Diagnostic Accuracy of Polymerase Chain Reaction Against Giemsa Staining on Tissue Biopsy for Cutaneous Leishmaniasis.

OBJECTIVE: To evaluate the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) kit targeting 18S rRNA against Giemsa-stained tissue slides in patients clinically suspected of cutaneous leishmaniasis (CL). STUDY DESIGN: Cross-sectional analytical study. Place and Duration of the Study: Department of Microbiology, Armed Forces Institute of Pathology / National University of Medical Sciences, Rawalpindi, Pakistan, from July to December 2022. METHODOLOGY: Samples of skin tissue in 98 patients suspected of CL were evaluated. These samples were subjected to Giemsa-staining for microscopy and real-time PCR. Sensitivity, specificity, and accuracy of the PCR were calculated keeping Giemsa-stained tissue slide microscopy as gold standard. RESULTS: Out of the 98 tissue samples, 37 were found positive for leishmaniasis on PCR while 13 were found Leishmania positive on microscopy of Giemsa-stained slides. The sensitivity, specificity, and accuracy of the PCR for the detection of Leishmania species were 100%, 71.8%, and 91.8%, respectively with 100% negative predictive value. CONCLUSION: This study demonstrates that the commercial PCR is a reliable diagnostic test for the diagnosis of CL. The ease, rapidity, and reliability of the PCR make it a dependable tool in diagnostic repertoire of CL. KEY WORDS: Giemsa stain, Leishmania spp., Polymerase chain reaction, Viasure.

DeepLeish: a deep learning based support system for the detection of Leishmaniasis parasite from Giemsa-stained microscope images.

BACKGROUND: Leishmaniasis is a vector-born neglected parasitic disease belonging to the genus Leishmania. Out of the 30 Leishmania species, 21 species cause human infection that affect the skin and the internal organs. Around, 700,000 to 1,000,000 of the newly infected cases and 26,000 to 65,000 deaths are reported worldwide annually. The disease exhibits three clinical presentations, namely, the cutaneous, muco-cutaneous and visceral Leishmaniasis which affects the skin, mucosal membrane and the internal organs, respectively. The relapsing behavior of the disease limits its diagnosis and treatment efficiency. The common diagnostic approaches follow subjective, error-prone, repetitive processes. Despite, an ever pressing need for an accurate detection of Leishmaniasis, the research conducted so far is scarce. In this regard, the main aim of the current research is to develop an artificial intelligence based detection tool for the Leishmaniasis from the Geimsa-stained microscopic images using deep learning method. METHODS: Stained microscopic images were acquired locally and labeled by experts. The images were augmented using different methods to prevent overfitting and improve the generalizability of the system. Fine-tuned Faster RCNN, SSD, and YOLOV5 models were used for object detection. Mean average precision (MAP), precision, and Recall were calculated to evaluate and compare the performance of the models. RESULTS: The fine-tuned YOLOV5 outperformed the other models such as Faster RCNN and SSD, with the MAP scores, of 73%, 54% and 57%, respectively. CONCLUSION: The currently developed YOLOV5 model can be tested in the clinics to assist the laboratorists in diagnosing Leishmaniasis from the microscopic images. Particularly, in low-resourced healthcare facilities, with fewer qualified medical professionals or hematologists, our AI support system can assist in reducing the diagnosing time, workload, and misdiagnosis. Furthermore, the dataset collected by us will be shared with other researchers who seek to improve upon the detection system of the parasite. The current model detects the parasites even in the presence of the monocyte cells, but sometimes, the accuracy decreases due to the differences in the sizes of the parasite cells alongside the blood cells. The incorporation of cascaded networks in future and the quantification of the parasite load, shall overcome the limitations of the currently developed system.

Leveraging code-free deep learning for pill recognition in clinical settings: A multicenter, real-world study of performance across multiple platforms.

