Staining Of Smears Research Articles

Aspects of Mannheimia haemolytica cryopreservation

Mannheimia haemolytica causes severe pneumonia and septicemia in ruminant animals. The study aims to create an effective protective media for the long‐term storage of microbial cultures of M. haemolytica in a veterinary laboratory. The protective nutrient media were based on Hottinger's nutrient broth. Glycerol and ethylene glycol were used as cryoprotectants. Two protective media were obtained. Medium #1 consisted of nutrient broth (90%) and glycerin (10%), and medium #2 had nutrient broth (90%) and ethylene glycol (10%) in it. A homogeneous suspension 1.0 McFarland units turbidity was prepared from a pure culture of microorganisms (3.0 x 108 CFU/ml). The suspension was added to ampoules with an appropriate protective medium. The inoculated media were placed in a cryogenic storage dewar in liquid nitrogen at ‐196 ° C. The viability of M. haemolytica was determined by inoculation on blood agar and counting the number of living bacteria. The initial inoculum concentration of M. haemolytica was 3.0x107 CFU/ml. After 18‐20 hours of cultivation, it becomes 7.0x109 CFU/ml. The survival and safety of bacteria depend on the cryoprotectant. The results of the experiment showed that 10% glycerol provides 98.6% survival of M. haemolytica. The number of viable cells after cryopreservation was 6.9x109 CFU/ml. Ethylene glycol showed a 95.7% survival rate. The number of viable cells after cryopreservation was 6.7x109 CFU/ml. Without cryoprotectant, M. haemolytica showed a 40.0% survival rate with only 2.8x109 CFU/ml viable cells after defrosting. M. haemolytica causes turbidity and sediment formation at the bottom of the tube on Hottinger broth. After growing on semi‐liquid media (semi‐liquid agar with the addition of glucose or serum), bacteria show bipolar staining in smears. On Hottinger broth, with the addition of 10% defibrinated ram blood M. haemolytica formed small dewy colonies with an even edge without a hemolysis zone. The final species identification was performed by the proteomic method using MALDI‐TOF‐SM. All used media keep microorganisms' cultures in a stable, viable state. Thus, they can be successfully used for the storage of bacteria at low‐temperature conditions.

Study of cytoplasmic staining in buccal smears using curcuma longa (turmeric) extract as an alternate to eosin as compared to H & E staining procedure

Eosin is an efficient synthetic dye belonging to xanthenes group. Eosin dyes are efficient but are hazardous to human and animal health. Owing to this, an alternative dye that has a good staining ability, eco-friendly, biodegradable & commercially available turmeric known as Curcuma longa was used. The intense yellow color of turmeric inspired us to explore its efficacy as an alternative for eosin in routine cytological procedure and compare it with routine H&E stain. The present study aims to explore the efficacy of turmeric extract as a cytoplasmic counter stain instead of eosin and a comparative assessment with routine H&E in cytological smear. A total of 30 individuals were initially included in the study, which comprised of 2 cytological smears were taken from each individual and each smear was stained with H&E and Curcuma longa respectively. All the slides were interpreted by 3 observers and findings were recorded separately. The intensity of staining was recorded as follow: 0 – Poor / no stain, 1 – Mild, 2 – Moderate, 3 – Intense. The results were analyzed using Spearsons correlation test.The results revealed that the staining intensity of Curcuma longa is similar to that of eosin.

Semi-Automated Microfluidic Device Combined with a MiniPCR-Duplex Lateral Flow Dipstick for Screening and Visual Species Identification of Lymphatic Filariae.

