Rapid Clinical Diagnosis Research Articles

Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR.

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

Designing luminescent ruthenium prodrug for precise cancer therapy and rapid clinical diagnosis

The effective design of a targeted drug delivery system could improve the therapeutic efficacy of anticancer drugs by reducing their undesirable adsorption and toxic side effects. Here, an RGD-peptide functionalized and bioresponsive ruthenium prodrug (Ru-RGD) was designed for both cancer therapy and clinical diagnosis. This prodrug can be selectively delivered to cervical tumor sites to enhance theranostic efficacy. The benzimidazole-based ligand of the complex is susceptible to acidic conditions so, after reaching the tumor microenvironment, ligand substitution occurs and the therapeutic drug is released. The deep-red emissions produced by both one-photon and two-photon excitation increases the potential of Ru-RGD for use in the deep tissue imaging of 3D tumor spheroids. The specific accumulation of the Ru prodrug in tumor sites allows for precise tumor diagnosis and therapy in vivo. Luminescence staining of 38 clinical patient specimens shows that Ru-RGD exhibits differences in binding capability between cervical cancer and normal tissue, with a sensitivity of 95% and a specificity of 100%. This study thus provides an approach for the effective design and application of targeted metal complexes in cancer therapy and clinical diagnosis.

Blood pressure waveform contour analysis for assessing peripheral resistance changes in sepsis

BackgroundThis paper proposes a methodology for helping bridge the gap between the complex waveform information frequently available in an intensive care unit and the simple, lumped values favoured for rapid clinical diagnosis and management. This methodology employs a simple waveform contour analysis approach to compare aortic, femoral and central venous pressure waveforms on a beat-by-beat basis and extract lumped metrics pertaining to the pressure drop and pressure-pulse amplitude attenuation as blood passes through the various sections of systemic circulation.ResultsValidation encompasses a comparison between novel metrics and well-known, analogous clinical metrics such as mean arterial and venous pressures, across an animal model of induced sepsis. The novel metric Ofe → vc, the direct pressure offset between the femoral artery and vena cava, and the clinical metric, ΔMP, the difference between mean arterial and venous pressure, performed well. However, Ofe → vc reduced the optimal average time to sepsis detection after endotoxin infusion from 46.2 min for ΔMP to 11.6 min, for a slight increase in false positive rate from 1.8 to 6.2%. Thus, the novel Ofe → vc provided the best combination of specificity and sensitivity, assuming an equal weighting to both, of the metrics assessed.ConclusionsOverall, the potential of these novel metrics in the detection of diagnostic shifts in physiological behaviour, here driven by sepsis, is demonstrated.

Ultrasensitive Detection of HIV DNA with Polymerase Chain Reaction-Dynamic Light Scattering.

Early diagnosis of HIV biomarkers or genes is the key to reducing acquired immunodeficiency syndrome (AIDS) mortality. In our work, we developed a novel polymerase chain reaction-dynamic light scattering (PCR-DLS) assay for one-step sensitive detection of HIV DNA based on the average-diameter change of gold nanoparticles (AuNPs). This is the first PCR assay that makes use of the DLS technique as a signal read-out, with the particle size measured by DLS increasing with the concentration of target DNA. With the help of the AuNP probes, this PCR-DLS assay can effectively improve the specificity of PCR reactions, which can greatly increase the detection sensitivity, with a detection limit of 1.8 aM (S/N = 3). In addition, the proposed strategy was successfully used to analyze target DNA in human serum samples, indicating that the PCR-DLS assay has a promising potential application for rapid and early clinical diagnosis of HIV infection.

A novel electrochemical biosensor for HIV-related DNA detection based on toehold strand displacement reaction and cruciform DNA crystal

A sensitive and specific electrochemical biosensor was developed for quantitative detection of HIV-related DNA by integrating toehold strand displacement reaction (TSDR) with cruciform DNA crystal. The TSDR system can be specifically triggered by HIV-related DNA generating plentiful double-stranded DNA products and releasing the target DNA to undergo an additional cycle of amplification. The hybridization reaction of double-stranded DNA products with capture probes on Au electrode surface makes the binding sites of cruciform DNA crystal available. After the attachment of cruciform DNA crystal and the employment of streptavidinylated alkaline phosphatase, a significantly amplified electrochemical signal is obtained. This strategy was applied to the highly sensitive detection of HIV-related DNA in a linear range from 1 pM to 100 nM and a detection limit as low as 0.21 pM was achieved. The proposed strategy can be accomplished without the employment of any enzymes or intensive purification. In addition, this method has good selectivity and anti-interference ability, which could be applied in complex samples with comparable analytical performance. For these advantages, the proposed strategy holds great potential for rapid and early clinical diagnosis of HIV infection.