BACKGROUND: Preventable patient harm, particularly medication errors, represent significant challenges in healthcare settings. Dispensing the wrong medication is often associated with mix-up of lookalike and soundalike drugs in high workload environments. Replacing manual dispensing with automated unit dose and medication dispensing systems to reduce medication errors is not always feasible in clinical facilities experiencing high patient turn-around or frequent dose changes. Artificial intelligence (AI) based pill recognition tools and smartphone applications could potentially aid healthcare workers in identifying pills in situations where more advanced dispensing systems are not implemented. OBJECTIVE: Most of the published research on pill recognition focuses on theoretical aspects of model development using traditional coding and deep learning methods. The use of code-free deep learning (CFDL) as a practical alternative for accessible model development, and implementation of such models in tools intended to aid decision making in clinical settings, remains largely unexplored. In this study, we sought to address this gap in existing literature by investigating whether CFDL is a viable approach for developing pill recognition models using a custom dataset, followed by a thorough evaluation of the model across various deployment scenarios, and in multicenter clinical settings. Furthermore, we aimed to highlight challenges and propose solutions to achieve optimal performance and real-world applicability of pill recognition models, including when deployed on smartphone applications. METHODS: A pill recognition model was developed utilizing Microsoft Azure Custom Vision platform and a large custom training dataset of 26,880 images captured from the top 30 most dispensed solid oral dosage forms (SODFs) at the three participating hospitals. A comprehensive internal and external testing strategy was devised, model's performance was investigated through the online API, and offline using exported TensorFlow Lite model running on a Windows PC and on Android, using a tailor-made testing smartphone application. Additionally, model's calibration, degree of reliance on color features and device dependency was thoroughly evaluated. Real-world performance was assessed using images captured by hospital pharmacists at three participating clinical centers. RESULTS: The pill recognition model showed high performance in Microsoft Azure Custom Vision platform with 98.7 % precision, 95.1 % recall, and 98.2 % mean average precision (mAP), with thresholds set to 50 %. During internal testing utilizing the online API, the model reached 93.7 % precision, 88.96 % recall, 90.81 % F1-score and 87.35 % mAP. Testing the offline TensorFlow Lite model on Windows PC showed a slight performance reduction, with 91.16 % precision, 83.82 % recall, 86.18 % F1-score and 82.55 % mAP. Performance of the model running offline on the Android application was further reduced to 86.50 % precision, 75.00 % recall, 77.83 % F1-score and 69.24 % mAP. During external clinical testing through the online API an overall precision of 83.10 %, recall of 71.39 %, and F1-score of 75.76 % was achieved. CONCLUSION: Our study demonstrates that using a CFDL approach is a feasible and cost-effective method for developing AI-based pill recognition systems. Despite the limitations encountered, our model performed well, particularly when accessed through the online API. The use of CFDL facilitates interdisciplinary collaboration, resulting in human-centered AI models with enhanced real-world applicability. We suggest that rather than striving to build a universally applicable pill recognition system, models should be tailored to the medications in a regional formulary or needs of a specific clinic, which can in turn lead to improved performance in real-world deployment in these locations. Parallel to focusing on model development, it is crucial to employ a human centered approach by training the end users on how to properly interact with the AI based system to maximize benefits. Future research is needed on refining pill recognition models for broader adaptability. This includes investigating image pre-processing and optimization techniques to enhance offline performance and operation on handheld devices. Moreover, future studies should explore methods to overcome limitations of CFDL development to enhance the robustness of models and reduce overfitting. Collaborative efforts between researchers in this domain and sharing of best practices are vital to improve pill recognition systems, ultimately enhancing patient safety and healthcare outcomes.

The study of the protective effect of mitochondrial uncouplers during acute toxicity of the fungicide difenoconazole in different organs of mice.

