Routine Microscopy Research Articles

Chronicle of migrant hyperinfective Strongyloides stercolaris larvae and associated bacteremia in a patient suffering from Idiopathic Thrombocytopenic Purpura

Background: Strongyloidiasis is a neglected nematode infestation and can lead to hyperinfection syndrome. The onset of strongyloides hyper-infection syndrome is associated with a myriad of seemingly unrelated symptoms including diarrhoea, abdominal pain, urticaria, anaemia, sepsis and acute respiratory distress syndrome. Case report: A 65-year-old female patient presenting with chief complaints of nausea, vomiting, abdominal cramps, diarrhea, fever and cough and was admitted to a teaching hospital on 2nd May 2019. The patient was a diagnosed case of idiopathic thrombocytopenic purpura on Azathioprine (50mg, BD) and oral corticosteroid therapy (Prednisolone 60mg, OD) and was on treatment from the same hospital. The patient developed these symptoms after 23 days of immunosuppressive therapy. Patient was continued with previous medication along with supportive management after admission. Blood and urine sample was received in the department of Microbiology on 3rd May 2019 for culture and sensitivity testing. Urine sample was sterile after 24 hours of aerobic incubation. Escherichia coli grew in blood culture and the isolate was susceptible to gentamicin, amikacin and colistin. Stool sample was received on 6th of May 2019 for routine microscopy. With wet mount preparation of stool specimen, numerous larvae of Strongyloides stercoralis were seen. Modified Ziehl-Neelsen staining was performed and oocysts of Cryptosporidium species were also seen. Wet mount preparation of sputum sample was also performed in which few larvae of S. stercoralis were seen. Conclusion: As corticosteroid is the mainstay of treatment in idiopathic thrombocytopenic purpura an early diagnosis and prompt specific anti-parasitic therapy is required to eradicate these infections

A Case of Malakoplakia Mimicking Prostatic Malignancy

Malakoplakia is a granulomatous inflammatory condition commonly involving the urogenital tract. Principally it involves the urinary bladder, prostate is a rare site. Clinical and radiological examination sometimes mimics malignancy. Histology remains confirmatory modality for the diagnosis. Hereby we report a case of prostatic malakoplakia in an elderly male who presented with lower urinary tract symptoms in the past fifteen days. Urine routine microscopy showed pus cells; culture report was positive for E. coli. Serum Prostate Specific antigen (PSA) was 4.6 ug/ml. Magnetic Resonance Imaging (MRI) showed a bulky prostate. A 12-core biopsy was done to rule out malignancy and the histopathology showed benign prostatic glands and the stroma with a predominant population of epithelioid histiocytes and the characteristic Michealis-Gutmann bodies. After a course of antibiotic Nitrofurantoin, transrectal ultrasound was repeated which showed reduction in the prostatic volume. Subsequently Trans-Urethral Resection of Prostate (TURP) was done to relieve the persistent lower urinary tract symptoms. Histology showed similar features. Special stains – Von kossa and Prussian Blue highlighted the Michealis-Gutmann bodies.

Apocrine Hidradenoma and Adenomyoepithelioma: Entities on a Biological Continuum of Adnexal Neoplasia.

