Direct Inoculation Research Articles

Urinary Metabolic Profiling in Volunteers Undergoing Malaria Challenge in Gabon.

The interaction of malaria parasites with their human host is extensively studied, yet only few studies reported how P. falciparum infection affects urinary metabolite profiles and how this is associated with immunity. We present a longitudinal study of the urinary metabolic profiles of twenty healthy Africans with lifelong exposure to malaria and five malaria-naïve Europeans, who were all challenged with direct venous inoculation of live P. falciparum sporozoïtes (PfSPZ) and followed up until they developed symptoms or became thick blood smear positive (TBS). Urine samples were collected before and at 2, 5, 9 and 11 days post challenge and were analysed. Upon infection, all Europeans became TBS positive, while Africans showed either a delay in time to parasitaemia or controlled infection. Our metabolic data showed that Europeans and Africans had distinct alterations in metabolite patterns, with changes mostly seen on days 5 and 9 post PfSPZ infection, and more prominently in Europeans. Within the African group, the levels of formate, urea, trimethylamine, threonine, choline, myo-inositol and acetate were significantly higher in TBS positive whereas the levels of pyruvate, 3-methylhistidine and dimethylglycine were significantly lower in individuals who remained TBS negative. Notably, before inoculation with PfSPZ, a group of metabolites including phenylacetylglutamine can potentially be used to predict parasitaemia control among Africans. Taken together, this study highlights the difference in urinary metabolic changes in response to malaria infection as a consequence of lifelong exposure to malaria and that change detectable before challenge might predict the control of parasitaemia in malaria-endemic areas.

Cutaneous Blastomycosis Presenting as a Nonhealing Wound in the Northeast United States: A Case Report.

Primary cutaneous blastomycosis is a rare presentation of infection caused by direct inoculation of a wound. We present a 61-year-old male with an extensive history of wound dehiscence and wound care noncompliance after a bite from a brown recluse spider on the left thigh while on vacation in Cape Cod in September of 2020. After antibiotic therapy and culture, treatment involved debridement, split thickness skin grafting, strict wound vacuum-assisted closure care, and oral itraconazole. This brief demonstrates a case of blastomycosis arising from trauma in a non-endemic region for infection and serves as an example of successful management of the longstanding wound.

Septic Pulmonary Embolism: A Contemporary Profile.

Septic pulmonary embolism (SPE) is a rare clinical entity that is distinct from the classic and more common non-septic thrombotic pulmonary embolism. SPE should be suspected in patients with a systemic acute inflammatory reaction or sepsis who develop signs and symptoms of pulmonary involvement. The diagnosis of SPE depends on the specific radiologic finding of multiple, peripheral, nodular, possibly cavitated lesions. SPE should prompt an immediate search for the primary source of infection; typically, right-sided infective endocarditis, cardiac implantable electronic devices, and septic thrombophlebitis as a complication of bone, skin, and soft tissue infection including Lemierre's syndrome, indwelling catheters, or direct inoculation via injection drug use. Invasive treatment of the infection source may be necessary; in thrombophlebitis, the efficacy and safety of anticoagulation remain undefined. Blood cultures may be negative, particularly among patients with recent antibiotic exposure, and broad-spectrum antimicrobial therapy should be considered. The in-hospital mortality of SPE ranges up to 20% in published case series. While trends in the incidence of SPE are unknown, the opioid epidemic, the growing use of cardiac implantable electronic devices worldwide, and the reported increase in cases of septic thrombophlebitis may be leading to an escalation in SPE cases. We provide a contemporary profile of SPE and propose a clinical management algorithm in patients with suspected or confirmed SPE.

