Infected Cultures Research Articles

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

The malaria parasite chaperonin containing TCP-1 (CCT) complex: Data integration with other CCT proteomes.

The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite's asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Phenotypic Evaluation of Nucleoside Analogues against Trypanosoma cruzi Infection: In Vitro and In Vivo Approaches.

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3'-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.

Impact of early empirical antifungal therapy on prognosis of sepsis patients with positive yeast culture: A retrospective study from the MIMIC-IV database.

Mortality and other clinical outcomes of culture-negative and culture-positive among patients with fungal sepsis have not been documented, and whether antifungal therapy prior to fungal culture reports is related to decreased mortality among patients remains largely controversial. This study aimed to determine the mortality and other clinical outcomes of patients with positive yeast cultures and further investigate the effects of initial empiric antifungal therapy. A retrospective study was conducted among septic patients using the Medical Information Mart for Intensive Care (MIMIC)-IV database. Patients with sepsis were divided into two groups based on first fungal culture status during intensive care unit (ICU) stay, and initial empirical antifungal therapy was prescribed based on physician's experience prior to fungal culture reports within 48 h. The primary outcome was in-hospital all-cause mortality. The secondary outcomes were 30-day all-cause mortality, 60-day all-cause mortality, length of ICU stay and length of hospital stay. Multivariate logistic regression, propensity score matching (PSM), subgroup analyses and survival curve analyses were performed. This study included 18,496 sepsis patients, of whom 3,477 (18.8%) had positive yeast cultures. Patients with positive yeast cultures had higher in-hospital all-cause mortality, 60-day all-cause mortality, and longer lengths of ICU stay and hospital stay than those with negative yeast cultures after PSM (all p < 0.01). Multivariate logistic regression analysis revealed that positive yeast culture was a risk factor for in-hospital mortality in the extended model. Subgroup analyses showed that the results were robust among the respiratory infection, urinary tract infection, gram-positive bacterial infection and bacteria-free culture subgroups. Interestingly, empiric antifungal therapy was not associated with lower in-hospital mortality among patients with positive yeast cultures, mainly manifested in stratification analysis, which showed that antifungal treatment did not improve outcomes in the bloodstream infection (odds ratio, OR 2.12, 95% CI: 1.16-3.91, p = 0.015) or urinary tract infection groups (OR 3.24, 95% CI: 1.48-7.11, p = 0.003). Culture positivity for yeast among sepsis patients was associated with worse clinical outcomes, and empiric antifungal therapy did not lower in-hospital all-cause mortality in the bloodstream infection or urinary tract infection groups in the ICU.

In vitro systems to study inborn errors of immunity using human induced pluripotent stem cells.

In the last two decades, the exponential progress in the field of genetics could reveal the genetic impact on the onset and progression of several diseases affecting the immune system. This knowledge has led to the discovery of more than 400 monogenic germline mutations, also known as "inborn errors of immunity (IEI)". Given the rarity of various IEI and the clinical diversity as well as the limited available patients' material, the continuous development of novel cell-based in vitro models to elucidate the cellular and molecular mechanisms involved in the pathogenesis of these diseases is imperative. Focusing on stem cell technologies, this review aims to provide an overview of the current available in vitro models used to study IEI and which could lay the foundation for new therapeutic approaches. We elaborate in particular on the use of induced pluripotent stem cell-based systems and their broad application in studying IEI by establishing also novel infection culture models. The review will critically discuss the current limitations or gaps in the field of stem cell technology as well as the future perspectives from the use of these cell culture systems.

Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization.

Hepatitis C virus (HCV) genotype 4 is highly prevalent in the Middle East and parts of Africa. Subtype 4d has recently spread among high-risk groups in Europe. However, 4d infectious culture systems are not available, hampering studies of drugs, as well as neutralizing antibodies relevant for HCV vaccine development. We determined the consensus 4d sequence from a chronic hepatitis C patient by next-generation sequencing, generated a full-length clone thereof (pDH13), and demonstrated that pDH13 RNA-transcripts were viable in the human-liver chimeric mouse model, but not in Huh7.5 cells. However, a JFH1-based DH13 Core-NS5A 4d clone encoding A1671S, T1785V, and D2411G was viable in Huh7.5 cells, with efficient growth after inclusion of 10 additional substitutions [4d(C5A)-13m]. The efficacies of NS3/4A protease- and NS5A- inhibitors against genotypes 4a and 4d were similar, except for ledipasvir, which is less potent against 4d. Compared to 4a, the 4d(C5A)-13m virus was more sensitive to neutralizing monoclonal antibodies AR3A and AR5A, as well as 4a and 4d patient plasma antibodies. In conclusion, we developed the first genotype 4d infectious culture system enabling DAA efficacy testing and antibody neutralization assessment critical to optimization of DAA treatments in the clinic and for vaccine design to combat the HCV epidemic.

