Following the success of nucleic acid-based detection in virology and bacteriology, multiplex real-time PCRs are increasingly used as first-line diagnostics in clinical parasitology, replacing ...microscopy. The detection and quantification of parasite-specific DNA in faeces is highly sensitive and specific and allows for cost-effective high-throughput screening. In this paper we discuss the clinical consequences of this radical change in diagnostic approach, as well as its potential drawbacks. In the Netherlands, routine diagnostic laboratories have been pioneering the implementation of multiplex real-time PCR for the detection of pathogenic intestinal protozoa and this has resulted in increased detection rates of Giardia lamblia and Cryptosporidium spp. As a consequence of this new diagnostic approach, expertise in the field of parasite morphology by conventional light microscopy seems to be disappearing in most of the high-throughput microbiological laboratories. As a result, to maintain a high standard of care, a formalized exchange of critical information between clinicians and laboratory staff is necessary to determine the most appropriate testing either in local laboratories or in reference centres, based on clinical signs and symptoms, exposure and immune status. If such a diagnostic algorithm is lacking, important infections in travellers, immigrants and immunocompromised patients may be missed.
Antibodies can prevent malaria by neutralizing the infectious Plasmodium falciparum sporozoites (SPZ) before they establish an infection in the liver. Circumsporozoite protein (CSP), the most ...abundant surface protein of SPZ is the leading candidate for passive (and subunit) immunization approaches against malaria. Comprehensive assessment of the parasite-inhibitory capacity of anti-CSP monoclonal antibodies (mAbs) is an important step in advancing CSP-based immunization strategies. In this study, we employed a quantitative imaging-based motility assay to quantify the effect of anti-CSP mAbs on SPZ motility, both in vitro and in human skin.Our assay provided a quantitative measure of mAb parasite-inhibitory capacity through measurement of the half-maximal motility inhibitory concentration (IC
) value for anti-CSP mAbs (IC
2A10: 24 nM, IC
3SP2: 71 nM). We found a sevenfold discrepancy between the IC
and the binding saturation concentration measured by ELISA, possibly related to the observed shedding of CSP-mAb complexes during SPZ movement. In a subset of SPZ (5%), in vitro motility was unaffected by the presence of 2A10 while 3SP2 was able to completely block movement. In our ex vivo skin explant model, SPZ proved less susceptible to anti-CSP mAbs compared to SPZ in an in vitro environment. By quantitatively assessing motility, we created a valuable tool that can be used for comprehensive assessment of anti-CSP mAb potency. Insight that will help deepen our understanding of anti-CSP mAb potency and guide selection of the most promising anti-CSP mAbs for downstream clinical development.
Schistosomiasis is a parasitic disease affecting over 200 million people in multiple organs, including the lungs. Despite this, there is little understanding of pulmonary immune responses during ...schistosomiasis. Here, we show type-2 dominated lung immune responses in both patent (egg producing) and pre-patent (larval lung migration) murine Schistosoma mansoni (S. mansoni) infection. Human pre-patent S. mansoni infection pulmonary (sputum) samples revealed a mixed type-1/type-2 inflammatory cytokine profile, whilst a case-control study showed no significant pulmonary cytokine changes in endemic patent infection. However, schistosomiasis induced expansion of pulmonary type-2 conventional dendritic cells (cDC2s) in human and murine hosts, at both infection stages. Further, cDC2s were required for type-2 pulmonary inflammation in murine pre-patent or patent infection. These data elevate our fundamental understanding of pulmonary immune responses during schistosomiasis, which may be important for future vaccine design, as well as for understanding links between schistosomiasis and other lung diseases.
Hookworms are soil-transmitted helminths that use immune-evasive strategies to persist in the human duodenum where they are responsible for anemia and protein loss. Given their location and immune ...regulatory effects, hookworms likely impact the bacterial microbiota. However, microbiota studies struggle to deconvolute the effect of hookworms from confounders such as coinfections and malnutrition. We thus used an experimental human hookworm infection model to explore temporal changes in the gut microbiota before and during hookworm infection. Volunteers were dermally exposed to cumulative dosages of 50, 100 or 150 L3 Necator americanus larvae. Fecal samples were collected for microbiota profiling through 16S rRNA gene amplicon sequencing at weeks zero, four, eight, fourteen and twenty. During the acute infection phase (trial week zero to eight) no changes in bacterial diversity were detected. During the established infection phase (trial week eight to twenty), bacterial richness (Chao1, p = .0174) increased significantly over all volunteers. No relation was found between larval dosage and diversity, stability or relative abundance of individual bacterial taxa. GI symptoms were associated with an unstable microbiota during the first eight weeks and rapid recovery at week twenty. Barnesiella, amongst other taxa, was more abundant in volunteers with more GI symptoms throughout the study. In conclusion, this study showed that clinical GI symptoms following N. americanus infection are associated with temporary microbiota instability and relative abundance of specific bacterial taxa. These results suggest a possible role of hookworm-induced enteritis on microbiota stability.
Large scale administration of the anthelminthic drug praziquantel (PZQ) to at-risk populations is the cornerstone of schistosomiasis control, although persisting high prevalence of infections in some ...areas and growing concerns of PZQ resistance have revealed the limitations of this strategy. Most studies assessing PZQ efficacy have used relatively insensitive parasitological diagnostics, such as the Kato-Katz (KK) and urine-filtration methods, thereby overestimating cure rates (CRs). This study aims to determine the efficacy of repeated PZQ treatments against Schistosoma mansoni infection in school-aged children in Côte d'Ivoire using the traditional KK technique, as well as more sensitive antigen- and DNA-detection methods.