Soil Actinomycetes of Vietnam Tropical Forests

Actinomycetes are an important group of bioactive hydrolytic bacteria in any ecosystem. However, the actinomycete biodiversity in tropical ecosystems, particularly in Vietnam, is still underexplored. The aim of this article is to analyze the abundance, taxonomic structure and ecophysiological features of actinomycete complexes of soils and litter in Vietnam’s protected areas. A total of 41 samples of soils, plant litter and suspended soils were collected from six of Vietnam’s national parks and nature reserves. The direct inoculation technique showed that the total abundance of actinomycetes varied from 2.0 × 104 to 1.0 × 108 CFU/g. According to the luminescent microscopy with acridine orange dye, the length of the actinomycete mycelium was as long as 1000 m/g in the litter of Xuan Son National Park. A total of 80 strains were isolated and tested for antagonistic activity against Bacillus subtilis, Aspergillus niger and Candida albicans. Inoculation on Getchinson’s medium showed high cellulolytic activity. The most active strains were isolated from alluvial brown soil, plant litter and suspended soil of the Pu Hoat Nature Reserve. In these samples, actinomycetes adapted to high temperatures and low pH were found to be predominant. High-throughput sequencing of the V3–V4 region of the 16S rRNA gene and bioinformatic analysis confirmed the high taxonomic diversity and high hydrolytic activity of actinomycete complexes of the Pu Hoat Nature Reserve samples.

Ejaculate for Microbiological Culture: To Wash or Not To Wash?

Bacteria can be associated with male infertility. Antibacterial substances (e.g., zinc-containing proteins, antimicrobial peptides) in ejaculates might impair the growth of bacteria in culture. We therefore wanted to test if removing antibacterial substances by washing the ejaculate could improve the detection of bacteria in culture. All ejaculates from patients ≥18 years old, which were obtained for routine diagnostics to assess male infertility were included in this study (no exclusion criteria were applied). Test samples were diluted with 2 mL sterile 0.45% saline, vortexed, and centrifuged (5 min; 7.5 × g). After the removal of 2 mL of the supernatant and resuspension, 10 μL of the pellet was used for aerobic and anaerobic culture. Control samples were cultured identically but without washing. Species identification was done with matrix-assisted laser desorption ionization-time of flight mass spectrometry. A total of 186 samples were included. The data set was stratified into five groups (Gram-negative rods [GNR], anaerobes [AN], Enterococcus spp. [EC], coagulase-negative staphylococci [CNS], and viridans streptococci [VS]). Compared to the control arm, the test arm revealed significant lower proportions for CNS (59.1% versus 44.6%, P < 0.01) and VS (53.8% versus 41.9%, P = 0.03). Similarly, slightly lower proportions of GNR (16.1% versus 15.1%, P = 0.89), AN (19.9% versus 17.2%, P = 0.5), and EC (25.3% versus 23.1%, P = 0.63) were observed. The medians of CFU were lower in test samples compared to the control samples (6.5 × 103 versus 2.5 × 103, P < 0.01) for any bacterial growth. Lower colony counts were also observed for individual bacterial groups. In conclusion, preculture washing of ejaculates results in a decrease in total bacteria count and culture-positive samples. IMPORTANCE This study compares two methods for processing ejaculate samples from men undergoing investigations for infertility. The method of sample washing and centrifugation was compared to the standard method of direct inoculation and culture. The study hypothesis was that preprocessing of samples may increase bacterial yield by removing bactericidal substances from semen. However, we found that washing ejaculate samples before microbiological culture did not improve the detection of bacteria and led to a reduction in colony counts.