The results of determining the indicators of the morphological composition of the blood of animals and individual biochemical indicators using a potential drug with a mixture of two active compounds: 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazole-3-yl)methyl)morpholine and 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol in milk thistle seed oil

The introduction of new domestic highly effective and safe medicines into medical and veterinary practice is an important task of modern pharmaceutical science. Continuation of the search and conduct of research to bring the biological activity of 1,2,4-triazole derivatives is one of the promising areas of such activity. At this stage of research, it is known that such 1,2,4-triazole derivatives as 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazole-3-yl)methyl)morpholine and 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol have a wide spectrum of biological activity. Interest for further thorough research is precisely the antimicrobial and antifungal effects. The introduction into medical and veterinary practice of newly created drugs for the treatment of fungal diseases on the skin of animals requires further studies of the properties of these compounds and their mixtures. The aim of the work was to determine the indicators of the morphological composition of the blood of animals and individual biochemical indicators when using a preparation with a mixture of two active compounds: 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazole-3-yl)methyl)morpholine and 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol in thistle seed oil. Materials and methods. The material for laboratory research was serum and blood plasma, stabilized by adding trilon-B, tissue biopsy. Indicators were determined in healthy animals and experimentally diseased animals on the third and tenth days after the onset of the disease according to the generally accepted method. During the research, in the course of treatment, general clinical examinations and local examinations of purulent wounds were carried out, as well as blood samples taken from each head of the sheep in the experimental and control groups before the beginning and on the third and tenth days of the experiment. The purulent-inflammatory process was caused by injuring the soft tissues in the groin area on the left side, with the help of a specially made template, followed by dilation of the muscle tissue with an angiotrip and implantation of aerobic-anaerobic surgical infection cultures taken from the bed. The resulting wounds had the following dimensions: 10 cm in length, 1 cm in width, and 2 cm in depth. As early as 24 hours after induction of experimental purulent inflammation, characteristic clinical signs appeared in sheep. The general condition of all 12 animals was somewhat depressed, the appetite was normal. The body temperature was in the range of 38.6–40.3 °C, the pulse was 76–82 beats, and the frequency of respiratory movements was 16–18 per minute. The frequency of scar contractions was 3–6 times in 2 minutes. During the examination, slight swelling was also found in the area of the wound and its edges. There was a small amount of serous-bloody exudate on the surface of the wound. On the 3rd day, the maximum manifestation of clinical signs of acute purulent inflammation was observed in all 12 sheep. In particular, the general condition of the animals is depressed. A partial loss of appetite was observed. General temperature 40.4–40.8 °C, pulse 75–82 beats, frequency of respiratory movements 18–22 per minute. The frequency of scar contractions was 3–6 times in 2 minutes. Visible mucous membranes are pale pink in color. When pressing on the area of the wound, a serous-purulent exudate was observed. Results. Laboratory indicators of blood and tissue biopsy that are more optimal than in the control group confirm positive changes in the clinical status of the animals in the experimental group. The use of a potential preparation with a mixture of two active compounds: 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazole-3-yl)methyl)morpholine and 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol in thistle seed oil leads to faster healing of purulent wounds, in relation to the use of “Vetmikoderm” liniment (control group). The difference in the areas of the wound defect on the 10–11th day was 17.5 %. Clinical studies were confirmed by laboratory studies and wound biopsies. Conclusions. The preparation with two compounds: 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazole-3-yl)methyl)morpholine and 5-(2-fluorophenyl)-4-((4-bromophenyl)ylidene)amino-1,2,4-triazole-3-thiol had pronounced pharmacokinetic parameters and was bioavailable.

