A Modified Basophil Activation Test for the Clinical Management of Immediate Hypersensitivity Reactions to Paclitaxel: A Proof-of-Concept Study.

Immediate hypersensitivity reactions (iHSRs) to taxanes are observed in 6% and 4% of gynecologic and breast cancer patients, respectively. Drug desensitization is the only option, as no comparable alternative therapy is available. Surfactants in the taxane formulation have been implicated in the immunopathogenesis of iHSRs, although sporadic skin test (ST) positivity and iHSRs to nab-paclitaxel have suggested the involvement of the taxane moiety and/or IgE-mediated pathomechanisms. In vitro diagnostic tests might offer insights into mechanisms underlying iHSRs to taxanes. The aim of the present study was to address this unmet need by developing a novel basophil activation test (BAT). The study included patients (n = 31) undergoing paclitaxel/carboplatin therapy. Seventeen patients presented with iHSRs to paclitaxel (iHSR-Taxpos), and eleven were tolerant (iHSR-Taxneg). Fourteen patients presented with iHSRs to carboplatin (iHSR-Plpos), and fourteen were tolerant (iHSR-Plneg). The BAT median stimulation index (SI) values were 1.563 (range, 0.02-4.11; n = 11) and -0.28 (range -4.88-0.07, n = 11) in iHSR-Taxpos and iHSR-Taxneg, respectively. The BAT median SI values were 4.45 (range, 0.1-26.7; n = 14) and 0 (range, -0.51-1.65; n = 12) in iHSR-Plpos and iHSR-Plneg, respectively. SI levels were not associated with iHSR severity grading. Comparing BAT results in iHSR-Taxpos and iHSR-Taxneg showed the area under the receiver operator characteristic (ROC) curve to be 0.9752 (p = 0.0002). The cutoff calculated by the maximized likelihood ratio identified 90.91% of iHSR-Taxpos patients and 90.91% of iHSR-Taxneg patients. Comparing BAT results for iHSR-Plpos and iHSR-Plneg showed the area under the ROC curve to be 0.9286 (p = 0.0002). The cutoff calculated by the maximized likelihood ratio identified 78.57% of iHSR-Plpos patients and 91.67% of iHSR-Plneg patients. Most iHSR-Taxpos patients for which ST was available (10/11) scored ST-negative and BAT-positive, whereas most iHSR-Plpos patients for which ST was available (14/14) scored both BAT- and ST-positive. This suggested the intervention of non-IgE-mediated mechanisms in iHSR-Taxpos patients. Consistent with this view, an in silico molecular docking analysis predicted the high affinity of paclitaxel to the degranulation-competent MRGPRX2 receptor. This hypothesis warrants further in vitro investigations. In conclusion, the present study provides preliminary proof-of-concept evidence that this novel BAT has potential utility in understanding mechanisms underlying iHSRs to taxanes.

Clostridioides difficile Infection in Children: A 5-Year Multicenter Retrospective Study.

While there are numerous studies regarding Clostridioides difficile infection (CDI) in adults, literature on the pediatric population is scarce. Therefore, we performed a 5-year retrospective study between January 2014 and December 2018 in two referral centers in Rome, Italy. There were 359 patients tested for CDI who were enrolled: 87 resulted in positive and 272 in negative. CDI children had a higher number of previous-day hospital admissions (p = 0.024), hospitalizations (p = 0.001), and total hospital admissions (p = 0.008). Chronic comorbidities were more frequent in the CDI group (66.7% vs. 33.3%). Previous use of proton pump inhibitors and antibiotics was associated with CDI (p < 0.001). Among the antibiotics, only fluoroquinolones were significantly associated with CDI. Also, CDI children were more frequently exposed to antibiotics during the episode of hospitalization when children were tested. Our study provides an updated clinical and epidemiological analysis of children with CDI compared with a control group of children who tested negative. Further prospective studies could better define risk factors and preventive methods.

Intestinal Parasitic Infections in Internationally Adopted Children: A 10-Year Retrospective Study.

BACKGROUND: Intestinal parasitic infections (IPIs) represent one of the leading causes of morbidity in the world. Children involved in international adoptions constitute a special group of subjects with specific problems and specific healthcare needs. Nevertheless, in current literature there are insufficient data on IPI in this subset of children. This study aims to evaluate the prevalence of IPI in a cohort of internationally adopted children and to investigate epidemiologic factors and clinical features related to IPIs. METHODS: A retrospective study involving internationally adopted children <18 years old for which results from 3 fecal parasitologic tests were available, evaluated between September 1, 2008 and April 31, 2018 at a tertiary level university hospital in Rome. Univariate and multivariate logistic regression analyses were carried out to identify demographic factors and clinical features associated with IPIs. Two comparisons were performed, the first one according to the positivity of the parasitologic examination of the feces and the second one according to the pathogenicity of the identified strains. RESULTS: Of 584 children evaluated, 346 (59.3%) had a positive parasitologic examination (143 pathogenic parasites and 203 nonpathogenic parasites) and 238 (40.8%) had a negative parasitologic examination. About 28.9% of children were positive for 2 or more parasites. A statistically significant positive association was found between IPIs and age, macroarea of origin (Africa and Latin America), living in institutions before adoption and vitamin D deficiency (P < 0.05). CONCLUSIONS: Intestinal parasites represent a widespread infection among internationally adopted children, especially in school-age children and those from Latin America and Africa. Importantly, the parasites found in adopted children were not pathogenic in most cases and did not cause significant alterations in growth, major micronutrient deficits or malnutrition.