ODP519 Thyroid Cyst Grows E. coli

Abstract Background Thyroid abscesses are extremely rare, representing less than 1% of all thyroiddiseases. The thyroid gland is very robust against infections due to its vastlymphatic drainage, dual blood supply, encapsulation, and iodine concentration. Immunocompromised patients are at greater risk of developing thyroid abscesses. 1 Case: A 51-year-old female presented to her primary care provider with acute onset ofpainful right neck mass with associated cough, hoarseness, fever, and fatigue. She was seen by an Otolaryngologist who aspirated 6 cc of brown purulentmaterial from a suspected thyroid mass. The thyroid fluid was sent for culture,and she was empirically started on Augmentin. CT neck demonstrated a cysticlesion of the right thyroid gland, retropharyngeal edema, and mass effect withleftward deviation of the larynx and trachea without airway obstruction. Due tothese findings, the patient was admitted to the hospital where initial two setsof blood cultures were negative but repeat blood cultures revealed 2 of 2positive for E. coli. Thyroid cyst fluid also grew E. coli. Urine culture hadno growth. She was found to have a suppressed TSH of 0.24 uIU/mL, elevated free T4 of 1.80 ng/dL, and suppressed total T3 of 0.52 ng/mL. Previous TSH was within normal limits, 0.82 uIU/mL. The suppressed TSH and suppressed T3 were thought to be secondary to euthyroid sick syndrome. She was maintained onCeftriaxone while admitted and discharged on Ciprofloxacin. Pathology from theright thyroid cyst revealed acute inflammatory cells only consistent with anabscess, negative for malignancy. The patient elected to undergo a righthemithyroidectomy with isthmusectomy. Discussion Thyroiditis is defined as inflammation of the thyroid gland and has a variety ofetiologies. Suppurative thyroiditis, which includes thyroid abscess as in thiscase we have described, is an infrequent cause of thyroiditis. It is usuallycaused by hematogenous spread or direct inoculation of a pathogen. Treatmentincludes systemic antibiotics targeting the causative organism; although thegold standard remains surgical drainage. Surgical management is the mainstay ofmanaging patients with thyroid abscesses. 2

Establishment of a Cynomolgus Macaque Model of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Direct Inoculation of Adult T-Cell Leukemia Patient-Derived Cell Lines for HTLV-1 Infection.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases have remained unclear, and effective vaccines for inhibiting the infection and the progression of pathogenesis have therefore not been developed. The use of a nonhuman primate (NHP) model is thought to be important for revealing the mechanisms of the progressive status and for the development of prevention procedures. In this study, we developed a cynomolgus macaque (CM) model of HTLV-1 infection by direct intravenous inoculation of HTLV-1-producing cells derived from ATL patients. The cell line used for infection, ATL-040, was selected as the most infectious one in our cell line library. CMs inoculated intravenously with 1 × 108 ATL-040 cells per animal became persistently infected with HTLV-1, as shown by the HTLV-1 provirus load (PVL) in peripheral blood mononuclear cells and HTLV-1-specific antibodies (2/2 animals). One CM inoculated intravenously with 1 × 107 ATL-040 cells did not have detectable PVLs despite the fact that anti-HTLV-1 antibodies were maintained for more than 2 years. Furthermore, immunological approaches, including CD8+ T cell depletion prior to infection (3/3 animals) and intrathecal inoculation (3/3 animals), led to increased proviral loads in the cynomolgus monkeys. The present method and the cynomolgus monkey model of HTLV-1 infection will be beneficial for immunological and virological studies on HTLV-1 aiming at the development of anti-HTLV-1 prophylactic vaccines and therapy drugs. IMPORTANCE HTLV-1 was discovered in the 1980s as the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases still remain unidentified. Thus, no effective vaccines to inhibit the infection and the progressive of pathogenesis have been developed. The use of appropriate animal models is essential for understanding HTLV-1 infection and pathogenesis. In order to establish a new nonhuman primate model for studies on HTLV-1 infection, cynomolgus monkeys were infected with HTLV-1 under a variety of experimental conditions. Our method, using a cell line generated from an ATL patient as a source of HTLV-1, was able to establish HTLV-1 infection in monkeys with a 100% success rate. This cynomolgus macaque model of HTLV-1 infection will contribute to the elucidation of HTLV-1 infection and its associated disease development.