Capture of red blood cells onto optical sensor for rapid ABO blood group typing and erythrocyte counting

ABO blood group typing and erythrocyte counting are essential for blood transfusion especially in the vital lifesaving procedures. Here, we developed a simple, rapid and reliable assay for ABO blood group typing and erythrocyte counting based on localized surface plasmon resonance biosensor. The optical biosensors were constructed by the deposition of gold nanoprisms (GNPs) on glass substrates, and the immobilization of antibodies on the GNPs surface. The sensors were further integrated into a microfluidic chip containing two independent sample cells with anti-isoagglutinins A and anti-B antibodies modified at the bottoms, respectively. The sensing chips coupled with ultraviolet-visible (UV–vis) spectrometer were applied for the identification of blood type A, B, AB and O, respectively; meanwhile, a simple proof-of-principle strategy for quantifying red blood cells (RBCs) through the optical biosensors were demonstrated with series of RBCs dilution, and observed by UV–vis spectra. The limit of detection for erythrocyte counting was as low as ∼104 cells/mL (R2 = 0.99). Additionally, we compared our sensing results with those from conventional gel column assay on 7 different blood samples, in which the accuracy of blood typing was 100%. The RBC concentrations estimated by our sensor chips were between 3.9–5.0 × 109 cells/mL, which locates in the normal range of RBCs concentration of human. The LSPR biosensors that we developed are capable of qualitative ABO blood typing and quantitative erythrocyte counting simultaneously, which have great potential to develop a preliminary and lab-training-free biomedical device for rapid clinical diagnosis.

A Case Report of Incidental Hepatic Cysts Found on Ultrasound Imaging: Implications for the Anesthesiologist.

Point-of-care ultrasound imaging is increasingly used by anesthesiologists to facilitate rapid clinical diagnosis and treatment in the perioperative period. With its growing popularity, inevitably, unfamiliar structures will be observed. We present a case of cystic liver lesions in a healthy model during a point-of-care workshop. We address the clinical, ethical, and legal implications of incidental findings in anesthesiology. We offer recommendations and an algorithmic approach to these occurrences.

Detection of biomarkers of acute myocardial infarction by high-throughput suspension array technology in serum sample.

In order to assist and support early diagnosis of acute myocardial infarction (AMI), suspension array technology was established for multiplexed, rapid and accurate measurement of AMI biomarkers in serum samples. Methodology & results: It was developed by coating AMI biomarkers on distinguishable microbeads and competing with free biomarkers for complementary antibodies. The limits of detection of three AMI biomarkers were 2.5- to 50-times lower than that of the previous methods and the working ranges were four to five orders of magnitude. Accuracy and stability also met satisfying acceptance criteria in both of the intra- and interbatch testing. The variation coefficients and relative standard deviations were all less than 10%. Suspension array technology is completely applicable for requirements of rapid clinical diagnosis in serum sample.

Same-day Detection of Methicillin Resistance in Staphylococcus aureus Isolates by StaResMet® Kit

Background: Staphylococcus aureus is an important cause of hospital-acquired infections. The most important issue with S. aureus is that the isolates are getting increasingly methicillin-resistant. Rapid differentiation between methicillin-resistant and methicillin-susceptible S. aureus species is necessary to optimize treatment and minimize costs. Objectives: The current study aimed at evaluating the StaResMet® kit for rapid detection of methicillin resistance in S. aureus isolates. Methods: A total of 217 methicillin-resistant S. aureus (MRSA) and 252 methicillin-susceptible S. aureus (MSSA) isolates were tested using the StaResMet® kit. The test was performed according to the manufacturer’s instructions. Results: The kit identified the MRSA isolates with 100% accuracy, and found that the minimum inhibitory concentrations (MICs) was > 32 µg/mL cefoxitin for 133 of the isolates, 32 µg/mL cefoxitin for 49, 16 µg/mL cefoxitin for 8, and 8 µg/mL cefoxitin for 11 of the isolates. Likewise, all 177 MSSA isolates were correctly identified using the kit, and the MICs against them were determined within the range of 1 to 4 µg/mL cefoxitin. The MIC of cefoxitin can be determined in 6 hours using the StaResMet® kit. Conclusions: The obtained results indicated that the StaResMet® kit allowed the detection of MRSA isolates rapidly and reliably, and could be a valuable tool for microbiology laboratories with limited facilities. Authors believed that the routine use of this time-saving and easy-to-use test contributes to rapid clinical diagnoses and treatments.

Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevices

Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection.