Pesticides represent a serious problem for agricultural workers due to their neurotoxic effects. The aim of this study was to evaluate the ability of pharmacological oxidative phosphorylation uncouplers to reduce the effect of the difenoconazole fungicide on mitochondrial DNA (mtDNA) of various organs in mice. Injections of difenoconazole caused cognitive deficits in mice, and the protonophore 2,4-dinitrophenol (2,4-DNP) and Azur I (AzI), a demethylated metabolite of methylene blue (MB), prevented the deterioration of cognitive abilities in mice induced by difenoconazole. Difenoconazole increased the rate of reactive oxygen species (ROS) production, likely through inhibition of complex I of the mitochondrial respiratory chain. After intraperitoneal administration of difenoconazole lungs, testes and midbrain were most sensitive to the accumulation of mtDNA damage. In contrast, the cerebral cortex and hippocampus were not tolerant to the effects of difenoconazole. The protonophore 2,4-DNP reduced the rate of ROS formation and significantly reduced the amount of mtDNA damage caused by difenoconazole in the midbrain, and partially, in the lungs and testes. MB, an alternative electron carrier capable of bypassing inhibited complex I, had no effect on the effect of difenoconazole on mtDNA, while its metabolite AzI, a demethylated metabolite of MB, was able to protect the mtDNA of the midbrain and testes. Thus, mitochondria-targeted therapy is a promising approach to reduce pesticide toxicity for agricultural workers.

Morphology and morphometry of sperm in Kurdish stallions, a local breed from western Iran.

The present work was designed for a thorough investigation into the sperm morphology and morphometry of Kurdish stallions. The semen samples were collected from 10 Kurdish stallions. Three preparations from each ejaculate were stained with eosin-nigrosin (EN), Diff-Quik (DQ) and Rose Bengal (RB). The area, perimeter, length and width of the sperm head as well as tail length and total sperm length were measured. The parameters ellipticity, elongation, roughness and regularity were calculated. The morphology of sperm was also investigated under scanning and transmission electron microscopes. DQ and RB provided more clarified images for examining sperm structures compared to the EN method. The head length, head width, area and perimeter in EN were significantly higher than those in DQ and RB (p ≤ .05). Furthermore, the difference in head width, head area and head perimeter between DQ and RB was not significant (p ≥ .05). The tail length and total sperm length in all methods were close together (p ≥ .05). The highest percentage of normal sperm was seen in DQ and RB methods (82.55 ± 2.88 and 88.31 ± 5.19) respectively. The highest values for ellipticity, elongation and regularity were found in RB, whereas the highest value for roughness was measured in EN. Tail defects including coiled tails, and folded midpieces were the most frequent. Scanning electron microscope revealed two types of head shapes: heads with round anterior border, and heads with flat anterior border. The results indicated that despite the routine use of EN for morphological assessment of stallion sperm, RB and DQ can be considered for more clarified details of sperm structure including acrosome and midpiece. Furthermore, the Kurdish stallion sperm has morphometric traits in the normal range established for stallions; yet, some traits were larger than those reported for other breeds. It seems that the sperm of the Kurdish stallion has a longer head and tail in comparison with other horse breeds.

Fluorescent periodic acid Schiff-like staining combined with standard cytologic staining of the same cytologic specimen may facilitate cytopathologic diagnosis.

BACKGROUND: Periodic acid Schiff (PAS) staining which detects glycogen and mucosubstances is frequently used as an ancillary method for an accurate cytopathologic diagnosis. Unfortunately, cytologic slides for PAS stain are not routinely prepared. Aqueous 7-amino-4-methylcoumarin (AMC) is colorless and transparent under bright field illumination but exhibits strong fluorescence under ultraviolet (UV) light and can be used as a Schiff reagent. We recently reported that combining [author: Please define (H&E) in the first occurrence if necessary.]H&E and AMC is useful for histopathologic diagnosis of various disease conditions. In this study, we investigated whether standard cytologic staining (Papanicolaou [Pap] and Giemsa) combined with AMC was useful for cytopathologic analysis. METHODS: Specimens of non-neoplastic human tissues and archived cytologic specimens of various disease conditions were stained with a combination of Pap and AMC (Pap/AMC) or Giemsa and AMC (Giemsa/AMC). RESULTS: The addition of AMC had no significant effect on Pap or Giemsa staining, and the cytomorphology under bright field microscopy was perfectly preserved. The AMC fluorescent signals observed under UV light were intense and the staining pattern was identical to that obtained by PAS staining. Diastase digestion differentiated glycogen from other AMC-positive elements. The efficacy of using Pap/AMC and Giemsa/AMC for archived cytologic specimens was demonstrated in several diseases including cases of endometrial carcinoma, adenoid cystic carcinoma, metastatic signet-ring cell carcinoma, candidiasis, and trichomoniasis. CONCLUSION: Pap/AMC and Giemsa/AMC are useful in aiding cytopathologic diagnosis especially when the information gained from PAS staining is critical and cytologic specimens for PAS are not available.

Romanowsky staining: history, recent advances and future prospects from a chemistry perspective.

Romanowsky staining was an important methodological breakthrough in diagnostic hematology and cytopathology during the late 19th and early 20th centuries; it has facilitated for decades the work of biologists, hematologists and pathologists working with blood cells. Despite more than a century of studying Romanowsky staining, no systematic review has been published that explains the chemical processes that produce the "Romanowsky effect" or "Romanowsky-Giemsa effect" (RGE), i.e., a purple coloration arising from the interaction of an azure dye with eosin and not due merely to their simultaneous presence. Our review is an attempt to build a bridge between chemists and biomedical scientists and to summarize the available data on methylene blue (MB) demethylation as well as the related reduction and decomposition of MB to simpler compounds by both light and enzyme systems and microorganisms. To do this, we analyze modern data on the mechanisms of MB demethylation both in the presence of acids and bases and by disproportionation due to the action of light. We also offer an explanation for why the RGE occurs only when azure B, or to a lesser extent, azure A is present by applying experimental and calculated physicochemical parameters including dye-DNA binding constants and electron density distributions in the molecules of these ligands. Finally, we discuss modern techniques for obtaining new varieties of Romanowsky dyes by modifying previously known ones. We hope that our critical literature study will help scientists understand better the chemical and physicochemical processes and mechanisms of cell staining with such dyes.

Photodynamic inactivation of methicillin-resistant Staphylococcus aureus by using Giemsa dye as a photosensitizer.

The rise of antibiotic-resistant bacteria calls for innovative approaches to combat multidrug-resistant strains. Here, the potential of the standard histological stain, Giemsa, to act as a photosensitizer (PS) for antimicrobial photodynamic inactivation (aPDI) against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains is reported. Bioassays were performed using various Giemsa concentrations (ranging from 0.0 to 20.0 µM) under 625 nm illumination at a light dose of 30 J cm-2. Remarkably, Giemsa completely inhibited the growth of MSSA and MRSA bacterial colonies for concentrations at 10 µM and higher but exhibited no inhibitory effect without light exposure. Partition coefficient analysis revealed Giemsa's affinity for membranes. Furthermore, we quantified the production of reactive oxygen species (ROS) and singlet oxygen (1O2) to elucidate the aPDI mechanisms underlying bacterial inactivation mediated by Giemsa. These findings highlight Giemsa stain's potential as a PS in aPDI for targeting multidrug-resistant bacteria.

Initial assessment of "Gissell's stain": A novel histopathological method for the identification of Helicobacter pylori.

INTRODUCTION AND OBJECTIVES: The histopathological identification of Helicobacter pylori using the routine method (haematoxylin-eosin) is not only very difficult but also has low sensitivity. Giemsa staining is often used in addition, but different protocols do not produce homogeneous results. Furthermore, the Gold Standard recommended by the European Helicobacter Pylori Study Group has been applied in very few studies, thus resulting in uncertain outcomes. Therefore, a new staining method is required to overcome these limitations. The aim of this study was to evaluate the diagnostic capacity and inter-observer agreement of "Gissell's stain". MATERIAL AND METHODS: A cross-sectional study evaluated 99 gastric paraffin blocks from a private laboratory. Three sections were prepared from each block, and haematoxylin-eosin (HE), Giemsa and "Gissell's stain" methods were applied. The kappa statistics, sensitivity, specificity, and predictive values were calculated. RESULTS: "Gissell's stain" obtained the highest inter-observer agreement (kappa=0.87) compared to the other two methods (HE, kappa=0.51; Giemsa, kappa=0.83). It also obtained the best sensitivity and negative predictive value (97.1% and 98.3%, respectively) compared with the other two methods (HE: 68.6% and 85.1%, respectively; Giemsa: 88.6% and 93.9%, respectively). CONCLUSIONS: Given its unique characteristics (fast, cheap, accessible, and easy to use), in addition to its statistical reliability, "Gissell's stain" has great potential for routine use in the identification of H. pylori.

Preliminary study of chromosome aberrations using Giemsa, two-colour fish, and micronucleus assays in lymphocytes of individuals living in elevated radon concentration areas.

The frequencies of unstable and stable chromosome aberrations and micronuclei were examined in peripheral blood samples from 10 individuals living in elevated radon concentration areas (Takandeang Village, Mamuju, Indonesia). Blood samples from 10 people living in Topoyo Village were used as a control group. For unstable chromosome aberration analysis, a dicentric chromosome assay was conducted using conventional Giemsa staining. Chromosomal painting of chromosomes 1 and 4 using the fluorescence in situ hybridisation technique was also applied to four subjects to assess the stable chromosome aberration. Our study showed no significant increases across all groups in dicentric and other unstable chromosome aberrations, such as rings and acentric fragments. Translocations were found in one person from Takandeang Village and two Topoyo Village inhabitants. The translocations found in the subjects from Takandeang Village were due more to aging factors than to radon exposure. The number of micronuclei per 1000 binucleus cells in Takandeang Village inhabitants was not significantly different than that in the control group (p = 0.943). A more comprehensive analysis should be conducted in a subsequent study by increasing the number of study donors and the number of metaphases to be analysed in both dicentric chromosome assay and fluorescence in situ hybridisation assays. Such research could provide valid information on the cytogenetic effects of elevated indoor radon exposure.

Tracking the geographical origin of Plasmodium falciparum causing a rare severe case of malaria imported into Palestine, a zero-indigenous case area.

BACKGROUND: Malaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system. CASE PRESENTATION: Three days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria [1, 2]. The double chromatin configuration (headphones) and accolé (applique) position are both indicative of Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer's instructions (Illumina® DNA Prep, (M) Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two. CONCLUSION: The travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like msp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination.

Diagnostic utility of special stains in defining the spectrum of maxillofacial pathologies.

Background: The Fine needle aspiration cytology (FNAC) is considered as a valuable and distinguished diagnostic test in the initial assessment of the patients presenting with a mass in the head and neck region or when a recurrence is suspected after previous treatment. Aims: This study was therefore designed to elucidate the efficacy of FNAC as an alternate diagnostic tool to histopathology in head and neck swellings and evaluation of staining efficacy of PAP and MGG stain over Haematoxylin and eosin (H and E) in routine cytopathological smears. Settings and Design: The study was conducted in the Department of Oral and Maxillofacial Pathology, where FNAC samples were collected from 150 patients with head and neck swellings. Materials and Methods: All the slides were stained with H and E, Papanicolaou (PAP), and May Grunewald Giemsa (MGG) stains. The cytopathological diagnosis was compared with histopathological diagnosis based on H and E stained sections obtained from paraffin-embedded formalin-fixed biopsy specimen of benign and malignant neoplasms. Statistical Analysis Used: The resulting data were analyzed using SPSS software version 19. Differences between the variables were analyzed using Pearson Chi-square test and Kruskal-Wallis test wherever applicable. Results: The FNAC as a diagnostic tool has sensitivity of 84.8%, 72.72%, and 78.78%, specificity of 62.5%, 75%, and 75%, and accuracy of 80.48%, 73.14%, and 78.04% in H and E, MGG, and PAP stain, respectively. PAP stain was the most efficient stain when all qualitative parameters are taken into consideration with maximum sensitivity and specificity for achieving definitive cytodiagnosis. Conclusions: The FNAC is an inexpensive and minimally invasive technique to diagnose different types of head and neck swellings and complement histopathological diagnosis.

Metagenomic next-generation sequencing for rapid detection of pulmonary infection in patients with acquired immunodeficiency syndrome.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is associated with a high rate of pulmonary infections (bacteria, fungi, and viruses). To overcome the low sensitivity and long turnaround time of traditional laboratory-based diagnostic strategies, we adopted metagenomic next-generation sequencing (mNGS) technology to identify and classify pathogens. RESULTS: This study enrolled 75 patients with AIDS and suspected pulmonary infections who were admitted to Nanning Fourth People's Hospital. Specimens were collected for traditional microbiological testing and mNGS-based diagnosis. The diagnostic yields of the two methods were compared to evaluate the diagnostic value (detection rate and turn around time) of mNGS for infections with unknown causative agent. Accordingly, 22 cases (29.3%) had a positive culture and 70 (93.3%) had positive valve mNGS results (P value < 0.0001, Chi-square test). Meanwhile, 15 patients with AIDS showed concordant results between the culture and mNGS, whereas only one 1 patient showed concordant results between Giemsa-stained smear screening and mNGS. In addition, mNGS identified multiple microbial infections (at least three pathogens) in almost 60.0% of patients with AIDS. More importantly, mNGS was able to detect a large variety of pathogens from patient tissue displaying potential infection and colonization, while culture results remained negative. There were 18 members of pathogens which were consistently detected in patients with and without AIDS. CONCLUSIONS: In conclusion, mNGS analysis provides fast and precise pathogen detection and identification, contributing substantially to the accurate diagnosis, real-time monitoring, and treatment appropriateness of pulmonary infection in patients with AIDS.

Identifying the function of genes involved in excreted vesicle formation in Acanthamoeba castellanii containing Legionella pneumophila.

BACKGROUND: Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free legionellae or Legionella-filled vesicles. The vesicles support Legionella to survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes of Acanthamoeba infected by Legionella (ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape of Legionella from the Acanthamoeba. METHODS: After ingestion of Escherichia coli and Legionella pneumophila, expression levels of target genes in Acanthamoeba were measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation of Legionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain. RESULTS: ACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion of Legionella in Acanthamoeba. ACA1_114460- and ACA1_091500-silenced Acanthamoeba failed to form the Legionella-containing excreted vesicles. Legionella was released as free legionellae from the Acanthamoeba. When the ACA1_362260 of Acanthamoeba was silenced, Legionella-containing excreted vesicles were fused with the lysosome. CONCLUSIONS: These results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba played important roles in the formation of Legionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.

C-Banding of Plant Chromosomes.

C-banding visualizes regions of chromosomes containing constitutive heterochromatin. It creates distinct patterns along the chromosome length and allows precise chromosome identification if C-bands are present in sufficient numbers. It is performed on chromosome spreads generated from fixed material, usually root tips or anthers. While there are numerous lab-specific modifications, all methods share the same steps: acidic hydrolysis, DNA denaturation in strong bases (usually saturated aqueous solution of barium hydroxide), washes in saline solution, and staining in Giemsa-type stain in a phosphate buffer. The method can be used for a wide range of cytogenetic tasks, from karyotyping, meiotic chromosome pairing analyses, to large-scale screening and selection of specific chromosome constructs.

Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects.

BACKGROUND: Large- and small-headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. METHODS: Six sperm staining methods, including Papanicolaou, Diff-Quik, Shorr, Hematoxylin-eosin (HE), Wright, and Wright-Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer-aided sperm morphological analysis system. RESULTS: The highest sperm head length and width were observed with the Wright-Giemsa and Wright staining, followed by the Diff-Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff-Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff-Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright-Giemsa staining. CONCLUSION: Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff-Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.

Evaluation of immunocytochemistry on destained giemsa stained smears as an alternative to conventional technique.