Optic Chiasmal Fungal Granuloma - Rare Cause of Blindness

Intracranial fungal infections are rare. Their presentation is mostly subtle, clinically atypical in presentation, and they are usually mistaken for tuberculosis or brain tumour. Aspergillosis is the most common cause of intracranial fungal infections. Fungal infection enters the brain directly from the paranasal sinuses, ear, skull bone, or haematogenous spread from the lungs and gastrointestinal tract, or direct inoculation during surgery or trauma. The pathology depends on the route of spread, host immunity, and type of fungus. Intracranially, fungal infection invades brain matter or blood vessels. [1]&#x0D; The rarity of brain fungal infection, with nonspecific clinical picture, and progression to a fatal outcome provides considerable challenges for both diagnosis and management. Invasive sino-orbital fungal infections are rarely reported in immunocompetent patients in literature. We could not find anything related to primary fungal granuloma of the optic tract reported in literature. We report a case of primary fungal granuloma of optic chiasm in an immunocompetent patient.[2]&#x0D; 13-year-old female patient presented to us with sudden onset of loss of vision in both eyes. There were no other complaints. On neurological examination, there was no vision in both eyes (no perception of light). On fundoscopy, there was bilateral optic atrophy. Rest of the neurological examination was normal. Patient was investigated for magnetic resonance imaging (MRI) of brain with contrast study. MRI of brain was suggestive of a 2.2 x 1.8 x 1.6 cm mass in suprasellar region with its epicentre in the optic chiasma and extending to both optic nerves and optic tract. The lesion was separate from the underlying pituitary gland. The lesion was iso-intense on T1 weighted images and hyper-intense on T2 weighted images. It showed homogenous post-contrast enhancement. Our primary differential diagnosis was optic nerve glioma.&#x0D; Patient underwent immediate surgery, right peritoneal craniotomy, for excision of lesion. Intra-operatively, a firm yellowish white colored lesion arising from the optic chiasm was seen. Safe subtotal excision was done. Post-operative, there was no improvement in vision.

Current Evidence Demonstrates That Monkeypox Is a Sexually Transmitted Infection.

Current Evidence Demonstrates That Monkeypox Is a Sexually Transmitted Infection.

Large pyomyoma after an urgent uterine artery embolization (UAE)

Leiomyomas, or fibroids, are a common, benign tumor of the smooth muscle of the uterus, with a cumulative incidence of 70% in white women and 80% in black women by age 50 [1]. Suppurative leiomyoma, also referred to as pyomyoma, is a rare disorder of infarction with subsequent infection of a leiomyoma, which must be distinguished from other degenerative changes [2,3]. Incidence of the disease is unknown but previously estimated under 1% of operated fibroids [3], and there have been approximately 100 cases reported since 1945. The modes of infection are thought to be via direct extension from the uterine cavity, a lymphatic or a vascular route of bacterial seeding [3,4]. Pregnancy, the post-partum period, and uterine instrumentation, including dilatation and curettage (D&amp;C) and C-section, are important risk factors for direct inoculation and account for the majority of pyomyoma cases that were reported [5,6]. Idiopathic pyomyomas also occur, such as in post-menopausal women; risk factors for ischemia, such as hypertension, diabetes, and atherosclerosis, increase the risk of pyomyoma [4,5]. Uterine Artery Embolization (UAE) is increasingly used as a non-invasive treatment for symptomatic fibroids and involves infarction of leiomyomas, which can rarely lead to pyomyoma [7,8]. We present a case of pyomyoma that developed after an urgent uterine artery embolization with a follow-up discussion of the relevant literature.

Highly efficient hairy root genetic transformation and applications in citrus.

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2-8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining "hard-to-get" transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Process Intensification Using a One‐Step Inoculum Production and High‐Seeded Fed‐Batch Processes

AbstractVarious approaches to process intensification are currently being investigated to ensure time and cost savings when producing biopharmaceuticals. In the present study, a one‐step inoculum production was established based on an ultra‐high cell density working cell bank with immunoglobulin G producing Chinese hamster ovary cells. Cryovials were used for direct inoculation of 1‐L wave‐mixed perfusion bioreactors. When reaching around 180 · 106 cells mL−1 in N−1 perfusion mode, low‐seed and high‐seed fed‐batch experiments in shake flasks were inoculated, as was the case for 250‐mL stirred single‐use bioreactors. Additionally, proof‐of‐concept runs at 50‐L and 200‐L scale were successfully performed. The intensification approach presented allows manufacturing capacity to be increased by up to 50 %.