Lymphatic filariasis (LF) is a leading cause of permanent disability worldwide that has been listed as a neglected tropical disease by the World Health Organization. Significant progress made by the Global Program to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decline in the population of the worm that causes LF infection. Diagnostic assays capable of detecting low levels of parasite presence are needed to diagnose LF. There is also a need for new tools that can be used in areas where multiple filarial species are coendemic and for mass screening or for use in a point-of-care setting. In the present study, we applied our previously developed semi-automated microfluidic device in combination with our recently developed mini polymerase chain reaction (miniPCR) with a duplex lateral flow dipstick (DLFD) (miniPCR-DLFD) for rapid mass screening and visual species identification of lymphatic filariae in human blood. The study samples comprised 20 Brugia malayi microfilariae (mf) positive human blood samples, 14 Wuchereria bancrofti mf positive human blood samples and 100 mf negative human blood samples. Microfilariae detection and visual species identification was performed using the microfluidic device. To identify the species of the mf trapped in the microfluidic chips, DNA of the trapped mf was extracted for miniPCR amplification of W. bancrofti and B. malayi DNA followed by DLFD. Thick blood smear staining for microfilariae detection was used as the gold standard technique. Microfilariae screening and visual species identification using our microfluidic device plus miniPCR-DLFD platform yielded results concordant with those of the gold standard thick blood smear technique. The microfluidic device, the miniPCR and the DLFD are all portable and do not require additional equipment. Use of this screening and visual species identification platform will facilitate reliable, cost-effective, and rapid surveillance for the presence of LF infection in resource-poor settings.

PREVALENCE OF POSITIVITY OF CBNAAT IN EXTRA PULMONARY TUBERCULOSIS

Background: TB is the 9th leading cause of death worldwide and the leading cause from a single infectious agent. It is estimated that 15 to 20% of all TB cases are extra pulmonary.
 Methods: This study conducted on Patients of EPTB diagnosed on the basis of clinico radiological basis attending the OPD. Patients with signs and symptoms and radiological examination suggestive of extra-pulmonary tuberculosis were included in this study.
 Results: In present study 16(64.00%) patients suggestive of TB by FNAC were CBNAAT positive and 9(36.00%) were CBNAAT negative. 5 patients (45.45%) were NOT suggestive of TB by FNAC were CBNAAT negative and 6(54.55%) were CBNAAT positive. This difference however is not found to be statistically significant (p=0.869).
 Conclusion: Present study revealed that more positivity rate by CBNAAT in comparision to smear staining in pleural fluid which indicate that it is a more sensitive technique as compared to conventional methods.
 Keywords: CBNAAT, Extrapulmonary tuberculosis, FNAC

Cell HE staining smears and paired cell paraffin sections in detection of epithelial growth factor receptor gene of pleural fluid specimens.

The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section. A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was. Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21. The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.

A New Method for the Sputum Cytology Test Without Direct Contact to Specimens During COVID-19 Pandemic.

BackgroundCoronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Specimens of blood, body fluids and excreta received in the department of pathology undoubtedly increased the risk of infection, especially in some hospitals that are short of professional protection capability. Here we provided a new simple way for the sputum cytology test during the COVID-19 pandemic.MethodsSputum samples from 30 patients with lung cancer were collected and divided into two groups, including the control group and the experimental group. Samples of the control group were processed in the biological safety cabinet, while the experimental group was put into the sealed specimen bag directly and pretreated with 75% medical alcohol. Then the cell morphology and tumor cell identification were analyzed by cell smears and liquid-based cell staining. The expression of cell antigens was determined by immunohistochemical staining.ResultOur result showed that both sputum samples in two groups exhibited complete cell structure and clear morphology according to the cell smear and liquid-based cell staining. In addition, the immunohistochemical result showed that cell antigens, including cytokeratin (CK), leukocyte common antigen (LCA), and thyroid transcription factor-1 (TTF1), were specifically expressed in the cell membrane, cytoplasm, and nucleus, respectively. The tumor cells were distributed diffusely, and cell antigens were located accurately after pretreatment with 75% medical alcohol and were consistent with that of the control group.ConclusionUsing 75% medical alcohol to pretreat sputum specimens has no obvious impact on cell morphology and antigens expression. Our study provided a new method for the sputum cytology test with no direct contact so as to protect medical staff against the virus during COVID-19 outbreak.

Differentiating necrotizing soft tissue infections from cellulitis by soft tissue infectious fluid analysis: a pilot study