Apocrine hidradenomas (AH) once believed to harbor myoepithelial cells are now considered pure epithelial neoplasms. They are categorized separately from adenomyoepitheliomas which consist of apocrine epithelial and myoepithelial components. Reports of myoepithelial tumors arising in AH have suggested a link between the 2. Our goal was to explore whether cases diagnosed on routine microscopy as AH harbored occult myoepithelial elements, which would be disclosed by an immunohistochemical evaluation. Twenty-nine such cases, derived from a teaching collection of one of the authors, formed the basis of the study. Clinical and demographic data were documented, and morphological details of the cases were recorded. A panel of immunohistochemistry (AE1AE3, CK8/18, epithelial membrane antigen, p63, S100 protein, glial fibrillary acid protein, calponin, alpha actin, and others), designed to identify myoepithelial cells, was used. The population consisted of 14 women and 15 men (mean age 55.8; range 26-82 years). The tumors, located on the head/neck (14), limbs (10), and trunk (5), were solid (2) and solid/cystic (27). They exhibited varied (often combined) cytological elements (clear, squamoid, polygonal, and mucinous cells). On immunohistochemistry, aggregates of myoepithelial cells were identified in 5 (17%) cases. Four were calponin+ and AE1AE3+; they occupied ≤30% of tumor volumes and exhibited fusiform cytomorphology. One was S100 protein+ and AE1AE3+; it occupied 70% of tumor volume and exhibited polygonal cytomorphology. The gradation in the volume of myoepithelial elements disclosed by immunohistochemistry in a subset of our cases suggests that AH and adenomyoepitheliomas exist on a biological continuum of adnexal neoplasia. The diagnostic categorization of lesions with dual elements requires further study, but we propose that the term adenomyoepithelioma be restricted to those in which myoepithelial cells constitute ≥25% of tumor volume.

Evaluation and management of measurable residual disease in acute lymphoblastic leukemia.

With standard chemotherapy regimens for adults with acute lymphoblastic leukemia, approximately 90% of patients achieve complete remission. However, up to half of patients have persistent minimal/measurable residual disease (MRD) not recognized by routine microscopy, which constitutes the leading determinant of relapse. Many studies in pediatric and adult populations have demonstrated that achievement of MRD negativity after induction chemotherapy or during consolidation is associated with significantly better long-term outcomes, and MRD status constitutes an independently prognostic marker, often superseding other conventional risk factors. Persistence of MRD after intensive chemotherapy is indicative of treatment refractoriness and warrants alternative therapeutic approaches including allogeneic stem cell transplantation, blinatumomab, or investigational therapies such as inotuzumab ozogamicin or chimeric antigen receptor T cells. Furthermore, the incorporation of novel monoclonal antibodies or potent BCR-ABL1 tyrosine kinase inhibitors, such as ponatinib into frontline treatment may have the advantage of achieving higher rates of MRD negativity while minimizing chemotherapy-related toxicities. Many studies are therefore ongoing to determine whether this strategy can improve cure rates without the need for allogeneic stem cell transplantation.

Clinical Probe of Cyp2C8*2 Mutants in a Malaria Hyperendemic Zone: Evidence from North-Central, Nigeria.

A tremendous level of success has been achieved since the introduction of chloroquine and the combination of amodiaquine and artemisinin for the treatment of both complicated and uncomplicated malaria infections in sub-Saharan Africa. However, the recent discovery of drug resistant strains of Plasmodium falciparum (P.f.) and the ability of the parasite to ingest CYP2C8 into its digestive vacuole is of great public health concern. This study probes the occurrence of CYP2C8*2 allelic mutant amongst malaria patients in North-Central Nigeria. Three hundred and eighty five (385) unrelated study participants were screened for current malaria episodes using routine microscopy and/or rapid diagnostic test strips (RDTs). Chelex extraction method was used for single nucleotide polymorphisms (SNPs) and identification of CYP2C8*2 (805A > T) variant respectively. Wild-type (A) and the defective allele (T) were differentiated with the use of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The results obtained were further validated with Sanger sequencing of a few samples and thereafter, the genotype data were statistically processed. All alleles obtained were in Hardy Weinberg equilibrium. Out of the 385 participants (45.5% Male and 54.5% Female) genotyped for SNPs, 75 (19.5%) had the autosomal recessive mutant trait. Occurrence of mutant traits was gender and ethnic independent (p > 0.05). Yoruba ethnic group recorded a reduction in proportion of genotypic defective CYP2C8*2 allele (T) (1 in every 8 persons) with a carrier percentage of 13.3% compared with Hausa (26.62%); Igbo (25.37%) and other minority ethnic groups (17.6%). A remarkable inter-ethnic differences in autosomal recessive CYP2C8*2 allele was observed. By implication, there is a gradual incursion of genetic drift for poor CQ and AQ-Artemisinin metabolizers among the inhabitants.