Chronic Osteomyelitis of the Navicular Bone: Case Report and Review of Literature

Osteomyelitis of the navicular bone is not common; the few reported cases are haematogenous osteomyelitis of the navicular bone and were mostly seen in children and another case seen in an adult. We are presenting a case of domestic accident resulting in chronic osteomyelitis of the navicular bone in an adult caused by direct inoculation of infection following traditional bone setter’s mismanagement and subsequent surgery on the same foot. Consent is duly obtained from the patient to present this case report. The aim of this case presentation is to highlight the need for high index of suspicion of osteomyelitis of the bones of the foot in adult which are not necessarily secondary to haematogenous spread as seen in children but due to direct inoculation of micro- organisms, the study also seeks to demonstrate a complete cure of chronic osteomyelitis following proper debridement of the sequestrum with adequate use of the relevant antibiotics for the specified durations of administration. The patronage of traditional bone setters by most patients in Africa is still worrisome because this results in delayed presentation and attendant complications as seen in this index patient. In conclusion, chronic osteomyelitis of navicular in adults tend to follow direct inoculation of micro-organism but with a radical debridement with appropriate administration of antibiotics can result in cure. However, health education is necessary to discourage patients’ patronage of traditional bone setters and encouraging early presentation with good outcomes.

Safety, Tolerability, and Parasite Clearance Kinetics in Controlled Human Malaria Infection after Direct Venous Inoculation of Plasmodium falciparum Sporozoites: A Model for Evaluating New Blood-Stage Antimalarial Drugs

Plasmodium falciparum sporozoite (PfSPZ) direct venous inoculation (DVI) using cryopreserved, infectious PfSPZ (PfSPZ Challenge [Sanaria, Rockville, Maryland]) is an established controlled human malaria infection model. However, to evaluate new chemical entities with potential blood-stage activity, more detailed data are needed on safety, tolerability, and parasite clearance kinetics for DVI of PfSPZ Challenge with established schizonticidal antimalarial drugs. This open-label, phase Ib study enrolled 16 malaria-naïve healthy adults in two cohorts (eight per cohort). Following DVI of 3,200 PfSPZ (NF54 strain), parasitemia was assessed by quantitative polymerase chain reaction (qPCR) from day 7. The approved antimalarial artemether-lumefantrine was administered at a qPCR-defined target parasitemia of ≥ 5,000 parasites/mL of blood. The intervention was generally well tolerated, with two grade 3 adverse events of neutropenia, and no serious adverse events. All 16 participants developed parasitemia after a mean of 9.7 days (95% CI 9.1-10.4) and a mean parasitemia level of 511 parasites/mL (95% CI 369-709). The median time to reach ≥ 5,000 parasites/mL was 11.5 days (95% CI 10.4-12.4; Kaplan-Meier), at that point the geometric mean (GM) parasitemia was 15,530 parasites/mL (95% CI 10,268-23,488). Artemether-lumefantrine was initiated at a GM of 12.1 days (95% CI 11.5-12.7), and a GM parasitemia of 6,101 parasites/mL (1,587-23,450). Mean parasite clearance time was 1.3 days (95% CI 0.9-2.1) and the mean log10 parasite reduction ratio over 48 hours was 3.6 (95% CI 3.4-3.7). This study supports the safety, tolerability, and feasibility of PfSPZ Challenge by DVI for evaluating the blood-stage activity of candidate antimalarial drugs.