BackgroundWe conducted this study to evaluate the characteristics of the infectious fluid in soft tissue infection and investigate the utility of the biochemical tests and Gram stain smear of the infectious fluid in distinguishing necrotizing soft tissue infection (NSTI) from cellulitis.MethodsThis retrospective cohort study was conducted in a tertiary care hospital in Taiwan. From April 2019 to October 2020, patients who were clinically suspected of NSTI with infectious fluid accumulation along the deep fascia and received successful ultrasound-guided aspiration were enrolled. Based on the final discharge diagnosis, the patients were divided into NSTI group, which was supported by the surgical pathology report, or cellulitis group. The t test method and Fisher’s exact test were used to compare the difference between two groups. The receiver–operator characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the discriminating ability.ResultsTotal twenty-five patients were enrolled, with 13 patients in NSTI group and 12 patients in cellulitis group. The statistical analysis showed lactate in fluid (AUC = 0.937) and LDH in fluid (AUC = 0.929) had outstanding discrimination. The optimal cut-off value of fluid in lactate was 69.6 mg/dL with corresponding sensitivity of 100% and specificity of 76.9%. The optimal cut-off value of fluid in LDH was 566 U/L with corresponding sensitivity of 83.3% and a specificity of 92.3%. In addition, albumin in fluid (AUC = 0.821), TP in fluid (AUC = 0.878) and pH in fluid (AUC = 0.858) also had excellent diagnostic accuracy for NSTI. The Gram stain smear revealed 50% bacteria present in NSTI group and all the following infectious fluid culture showed bacteria growth.ConclusionsThe analysis of infectious fluid along the deep fascia might provide high diagnostic accuracy to differentiate NSTI from cellulitis.

Virtual Staining, Segmentation, and Classification of Blood Smears for Label-Free Hematology Analysis.

Objective and Impact Statement. We present a fully automated hematological analysis framework based on single-channel (single-wavelength), label-free deep-ultraviolet (UV) microscopy that serves as a fast, cost-effective alternative to conventional hematology analyzers. Introduction. Hematological analysis is essential for the diagnosis and monitoring of several diseases but requires complex systems operated by trained personnel, costly chemical reagents, and lengthy protocols. Label-free techniques eliminate the need for staining or additional preprocessing and can lead to faster analysis and a simpler workflow. In this work, we leverage the unique capabilities of deep-UV microscopy as a label-free, molecular imaging technique to develop a deep learning-based pipeline that enables virtual staining, segmentation, classification, and counting of white blood cells (WBCs) in single-channel images of peripheral blood smears. Methods. We train independent deep networks to virtually stain and segment grayscale images of smears. The segmented images are then used to train a classifier to yield a quantitative five-part WBC differential. Results. Our virtual staining scheme accurately recapitulates the appearance of cells under conventional Giemsa staining, the gold standard in hematology. The trained cellular and nuclear segmentation networks achieve high accuracy, and the classifier can achieve a quantitative five-part differential on unseen test data. Conclusion. This proposed automated hematology analysis framework could greatly simplify and improve current complete blood count and blood smear analysis and lead to the development of a simple, fast, and low-cost, point-of-care hematology analyzer.

Genotypes Diversity and Virulence Factor screening of Trichomonas vaginalis Isolated from Pregnant Women in Mosul (North of Iraq)

Trichomonas vaginalis is a causative agent of trichomoniasis , one of the most common non-viral sexually transmitted disease (STD) over all the world, especially in immunocompromised women such as pregnant. Wet smear and Giemsa stain are the current methods used in hospital to diagnosis trichomoniasis. DNA based diagnosis is still to be validated to diagnose the local isolates, the objective of the present study was to compare the conventional methods of disease diagnosis with the DNA-based method to diagnose Trichomonas incidence in local isolates. In the present study, 105 samples were collected from outpatient women (18-45 years) of Maternity hospital in Mosul who showed a classical presentation of Trichomonas infection including foul-smelling vaginal discharge with a pH exceeding 4.5. Samples underwent microscopic examination and nucleic acid detection of AP65-1 gene, the wet smear test showed that 16.18% of the collected specimen were positive while the parasite appeared only in 8.9% of the samples stained with Giemsa dye, the molecular screening for AP65-1gene that encodes for the surface attachment protein showed high sensitivity level of 100 at 50 specificity level in compare with other routine methods, the algorithm was evaluated according to specificity, sensitivity, and predictive values. Random Amplification Polymorphic DNA (RAPD) was achieved to estimate genetic indices among isolated strains. Phylogenetic analysis was performed using PAST (Paleontological statistics software) and dendrogram with neighboring clusters was constructed. According to the outcomes of research, we recommended to utilize a probiotic vaccines and molecular silencing vaccine (like miRNA sense and antisense oligonucleotides) as a treatment in.