Corneal endothelial density: An indicator for primary glaucoma progression

Purpose: Glaucoma is a progressive optic neuropathy causing death of retinal ganglion cells and their axons resulting in visual morbidity. This study was aimed to evaluate the impact of primary glaucoma on retinal nerve fiber layer (RNFL) thickness and corneal endothelial damage and to establish any correlation between these two. Materials and Methods: A total of 104 patients (179 eyes) diagnosed as primary open-angle glaucoma (POAG), primary angle-closure glaucoma (PACG), and normotensive glaucoma (NTG) were included in this study. Secondary glaucoma, postviral infection, ocular trauma, ocular surgery, and diabetes mellitus were criteria for exclusion. Average retinal nerve fiber layer (AvRNFL) thickness and endothelial cell count (cell density [CD]) were measured. Statistical analysis was done using Spearman's correlation coefficient. Results: The AvRNFL thickness in POAG (n = 120) was 83.83, PACG (n = 46) was 81.45, and Normotensive (NTG) (n = 13) was 71.07 μ. The mean CD values were 2416.68 cells/mm2, 2622.543 cells/mm2, and 2482.69 cells/mm2, respectively. A significant positive correlation between these two values was observed in POAG (P = 0.004) but not seen in PACG or NTG. Conclusion: Corneal endothelium loss may be an indicator of disease progression and RNFL thinning in patients of POAG. Routine specular microscopy evaluation of these patients can be helpful, and low endothelial counts indicate RNFL damage.

Implication of Perifollicular Clusters and Folliculotropic Distribution of Dendritic Cells in the Pathogenesis of Seborrhoeic Dermatitis.

The pathogenesis of seborrhoeic dermatitis is controversial and remains unclear. Malassezia is considered to be a commensal fungi and is found not only in the stratum corneum but also in hair follicles. It is an important pathogenic factor in seborrhoeic dermatitis. The aim of this study was to clarify the pathogenesis of seborrhoeic dermatitis, morphologically, through comparison with psoriasis vulgaris. Fifteen cases of seborrhoeic dermatitis, 7 of psoriasis, and 6 of normal skin were examined using routine histopathology, immunohistochemistry, and electron microscopy. Macrophages were found to be diffusely distributed in the upper dermis of seborrhoeic dermatitis and psoriasis. In contrast, a significant increase in the number of dendritic cells in the follicular epithelium and dendritic cell clusters in the perifollicular dermis were found only in seborrhoeic dermatitis. Ultrastructural examination of the clusters demonstrated that dendritic cells interacted with lymphocytes, macrophages, and other dendritic cells. In conclusion, folliculotropic distribution of dendritic cells as well as dendritic cell-immune cell clusters play an important role in the pathogenesis of seborrhoeic dermatitis.

Airyscan Superresolution Microscopy to Study Trypanosomatid Cell Biology.

The recent introduction by Carl Zeiss Ltd. of the Airyscan detector module for their LSM880 confocal laser-scanning microscope has enabled routine superresolution microscopy to be combined with the advantages of confocal-based fluorescence imaging. Resulting enhanced spatial resolution in X, Y, and Z provides tractable opportunity to derive new insight into protein localization(s), organelle dynamics, and thence protein function within trypanosomatids or other organisms. Here, we describe methods for preparing slides, cells, and basic microscope setup for fluorescence imaging of trypanosomatids using the LSM-880 with Airyscan platform.