57 Ocular tuberculosis: a differential for chronic panuveitis

Background Mycobacterium tuberculosis is an organism responsible for causing chronic granulomatous inflammation leading to active or latent tuberculosis (TB). Both pulmonary and extrapulmonary infections; including the skin, the eye, the cardiovascular, genitourinary and gastrointestinal systems can occur. Within the eye, it can cause global disease ranging from anterior or posterior uveitis to retinal TB. Within this abstract we demonstrate a case of chronic panuveitis with a diagnosis of ocular TB.MethodologyRetrospective chart reviewResultsWe reviewed a 9-year-old- male in the rheumatology outpatient clinic who was on treatment for ocular TB with methotrexate 2.5 mg once a week, prednisone 5 mg once a day, maxitrol eye drops once a day and prednisolone eye drops twice a day.He presented with a red, painless left eye with deteriorating vision 2 years ago. Functional enquiry was unremarkable. He was noted to have a fixed dilated left pupil at 3-4mm but other examinations were normal. Extensive investigations were carried out, including an Interferon-gamma release assay (IGRA)- which was positive. Toxoplasma antibody test, Treponema pallidum Haemagglutination test, and chest X-ray were all negative. He was then started on the above-mentioned therapies with marked improvement.A decision on increasing the methotrexate dose if steroid tapering showed no improvement was undertaken and a plan on reassessing the patient with ANA, ACE and HLA B51 was made, to ensure complete workup.Discussion and conclusionIntra or extra ocular TB (OTB) manifestations may result as a direct inoculation of the organism in ocular fluid or as a systemic immune response initiated by an infection from a distant site. There is a myriad of ocular manifestations of OTB which make forming a clinical diagnostic criterion difficult. The gold standard for diagnosis is an ocular fluid culture showing organisms but the yield may be low and the risk of damage to surrounding structures may be high. PCR tests done on the sample may yield clinical information but non ocular tests, such as the IGRA and tuberculin tests may also be employed. Therapy depends on active vs latent disease and mainly involves anti-inflammatory agents and anti-TBs.Ocular TB may lead to sudden and irreversible blindness with high socioeconomic implications and hence, an index of suspicion in areas with a high disease burden should be kept as diagnosis poses a challenge.

Rift Valley Fever Virus Infects the Posterior Segment of the Eye and Induces Inflammation in a Rat Model of Ocular Disease.

ABSTRACTPeople infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes.IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.

BURKHOLDERIA BACTEREMIA PRESENTING AS SEPTIC SHOCK AND MASSIVE HEMOPTYSIS IN A PATIENT WITH SICKLE CELL DISEASE

BURKHOLDERIA BACTEREMIA PRESENTING AS SEPTIC SHOCK AND MASSIVE HEMOPTYSIS IN A PATIENT WITH SICKLE CELL DISEASE

S2.2a African histoplasmosis

S2.2 Histoplasmosis and talaromycosis, September 21, 2022, 3:00 PM - 4:30 PM African histoplasmosis caused by Histoplasma carpsulatum var dubosii is endemic in Africa with few cases reported from outside Africa usually attributed to travelling or visits to sub-Saharan Africa. The pathogenesis is yet unclear. Infection can be acquired via inhalation of microconidia or by direct inoculation. African histoplasmosis commonly presents with papules, nodules, ulcers, swellings, lymph node enlargement, eczematoid, or psoriosiform skin lesions. Subcutaneous abscesses may also develop with discharging sinuses containing yeast cells of the fungus. Although it is generally believed to be acquired through inhalation, the lungs are usually spared. Disseminated forms are usually characterized by the involvement of bones and other organs including the gastrointestinal tract. As a result of limited availability of diagnostics, data on its prevalence and epidemiology are scarce. As with classical histoplasmosis, African histoplasmosis also mimics other clinical entities including TB and neoplasms. More awareness and a high index of suspicion on the part of clinicians will lead to early diagnosis and invariably improve clinical outcomes. An extensive review of literature revealed 365 cases of African histoplasmosis reported globally; 236 cases from Africa and 38 cases from other geographical regions including an indigenous case report from India; the location of the remaining cases was not found, positive HIV status was found in 75 cases only. No statistically significant relationship was observed when comparing the relationship between positive HIV status and fatal outcomes (P >.05, Fisher's exact test). A case report from the UK mentioned 21 cases of African histoplasmosis previously identified in the region and all were associated with travelling to Africa. Out of the 365 identified cases, diagnostic modality was specified in 264 cases; histopathology (87.9%, n = 232), culture (20.8%, n = 55), microscopy (7.2%, n = 19), serology (2.7%, n = 7), cytology (n = 1, 0.4%), polymerase chain reaction (n = 43, 16.3%), and peripheral blood film (n = 1, 0.4%). Amphotericin B (n = 53), itraconazole (n = 37), and ketoconazole (n = 26) were the predominant antifungals used for treatment. More studies are required to ascertain the true burden and epidemiology of African histoplasmosis and to determine whether these cases reported outside Africa were autochthonous or imported from Africa. Diagnosis by culture, although the gold standard is also not routinely achieved due to lack of biosafety level 3 cabinet especially in resource-limited settings, leading to significant under diagnosis. It is imperative that capacity building and strengthening is instituted to aid the diagnosis and management of African histoplasmosis. There is a need for more studies to ascertain whether there are additional phylogenetic species within the African clade.

Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system.

The low efficiency of genetic transformation and gene editing across diverse cultivars hinder the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9-expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV-sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high-expressing locus of Cas9 into adapted spring and winter cultivars by marker-assisted introgression and use of the BSMV-sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis-regulatory diversity or large-scale editing of multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes, and accelerating gene discovery and trait improvement efforts.

Efficient infection of non-human primates with purified, cryopreserved Plasmodium knowlesi sporozoites

BackgroundPlasmodium falciparum (Pf) sporozoite (SPZ) vaccines are the only candidate malaria vaccines that induce > 90% vaccine efficacy (VE) against controlled human malaria infection and the only malaria vaccines to have achieved reproducible VE against malaria in adults in Africa. The goal is to increase the impact and reduce the cost of PfSPZ vaccines by optimizing vaccine potency and manufacturing, which will benefit from identification of immunological responses contributing to protection in humans. Currently, there is no authentic animal challenge model for assessing P. falciparum malaria VE. Alternatively, Plasmodium knowlesi (Pk), which infects humans and non-human primates (NHPs) in nature, can be used to experimentally infect rhesus macaques (Macaca mulatta) to assess VE.MethodsSanaria has, therefore, produced purified, vialed, cryopreserved PkSPZ and conducted challenge studies in several naïve NHP cohorts. In the first cohort, groups of three rhesus macaques each received doses of 5 × 102, 2.5 × 103, 1.25 × 104 and 2.5 × 104 PkSPZ administered by direct venous inoculation. The infectivity of 1.5 × 103 PkSPZ cryopreserved with an altered method and of 1.5 × 103 PkSPZ cryopreserved for four years was tested in a second and third cohort of rhesus NHPs. The lastly, three pig-tailed macaques (Macaca nemestrina), a natural P. knowlesi host, were challenged with 2.5 × 103 PkSPZ cryopreserved six years earlier.ResultsIn the first cohort, all 12 animals developed P. knowlesi parasitaemia by thick blood smear, and the time to positivity (prepatent period) followed a non-linear 4-parameter logistic sigmoidal model with a median of 11, 10, 8, and 7 days, respectively (r2 = 1). PkSPZ cryopreserved using a modified rapid-scalable method infected rhesus with a pre-patent period of 10 days, as did PkSPZ cryopreserved four years prior to infection, similar to the control group. Cryopreserved PkSPZ infected pig-tailed macaques with median time to positivity by thin smear, of 11 days.ConclusionThis study establishes the capacity to consistently infect NHPs with purified, vialed, cryopreserved PkSPZ, providing a foundation for future studies to probe protective immunological mechanisms elicited by PfSPZ vaccines that cannot be established in humans.