In vitro antiprotozoal effect of alcoholic extract of hemolymph of Galleria mellonella larva against Trichomonas gallinae

Trichomonas gallinae is a protozoan parasite that lives in the upper gastrointestinal tract of birds. Recently, the hemolymph of Galleria mellonella has been widely used as an anti-parasitic drug. The present work aimed to investigate the inhibitory activity of an alcoholic extract of G. mellonella hemolymph (AEGmH) on T. gallinae in vitro compared with that of metronidazole using wet smear and vital stain acridine orange (AO). The results showed that the degree of growth inhibition of Trichomonas was based on the AEGmH concentration. Furthermore, the highest reduction of parasitic growth (100%) was observed in cultures treated with 25, 12.5, and 6.25mg/mL AEGmH before 24h. Moreover, the same results were detected in cultures treated with 3.1 and 1.6mg/mL AEGmH, but after 48h, and doses of AEGmH (0.8 and 0.4mg/mL) after 96h. In comparison, complete inhibition of parasite growth (100%) was obtained by metronidazole at 25mg/mL after 72h. Besides, light microscopy observations revealed changes in trophozoites' morphology in culture treated with AEGmH. We concluded that the alcoholic extract of hemolymph of G. mellonella is suitable to counter trichomoniasis in vitro and showed antiprotozoal potential.

Infection Control of Gastrointestinal Endoscopic Procedures Performed by Nurses at Hawler and Rizgary Teaching Hospitals in Erbil City /Iraq

Background and objectives: Gastrointestinal endoscopy is a valuable diagnostic and therapeutic tool for the care of patients with gastrointestinal and pancreaticobiliary disorders. Compliance with accepted guidelines for the reprocessing of gastrointestinal endoscopes between patients is critical to the safety and success of their use. This study aimed to assess the nurses' infection control practice and identify the prevalence of fungal and bacterial growth on gastroscopy and colonoscopy instruments. Methods: A descriptive study was carried out on a purposive (non-probability) sample of all nurses who worked in the gastrointestinal endoscopy units at Hawler and Rizgari Teaching Hospitals in Erbil City from January to June 2016 from where the sample specimens were also taken. The questionnaire consisted of three parts. The first part included sociodemographic data, the second part contained nurses' infection control practice before, during and after the endoscopy procedure including pre-cleaning, leak testing, manual cleaning rising, disinfection, rinsing, drying and storing instruments. The third part of the questionnaire was used to document 80 endoscopic specimens, which were collected from the endoscopic instruments. Results: The findings of the study revealed that the majority of nurses (38.5%) were within the 25-35 years age group, 61.5% were males and having less than 5 years experience. The infection control practices for endoscopy procedure showed that a majority (69.2%) of nurses practised at a fair level. The Grams stain smear results showed positivity of 65.62% for fungus and 34.375% for bacteria. All the fungi isolated belonged to the Candida species. Conclusion: The study concluded that the infection control of endoscopic procedures performed by nurses was inadequate. The swab cultures from the automated endoscope reprocessors, in combination with other bacterial isolates, showed that the Candida species were present in the endoscopic specimens.

Acanthoamoeba Keratitis Induced by a Therapeutic, Soft Contact Lens: Diagnosis via Gram Staining

Purpose: We report two cases of &lt;i&gt;Acanthamoeba&lt;/i&gt; keratitis diagnosed by Gram staining in patients who had recently worn therapeutic, soft contact lenses and had no history of lens use for visual correction.Case summary: The first patient was initially diagnosed with suspected mixed bacterial or fungal keratitis before a final diagnosis of &lt;i&gt;Acanthamoeba&lt;/i&gt; keratitis was confirmed by Gram staining of a corneal smear. The second patient was initially diagnosed with a persistent epithelial defect caused by an earlier lid injury inflicted by a metallic foreign body, and then with a suspected mixed infection combined with herpetic uveitis. The patient was finally diagnosed with &lt;i&gt;Acanthamoeba&lt;/i&gt; keratitis by Gram staining of a corneal smear. Both cases were treated with polyhexamethylene biguanide and chlorhexidine.Conclusions: Therapeutic, soft contact lenses are used to enhance corneal, epithelial wound healing in conjunction with antimicrobial prophylaxis. However, application of such a lens to a diseased cornea may predispose to the development of microbial keratitis caused by microorganisms resistant to the usual, prophylactic, antimicrobial eye drops. Therapeutic, soft contact lenses are associated with a risk of &lt;i&gt;Acanthamoeba&lt;/i&gt; keratitis; early diagnosis is important. Gram staining of a corneal smear is useful in this context. &lt;i&gt;Acanthamoeba&lt;/i&gt; is not eradicated by empirical broad-spectrum antimicrobials.