Comparative Morphologic and Morphometric Study on the Developmental Aspects of In Vitro and In Vivo Reared Echino-coccus granulosus Sensu Stricto Using Differential Interference Contrast (DIC)/Nomarski and Phase Contrast Microscopy

Echinococcus granulosus is a zoonotic parasite with worldwide distribution. The present study focused on comparative morphologic and morphometric observations on the developmental aspects of whole body, more special the reproductive structures of in vitro reared adult worms (RAW) and in vivo reared adult worms in definitive host (AWIDH) using differential interference contrast (DIC)/Nomarski, phase contrast and routine optical microscopy. A total number of 10 in vitro and 10 in vivo reared adult worms of E. granulosus sensu stricto, G1 strain were selected. The worms were processed by Formaldehyde-Alcohol-Azocarmine-Lactophenol (FAAL). The details of morphological factors and reproductive structures of each worm including 25 biometrical parameters were studied by routine optical, phase contrast and Nomarski microscopy. The details of the samples were photographed, measured and analyzed. The fine structures of the parasite including the details of cirrus sac and developmental stages in different strobila were more obvious observing by Nomarski microscopy. The morphometric characters in the RAW and AWIDH showed that length of immature proglottid, length of mature proglottid, length of suckers are larger in RAW than AWIDH worms with statistical difference. Characters in E. granulosus of RAW and AWIDH showed that total number of segments, number of mature segments and the total number of testes were greater in RAW than AWIDH worms; while only the number of mature segments was statistically different is two groups. Application of DIC/Nomarski and phase contrast microscopy together with morphometric criteria are useful means for comparing the developmental aspects of in vitro and in vivo reared adults of E. granulosus.

Human Adipose Tissue: Purinergic Receptor Involvement

Adipose tissue is derived from the pluripotent mesenchymal cells and further on from cells of the reticular connective tissue, which can produce lobes of fatty tissues like grapes. The reticulum cells store fat droplets that will finally conjoin to one large drop. In the process, the cells become rounder. Extrinsic signaling mechanisms control the cell fate and determination of progenitor cells. Extrinsic signals such as neurotransmitters and neurotrophins have a significant impact on the production and fate or progenitor cells. In addition to intrinsic regulators, extrinsic signaling systems such as neurotransmitters, play a fundamental role in the control of cell proliferation. Purinergic receptors are actively seen at an early stage of development in the germal layer at the embryonic stage. Aim: This project aims to investigate the presence of purinergic signaling on human adipose tissue. Examine the essential histology of the human adipose tissue through routine histology and scanning electron microscopy. Materials and Methods: Human adipose tissues were obtained from routine autopsy at AL-Hilla mortuary. Hematoxyline and eosin staining were processed on microtome sectioned slides. Tissues were also examined using scanning electron microscopy SEM. Immunohistochemistry of purinergic receptors (P2Y1, P2Y2, P2Y4) was examined in human adipose tissue. Results: Histological examination/n of adipose tissue shows mature adipocytes with the lateral location of the nucleus. Purinergic receptors P2Y1, P2Y2, and P2y4 are seen on membranes of adipocytes. PCR product of digested adipose tissues also shows clear bands for all three purinergic receptors in human adipose tissue. Conclusion: This study shows that mature adipocytes are an excellent source of pluripotent stem cells that are capable of proliferating and differentiating into other cell lineages through purinergic receptor activation using various purine pharmacology. Purinergic receptors are a good indicator of the presence of mesenchymal stem cells.

Urinary Tract Infection among Patients Visiting Ganesh Man Singh Memorial Hospital and Research Center, Lalitpur, Nepal

Objectives: The objective of this study was to determine the prevalence of urinary tract infection (UTI) and antibiotic sensitivity pattern among the suspected UTI cases visiting at Ganeshman Singh Memorial Hospital Lalitpur, Nepal.
 Methods: A total of 300 mid-stream urine, catheter and suprapubic aspirate from UTI suspected patients were included and processed for routine microscopy and culture and then identified by standard microbiological methods. Antibiotic susceptibility test was performed by Kirby- Bauer disc diffusion method.
 Results: Out of 300 samples, 55(84.6%) mid-stream urine and 10(15.4%) catheter sample had significant bacterial growth. E. coli (32,49.2%) was the most common isolate followed by Staphylococcus aureus (10,15.3%), Enterobacter spp. (8,12.3%), Klebsiella spp. (7,10.7%), Pseudomonas aeruginosa (3,4.6%), Proteus spp. (3,4.6%), Acinetobacter spp. (1,1.5%) and Enterococcus spp. (1,1.5%). Most of the Gram-negative bacterial isolates were sensitive to Ceftriaxone (88.8%) followed by Gentamicin (72.2%), and Nitrofurantoin (64.8%) and resistant to Amoxicilin (68.5%) followed by Nalidixic Acid (53.7%). Gram positive isolates were sensitive to Amikacin (72.7%) followed by Imipenem (63.6%) and Gentamicin (63.6%) whereas resistant to Amoxycilin (72.7%) and Ciprofloxacin (63.63%).
 Conclusion: The main cause of the UTIs was found as Gram negative bacteria. Prescription of antibiotics based on susceptibility tests would help in reduction of antibiotic resistance.

INCIDENCE OF MALIGNANT KIDNEY TUMOURS IN PAEDIATRIC AGE GROUP

Background: Cancer is essentially a disease of adults, yet it is one of the common killers in childhood. In western countries cancer is next only to trauma as a cause of mortality in children under 20 years of age In India.
 Methods: The material for present study is obtained from S. P. Medical College, Bikaner and referred cases for a period of five years from 2011-2014. The clinical history regarding duration of the disease, mode of presentation, symptoms and signs are recorded from the case papers, request forms, patient’s history, clinical data along with relevant details obtained from available hospital and departmental records. The histopathology slides and paraffin blocks are reviewed. Gross examination is done carefully noting the size, shape, extent and configuration, nodularity, consistency (solid, cystic or mixed). A minimum of 4-5 bits are selected from the representative areas of tumor. The tissue for routine microscopy is preserved and fixed in 10% neutral buffered formalin for 24 hours and processed in automatic tissue processor (Histokinette) and embedded in paraffin. The sections 3-5 μ thick, are cut and stained by haematoxylin and eosin in all cases and special stains like PAS, MTS, and RT done where ever feasible
 Results: The incidence of malignant tumour in our study was 6.66% found.
 Conclusion: The present study provides a fair insight into the histological patterns of lesion in paediatric age group. Most common incidence was that of wilms tumour in paediatric age group followed by renal cell carcinoma. Imaging and histologic characterization are crucial to avoid any delay in management.
 Keywords: Malignant, Wilms, Histopatological tumour.

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden

Background: Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or Cryptosporidium spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of Giardia intestinalis, Dientamoeba fragilis, Cryptosporidium spp., Entamoeba histolytica and Entamoeba dispar from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results.Methods: SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region Jönköping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl–Neelsen staining on patients with certain symptoms and real-time PCR.Results: We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR.Conclusion: In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and Cryptosporidium spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.

ES04.02 Pathology Overview for Carcinoid and NE Spectrum

ES04.02 Pathology Overview for Carcinoid and NE Spectrum

Glomerular Immunodeposits of Patients with IgA Nephropathy Are Enriched for IgG Autoantibodies Specific for Galactose-Deficient IgA1

