Direct Staining Research Articles

Characterization of peripheral cytokine-secreting cells responses in HIV/TB co-infection.

Currently the responses of peripheral cytokine-secreting cells in the natural course of human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection haven't been fully elucidated. The function of peripheral proinflammatory, regulatory and cytotoxic cytokine-secreting cells were investigated by direct intracellular cytokine staining (ICS) and flow cytometry, additionally, the absolute numbers of different cytokine-secreting cells were measured among patients with HIV/TB co-infection (HT group), and compared them with the healthy controls (HC group), patients with TB (TB group) and patients with HIV infection (HIV group). After one week's anti-TB treatment, the changes of the percentages of cytokine-secreting cells were further evaluated in TB and HT groups. Totally 26 individuals in the HC group, 51 in the TB group, 26 in the HIV group and 29 in the HT group were enrolled. The HT. HT group exhibited significantly lower absolute numbers of IFN-γ+CD4+, IFN-γ+CD8+, TNF-α+CD4+, IL17A+CD4+ T cells and TNF-α+CD14+ monocytes than the TB and HIV groups. Compared with the TB group, the percentages of CD8+ T cells secreting IFN-γ and perforin (p=0.010; p=0.043) were significantly lower among the HT group. Compared with the HIV group, the percentages of CD4+, CD8+ T cells and CD14+ monocytes secreting TNF-α (p=0.013; p=0.001; p<0.001) were significantly decreased, and the percentage of CD8+ T cells secreting IL-17A (p=0.015) was significantly increased among the HT group. Both the percentages of CD4+ T cells secreting TGF-β (p<0.001; p=0.001), and CD4+ and CD8+ T cells secreting granzyme A (all p<0.001), were significantly higher among the HT group than among the TB group and HIV group. After one week's anti-TB treatment, an increased percentage of CD4+ T cells secreting TNF-α (p=0.003) was found in the TB group, and an increased percentage of CD8+ T cells secreting TNF-α (p=0.029) was found in the HT group. Significantly different functional profiles of peripheral proinflammatory, regulatory, and cytotoxic cytokine-secreting cells were observed in the natural course of HIV/TB co-infection compared to TB and HIV infection alone, even though the absolute numbers of those cells were significantly lower in HIV/TB co-infection. TNF-α-secreting CD8+ T cells may be a more sensitive marker for early evaluation of anti-TB treatment efficacy in patients with HIV/TB co-infection.

Diagnosis of Legionnaires' Disease Assisted by Next-Generation Sequencing in a Patient with COVID-19.

Coinfection in COVID-19 patients is associated with worsening outcome. Among patients with COVID-19, Legionella pneumophila, a common cause of pneumonia, has been reported as a co-occurring respiratory infection. A nonspecific clinical presentation, however, makes an early diagnosis difficult. Bronchoalveolar lavage fluid was collected from a patient suffering from COVID-19 and presenting with pneumonia and sent for metagenomic analysis. Differential abundance analysis was carried out by comparing each taxon reads per million between the bronchoalveolar lavage fluid sample and the negative control. Two replicates of metagenomic sequencing were conducted on bronchoalveolar lavage fluid samples. Within each replicated sequencing, one negative control was sequenced for comparison of taxon abundance in the BALF sample. In both replicates, Legionella pneumophila was the only taxon with significantly higher abundance when compared with the negative control. PCR of the bronchoalveolar further confirmed the presence of L. pneumophila. Several studies have estimated that the incidence of Legionnaires' disease co-infection in patients with COVID-19 infection is approximately 0% to 1.5%. There are some common characteristics of COVID-19 and co-infection with Legionnaires' disease, making it difficult to diagnose bacterial infection early. The diagnosis of these cases is important due to the different treatments used. Current diagnostic tests for Legionnaires' disease include conventional culture, urinary antigen for L. pneumophila serogroup 1, polymerase chain reaction, direct fluorescent antibody stain, and paired serology. The current study demonstrated that metagenomics is a promising approach that facilitated the diagnosis of Legionnaires' disease.

Phenotype and function of peripheral blood γδ T cells in HIV infection with tuberculosis.

Although γδ T cells play an essential role in immunity against Human Immunodeficiency Virus (HIV) or Mycobacterium tuberculosis (MTB), they are poorly described in HIV infection with tuberculosis (TB). The phenotypic and functional properties of peripheral blood γδ T cells in patients with HIV/TB co-infection were analyzed compared to healthy controls and patients with HIV mono-infection or TB by direct intracellular cytokine staining (ICS). The percentage of Vδ1 subset in HIV/TB group was significantly higher than that in TB group, while the decreased frequency of the Vδ2 and Vγ2Vδ2 subsets were observed in HIV/TB group than in TB group. The percentage of CD4+CD8- Vδ2 subset in HIV/TB group was markedly lower than in TB group. However, the percentage of CD4+CD8+ Vδ2 subset in HIV/TB group was markedly higher than HIV group or TB group. A lower percentage TNF-α and a higher percentage of IL-17A of Vδ2 subset were observed in HIV/TB group than that in HIV mono-infection. The percentage of perforin-producing Vδ2 subset was significantly lower in HIV/TB group than that in HIV group and TB group. Our data suggested that HIV/TB co-infection altered the balance of γδ T cell subsets. The influence of HIV/TB co-infection on the function of γδ T cells to produce cytokines was complicated, which will shed light on further investigations on the mechanisms of the immune response against HIV and/or MTB infection.

Quantifying multiple stain distributions in bioimaging by hyperspectral X-ray tomography

Chemical staining of biological specimens is commonly utilised to boost contrast in soft tissue structures, but unambiguous identification of staining location and distribution is difficult without confirmation of the elemental signature, especially for chemicals of similar density contrast. Hyperspectral X-ray computed tomography (XCT) enables the non-destructive identification, segmentation and mapping of elemental composition within a sample. With the availability of hundreds of narrow, high resolution (~ 1 keV) energy channels, the technique allows the simultaneous detection of multiple contrast agents across different tissue structures. Here we describe a hyperspectral imaging routine for distinguishing multiple chemical agents, regardless of contrast similarity. Using a set of elemental calibration phantoms, we perform a first instance of direct stain concentration measurement using spectral absorption edge markers. Applied to a set of double- and triple-stained biological specimens, the study analyses the extent of stain overlap and uptake regions for commonly used contrast markers. An improved understanding of stain concentration as a function of position, and the interaction between multiple stains, would help inform future studies on multi-staining procedures, as well as enable future exploration of heavy metal uptake across medical, agricultural and ecological fields.

Comparison of Examination Results of Acid-Fast Bacilli (AFB) Ziehl Neelsen (ZN) Method on BTA Preparations that are Directly Stained with Delayed Preparations

Tuberculosis is a direct infectious disease caused by the bacterium Mycobacterium tuberculosis. Most tuberculosis germs attack the lungs, which is known as Pulmonary Tuberculosis (pulmonary TB), but can also affect other organs, which is called extrapulmonary TB. Pulmonary TB germs in the form of rods, have special properties, namely acid resistance to staining. The purpose of this study was to determine the results of examination of acid fast bacilli (AFB) by the Ziehl Neelsen method on smear preparations which were immediately stained and left to stand for three days. The research was conducted using a descriptive approach with a sample of 10 people using a purposive sampling technique. In this study, staining was carried out twice, namely direct staining and delayed staining for 3 days and examined under a microscope. The results showed that there was no difference between the preparedness that was immediately colored and the one that was postponed for three days.

Dynamic analysis of tuberculosis specific mononuclear cells in peripheral blood of HIV patients with tuberculosis

Objective: To evaluate the response of peripheral blood mononuclear cells (PBMCs) in patients with human immunodeficiency virus (HIV) combined with active tuberculosis (TB) to TB-specific antigen stimulation. Methods: From January to December, 2018, individuals infected with both HIV and TB (HIV/TB group) were taken as the study subjects. Individuals infected with HIV alone (HIV group), individuals infected with TB alone (TB group) and healthy people (Health control group, HC group) were taken as the control groups. PBMCs were isolated and stimulated with purified protein derivative of bacillus calmette-guerin (BCG-PPD). The expression of surface molecules in T cells (CD3+, CD4+, CD8+) and monocytes (CD14+) and the percentages of Interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected by cell surface molecular staining, direct intracellular cytokine staining and flow cytometry (CD3- lymphocytes were mainly B lymphocytes and NK cells). Analysis of non-parametric data was used to compare the data between the two groups, and paired t-test was used to compare the data before and after PPD stimulation in each group. Results: Before PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the peripheral blood of HIV/TB group(mean 0.52%) was significantly lower than that in TB group(mean 0.94%, P=0.010). The TNF-α+cell percentages in CD3+, CD4+, CD8+, or CD14+ cells in the HIV/TB group(mean 19.2%) were significantly lower than those in the HIV group(mean 31.9%, P=0.002). The percentage of TNF-α secreted by monocytes in the HIV group was significantly lower than that in the HC group. The percentages of IFN-γ+ CD8+ and IFN-γ+ CD3- cells in the peripheral blood of the TB group (mean 0.94%) were significantly higher than thoset in the HC group(mean 0.51%, P=0.020), while the percentages of TNF-α+ cells in each subsets of PBMCs were significantly lower than those in the HC group. After PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the HIV/TB group was significantly lower than that in the TB group(P=0.008), and the change was more marked than that before stimulation. The percentage of IFN-γ+ CD8+ cells in the HIV group(mean 0.20%) was lower than that in the HC group (mean 0.52%, P=0.044). The percentage of IFN-γ+ CD3- in the TB group was significantly higher than in the HC group. There were no significant differences in TNF-α+ cell percentages in the 3 groups compared with the control group after PPD stimulation. The percentages of IFN-γ+ CD4+ cells in the HC and the TB groups were significantly increased after PPD stimulation in each group (P=0.002, P=0.001, respectively). However, there were no significant differences of IFN-γ+ CD4+ cell percentages in the HIV/TB group and the HIV group. The percentages of TNF-α production by monocytes were significantly increased after PPD stimulation in all groups. Conclusions: Chronic Mycobacterium tuberculosis (MTB) infection reduced the ability of PBMCs to produce TNF-α. For patients with TB infection, the production of TNF-α was reduced when combined with HIV infection. The capacity of CD8+ and CD3- lymphocytes to produce IFN-γ was increased in TB patients, while the capacity of CD8+ T cells to produce IFN-γ was decreased with co-infection of HIV. Infection of HIV weakened the immune response to MTB infection, which made the clinical diagnosis and treatment of TB more difficult.

Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Performance evaluation of UF-4000 body fluid mode for detection of bacteria in body fluids

BackgroundMicrobiological analysis of body fluids (BF) provides important information for diagnosis of infection. We evaluated the analytical performance of bacterial count by UF-4000 BF mode for ascitic, cerebrospinal, pleural, synovial and continuous ambulatory peritoneal dialysis fluids compared to classical microbiological procedure (direct Gram staining and culture). Materials and methodsFor the 1,734 BF analyzed, distribution of UF-4000 bacterial count was analyzed according to the level of growth culture and results were compared using Mann-Whitney test. ROC curves analysis allowed to define the best cut-off value to predict or exclude positive culture for each type of BF. ResultsUF-4000 bacterial counts were significantly lower in sterile than in infected BFs (p < 0.00001) and correlated with the level of growth on culture. The ROC curves of bacteria/μL and culture positivity yielded area under the curve >0.80 for each type of BF. Optimal cut-offs were chosen with excellent statistical parameters (sensitivity ranging from 0.70 to 0.86, specificity from 0.78 to 0.98, negative predictive value >0.95 and Youden index >0.55). ConclusionFor BF, UF-4000 bacterial count correlate with culture results and is a discriminative method enhancing detection of microbiological etiology. It could be used as a screening method based on the cut-off values proposed in this study.

Cytauxzoon felis in salivary glands of Amblyomma americanum

Cytauxzoon felis is a tick-borne piroplasmid hemoparasite that causes life-threatening disease in cats. Despite the critical role that ticks play in pathogen transmission, our knowledge regarding the C. felis life cycle remains limited to the feline hosts. Specific life stages of C. felis within the tick host have never been visualized microscopically and previous investigations have been limited to molecular detection by polymerase chain reaction (PCR). Sporozoites are the infectious stage of piroplasmids that are transmitted by ticks. In other tick-borne piroplasmids, sporozoite-based vaccines play a key role in disease prevention and management. We believe sporozoites have similar potential for cytauxzoonosis. Therefore, the objective of this study was to use different molecular and microscopic techniques to detect and evaluate C. felis sporozoites in tick salivary glands (SG). A total of 140 Amblyomma americanum adults that were fed on C. felis-infected cats as nymphs were included for this study. Specifically, dissected SGs were quartered and subjected to C. felis RT-PCR, RNAscope® in situ hybridization (ISH), histology, direct azure staining, and transmission electron microscopy (TEM). Cytauxzoon felis RT-PCR was also performed on half tick (HT) carcasses after SG dissection. Cytauxzoon felis RNA was detected in SGs of 17/140 ticks. Of these, 7/17 ticks had microscopic visualization via ISH and/or TEM. The remaining 10/17 ticks had only molecular detection of C. felis in SGs via RT-PCR without visualization. Cytauxzoon felis RNA was detected solely in HT carcasses via RT-PCR in 9/140 ticks. In ISH-positive tick SGs, hybridization signals were present in cytoplasms of SG acinar cells. TEM captured rare C. felis organisms with characteristic ultrastructural features of sporozoites. This study describes the first direct visualization of any developing stage of C. felis in ticks. Forthcoming studies should employ a combination of molecular and microscopic techniques to investigate the C. felis life cycle in A. americanum.

S3170 Syphilitic Hepatitis With Cholestatic Liver Injury

Introduction: The incidence of syphilis in the United States has increased, predominantly among men who have sex with men. Very few patients with early syphilis meet the criteria for syphilitic hepatitis, but many are HIV+ and engage in high-risk sexual behavior. Case Description/Methods: A 30-year-old man who identified as a MSM presented with yellowing of his eyes and hyperbilirubinemia, preceded by one month with a rash and photosensitivity and redness of his right eye. He denied any penile lesions, history of liver disease or alcohol use. A physical examination revealed multiple hyperpigmented macular lesions over his torso, arms, palms, and the soles of his feet and scleral icterus. A slit lamp examination indicated anterior uveitis in both eyes and chorioretinitis/placoid retinitis in the right eye. CBC and iron studies showed mild anemia of chronic disease (Hb 11.7 g/dL). BMP was within normal limits. LFTs were as mentioned in Table 1. Blood tests showed positive HBsAb and T. pallidum antibody with RPR 1:128. Serum markers for viral hepatitis and HIV were negative. CSF from a lumbar puncture had an elevated WBC count (9/mm3). No biliary obstruction or masses were noted during on US and MRCP. Liver biopsy showed bland perivenular cholestasis (zone 3) (Fig. 1A) without necroinflammatory changes, mild to minimal portal inflammation (Fig. 1B), bile duct damage, and ductular reaction. The patient was treated with penicillin G for neurosyphilis, prednisolone and cyclopentolate for ocular syphilis, and ursodiol for cholestasis. Total bilirubin levels decreased to 1.7 mg/dL after 8 weeks of follow-up, and liver enzymes normalized. Discussion: This patient met the criteria for syphilitic hepatitis (elevated liver enzymes, serologic evidence of syphilis, exclusion of other etiology, and improvement following appropriate therapy). He also had bilateral anterior uveitis and placoid retinitis of the right eye, suggesting ocular syphilis. Serum chemistries, testing for T. pallidum and other serologies (including positive anti-smooth muscle antibody) supported hepatic involvement, and CSF studies were consistent with neurosyphilis. Liver biopsy showed a bland cholestatic pattern without evidence of other causes of liver disease. The biopsy results are compatible with syphilitic hepatitis, though histologic features can vary and direct staining for spirochetes has limited sensitivity. These findings, together with his rapid improvement with penicillin therapy, confirmed a diagnosis of syphilitic hepatitis.Figure 1.: (A) Liver specimen (x40 magnification, hematoxylin and eosin stain) showing bland perivenular cholestasis (zone 3 with rare apoptotic hepatocytes. No necroinflammatory activity is present. (B) Liver specimen (x40 magnification, hematoxylin and eosin stain) showing periportal inflammation and bile duct damage with mild bile ductular reaction. Table 1. - Liver function test results AST 41 IU/L ↑ (ULN < 38 IU/L) ALT 53 IU/L ↑ (ULN < 42 IU/L) ALP 210 IU/L ↑↑ (ULN < 129 IU/L) Total bilirubin 11.6 mg/dL ↑↑ (ULN < 1.3 mg/dL) Direct bilirubin 8.8 mg/dL ↑↑ (ULN < 0.4 mg/dL) GGT 53 (ULN < 61 IU/L) Albumin 3.8 g/dL INR 1.0 AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase; INR, international normalized ratio; UNL, upper limits of normal.

Evaluation of modified bleach technique for the detection of acid fast bacilli in lymph node aspirate at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia

BackgroundTuberculous Lymphadenitis (TBLN) is the commonest form of extrapulmonary tuberculosis. Definitive diagnosis of TBLN is difficult due to the low efficacy of the routine diagnostic techniques as compared to culture. ObjectiveTo determine whether prior bleach concentration can improve the detection of acid-fast bacilli when compared with conventional Ziehl-Neelsen (ZN) staining by using culture as the gold standard. MethodsCross-sectional study was conducted from March 01, 2015 to May 30, 2015. The study subjects were clinically suspected TBLN patients. Fine needle aspiration (FNA) was performed. Two different smears were prepared directly from the first needle pass for cytology and ZN staining. The remaining aspirate was used for the modified bleach concentration. The second needle pass aspirate was transferred into a sterile container containing sterile normal saline. The sediment was inoculated on the Lowenstein Jensen (LJ) medium. Measurement parameters for reliability and validity were used to analyze the results. ResultA total of 93 participants were included in the study. Fifty-six out of the 93 (60.2 %) cases were positive for mycobacterium tuberculosis complex on LJ culture. The detection rates of direct ZN staining and modified bleach method were 20.4 % and 44.1 %, respectively. 73.1 % of the cases showed cytomorphological features consistent with TBLN by cytologic examination. The sensitivities of direct ZN staining and modified bleach method and cytomorphology were 32.0 %, 67.8 %, and 92.8 %, respectively. ConclusionImplementation of bleach concentration increases the detection rate of AFB over the direct ZN method. The bleach method can also be easily performed and provide a safe working environment by reducing infections.

Diagnosis of Gardnerella Vaginalis Isolated from Vaginal Discharge of Women in Babylon Province

Bacterial vaginitis is a prevalent condition that affects women all over the world and is the most common cause of vaginitis that has been recognized. Gardnerella vaginalis was found to be the most often identified pathogen in the vaginal discharge samples taken from patients diagnosed with vaginal infections. Two hundred (100 for Amsel test, 100 for culture) vaginal samples were taken from 100 women (two swaps from each woman) by the gynecologist to study the identification of G. vaginalis by Wet smear, Direct staining, Amsel test, Culture identification, and Molecular identification by 16sRNA. The result of Amsel test presents 10(18%) samples positive to four Amsels criteria and other samples positive to three of Amsel’s criteria from the 56 samples for positive Gardnerella vaginalis samples. The result of culture, biochemical, and Gram stain present the percentage of samples positive for G. vaginalis was 56 (56%). Electrophoresis of the PCR results of G. vaginalis DNA extracted from vaginal samples revealed the presence of a band with a size of 300 base pairs in only 12 of the 56 positive culture samples. As a result, it is imperative that diagnostic techniques for the detection of this illness be improved, particularly in terms of differentiating BV from other potential causes of vaginal infections Keywords: Bacterial vaginitis, Amsel test, Polymerase chain reaction.

Bioaerosol Seasonal Variation and Contribution to Airborne Particulate Matter in Huangshi City of Central China

Ambient bioaerosols affect ecosystems and public health, but their seasonal variations and their contributions to aerosol particles are limitedly understood. Ambient bioaerosols in PM2.5 and PM10 samples were measured in Huangshi City, Hubei Province of China from April 2018 to December 2018. Bioaerosols were measured using a fluorescence microscope after staining with 4′, 6-diamino-2-phenylindole dihydrochloride (DAPI) following a direct staining technique. The bioaerosol number concentrations ranged from 0.12 to 15.69 # cm−3 for PM2.5 and 0.22 to 18.20 # cm−3 for PM10, with averages of 2.79 # cm−3 and 4.66 # cm−3, respectively. The bioaerosol concentrations of PM2.5 and PM10 varied significantly by seasons and were arranged in the following descending order: spring &gt; fall &gt; winter &gt; summer. Bioaerosol numbers were dominated by fine particles of 0.37–2.5 μm diameter, while the spring bioaerosol particles were detected at the peak concentration of 0.56–1 μm diameter. Bioaerosol fractions accounted for 18.3 ± 10.6% PM10 mass and 13.7 ± 12.5% PM2.5 mass. Bioaerosol concentrations were increased during the haze event, but the increased amounts were not as large as those of the dust event, and higher bioaerosol contributions to PM were observed in the dust event than in the haze event. As enhanced emission controls have reduced PM concentrations in China, bioaerosols can be important contributors to PM mass.

Progressive ascending telangiectasias

Progressive ascending telangiectasias

The Role of CD4+CD8+ T Cells in HIV Infection With Tuberculosis

BackgroundTuberculosis (TB) is an important opportunistic infection in acquired immunodeficiency diseases (AIDS). Although the frequency of CD4+CD8+ double-positive (DP) T cells has been observed to increase in pathological conditions, their role (phenotypic and functional) is poorly described, especially in human immunodeficiency virus (HIV) infection with TB (HIV/TB (HT) coinfection).MethodsThe percentage and phenotypic and functional properties of peripheral blood DP T cells in patients with HT coinfection in comparison to uninfected controls and to patients with HIV or TB mono-infection were analyzed by direct intracellular cytokine staining (ICS).ResultsTotal and CD4lowCD8high DP T cells were significantly increased in patients with both HIV and TB mono-infection, especially in patients with HT coinfection. Compared with healthy controls (HCs), the percentage of DP T cells expressing chemokine receptor 5 (CCR5) in patients with HT coinfection was significantly higher. Compared with HCs and patients with TB, a lower percentage of tumor necrosis factor α (TNF-α) secreting DP T cells and a higher percentage of granzyme A-secreting DP T cells were observed in patients with HIV mono-infection and HT coinfection, respectively. In addition, DP T cells expressed more cytolytic markers (granzyme A and perforin) than CD4+ T cells, but similarly to CD8+ T cells in patients with HT coinfection.ConclusionsOur data suggested that HT coinfection resulted in a marked increase in DP T cells, especially the CD4lowCD8high subpopulation. DP T cells may be susceptible to HT coinfection, and have the same cytotoxic function as CD8+ T cells.

Massive eosinophilia despite severe aplastic anemia.

A 47-year-old nonsmoking man developed very severe aplastic anemia (AA) (hemoglobin 56 g/L, neutrophils 110/μl, eosinophils 0/μl, platelets 4000/μl) with fatty marrow (Figure 1A: hematoxylin–eosin stain, ×4 objective). He had a history of bronchial asthma for 3 years and had been treated with inhaled corticosteroid in combination with long-acting β2-agonist (budesonide/formoterol fumarate). In addition, he had developed eosinophilic pneumonia requiring systemic administration of prednisolone (<30 mg/day) twice in the last 1.5 years. His family history was not significant for any serious diseases. Treatment with antithymocyte globulin, eltrombopag olamine, and cyclosporine (CsA) (500 mg/day) eliminated the need for blood transfusions and the CsA dose was gradually reduced. While pancytopenia continued (hemoglobin <97 g/L, neutrophils <1400/μl, platelets <30,000/μl), eosinophilia (>500/μl) appeared 17 months after the start of treatment. This increased to 4550/μl in 4 months after discontinuation of CsA treatment at 33 months. In addition, he presented with a rash with purpura on the legs, for which prednisolone (<20 mg/day) was not effective. Bone marrow examination showed mild hypocellular marrow with eosinophil expansion (43.8%) (Figure 1B: hematoxylin–eosin stain, ×4 objective; Figure 1C: hematoxylin–eosin stain, ×40 objective; Figure 1D: direct Fast Scarlet stain, ×40 objective). Increased blasts and significant dysplasia were not observed. G-banding and fluorescence in situ hybridization for the FIP1L1–PDGFRA re-arrangement detected no chromosomal aberrations. Moreover, skin biopsy revealed eosinophilic vasculitis and panniculitis, compatible with eosinophilic granulomatosis with polyangiitis (Figure 1E: hematoxylin–eosin stain, ×20 objective; Figure 1F: direct Fast Scarlet stain, ×40 objective). After re-administration of CsA (150 mg/day), eosinophilia and skin lesions disappeared in 5 weeks. This case indicates that massive reactive eosinophilia can occur even in AA patients with eosinophil-associated diseases, especially during dose reduction, or after the discontinuation of CsA. None. The authors declare that there is no conflict of interest. Aya Takahashi and Tsutomu Shinohara designed the research, drafted the manuscript, and created the figure. Aya Takahashi performed the skin biopsy. Yoshihito Iwahara, Aya Takahashi, Hisanori Machida, Keishi Naruse, Eiji Takeuchi, and Tsutomu Shinohara analyzed the data. Keishi Naruse contributed to pathological assessment. All authors revised the manuscript critically and approved the final manuscript.

Best Practices for the Analytic Phase of Anaerobic Bacteriology

The analytic process for identification of anaerobic bacteria involves direct specimen Gram stains and workup of growth in culture. Direct Gram stains help to develop the initial differential, aiding in guiding both the extent of laboratory workup and clinician decision making for patient management. Workup of anaerobic growth should involve an aerotolerance test to assess aerobic growth and to compare growth rates in anaerobic versus aerobic environments. While matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become a routine means of anaerobe identification for many clinical laboratories, spot biochemical tests, such as indole and catalase, should be available in every laboratory that identifies anaerobes. If MALDI-TOF MS and biochemical methods are unsuccessful at providing an identification, 16S rRNA gene PCR and/or sequencing may be performed if the information impacts clinical care. The extent of culture workup to the level of presumptive identification versus definitive identification depends upon several factors, including the specimen source, direct Gram stain results, and the presence or absence of mixed microbiota. In addition to discussing the current best practices for anaerobic culture workup, commonly encountered anaerobic organism Gram stains and culture growth are detailed.

Prevalence of Oral Candidiasis among Children Caused by Different Candida Species

Introduction: Oral candidiasis, which is caused by Candida species by the involvement of hard and soft palates, tongue, buccal mucosa, and floor of the mouth, is becoming a very common threat to oral health. Although in the majority of the cases, this infection is caused by Candida albicans which is a normal commensal organism in humans, however, it might be also caused by other Candida species, such as C. glabrata, C. tropicalis, and C. krusei. Objectives: This study aimed to evaluate the prevalence of oral candidiasis among children and to compare the type of causative candida species among them. Materials and Methods: This prospective observational study was conducted in the Shishu Hospital, Shamoly, Dhaka, spanned January 2016 through December 2016. A total of 286 oral swab samples were collected, and three methods including direct wet mount smear, staining, and culture were used for laboratory investigation of Candida infections. Results: Among 286 respondents, 161 (56.29%) were culturally and microscopically confirmed to have oral candidiasis. Among all of the detected species, C. Albicans was by far the highest species with 139(86.34%) followed by C. tropicalis with 14 (8.68%). Whereas 05(3.12%) cases were infected by C. krusei. On the other hand, the least detected species were C. glabrata and C. guilliermondii with 01(0.62%) and 02(1.24%) respectively. Conclusion: These days accurate and prompt identification of the infectious candida strains is crucial because isolates of Candida species differ extremely, both in their ability to produce infection and also in their susceptibility to antifungal agents. Medicine Today 2022 Vol.34(1): 57-60

Gastrointestinal parasites in immunocompromised patients; A comparative cross-sectional study

Gastrointestinal parasites (GIPs), including helminths and protozoa species, are a major health problem in many parts of the world. About 3.5 billion people are affected by the parasites worldwide. GIPs are one of the leading causes of death among immunocompromised individuals and can cause serious clinical complications, especially in people with Human Immunodeficiency Virus (HIV)/AIDS, hemodialysis patients, and transplant recipients. This study aimed to compare the prevalence of GIPs among immunocompromised patients and immunocompetent individuals in Lorestan province, West Iran. In the current study, with a sampling of 232 participants (114 hemodialysis, AIDS, and organ transplantation immunocompromised patients and 118 immunocompetent individuals as the control group), demographic characteristics and risk factors for GIPs were collected through a pre-designed questionnaire. Stool samples of patients and the control group were examined for GIPs using different diagnostic methods including direct smear (saline and Lugol's iodine), Ziehl-Neelsen staining, agar-plate culture, and concentration method (formalin ether sedimentation). To evaluate the relative status of the immune system, TCD4+ cells were counted in the blood samples of the subjects by flow cytometry. The results were analyzed using SPSS 21 software, Fisher exact, and chi-square statistical tests. Multivariate modeling of the data was performed using logistic regression. The prevalence of GIPs in immunocompromised patients was more than twice that of immunocompetent individuals in the control group (42.06% vs. 17.79%). The most prevalent parasites identified among immunocompromised patients were Cryptosporidium sp. (27.1%), Blastocystis sp. (16.7%), and Entamoeba coli (14.6%) respectively. Cryptosporidium sp. had the highest frequency among hemodialysis patients (6.49%), AIDS patients (26.92%), and transplant recipients (18.18%) respectively. Patients with AIDS had the highest positive results for Cryptosporidium sp. followed by Microsporidia sp. (23.7%). In immunocompetent individuals, the highest prevalence of GIPs was related to Blastocystis sp and Trichomonas hominis (28.57%). Statistical analysis of the data showed that there was a statistically significant difference between various age groups regarding infection with GIPs so the highest rate of GIPs infection was observed in the age group lower than 50 years (P = 0.035). The statistical difference between the variable of location and infection with GIPs was insignificant but remarkable (P = 0.070). According to the results, it can be concluded that GIP is more common in immunocompromised patients than in immunocompetent individuals with cryptosporidium sp. predominance. Due to the favorable conditions of immunocompromised patients for GIPs and considering them as one of the important sources of parasitic infections and parasite transmission in society, control, prevention, and monitoring of their social behaviors along with health issues are inevitable.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.