A portable microreactor with minimal accessories for polymerase chain reaction: application to the determination of foodborne pathogens

The authors describe a microreactor for performing a multiplexed polymerase chain reaction (PCR) which is operated with minimal accessories such as a single heater for gene amplification and a hand-held syringe for sample actuation. It was fabricated by wrapping a polytetrafluoroethylene (PTFE) tube around a rectangular poly(dimethylsiloxane) (PDMS) block having a predetermined thickness. The resulting portable microreactor was applied to PCR using a single heater because the rectangular PDMS block spatially segregated the upper and lower arrays of the PTFE tube. This warrants the adjustment of distinct temperatures inside the upper and lower tubes. A hand-held plastic syringe was connected to the inlet of the PTFE tube and used as a portable pump to achieve a homogeneous transport of a sample liquid inside the microreactor. The use of gas-impermeable PTFE prevents the formation of bubbles during thermal cycling. The microreactor was successfully applied to the amplification of typical DNA fragments of three foodborne pathogens in less than 30 min. In our perception, this method paves the way to the construction of a truly portable PCR chip that is applicable to rapid clinical diagnosis and the detection of foodborne pathogens.

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope

ObjectiveTo develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope. MethodsThe lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. ResultsBased on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. ConclusionThe new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

High Efficiency Binding Aptamers for a Wide Range of Bacterial Sepsis Agents.

Sepsis is a major health problem worldwide, with an extremely high rate of morbidity and mortality, partly due to delayed diagnosis during early disease. Currently, sepsis diagnosis requires bacterial culturing of blood samples over several days, whereas PCR-based molecular diagnosis methods are faster but lack sensitivity. The use of biosensors containing nucleic acid aptamers that bind targets with high affinity and specificity could accelerate sepsis diagnosis. Previously, we used the systematic evolution of ligands by exponential enrichment technique to develop the aptamers Antibac1 and Antibac2, targeting the ubiquitous bacterial peptidoglycan. Here, we show that these aptamers bind to four gram-positive and seven gram-negative bacterial sepsis agents with high binding efficiency. Thus, these aptamers could be used in combination as biological recognition elements in the development of biosensors that are an alternative to rapid bacteria detection, since they could provide culture and amplification-free tests for rapid clinical sepsis diagnosis.

Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.

Glass capillary based microfluidic ELISA for rapid diagnostics.

Enzyme-linked immunosorbent assay (ELISA) is widely used in medical diagnostics and fundamental biological research due to its high specificity and reproducibility. However, the traditional 96-well-plate based ELISA still suffers from several notable drawbacks, such as long assay time (4-6 hours), burdensome procedures and large sample/reagent volumes (∼100 μl), which significantly limit traditional ELISA's applications in rapid clinical diagnosis and quasi-real-time prognosis of some fast-developing diseases. Here, we developed a user friendly glass capillary array based microfluidic ELISA device. Benefiting from the high surface-to-volume ratio of the capillary and the rapid chemiluminescent photo-imaging method with a commercial camera, our capillary based ELISA device significantly reduced the sample volume to 20 μL and shortened the total assay time to as short as 16 minutes (including detection time), which represent approximately 10-fold and 5-fold reduction in assay time and sample volume, respectively, in comparison with the traditional plate-based method. Furthermore, through the double exposure method, a nearly 10-fold increase in the detection dynamic range was achieved over the traditional well-based ELISA. Our device can be broadly used in rapid biochemical analysis for biomedicine and research/development laboratories.

Discrimination between Legionnaires' Disease and Pneumococcal Pneumonia Based on the Clinical and Laboratory Features: A Quantitative Approach Using the Modified Winthrop-University Hospital Weighted Point System.

Objective Legionnaires' disease (LD) is a common form of lobar pneumonia, but the optimum diagnostic modality has long been a subject of debate due to incomplete sensitivity and specificity. A delay in the initiation of specific therapy for LD is associated with increased mortality. The decision to treat a patient for Legionella must be made quickly. The purpose of this study was to evaluate the ability of the modified Winthrop-University Hospital WUH system to identify LD while discriminating against pneumococcal pneumonia at the time of hospitalization for community-acquired pneumonia. Methods Five patients with LD and 13 patients with pneumococcal pneumonia were retrospectively analyzed. Results The WUH system identified 4 of 5 patients with LD (sensitivity, 80%) while excluding legionellosis in 12 of 13 patients with pneumococcal pneumonia (specificity, 92%). The positive and negative likelihood ratios were 10.4 and 0.2. The area under the receiver operating characteristic curve was 0.969. Conclusion The WUH system is useful for obtaining a rapid presumptive clinical diagnosis of LD. Further investigation with a larger number of patients is strongly recommended.

Diagnostic Utility and Pitfalls of Tzanck Smear Cytology in Diagnosis of Various Cutaneous Lesions.