A large spectrum of alpha and beta papillomaviruses are detected in human stool samples.

Human papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through faeces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analysed 103 faecal specimens collected from hospitalized patients with diarrhoea using validated primers able to detect α, ß and γ HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the faecal samples. Thirteen samples (12.6 %) were positive for nine genotypes belonging to the α and ß genera: HPV32 (LR, α1), HPV39 (HR, α7), HPV44 (LR, α10), HPV8 (ß1), HPV9, HPV23, HPV37, HPV38 and HPV120 (ß2). Two putative novel genotypes of the ß genus, species 1 and 2, were also detected. The tissue(s) of origin is unknown, since faeces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in faecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a consequence of shedding in the faeces. This highlights the need for further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.

Detection of rare G3P[19] group A rotavirus in human patient, Italy.

Infection with a rare G3P[19] rotavirus A strain was identified in an immunosuppressed patient in Italy. The strain showed a P[19] viral protein 4 gene and a complete AU-1-like genomic constellation. Phylogenetic analyses showed high nucleotide identity between this strain and G3P[19] rotavirus A strains from Asia, indicating possible reassortment events.

Entamoeba dispar: A Rare Case of Enteritis in a Patient Living in a Nonendemic Area.

Entamoeba dispar, a common noninvasive parasite, is indistinguishable in its cysts and trophozoite forms from Entamoeba histolytica, the cause of invasive amebiasis, by microscopy. To differentiate the two species seems to be a problem for laboratory diagnosis. Recent experimental studies showed that E. dispar can be considered pathogenic too. We present a rare case of enteritis due to E. dispar.

Effective use of nitrocellulose-blotted antigens for phage display monoclonal antibody selection.

The combinatorial phage display library approach to antibody repertoire cloning offers a powerful tool for the isolation of specific antibodies to defined target antigens. Panning strategy is often a very critical point for selecting antibody displayed on the surface of bacteriophages. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well defined antigen source. However, when the antigen is difficult to purify by means of laborious and time-consuming chromatography procedures, panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental complications. In the present work, we successfully generated a mouse monoclonal antibody fragment from a phage display library directed against protein E7 of HPV18 avoiding antigen purification as for immunizing mice as for antibody library selection. Our work demonstrates the feasibility of phage antibody selections on antigens transferred to a nitrocellulose membrane as solid support, using one-dimensional polyacrylamide gel electrophoresis system as the only practice to separate a given antigen present in bacterial crude cell lysate.

Human antibodies from phage display libraries: expression of recombinant full length immunoglobulin G specific to the hepatitis C virus E2 glycoprotein.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 glycoprotein (HCV/E2) represents a major target antigen involved in the containment and resolution of naturally occurring HCV infection. Antibody phage display allows the molecular cloning of cDNA sequences encoding antibody fragments specific to a wide range of diverse antigens. These antibodies may be produced in bacteria as Fab or converted into full length IgG. The latter have a higher serum half life and display Fc encoded function. Using a library prepared from an HCV-infected individual, we selected a panel of Fab fragments for binding to invariant epitopes of the E2 glycoprotein. This work describes a technique used to convert the selected Fab fragments into full length IgG and to express these antibodies in eukaryotic cells. All the recombinant antibodies retained the binding specificity of the parental Fab showing an increase in apparent relative affinity for E2.

First Italian case of cyclosporiasis in an immunocompetent woman: local acquired infection.

Cyclospora cayetanensis is a coccidian agent of chronic diarrhea in humans with a worldwide distribution. We report the first documented case of acquired Cyclosporiasis in Italy. The patient was an immunocompetent woman with no recent history of travel outside the country. Microscopy detected Cyclospora oocysts in a feces sample. PCR detected the pathogen in a second sample, which had tested negative by microscopy. The patient was investigated to detect other microorganisms in feces, such as Salmonella spp., Shigella spp, Campylobacter spp., Yersinia spp, and enteroviruses: all were negative. All symptoms disappeared 72 h after the beginning of therapy.

Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

Multiple malaria infection in a pregnant woman from Nigeria: detection by multiplex PCR.

For the last 100 years, diagnosis of malaria has been based on examination of Giemsa-stained thick and thin blood films under the microscope. This is a time-consuming procedure which often fails to correctly diagnose the infecting species--especially when carried out by inexperienced technicians or when blood levels of parasite are low. Rapid Diagnostic Tests (RDTs) for antigen detection can distinguish between Plasmodium falciparum and Plasmodium vivax but cannot identify the species present in mixed infections. In the case reported here we used multiplex PCR to investigate suspected mixed infection in a pregnant woman from Nigeria. The results suggest that the method used is highly specific and can be very sensitive and that it has several advantages with respect to microscopy and RTDs.

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens.

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.

Human monoclonal antibody fragment specific for glycoprotein G in herpes simplex virus type 2 with applications for serotype-specific diagnosis.

A combinatorial library was used to select a human monoclonal antibody fragment (Fab) with high affinity for G glycoprotein in herpes simplex virus type 2 (HSV-2). Tests with 112 clinical specimens demonstrated successful discrimination between HSV-2 and HSV-1, showing the potential of Fab as a low-cost tool for HSV subtyping in clinical diagnosis.