Increased Bone Marrow Iron at Diagnosis Is Associated with Inferior Prognosis in Patients with Myelodysplastic Syndromes

Introduction: Iron storage in patients (pts) with myelodysplastic syndromes at the time of diagnosis may vary from normal to iron overload. Even before the first blood transfusion, storage iron can be increased due to down-regulation of hepcidin and subsequent increase in duodenal iron uptake. Iron overload is known to worsen the prognosis of MDS patients, partly due to iron-related organ damage after long-term transfusion therapy, and partly due to an increased risk of infections. However, it is unclear whether increased storage iron at the time of diagnosis already has a prognostic influence. We assessed bone marrow iron stores at the time of MDS diagnosis and correlated them with clinical outcome.Methods: In a retrospective analysis of 3762 adult MDS patients from the Düsseldorf MDS Registry, Prussian blue staining of marrow smears was performed in our cytology lab to assess iron stores according to the following categories: normal or decreased iron stores versus increased iron stores versus iron overload. Patients were followed up for survival and AML evolution until June 2021. Median time of follow-up was 20 months. 67.4% of the patients died during the course of the disease.Results: The study included 3.762 adult patients who received their initial diagnosis of MDS between 1970 and 2021. 58% were diagnosed as non-blastic MDS ( MDS SLD (RS) (n=240), MDS MLD (RS) (n=350), MDSdel(5q) (n=107), and MDS-U (n=25). Iron stores were decreased in 8% of the patients, normal in 44%, increased in 41%, and strongly increased in 7% (massive iron overload).In 282 cases, histologic assessment of storage iron was available. When comparing cytologic and histologic assessment, we found a strong correlation (p<0.0005), since 87% of the patients with increased iron on cytomorphology also showed increased iron as assessed by histopathology. However, 37% of the patients who cytologically showed normal iron stores, were reported to have slightly increased iron as assessed by histopathology. Median and mean serum ferritin values of patients with normal or decreased iron stores were 295 and 629 µg/l, respectively, as compared to 548 and 902 µg/l, respectively, in patients with increased iron stores.The cumulative risk of AML evolution was not associated with the results of iron staining. Regarding survival, we found that patients with decreased or normal storage iron had a median survival of 31 months, whereas those with increased iron had a median survival of 28 months (p=0.007). Focusing on patients with non-blastic MDS, the difference was not significant (46 vs 44 ms). However, patients who presented as EB I (n=435), EBII (n=510), AML MRC (n=264), CMML I (n=254), or CMML II (n=77), showed a prognostic impact of storage iron; patients with increased iron had a median survival of 11 months, as compared to 16 months in patients with normal or decreased iron (p<0.0005).Conclusion: Increased tissue iron in the bone marrow at the time of diagnosis is associated with inferior survival in patients with MDS, primarily in patients with higher risk MDS. At diagnosis, patients are not yet transfusion-dependent. This suggests that increased iron reflects a prolonged period of increased duodenal iron uptake as a consequence of ineffective erythropoiesis. Therefore, increased marrow iron at the time of MDS diagnosis seems to be a surrogate parameter of hematopoietic insufficiency, which is the real cause of inferior prognosis. DisclosuresNachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Gattermann: Novartis: Honoraria; Takeda: Research Funding; Celgene: Honoraria. Germing: Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Research Funding.

Deep Learning-Based Cell Detection and Extraction in Thin Blood Smears for Malaria Diagnosis.

Malaria is a major health threat caused by Plasmodium parasites that infect the red blood cells. Two predominant types of Plasmodium parasites are Plasmodium vivax (P. vivax) and Plasmodium falciparum (P. falciparum). Diagnosis of malaria typically involves visual microscopy examination of blood smears for malaria parasites. This is a tedious, error-prone visual inspection task requiring microscopy expertise which is often lacking in resource-poor settings. To address these problems, attempts have been made in recent years to automate malaria diagnosis using machine learning approaches. Several challenges need to be met for a machine learning approach to be successful in malaria diagnosis. Microscopy images acquired at different sites often vary in color, contrast, and consistency caused by different smear preparation and staining methods. Moreover, touching and overlapping cells complicate the red blood cell detection process, which can lead to inaccurate blood cell counts and thus incorrect parasitemia calculations. In this work, we propose a red blood cell detection and extraction framework to enable processing and analysis of single cells for follow-up processes like counting infected cells or identifying parasite species in thin blood smears. This framework consists of two modules: a cell detection module and a cell extraction module. The cell detection module trains a modified Channel-wise Feature Pyramid Network for Medicine (CFPNet-M) deep learning network that takes the green channel of the image and the color-deconvolution processed image as inputs, and learns a truncated distance transform image of cell annotations. CFPNet-M is chosen due to its low resource requirements, while the distance transform allows achieving more accurate cell counts for dense cells. Once the cells are detected by the network, the cell extraction module is used to extract single cells from the original image and count the number of cells. Our preliminary results based on 193 patients (including 148 P. Falciparum infected patients, and 45 uninfected patients) show that our framework achieves cell count accuracy of 92.2%.

A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection.

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

Diagnostic yield of bronchoalveolar lavage GeneXpert in smear-negative and sputum-scarce pulmonary tuberculosis

Background Tuberculosis remains a common public health hazard in the underdeveloped world. Initial approach to diagnosis is sputum smear staining for acid-fast bacilli, however only 40% of patients are smear positive. Aim and objectives To assess the diagnostic yield of GeneXpert in bronchoalveolar lavage in a smear negative or sputum scarce suspected case of pulmonary tuberculosis. Methods This analytical cross-sectional study included 50 patients who had clinical and radiological signs consistent with pulmonary tuberculosis and three negative Ziehl–Neelsen sputum smears or unobtainable sputum. All patients underwent fiberoptic bronchoscopy guided bronchoalveolar lavage that was sent for evaluation with GeneXpert and mycobacterial culture (Löwenstein–Jensen). Sensitivity analysis was carried out using culture as the gold standard. Results There is a statistically significant increase of positive TB cases diagnosed by bronchoalveolar lavage GeneXpert, as all cases were positive (100%) including 17 patients that were sputum scant, while all cases diagnosed by smear acid-fast bacilli were negative (100%) (P&lt;0.05) However, there is statistically insignificant difference of positive TB cases diagnosed by bronchoalveolar lavage GeneXpert (100%) and bronchoalveolar lavage L.J culture (100%) (P&gt;0.05). Conclusion Bronchoalveolar lavage GeneXpert has superior diagnostic yield than acid-fast bacilli smear microscopy, and the yield is as effective as mycobacterial cultures, but much faster and time saving. It can be used as a gold standard for TB diagnosis.

A new single run polymerase chain reaction assay for cyclosporiasis in immunocompromised patients

causes human intestinal cyclosporiasis. It is more common in the immunocompromised patients and mainly seen in people living with HIV/AIDS (PLHA), post-renal transplant (PRT) patients and immunocompromised children (IC). Diagnostic microscopy for the oocysts of the parasite is less sensitive, requiring examination of multiple stool samples. Here we developed a new single run polymerase chain reaction (PCR) assay for the detection of and it was used to know the hospital based prevalence of cyclosporiasis. A cross-sectional study was conducted from June 2016 to October 2020 in a tertiary care teaching hospital. A new single run amplification PCR-based diagnostic assay was developed for . Stool samples were collected from 121 PLHA, 135 PRT and 79 immunocompromised children (IC) other than PLHA and PRT. All stool samples were examined for the presence of oocysts as well as tested with new PCR assay. Modified Ziehl-Neelsen staining of the concentrated stool smear did not reveal oocysts of species in any stool specimen. However, new PCR assay detected in 2 stool specimens – one from a PLHA patient and another from a PRT patient, giving a prevalence of 0.6% (2/335), 0.8% (1/121) in PLHA and 0.7% (1/135) in PRT. It was not detected in IC. Cyclosporiasis is infrequent in southern part of India. The new single run PCR assay developed by us is simple and cost effective molecular assay for the detection of .