Aims:Tzanck smear is an old but useful test for diagnosis of cutaneous dermatoses. The aim of this study was to highlight the potential usefulness and diagnostic pitfalls of Tzanck smear for diagnosis of cutaneous dermatoses and infections.Materials and Methods:This hospital based cross-sectional study was carried out on all Tzanck smears received for a period of twenty months (January 2014–August 2015). The smears were assessed to establish the utility of Tzanck smears in corroborating or excluding a diagnosis of immunobullous lesion or herpetic infection. Cases with discrepant diagnosis on histopathology were reviewed to identify additional cytomorphological features.Results:A total of 57 Tzanck smears were performed during the study period. Out of the 18 clinically suspected cases of immunobullous disorders, Tzanck smear findings corroborated the clinical diagnosis in 7/18 cases, one case was diagnosed as cutaneous candidiasis, and diagnosis of immunobullous lesions could be excluded in 5/18 cases. Out of the 19 suspected cases of herpetic infections, viral cytopathic effect was observed in 8/19 cases. Besides immunobullous lesions and herpetic infections, acantholytic cells were also observed in spongiotic dermatitis and genodermatosis. Dyskeratotic keratinocytes seen in vacuolar interface dermatitis were not easily distinguishable from acantholytic cells on Tzanck smear.Conclusions:Tzanck smear test is an inexpensive and useful diagnostic tool for certain skin diseases. It can aid in establishing a rapid clinical diagnosis and can serve as a useful adjunct to routine histological examination. We recommend the use of Tzanck smear as a first-line investigation for vesiculobullous, erosive, and pustular lesions.

A Case Report on Primary Hepatic Amyloidosis

Background: Hepatic amyloidosis is a rare disease that presents as an infiltrative disease involving the liver. Amyloidosis is a systemic disease characterized by extracellular deposition of amyloid protein in many organs. Progressive organ involvement leads to organ malfunction and death usually resulting from renal and/or cardiac involvement. Liver and spleen are major sites of involvement. The wide range of presenting symptoms encountered makes rapid clinical diagnosis difficult. None of the present imaging techniques is capable of specifically demonstrating the presence of amyloid. Even when suspected clinically and radiologically, the diagnosis of amyloidosis depends on a tissue biopsy to confirm the presence of amyloid deposits. Case Report: A 38year old female was admitted with the complaint of bilateral pedal edema for the last one month. It was associated with loss of weight (10 kg). There was no history of decreased urine output, shortness of breath, abdominal distension or PND episodes. On examination their was a large palpable liver which was firm in consistency. Her CBP, RFTs, ECG, 2D-Echo were completely normal. LFTs showed elevated SGOT (88 U/L) and SGPT (96 U/L) with other parameters being completely normal. USG of abdomen confirmed hepatomegaly (17 cm) with normal echogenicity. CECT-abdomen confirmed similar findings. Liver biopsy was performed with an 18 G Bard gun-biopsy needle. Microscopically, diffuse amyloid deposits were found and the Congo red stain was positive. Additional examinations were done to evaluate the extent of amyloidosis involvement. Bone marrow aspirate showed a small increase in the percentage of plasma cells 8%. Colonic mucosal biopsy also showed amyloidosis. These examinations confirmed the diagnosis of AL, which was later, classified as primary systemic AL. The authors have none to declare.

Toward a comprehensive care of HIV patients: finding a strategy to detect depression in a Spanish HIV cohort

ABSTRACTDepression is a common but frequently undiagnosed feature in individuals with HIV infection. To find a strategy to detect depression in a non-specialized clinical setting, the overall performance of the Hospital Anxiety and Depression Scale (HADS) and the depression identification questions proposed by the European AIDS Clinical Society (EACS) guidelines were assessed in a descriptive cross-sectional study of 113 patients with HIV infection. The clinician asked the two screening questions that were proposed under the EACS guidelines and requested patients to complete the HADS. A psychiatrist or psychologist administered semi-structured clinical interviews to yield psychiatric diagnoses of depression (gold standard). A receiver operating characteristic (ROC) analysis for the HADS-Depression (HADS-D) subscale indicated that the best sensitivity and specificity were obtained between the cut-off points of 5 and 8, and the ROC curve for the HADS-Total (HADS-T) indicated that the best cut-off points were between 12 and 14. There were no statistically significant differences in the correlations of the EACS (considering positive responses to one [A] or both questions [B]), the HADS-D ≥ 8 or the HADS-T ≥ 12 with the gold standard. The study concludes that both approaches (the two EACS questions and the HADS-D subscale) are appropriate depression-screening methods in HIV population. We believe that using the EACS-B and the HADS-D subscale in a two-step approach allows for rapid, assumable and accurate clinical diagnosis in non-psychiatric hospital settings.

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories.