Lupus vulgaris with tubercular lymphadenitis and IgA nephropathy

A 14-year-old girl presented with a 10-year history of a large crusted plaque over the right thigh for 10 years and small reddish plaque over the left upper back for 3 months. On routine evaluation, she was found to have hematuria. Skin biopsy from the lesion was suggestive of skin tuberculosis (lupus vulgaris), and kidney biopsy showed features of IgA nephropathy (IgAN). Fine-needle aspiration from the inguinal lymph node was consistent with granulomatous disease. The patient has been on anti-tubercular treatment, and the hematuria has subsided.     

Intralesional immunotherapy with killed Mycobacterium w vaccine for the treatment of ano-genital warts: an open label pilot study

Background  Intralesional immunotherapy with skin test antigens and vaccines has been found to be effective in the management of genital and extragenital warts.
Objective  To evaluate the efficacy and safety of intralesional Mycobacterium w (M w ) vaccine monotherapy for the treatment of ano-genital warts.
Patients and methods  Ten patients clinically diagnosed to have external ano-genital warts, including three with giant ano-genital warts (Buschke Löwenstein tumour), were included in this open-label pilot study. Two patients were human immunodeficiency virus seropositive, and one was on iatrogenic immunosuppression for renal transplantation. M w vaccine (0.1 mL) was initially injected intradermally in the deltoid region on both the sides, followed 2 weeks later by intradermal intralesional injection into the genital warts. Intralesional injections were repeated weekly until either complete clearance or a maximum of 10 injections was achieved.
Results  One patient was lost to follow-up after the first intralesional injection. In 8 out of remaining 9 patients (88.9%), the genital warts cleared completely. In one patient with giant perianal wart, the lesion was reduced to less than 5% of its volume after 10 intralesional injections, which was later electrosurgically excised. The treatment was well tolerated by the majority of the patients. The adverse reactions were noted in four patients, which were reversible. No recurrence was seen after a mean follow-up of 5.1 months.
Conclusion  Intralesional immunotherapy of ano-genital warts with M w vaccine seems to be a promising new approach, which needs to be evaluated in the randomized controlled trials.     

Lesional T-cell subset in post-kala-azar dermal leishmaniasis

BACKGROUND: The pathogenesis of post-kala-azar dermal leishmaniasis (PKDL) is ill understood. This study was carried out to find the percentage of T-helper and T-suppressor cells in lesional tissue and their probable role in the pathogenesis of PKDL. METHODS: An immunoperoxidase monoclonal antibody technique was used to characterize and quantify the subsets of T lymphocytes in the infiltrate in 25 patients with PKDL. RESULTS: The ratio of T-helper to T-suppressor cells was 0.87 in hypopigmented macules and 0.85 in nodule and/or plaque lesions of PKDL. CONCLUSIONS: In this study, there was a definite preponderance of T-suppressor cells over T-helper cells in both types of skin lesions of PKDL. Further studies should be undertaken on larger numbers of patients to compare T-cell subsets both in skin lesions and the circulation, in order to determine the pathogenesis of PKDL.     

Meconium peritonitis presenting as abdominal calcification: three cases with different pathology

Intra-abdominal calcification is uncommon in newborns and has several causes of which meconium peritonitis is the most frequent. Three neonates with intra-abdominal calcification as a complication of meconium peritonitis are presented. The types of meconium peritonitis were cystic, meconium pseudocyst and meconium ascites. Two required surgical intervention. Meconium peritonitis should be considered in newborns with intra-abdominal calcification.     