An open-label, randomised controlled trial will be conducted in school-aged children (5 to 18 years) from the region of Taabo, Côte d'Ivoire, an area endemic for S. mansoni. This 8-week trial includes four two-weekly standard doses of PZQ in the "intense treatment" intervention group and one standard dose of PZQ in the "standard treatment" control group. The efficacy of PZQ will be evaluated in stool samples using the KK technique and real-time PCR as well as in urine using the point-of-care circulating cathodic antigen test and the up-converting phosphor, lateral flow, circulating anodic antigen assay. The primary outcome of the study will be the difference in CR of intense versus standard treatment with PZQ on individuals with a confirmed S. mansoni infection measured by KK. Secondary outcomes include the difference in CR and intensity reduction rate between the intense and standard treatment groups as measured by the other diagnostic tests, as well as the accuracy of the different diagnostic tests, and the safety of PZQ.
This study will provide data on the efficacy of repeated PZQ treatment on the clearance of S. mansoni as measured by several diagnostic techniques. These findings will inform future mass drug administration policy and shed light on position of novel diagnostic tools to evaluate schistosomiasis control strategies.
The study is registered at EudraCT (2016-003017-10, date of registration: 22 July 2016) and ( NCT02868385 , date of registration: 16 August 2016).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pre-targeting is a proven strategy for in vivo delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is based ...on copper-free "click" chemistry. Copper-free click reactions have shown in vivo potential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the in vivo efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and Staphylococcus aureus bacteria.
MAA microspheres (diameter 10-90 μm) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N3), to generate MAA-Cy5-N3. S. aureus (diameter ∼1 μm) were functionalized with 99mTc-UBI29-41-Cy5-N3, generating S. aureus-99mTc-UBI29-41-Cy5-N3. In situ and in vitro click conjugation on the -N3 moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For in vivo validation, both primary entities, radiolabeled with 99mTc, were deposited into the microvasculature of the liver via intrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (111In-DTPA-DBCO). To assess click reaction efficiency in vivo, 99mTc and 111In-biodistributions were measured (SPECT and %ID g-1). Use of 111In-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized S. aureus served as controls. Ex vivo confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria.
In vitro data confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (p < 0.05) increased accumulation of 111In-DTPA-DBCO at the sites where MAA-Cy5-N3 (7.5 ± 1.5%ID g-1vs. 3.5 ± 0.5%ID g-1 in control mice) and S. aureus-99mTc-UBI29-41-Cy5-N3 (9.3 ± 1.3%ID g-1vs. 6.0 ± 0.5%ID g-1 in control mice) resided. Ex vivo fluorescence imaging confirmed the presence of either functionalized MAA or S. aureus in excised spleens and livers of mice.
Copper-free click chemistry between a DBCO moiety and Cy5-N3-functionalized microspheres or bacterial entities in the liver can be used to realize in vivo imaging and targeting.
Malaria vaccine candidates based on live, attenuated sporozoites have led to high levels of protection. However, their efficacy critically depends on the sporozoites' ability to reach and infect the ...host liver. Administration via mosquito inoculation is by far the most potent method for inducing immunity but highly impractical. Here, we observed that intradermal syringe-injected
sporozoites (
SPZ) were 3-fold less efficient in migrating to and infecting mouse liver than mosquito-inoculated sporozoites (
SPZ). This was related to a clustered dermal distribution (2-fold-decreased median distance between
SPZ and
SPZ) and, more importantly, a 1.4-fold (significantly)-slower and more erratic movement pattern. These erratic movement patterns were likely caused by alteration of dermal tissue morphology (>15-μm intercellular gaps) due to injection of fluid and may critically decrease sporozoite infectivity. These results suggest that novel microvolume-based administration technologies hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.
Malaria still causes a major burden on global health and the economy. The efficacy of live, attenuated malaria sporozoites as vaccine candidates critically depends on their ability to migrate to and infect the host liver. This work sheds light on the effect of different administration routes on sporozoite migration. We show that the delivery of sporozoites via mosquito inoculation is more efficient than syringe injection; however, this route of administration is highly impractical for vaccine purposes. Using confocal microscopy and automated imaging software, we demonstrate that syringe-injected sporozoites do cluster, move more slowly, and display more erratic movement due to alterations in tissue morphology. These findings indicate that microneedle-based engineering solutions hold promise for replicating the success of mosquito-inoculated live, attenuated sporozoite vaccines.
Summary
Studies in animal models suggest that protection against malaria induced by intradermal (ID) administration of sporozoites is less effective compared to intravenous injection (IV). We ...investigated in a murine model the protective efficacy and immune responses after ID or IV immunization of sporozoites. Mice were immunized via either IV or ID route with Plasmodium berghei sporozoites in combination with chloroquine treatment (CPS) (allowing full liver stage development) or by γ‐radiation‐attenuated sporozoites (RAS) (early liver stage arrest). While IV immunization with both RAS and CPS generated 90–100% protection, ID immunization resulted in reduced levels of protection with either immunization strategy in both Balb/cByJ (50%) and C57BL/6j mice (7–13%). Lower protection by ID routing associated with a 30‐fold lower parasite liver load P < 0.001 (χ2 = 49.08, d.f. = 1) assessed by real‐time in vivo imaging of bioluminescent P. berghei parasites. Unlike IV, ID immunization did not result in expansion of CD8+ T cells with effector memory phenotype and showed lower IFNγ responses irrespective of the immunization regime. In conclusion, protection against sporozoite infection is likely dependent on parasite liver infection and subsequently generated cellular immune responses.
Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory ...syndrome coronavirus 2 (SARS-CoV-2) replicates
. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c
natural killer (NK) cells and CD4
T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DR
monocytes, CD38
PD1
CD4
T effector (T
) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127
granulocytes and CD38
CD8
tissue-resident memory T cells (T
). SARS-CoV-2-specific CD8
T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.
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