by organ transplantation.

BACKGROUND: In December 2003 and April 2005, signs and symptoms suggestive of infection developed in two groups of recipients of solid-organ transplants. Each cluster was investigated because diagnostic evaluations were unrevealing, and in each a common donor was recognized. METHODS: We examined clinical specimens from the two donors and eight recipients, using viral culture, electron microscopy, serologic testing, molecular analysis, and histopathological examination with immunohistochemical staining to identify a cause. Epidemiologic investigations, including interviews, environmental assessments, and medical-record reviews, were performed to characterize clinical courses and to determine the cause of the illnesses. RESULTS: Laboratory testing revealed lymphocytic choriomeningitis virus (LCMV) in all the recipients, with a single, unique strain of LCMV identified in each cluster. In both investigations, LCMV could not be detected in the organ donor. In the 2005 cluster, the donor had had contact in her home with a pet hamster infected with an LCMV strain identical to that detected in the organ recipients; no source of LCMV infection was found in the 2003 cluster. The transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within three weeks after transplantation. Diarrhea, peri-incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died, 9 to 76 days after transplantation. One recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. CONCLUSIONS: We document two clusters of LCMV infection transmitted through organ transplantation.