A high prevalence of Cutibacterium acnes infections in scoliosis revision surgery, a diagnostic and therapeutic dilemma

PurposeTo investigate if serum inflammatory markers or nuclear imaging can accurately diagnose a chronic spinal instrumentation infection (SII) prior to surgery.MethodsAll patients who underwent revision of spinal instrumentation after a scoliosis correction between 2017 and 2019, were retrospectively evaluated. The diagnostic accuracy of serum C-reactive protein (CRP) and Erythrocyte Sedimentation Rate (ESR), 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG–PET/CT) and Technetium-99m-methylene diphosphonate (99mTc-MDP) 3-phase bone scintigraphy (TPBS) to diagnose infection were studied. Patients with an acute infection or inadequate culture sampling were excluded. SII was diagnosed if ≥ 2 of the same microorganism(s) were isolated from intra-operative tissue cultures.Results30 patients were included. The indication for revision surgery was pseudoarthrosis in the majority of patients (n = 15). 22 patients (73%) were diagnosed with SII. In all infected cases, Cutibacterium acnes was isolated, including 5 cases with a polymicrobial infection. The majority of patients had low inflammatory parameters preoperatively. For CRP > 10.0 mg/L, the sensitivity was 9.1% and specificity 100%; for ESR > 30 mm/h, the sensitivity was 9.1% and specificity 100%. The diagnostic accuracy for nuclear imaging was 64% for the FDG–PET/CT and 67% for the TPBS to diagnose infection.ConclusionsThe prevalence of SII in patients undergoing revision spinal surgery is high, with Cutibacterium acnes as the main pathogen. No diagnostic tests could be identified that could accurately diagnose or exclude SII prior to surgery. Future studies should aim to find more sensitive diagnostic modalities to detect low-grade inflammation.

C-Reactive Protein, Procalcitonin, and a Novel Pathogenesis and Therapeutic Target of Thrombocytopenia in Sepsis.

Objective The aim of the study is to analyze the clinical characteristics, pathogen distribution, and drug sensitivity information of adult sepsis, and to provide reference for empirical clinical use; to explore the relationship between C-reactive protein (CRP) and calcitonin (PCT) The clinical value in the diagnosis of adult sepsis. Methods We collected 455 cases of hospitalized patients with positive blood culture, including 352 cases with sepsis and 103 cases without sepsis; 1609 cases of hospitalized patients with suspected infection and negative blood culture, including 287 cases of sepsis, and 518 cases of non-infectious systemic inflammatory response syndrome (SIRS) and 804 cases of local infection. Age, gender, route of admission, admission status, CRP, PCT, and white blood cell (WBC) levels were collected from the patients. The differences between the factors were statistically analyzed, and the receiver operating characteristic curve (ROC curve) was plotted to obtain the optimal cut-off values of CRP and PCT and their area under the curve (AUC), and to compare the CRP, PCT and PCT, and the CRP + PCT tandem to diagnose sepsis sensitivity and specificity. Results (1) 387 pathogenic strains were isolated from blood cultures of patients with sepsis, 71.06% Gram-negative, 26.87% Gram-positive and 2.07% fungi. (2) Among Gram-positive bacteria, Staphylococcus aureus was 87.5% resistant to penicillin and sensitive to vancomycin, milantropine, and teicoplanin; among Gram-negative bacteria, Escherichia coli was resistant to piracillin The resistance rate was 73.1%, fully susceptible to viraemia (100%) and resistant to imipenem, amikacin, and lacillin/tazole. (3) Among patients with positive blood cultures, CRP and PCT levels were higher in patients with sepsis than in those without sepsis. Pairwise comparison of ROC curves showed that the diagnostic value of PCT was greater than that of CRP (P=0.016). Conclusion CRP and PCT have a good reference value for diagnosis of sepsis patients and determination of the degree of infection in septic patients, especially PCT is more valuable for diagnosis of sepsis.

A FATAL CASE OF COMPLICATED SALMONELLA AORTITIS

A FATAL CASE OF COMPLICATED SALMONELLA AORTITIS

Azathioprine Induced Pancytopenia in a Patient with Vogt-Koyanagi-Harada Disease: A Case Report

case reports; pancytopenia; steroid.

Mycobacterium genavense Infections in Immunocompromised Patients Without HIV: Case Series of Solid Organ Transplant Patients and Literature Review.

Background Mycobacterium genavense infection is rare and can occur in immunocompromised patients without human immunodeficiency virus (HIV).MethodsWe describe 2 cases of M genavense infection in solid organ transplant (SOT) recipients, and we performed a literature review of immunocompromised patients without HIV.ResultsFifty-two cases are reported. Predisposing factors were receipt of SOT (40.4%) and autoimmune disease (36.5%). Infection was disseminated in 86.5% of cases. Organs involved were lymph nodes (72.3%), gastrointestinal tract (56.5%), lung (35.5%), and bone marrow (28.8%). Most patients were treated with at least 3 antimycobacterial agents (98%), with a clinical cure achieved in 54.9%. In multivariate analysis, lack for cure was associated with age of the time infection (odds ratio [OR], 15.81 [95% confidence interval {CI}, 2.92–152.93]; P = .011) and positive bone marrow culture (OR, 1.05 [95% CI, 1.01–1.12]; P = .042).Conclusions Mycobacterium genavense infection is a rare and generally disseminated disease with a poor prognosis. Optimal treatment regimen and its duration remain to be defined.

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.

ABSTRACTEnterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells.IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Serial Measurement of Interleukin-6 Enhances Chance to Exclude Early-Onset Sepsis in Very Preterm Infants.

The aim of this retrospective study was to evaluate the reliability of peak interleukin-6 (IL-6) level within 24 hours after delivery as a predictor for early-onset sepsis (EOS) in very preterm neonates. Interleukin-6 was assessed at 2 hours and at 12 to 24 hours after delivery. The highest level was considered a peak value. The definition of EOS was based on positive blood culture and clinical signs of infection or negative blood culture, clinical signs of infection, and C-reactive protein >10 mg/L. Among 445 enrolled infants, 53 developed EOS. A peak IL-6 level of more than 200 ng/L had a sensitivity of 89% and specificity of 77% for the presence of EOS. The negative predictive value was 98%. Receiver operating characteristics curve had area under the curve of 0.92. Peak IL-6 is a reliable marker of systemic inflammatory response and might be useful to exclude EOS within the first 24 hours.

P203 Chronic pulmonary aspergillosis (CPA) in post tuberculosis sequele— aclinical experience from tertiary care

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMIntroductionChronic pulmonary aspergillosis (CPA) is a spectrum of illnesses clinically presenting as a persistent cough, dyspnea, hemoptysis, fatigue, and weight loss and radiologically can range from single aspergilloma, Aspergillus nodule, or chronic cavitary pulmonary aspergillosis (CCPA) which can progress to chronic fibrosing pulmonary aspergillosis if left untreated.1 CPA has high morbidity, burden in India estimated to be in a 5-year prevalence of 24/100 000.2 The commonly used criteria for diagnosing CPA include cough or hemoptysis for 1 month, raised Aspergillus-specific IgG, absence of positive GeneXpert test for Mycobacterium tuberculosis and either paracavitary fibrosis or a fungal ball on imaging of the thorax or progressive cavitation (either new cavitation or deterioration of pre-existing cavitation) on serial chest radiographs. Pulmonary tuberculosis (PTB) is the important predisposing risk factor for CPA,3 India being an endemic country, incidence of CPA may be underestimated or it may be misdiagnosed as smear-negative tuberculosis. Microbiologically, diagnosis by direct confirmation of Aspergillus spp infection (microscopy or culture from respiratory samples) may not be always positive, in such a scenario the immune response to Aspergillus spp. by measuring Aspergillus specific Immunoglobulin Ig G in clinically suspected cases may be used for diagnosis of CPA.MethodThis is a cross-sectional conducted in a tertiary care hospital, New Delhi, India. The patients with previous history of pulmonary tuberculosis who presented with symptoms of cough, hemoptysis, fever, shortness of breath, chest pain, and weight loss of ˃12-week duration were enrolled in the study. Relevant investigations including blood tests, chest imaging, sputum examination for bacterial infections, fungal (KOH mount), and tuberculosis (AFB smear, CBNAAT, mycobacterial cultures) were done. Microbiological evidence included a positive Aspergillus-specific IgG (cut off >8 units/ml) or positive serum galactomannan index (GMI) (cut off >1 according to EORTC/MSG guidelines) or KOH mount on sputum showing branching hyaline septate hyphae morphologically suggestive of Aspergillus spp. Patients who were diagnosed with CPA according to criteria were treated and followed up.ResultsA total of 15 patients were screened in the study, 4 patients who had concurrently detected pulmonary tuberculosis detected by Genexpert, were excluded from the study. Majority of patients presented with complaints of recurrent episodes of cough and hemoptysis. Imaging features included cavitation, bronchiectasis, pleural thickening, and fungal ball. Sputum microscopy for fungal elements was positive only in 10 patients. The serum Aspergillus Ig G (values ranged from 19.8-200 u/mL) was raised in all patients while serum GMI above cut-off was present in only 5 patients. All confirmed CPA patients were managed with voriconazole for 4 months. Following 4 months of treatment, all patients had favorable outcomes in terms of radiological improvement and clinical cure.ConclusionCPA is an underestimated post-PTB sequel and should be considered as differentials in patients with respiratory symptoms in post TB patients. Aspergillus Ig G and chest imaging are recommended as initial diagnostic tools for diagnosing CPA.Sources:Denning DW, Cadranel J, Beigelman-Aubry C et al. European Society for Clinical Microbiology and Infectious Diseases and European Respiratory Society. Chronic pulmonary aspergillosis: rationale and clinical guidelines for diagnosis and management. Eur Respir J. 2016; 47(1): 45-68.Agarwal R, Denning DW, Chakrabarti A. Estimation of the burden of chronic and allergic pulmonary aspergillosis in India. PLoS One. 2014; 9(12): e114745.Page ID, Byanyima R, Hosmane S et al. Chronic pulmonary aspergillosis commonly complicates treated pulmonary tuberculosis with residual cavitation. Eur Respir J. 2019; 53: 1801184

Characterization of Chlamydia muridarum TC0668 Protein: Localization, Expression, and Inflammation-Inducing Effects on Host Cell

The objective of this study is to elucidate the basic biological properties and function of TC0668 in vitro. Laser confocal microscopy and immune-electron microscopy were used to detect localization of TC0668 in Chlamydia-infected human epithelial cells, while the expression phase was investigated by qRT-PCR and western blot analysis. Protein array technology was employed to evaluate differences in cytokine secretion between cells infected with tc0668 single mutants and those infected with tc0668 null mutants. We found that TC0668 is restricted to the chlamydial inclusion. Translation and transcription of TC0668 were detected at 4 h and peaked at 16 h during the life cycle of Chlamydia in vitro. The cytokines produced by tc0668 single mutant infected cultures compared with tc0668 null mutant group indicated that 36 cytokines were downregulated, while 10 were up-regulated significantly. C. muridarum bearing a single tc0668 gene mutation have decreased urogenital pathogenicity that is explained by the effects of the mutation on the regulation of inflammation-related cytokine secretion.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-022-03018-w.

INHIBITORY ACTIVITY OF ARTEMISIA ANNUA L. EXTRACTS AGAINST SARS-COV-2 CORONAVIRUS

The article describes preparing extracts of Artemisia annua L. and the use of the extracts to compare the virus-inhibiting activity against the coronavirus SARS-CoV-2.&#x0D; The extracts were prepared using hot water, water-alcohol mixture and butanol. The extracts were tested in screening-format tests on Vero E6 cells to measure the cytotoxicity and antiviral activity. Values of 50% inhibitory concentration (IC50) which measure the cytotoxicity were as follows: for the hot-water extract - 1587 µg/ml, for butanol extract - 713 µg/ml, and for water-alcohol extract &gt;2000 µg/ml. The quite high IC50 values indicate that the hot-water and butanol extracts have little cytotoxicity, and the water-alcohol extract is not toxic in vitro.&#x0D; Protection from the virus-induced cytopathic effect was observed in infected cultures upon addition of the extracts. Concentrations at which the extracts demonstrate the highest protection were measured by counting densities of live cells (in infected cultures)using the MTT-test. The results of the MTT-testin a form of dependence of optical densities from extracts’ concentrations are presented in the paper. All three extracts show the ability to inhibit the replication of the SARS-CoV-2 virus. For hot-water and butanol extracts, the peak density of live cells in infected cultures was observed when the extracts were present at the concentration of 667 μg/ml;however at the higher concentration (2000 µg/ml), both extracts inhibit cell growth. However, none of the three extracts at the optimal concentration completely suppress viral replication.The work needs to be continued to produce an effective herbal drug against the SARS-CoV-2 virus.

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.