Introduction: Protocol for immunocytochemical (ICC) staining in May-Grünwald Giemsa (MGG)-stained smears has been difficult to establish. It is the need of the hour to be able to use prestained slides for ICC in specific cases to deliver timely diagnoses and reduce inconvenience to patients. Aims and Objectives: To evaluate and compare the use of MGG-stained smears for the purpose of ICC, after de-staining and saline rehydration to that of routine standard ICC. Materials and Methods: A prospective study was conducted on 40 FNAC samples: 25 cases of breast disease and 15 cases of reactive lymphoid hyperplasia known to express pancytokeratin and leukocyte common antigen (LCA)/CD45, respectively. Air-dried smears of each case were stained by standard MGG stain and after the report was dispatched, one smear was selected and sent for ICC. The smears were analyzed to determine the overall result and grade each smear semi-quantitatively with respect to staining-intensity, stain-localization, staining-uniformity, counter-staining, and background-staining. Observations and Results: The proposed protocol was inferior to conventional ICC in all the parameters, more pronounced in pancytokeratin than LCA/CD45. Only 8% of air-dried smears stained for pancytokeratin showed optimal stain intensity (as opposed to 44% of wet-fixed smears), whereas only 14.3% of air-dried smears were optimally stained for LCA (as opposed to 85.7% of wet-fixed smears). Conclusion: The proposed protocol of de-stained Giemsa smears as an alternative to conventional technique for ICC was unsuccessful in giving satisfactory results.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Detection of Helicobacter pylori in Gastric Biopsies Among Sudanese Patients with Confirmed Gastric Disorders Using Three Diagnostic Methods.

BACKGROUND: Helicobacter pylori (H. pylori) infection represents one of the most common chronic bacterial infections in developing countries. However, in Sudan, the infection is not well diagnosed with standard laboratory methods in many parts of the country. This study aimed to detect H. pylori in gastric biopsies of patients with gastric disorders, using three diagnostic methods. MATERIALS AND METHODS: A cross-sectional study was conducted among 100 patients in Gezira state, central Sudan. Giemsa stain for histopathological examination (HPE), rapid urease test (RUT), and polymerase chain reaction (PCR) techniques were performed to detect H. pylori from the gastric biopsy samples as per standard assays. RESULTS: Most of the patients were males (66%), from rural areas (72%) and in the age group 31 to 50 years. H. pylori were identified in 85% of the samples by at least one of the three tests. The highest positivity was detected by HPE (83%), followed by PCR (67%) and RUT (63%), while 59% were positive by the three diagnostic methods. PCR showed higher sensitivity (80.72% vs. 73.49%) and specificity (100% vs. 88.24%) than RUT. Positive predictive values were reported as 100% for PCR and 96.83% for RUT. Considering PCR as a gold standard method, HPE revealed higher sensitivity (100%) than RUT (88.06%). On the contrary, RUT showed higher specificity (87.88%) than PCR (51.52%). There were no significant associations between H. pylori infection patients' gender (p = 0.747). Loss of weight (p = 0.007) and nausea (p = 0.032) were significantly associated with H. pylori infection. CONCLUSIONS: There was a high prevalence of H. pylori infection in central Sudan. This highlights the need to analyze epidemiological status, virulence factors, and strain characteristics to control disease transmission. PCR is a reliable and valuable technique in detecting H. pylori infection from gastric biopsy samples.

Performance of Field's Stain Compared with Conventional Giemsa Stain for the Rapid Detection of Blood Microfilariae in Gabon.

Filarial infections caused by Loa loa and Mansonella perstans are a considerable public health burden in rural regions of Central Africa. Rapid diagnostic tools for the detection of microfilariae in the blood are needed. Field's stain is a rapid staining technique for microscopic slides originally established for malaria diagnostics. It requires less than 1 minute of staining compared with conventional staining protocols requiring at least 15 to 20 minutes for staining and could thus significantly accelerate diagnostics for human filariasis. Here we evaluated Field's stain as a rapid staining technique in comparison to Giemsa stain for the detection of microfilariae in peripheral blood. Blood smears were collected from 175 participants residing in the region of Lambaréné and Fougamou, Gabon. Each participant's samples were stained in parallel with Field's stain and conventional Giemsa stain. Slides were then microscopically assessed and compared for qualitative and quantitative results by a blinded assessor for the two endemic filarial blood pathogens M. perstans and L. loa. Field's stain shows excellent diagnostic performance characteristics for L. loa microfilariae compared with Giemsa staining. Concordance was favorable for M. perstans although lower than for L. loa. Field's stain offers a rapid alternative to Giemsa stain for detection of L. loa microfilariae in thick blood smears. This could help accelerate diagnostics of blood filarial pathogens in mass screening programs or resource constrained health care institutions with high patient load.