Solubility Prediction of Satranidazole in Propylene Glycol-Water Mixtures Using Extended Hildebrand Solubility Approach

Extended Hildebrand solubility approach is used to estimate the solubility of satranidazole in binary solvent systems. The solubility of satranidazole in various propylene glycol-water mixtures was analyzed in terms of solute-solvent interactions using a modified version of Hildebrand-Scatchard treatment for regular solutions. The solubility equation employs term interaction energy (W) to replace the geometric mean (δ₁δ₂), where δ₁ and δ₂ are the cohesive energy densities for the solvent and solute, respectively. The new equation provides an accurate prediction of solubility once the interaction energy, W, is obtained. In this case, the energy term is regressed against a polynomial in δ₁ of the binary mixture. A quartic expression of W in terms of solvent solubility parameter was found for predicting the solubility of satranidazole in propylene glycol-water mixtures. The expression yields an error in mole fraction solubility of ~3.74%, a value approximating that of the experimentally determined solubility. The method has potential usefulness in preformulation and formulation studies during which solubility prediction is important for drug design.     

Improving Parent Participation at Pediatric Diabetes and Sickle Cell Appointments Using a Brief Intervention

We conducted two separate experiments designed to increase parents' question-asking at pediatric diabetes and sickle cell appointments using a brief intervention. Sixty-two parent-child dyads attending pediatric diabetes appointments and ninety-two parent-child dyads attending sickle cell appointments were included in the study. In experiment 1, parents of pediatric diabetes patients were randomly assigned to either an oral instruction or written instruction condition. Participants in the oral instruction condition were asked by a psychology resident if they had any questions that they planned to ask the pediatrician and encouraged to ask all questions. Participants in the written instruction condition were treated identically to the oral instruction condition participants except that their questions were recorded on 3 5 index cards and given to them as reminders. Results showed that parents in the written instruction condition asked more questions than parents in the oral instruction condition. A second experiment was conducted with parents of sickle cell patients to confirm these results. The results of the second experiment were less definitive, and are discussed in relationship to visit characteristics that may account for the differences in results.     

Synthesis, kinetic studies and pharmacological evaluation of mutual azo prodrug of 5-aminosalicylic acid for colon-specific drug delivery in inflammatory bowel disease

Mutual azo prodrug of 5-aminosalicylic acid with l-tyrosine was synthesized by coupling l-tyrosine with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in rat fecal matter showed 87.18% release of 5-aminosalicylic acid with a half-life of 140.28min, following first order kinetics. Therapeutic efficacy of the carrier system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. Myeloperoxidase activity was determined by the method of Krawisz et al. The synthesized prodrug was found to produce comparable mitigating effect as that of sulfasalazine on colitis in rats.     