Emerging cases of mucormycosis in post Covid-19 disease patients

Mucormycosis (MC) is a serious threat in this covid-19 pandemic situation; cases of MC continue to increases as post covid-19 disease affliction in India. It is a life-threatening disease that happens due to black mold. Mucormycosis is described as a potentially lethal infection amongst immune-compromised (IC) hosts, particularly in those with diabetes, leukemia, and lymphoma. Mortality rate of MC just doubles in IC host, delayed diagnosis increase the rate of mortality. Mucormycosis is difficult to diagnose which affects outcomes and results in a poor prognosis. The main objective of the study was to detect MC in the clinical species received during or post covid-19 treatment in our laboratory.Basic microbiological methods such as gram stain and KOH smear were used for the detection of MC in the received clinical specimen and morphology was seen in the microscope. The is a cross-sectional observational study conducted in our microbiology laboratory for one month periods during May 15, 2021 to June 15, 2021 in a tertiary care hospital of central India. During study period our microbiology lab received n=35 suspected clinical specimens from N= 27 post covid-19 patients for MC diagnosis over one month period. Out of n=35 specimens, n=8 specimens (obtained from N=5 patients) were positive for MC by gram and KOH smear method and we saw filamentous fungi by conventional microscopic method. The present study concluded that the cases of life threatening MC increase day by day in central India as post complication of covid-19 disease.

Scrofuloderma: A Rare Case Report of Sequelae of Intestinal Tuberculosis

Abstract Introduction: Scrofuloderma is caused by Mycobacterium tuberculosis and is the most common form of cutaneous tuberculosis. It remains a significant medical challenge because its clinical manifestations mimic those of various other conditions, making diagnosis difficult. Here, we report a 28-year-old man who had been diagnosed with intestinal tuberculosis but discontinued treatment. Case presentation: He subsequently presented with a 5-month history of multiple non-healing ulcerative lesions on the abdomen with no other significant clinical findings. Ziehl-Neelsen staining of an impression smear taken from the lesions was performed as the preliminary investigation and confirmed the presence of acid-fast bacilli. Histopathologic examination of a skin biopsy specimen, PCR, and the Mantoux test also confirmed the presence of Mycobacterium tuberculosis. The patient began treatment with anti-tubercular drugs and his lesions gradually regressed. Discussion: The most common finding of scrofuloderma is a chronic, painless ulcerative lesion with purulent discharge. Cervical lymph nodes are frequently involved. These clinical features mimic several other bacterial and fungal conditions. However, Ziehl-Neelsen staining of the discharging ulcers can be used as a preliminary test to demonstrate acid fast bacilli in most of the cases. Histopathologic examination of skin biopsy specimen, culture on Lowenstein-Jensen media and PCR can confirm the diagnosis. Conclusion: Although Scrofuloderma is a rare cutaneous manifestation, it is curable with early diagnosis and prompt intervention of antitubercular drugs.

Prevalence of Intestinal Parasitic Infections Among Diarrheic Patients from Ahvaz, Southwestern Iran.

Intestinal parasitic infections are among the most common infections in humans, particularly in resource-poor communities. Gastrointestinal parasites, specially protozoa, can lead to diarrhea, malabsorption, and anemia. The majority of parasitic diarrhea is caused by Entamoeba histolytica, Giardia duodenalis, and Cryptosporidium spp. The present study aimed to evaluate the prevalence of intestinal parasites among diarrheic patients referred to the Shahid Rajaee Polyclinic, Ahvaz, southwestern Iran. A cross-sectional study was conducted to determine the prevalence of intestinal parasites among 250 diarrheic patients using direct smear, formalin-ether concentration, Ziehl-Neelsen and trichrome staining. The results indicated that 34.4% (86/250) of the patients were infected with pathogenic parasites. Giardia duodenalis with an occurrence of 18.8% (47/250) and Cryptosporidium spp. with a frequency of 2.8% (7/250) had the highest and lowest infection rates, respectively. Blastocystis hominis with a frequency of 15.2% (38/250) showed the highest prevalence rate after G. duodenalis. Entamoeba histolytica/dispar was observed in 3 (1.2%) of diarrheic patients. The age group 1-10 years old was the most frequently infected group (27.9%). We could not find a significant association between the source of drinking water and intestinal parasitic infections (p= 0.912). This study demonstrated that G. duodenalis was the predominant parasite found among the patients. The results revealed that intestinal parasites were one of the main health problems in the region.