Delayed diagnosis among patients with visceral leishmaniasis.

We aimed to estimate the diagnostic latency of patients with visceral leishmaniasis (VL). A monocentric retrospective observational study was conducted including all confirmed cases of VL diagnosed from January 2005 to March 2022. Epidemiological and clinical characteristics of patients with VL were collected. The diagnostic latency was defined as the number of days between the first contact with a health-care provider for signs and/or symptoms referable to VL and the laboratory diagnosis of leishmaniasis. Twenty-four cases of VL were included in the study, mostly male (75%) and Italians (79.2%), with a median age of 40 years [Inter Quartile Range (IQR 30-48)]. Fourteen (58.3%) VL cases were people living with HIV (PLWH) and 4 (16.6%) subjects were on immunosuppressive therapy. For VL the median diagnostic latency was 54 days (IQR 28-162). The shorter diagnostic latency was observed in PLWH [31 days (IQR 20-47)] followed by immunocompetent patients [160 days (IQR 133-247)] and those on immunosuppressive therapy [329 days (IQR 200-678)]. Twelve patients (50%) reported at least one medical encounter before the diagnosis of VL and 6 patients received a wrong therapy. Diagnostic delay in VL was significant in patients under immune suppressive treatment.

Chagas disease prevalence among migrants from El Salvador in Milan: a cross- sectional study of an often-overlooked population.

BACKGROUND: Chagas disease (CD) is considered to be highly endemic in El Salvador, where its prevalence is estimated to be 1.3-3.7%. Although more than 40,000 migrants from El Salvador are currently living in Europe (particularly in Spain and Italy), there are few data regarding the prevalence of CD in this population. The aim of this study was to evaluate the prevalence of CD among Salvadorans living in Italy. METHODS: A cross-sectional serological survey of CD among Salvadorans living in the metropolitan area of Milan was carried out between October 2017 and December 2019. The participants' blood samples were tested for Trypanosoma cruzi antibodies using two different serological assays. The collected demographic data included their biological sex, province of origin, the type of housing in their country of origin, and family history of CD. RESULTS: Of the 384 subjects who voluntarily participated in the study, five (1.3%, most coming from La Paz) were positive to both serological assays and therefore conclusively diagnosed as having CD. Five other subjects had discrepant serological results but were not positive to a third assay. Three of the five subjects with a diagnosis of CD completed medical staging, one of whom had chronic disease (digestive and cardiac involvement). CONCLUSIONS: The prevalence of CD among Salvadorans living in Milan is similar to that estimated by the WHO in 2010. Although they are often overlooked in CD surveys, Salvadoran migrants should be included in CD control programs in countries in which the disease is not endemic.

Family cluster of Chagas disease among Bolivian immigrants in Italy: High rate of maternal-fetal transmission.

BACKGROUND: Chagas disease (CD) or American trypanosomiasis is a neglected anthropozoonosis caused by Trypanosoma cruzi that affects 6-8 million people worldwide (mainly in Latin America), 30-40% of whom develop cardiac or digestive complications. Once confined to endemic areas of Latin America, CD has more recently become a global disease as a result of migration flows from endemic to non-endemic regions, particularly in northern America and Europe. Congenital transmission is a particular challenge as it may be sustained for multiple generations and perpetuate the infection even in non-endemic countries. METHODS: Subjects were identified during a cross-sectional survey of CD among Latin American people living in Milan, Italy. Serology was carried out using tests based on either a lysate and a recombinant antigen of Trypanosoma cruzi. They were also tested by a conventional Polymerase Chain Reaction (PCR) targeting the 330 bp variable region of the T. cruzi kinetoplast minicircle genome and a commercial real-time PCR. RESULTS: We here describe a Bolivian family cluster with seven affected people with at least two autochthonous congenital T. cruzi infection which was identified during the course of a CD screening programme. We also review the epidemiology, diagnosis and control of congenital CD, with particular emphasis on the challenges facing the control and management of such a complex and still largely hidden disease. CONCLUSIONS: Our experience confirms the need to screen for CD all family members once a case is diagnosed and shows the possible high rate of congenital CD also in non-endemic areas.

Elucidating the 3D Structure of a Surface Membrane Antigen from Trypanosoma cruzi as a Serodiagnostic Biomarker of Chagas Disease.

Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.

The value of lamp to rule out imported malaria diagnosis: a retrospective observational study in Milan, Italy.

