Simplified MLST scheme for direct typing of Leptospira human clinical samples

Abstract Background Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA. Results In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. Conclusions The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

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Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines

PLoS ONE ◽

10.1371/journal.pone.0258891 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258891

Author(s):

Marjo V. Mendoza ◽

Windell L. Rivera

Keyword(s):

Disease Surveillance ◽

The Philippines ◽

Pathogen Transmission ◽

23S Rrna ◽

Clinical Samples ◽

Genetic Profile ◽

Serological Tests ◽

Clinical Specimens ◽

Mlst Scheme ◽

Pcr Targeting

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

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Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128821 ◽

1987 ◽

Vol 45 ◽

pp. 908-909

Author(s):

Alain R. Trudel ◽

M. Trudel

Keyword(s):

Electron Microscopy ◽

Fold Increase ◽

Observation Time ◽

Clinical Samples ◽

Maximum Speed ◽

Viral Particles ◽

Structural Details ◽

Latex Spheres ◽

Increased Sensitivity ◽

Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

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External Validity of SCORS-G Ratings of Thematic Apperception Test Narratives in a Sample of Outpatients and Inpatients

Rorschachiana Journal of the International Society for the Rorschach ◽

10.1027/1192-5604/a000057 ◽

2015 ◽

Vol 36 (1) ◽

pp. 58-81 ◽

Cited By ~ 10

Author(s):

Michelle B. Stein ◽

Jenelle Slavin-Mulford ◽

Caleb J. Siefert ◽

Samuel Justin Sinclair ◽

Michaela Smith ◽

...

Keyword(s):

Life Events ◽

Psychiatric Hospitalization ◽

Clinical Care ◽

Thematic Apperception Test ◽

Clinical Samples ◽

Cross Sectional ◽

Conduct Disordered ◽

Global Ratings ◽

Thematic Apperception ◽

History Of

Abstract. The Social Cognition and Object Relations Scale-Global Ratings Method (SCORS-G; Stein, Hilsenroth, Slavin-Mulford, & Pinsker-Aspen, 2011 ) is a reliable system for coding narrative data, such as Thematic Apperception Test (TAT) stories. This study employs a cross-sectional, correlational design to examine associations between SCORS-G dimensions and life events in two clinical samples. Samples were composed of 177 outpatients and 57 inpatients who completed TAT protocols as part of routine clinical care. Two experienced raters coded narratives with the SCORS-G. Data on the following clinically relevant life events were collected: history of psychiatric hospitalization, suicidality, self-harming behavior, drug/alcohol abuse, conduct-disordered behavior, trauma, and education level. As expected, the clinical life event variable associated with the largest number of SCORS-G dimensions was Suicidality. Identity and Coherence of Self was related to self-harm history across samples. Emotional Investment in Relationships and Complexity of Representations were also associated with several life events. Clinical applications, limitations of the study, and future directions are reviewed.

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Are Three Items Sufficient to Measure Sense of Coherence?

European Journal of Psychological Assessment ◽

10.1027/1015-5759/a000337 ◽

2018 ◽

Vol 34 (4) ◽

pp. 229-237 ◽

Cited By ~ 2

Author(s):

Francesca Chiesi ◽

Andrea Bonacchi ◽

Caterina Primi ◽

Alessandro Toccafondi ◽

Guido Miccinesi

Keyword(s):

Sense Of Coherence ◽

Convergent Validity ◽

Clinical Sample ◽

Clinical Samples ◽

Depression Symptoms ◽

Time Saving ◽

Positive Role ◽

Patients With Cancer ◽

Measure Sense

Abstract. The present study aimed at evaluating if the three-item sense of coherence (SOC) scale developed by Lundberg and Nystrom Peck (1995) can be effectively used for research purpose in both nonclinical and clinical samples. To provide evidence that it represents adequately the measured construct we tested its validity in a nonclinical (N = 658) and clinical sample (N = 764 patients with cancer). Results obtained in the nonclinical sample attested a positive relation of SOC – as measured by the three-item SOC scale – with Antonovsky’s 13-item and 29-item SOC scales (convergent validity), and with dispositional optimism, sense of mastery, anxiety, and depression symptoms (concurrent validity). Results obtained in the clinical sample confirmed the criterion validity of the scale attesting the positive role of SOC – as measured by the three-item SOC scale – on the person’s capacity to respond to illness and treatment. The current study provides evidence that the three-item SOC scale is a valid, low-loading, and time-saving instrument for research purposes on large sample.

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Minimizing Carry-Over Effects After Treatment Failure and Maximizing Therapeutic Outcome

Zeitschrift für Psychologie ◽

10.1027/2151-2604/a000180 ◽

2014 ◽

Vol 222 (3) ◽

pp. 171-178 ◽

Cited By ~ 3

Author(s):

Mareile Hofmann ◽

Nathalie Wrobel ◽

Simon Kessner ◽

Ulrike Bingel

Keyword(s):

Treatment Failure ◽

Human Subjects ◽

Subsequent Treatment ◽

Clinical Samples ◽

Administration Route ◽

Healthy Human ◽

Negative Experiences ◽

Analgesic Treatment ◽

Balanced Design ◽

Carry Over

According to experimental and clinical evidence, the experiences of previous treatments are carried over to different therapeutic approaches and impair the outcome of subsequent treatments. In this behavioral pilot study we used a change in administration route to investigate whether the effect of prior treatment experience on a subsequent treatment depends on the similarity of both treatments. We experimentally induced positive or negative experiences with a topical analgesic treatment in two groups of healthy human subjects. Subsequently, we compared responses to a second, unrelated and systemic analgesic treatment between both the positive and negative group. We found that there was no difference in the analgesic response to the second treatment between the two groups. Our data indicate that a change in administration route might reduce the influence of treatment history and therefore be a way to reduce negative carry-over effects after treatment failure. Future studies will have to validate these findings in a fully balanced design including larger, clinical samples.

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A Comparison of Abuse Experiences in USA and German Clinical Samples

PsycEXTRA Dataset ◽

10.1037/e609232012-020 ◽

2002 ◽

Author(s):

Kim R. Tousignant

Keyword(s):

Clinical Samples

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The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650659 ◽

1996 ◽

Vol 76 (05) ◽

pp. 774-779 ◽

Cited By ~ 14

Author(s):

John T Brandt ◽

Carmen J Julius ◽

Jeanne M Osborne ◽

Clark L Anderson

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Complement Activation ◽

Thromboembolic Disease ◽

Clinical Samples ◽

Hla Antigen ◽

Aggregation Response ◽

Immune Mediated ◽

Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

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