IgA nephropathy (IgAN) is the leading primary GN worldwide. The disease is thought to result from glomerular deposition of circulating immune complexes of IgG bound to galactose-deficient IgA1 (Gd-IgA1). However, routine immunofluorescence microscopy fails to detect IgG in many kidney biopsies from patients with IgAN and the specificity of IgG in immunodeposits has not been tested. We used remnant frozen kidney-biopsy specimens from 34 patients with IgAN; 14 were IgG-positive and 20 were IgG-negative by routine immunofluorescence microscopy. Six patients with primary membranous nephropathy (MN) and eight with lupus nephritis (LN) served as controls. IgG in the kidney tissue was extracted and its amount determined by ELISA. IgG molecular integrity was assessed by SDS-PAGE immunoblotting. Antigenic specificity of extracted IgG was determined by ELISA using phospholipase A2 receptor (PLA2R) or Gd-IgA1 as antigen. In addition, ten other IgAN cases, six IgG-positive and four IgG-negative by routine immunofluorescence, were used for colocalization studies by confocal microscopy. IgG extracted from MN but not IgAN immunodeposits reacted with PLA2R. Conversely, IgG extracted from IgAN but not MN or LN immunodeposits reacted with Gd-IgA1. Even IgAN kidney-biopsy specimens without IgG by routine immunofluorescence microscopy had IgG specific for Gd-IgA1. Confocal microscopy confirmed the presence of IgG in the IgAN biopsies with colocalization of glomerular IgA and IgG. These results reveal for the first time that IgAN kidney biopsies, with or without IgG by routine immunofluorescence, contain Gd-IgA1-specific IgG autoantibodies. These findings support the importance of these autoantibodies in the pathogenesis of IgAN.

New possibilities to diagnose pulmonary tuberculosis at a pulmonology inpatient department

The aim of the study was to investigate new methods for pulmonary tuberculosis (TB) detection at a pulmonology inpatient department.Methods. Pulmonary TB cases (n = 49) newly diagnosed at a pulmonology department of a hospital were involved in the study; among them, 69.4% of patients were infected with human immunodeficiency virus (HIV). All patients were asked if he/she had previously contacted with other TB patients, and if he/she was diagnosed with TB previously. The patients were also about family history. Posteroanterior and lateral chest X-ray and laboratory analyses were performed in all patients. Mycobacteria tuberculosis (MBT) were searched using smear microscopy, GeneXpert MTB/RIF molecular method and culturing (BACTEC MGIT) of sputum and bronchoalveolar lavage fluid (BALF) obtained during bronchoscopy. Patients with positive results only were included in the analysis in order to investigate and to compare diagnostic value of the methods.Results. Questioning patients at admission was useful to diagnose TB in 4% of patients. Sputum smears were positive in 12 patients (27.3%). BALF was obtained in 18 patients (36.7%); among them, MBT was identified in 52 (5%). Molecular testing was positive in all 49 patients (100%); among them, BACTEC MGIT culturing yielded MBT in 48 patients (97.9%). In patients with negative sputum smears during the first 3 days after admission, GeneXpert required 10.4 ± 5.3 days.Conclusion. GeneXpert method was highly useful to diagnose TB and was comparable with BACTEC. Routine sputum and BALF smear microscopy was significantly less rapid and less sensitive method. The diagnostic accuracy could be increased with the use of molecular methods in patients with negative sputum smears obtained at a general hospital. Therefore, molecular methods could be used in patients highly suspected for TB.

Detection of Extravascular <em>Trypanosoma</em> Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Routine Microscopy in Quantum Dot Industry

Routine Microscopy in Quantum Dot Industry

Conversion of organic biomedical waste into potential fertilizer using isolated organisms from cow dung for a cleaner environment.

Management of organic biomedical waste is a global quandary, and it is becoming difficult to confront day by day. Conversion of organic biomedical waste into fertilizer is of great concern. In the present research, organic biomedical waste samples (blood swabs, dressing swabs, and used cotton) were collected then after cow dung was collected in sterile container and immediately transported to the laboratory and screened for any gastrointestinal infection by using routine microscopy for intestinal parasitic infection, routine bacterial culture, and fecal occult blood for any intestinal bleeding. Then after, the pure culture of organisms and fungus were prepared, and further samples were subjected to degradation for 288 h by using various organisms and fungus. Then after, the specific quantity of biomedical waste was subjected for incineration. The physicochemical parameters of biomedical waste samples were analyzed. Then treated samples were mixed with soil to confirm a role as potential fertilizer. Then after, tomato plantation was done and phytochemical parameters of tomato plant were analyzed. This study states that organic biomedical waste produces a sanitary and stable fertilizer.

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.