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung,Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.In modern oncologic practice, patients with advanced-stage non-small cell lung cancer (NSCLC),^{1, 2} melanoma,^{3, 4} and colorectal adenocarcinoma^{5, 6} are often treated with targeted therapies as standard of care or after enrollment in clinical trials. Molecular mutation analysis is the preferred testing modality to guide therapeutic decision making and/or eligibility for biological studies. Therefore, laboratory-developed mutation assays require robust workflows that produce high-quality sequence information from routine clinical specimens, namely formalin-fixed, paraffin-embedded (FFPE) samples. As molecular testing transitions from an ancillary tool to a seminal requirement for optimal oncologic patient management, multiplex sequencing assays with clearly defined content and bioinformatics workflows are essential for accurate and consistent results, reporting, and patient management.Published guidelines endorse which genes to test in a particular tumor type and provide timeframes for receipt of actionable results, but they also grant individual laboratories autonomy to perform mutation testing using any suitable validated method.² Historically at our institution, single-gene mutation analysis for clinically relevant genes was performed either in-house or at a Clinical Laboratory improvement Amendment–certified reference laboratory. Depending on the result, reflex testing was performed for additional genes per mutation frequency or designated algorithms. Unfortunately, this approach introduced considerable turn-around time delays and unnecessary cost, particularly when send-out testing was required. Therefore, we sought testing modalities that could analyze multiple clinically relevant mutations simultaneously, accurately, and expeditiously.Next-generation sequencing (NGS) technologies have revolutionized genomic medicine by allowing high-throughput, parallel sequencing of the human genome.⁷ Currently, however, a large proportion of clinical NGS endeavors are supported by larger academic institutions with shared access to established genomic and bioinformatics research infrastructures, and routine clinical implementation of NGS is complicated by mitigating factors, such as clinical performance, laboratory expertise, lengthy turn-around times, and cost.⁸ Thus, we investigated affordable methods to detect clinically relevant somatic mutations in NSCLC, melanoma, and gastrointestinal (GI) malignancies that generated high-quality sequencing data from FFPE samples, and offered manageable turn-around times. Targeted amplicon-based library preparation methods combined with parallel sequencing offered a practical solution, and recent studies have demonstrated the utility of this approach.^{9, 10}Reversible terminal dideoxynucleotide sequencing chemistry by Illumina (San Diego, CA) consistently generates accurate and reproducible sequencing data.^{11, 12} To use this chemistry for clinical testing, we purchased the bench-top NGS sequencer, the Illumina MiSeq, and paired it with the MiSeq-compatible Illumina TruSight tumor (TST) 26-target amplicon-based library preparation kit. TST targets 26 genes and 174 amplicons selected from College of American Pathologists/National Comprehensive Cancer Network guidelines, relevant publications, and late-phase pharmaceutical clinical trials (Supplemental Table S1). TST offered several advantages over other commercially available mutation testing kits, such as bidirectional targeting of the positive and negative DNA strands, full-exon coverage as opposed to hotspot analysis, and robust vendor-supplied bioinformatics techniques optimized for somatic variant detection. More important, TST library preparation is optimized for FFPE samples, multiple safeguards exist to detect FFPE variant artifacts, and deep sequencing of TST libraries consistently yields high depths of coverage of targeted regions.Somatic mutation testing for many of the TST genes has clinical utility in a wide variety of solid tumors. For example, testing for CTNNB1 exon 3 is performed clinically for diagnostic and prognostic purposes in pediatric desmoid tumors, select PIK3CA hotspot mutations are positive prognostic factors for breast carcinoma, and multiple exons in PDGFRA and KIT are routinely tested in GI stromal tumors to predict response to targeted therapies. More important for our intended validation purposes, all of the clinically relevant genes and regions mutated in NSCLC, melanoma, and colonic adenocarcinoma that were tested in our routine clinical practice were represented. In addition, we could easily incorporate the TST NGS into a 5 business day workflow model, and a cost-analysis demonstrated a reasonable cost per test.Last, TST NGS data are processed from raw sequence (FASTQ) to called variants with on-board MiSeq Reporter software version 2.3, and variant annotations can be performed with Illumina''s VariantStudio software version 2.1 software using standard desktop and laptop computers. The ease of library preparation, sequencing, and data analysis with tools provided by a single vendor best fit our clinical priorities and the resources available at our academic molecular pathology laboratory.Herein, we present our results from the clinical validation of TST NGS using 80 sequenced samples that were selected from 100 FFPE patient samples (40 NSCLCs, 30 melanomas, and 30 GI malignancies). During our validation, we achieved high depths of coverage for multiple clinically relevant variants when multiplexing 8 to 12 samples on a single MiSeq flow cell. TST NGS consistently demonstrated sensitivities comparable to reference assays, showed 100% concordance with known variants, detected novel variants in many samples, and uncovered variants missed by less-sensitive testing modalities. The TST variant-calling pipeline was robust and showed high concordance when compared with an alternative analysis pipeline, and we used an in-house custom Java program to assess laboratory-defined quality control (QC) metrics and streamline clinical reporting (developed by G.H.S., Emory University, http://github.com/ghsmith/coverageQc). More important, although the results detailed herein represent the experience of a single institution, the data and validation strategies shown herein are broadly applicable to most clinical molecular laboratories interested in offering NGS for NSCLCs, melanomas, and GI malignancies as well as many other solid tumors.