BACKGROUND: The diagnosis of malaria in returning travellers could be a challenge in non-endemic settings. We aimed to assess the performance of LAMP in comparison with standard conventional diagnostic methods using real-time-polymerase chain reaction (PCR) in case of discordant results. METHODS: All travellers returning from malaria-endemic areas who presented to our Emergency Department (ED) from January 2017 to December 2020 with signs and symptoms suggestive for malaria were included. Blood microscopy was the reference diagnostic method applied at our laboratory with LAMP implemented as an additional method to aid in malaria diagnosis. PCR was employed only in case of between test's discordant results. Sensitivity and specificity of microscopy compared to LAMP were calculated with the confidence interval of 95%. RESULTS: Four-hundred and eight patients (55.6% male, median age 42 years) were screened for malaria. The diagnosis was confirmed in 49 cases (12%): 44 cases (90%) caused by Plasmodium falciparum. Peripheral blood smear missed to identify three malaria cases, which tested positive with LAMP and PCR. One case of malaria caused by P. malariae in a naive tourist, one case by P. falciparum in a semi-immune pregnant women and one case by P. falciparum in a previously treated semi-immune patient. All the discordant cases were characterized by a very low parasitaemia. Microscopy when compared to LAMP showed a sensitivity of 93.9% (95% confidence interval (CI) 83.1-98.7%) and a specificity of 100% (95% CI 98.9-100%). CONCLUSIONS: In our non-endemic setting LAMP was able to identify malaria cases with low-level parasitaemia otherwise missed by blood microscopy.

A Lab-On-chip Tool for Rapid, Quantitative, and Stage-selective Diagnosis of Malaria.

Malaria remains the most important mosquito-borne infectious disease worldwide, with 229 million new cases and 409.000 deaths in 2019. The infection is caused by a protozoan parasite which attacks red blood cells by feeding on hemoglobin and transforming it into hemozoin. Despite the WHO recommendation of prompt malaria diagnosis, the quality of microscopy-based diagnosis is frequently inadequate while rapid diagnostic tests based on antigens are not quantitative and still affected by non-negligible false negative/positive results. PCR-based methods are highly performant but still not widely used in endemic areas. Here, a diagnostic tool (TMek), based on the paramagnetic properties of hemozoin nanocrystals in infected red blood cells (i-RBCs), is reported on. Exploiting the competition between gravity and magnetic forces, i-RBCs in a whole blood specimen are sorted and electrically detected in a microchip. The amplitude and time evolution of the electrical signal allow for the quantification of i-RBCs (in the range 10-105 i-RBC µL-1) and the distinction of the infection stage. A preliminary validation study on 75 patients with clinical suspect of malaria shows on-field operability, without false negative and a few false positive results. These findings indicate the potential of TMek as a quantitative, stage-selective, rapid test for malaria.

Structure, Immunoreactivity, and In Silico Epitope Determination of SmSPI S. mansoni Serpin for Immunodiagnostic Application.

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.

Loop-mediated isothermal amplification (LAMP) assay for the diagnosis of imported malaria: a narrative review.

Loop-mediated isothermal amplification (LAMP) is a molecular method to detect malaria recently introduced in the market. LAMP is simple to perform and does not require advanced equipment and training thus satisfying the qualification as a point-of-care diagnostic screening test. In this narrative review, we focus on the role of LAMP for malaria diagnosis in non-endemic settings. We searched PubMed, Embase, Scopus, and Google Scholar, using the following search terms: 'Malaria LAMP' in combination with 'imported malaria' or 'travellers' malaria' or 'non-endemic setting' or 'non-endemic region' or 'malaria screening' or 'malaria diagnosis'. References of each article were also reviewed for possible studies or reports not identified in our search. Overall, 18 studies encompassing 6289 tested samples with 1663 confirmed malaria diagnoses were retrieved. Most of these studies (13/18, 72.2%) were conducted in Europe, and almost half were retrospective. Fourteen studies (77.8%) employed real-time or nested-polymerase chain reaction as the reference method for confirming malaria diagnosis. Sensitivity of LAMP ranged from 93.9 to 100% and specificity from 93.8 to 100% with a negative predictive value of 99.6%-100%. The rate of reported invalid results requiring repeat of the test varied from 0.01% to 5.7%, but they were solved in the majority of cases with a secondary analysis. In non-endemic countries the adoption of LAMP malaria assay as the screening test for malaria diagnosis seems to perform better than conventional methods. However, blood microscopy remains essential to either identify Plasmodium species and quantify parasitaemia and adequately managing malaria cases.

A case of crusted scabies with a delayed diagnosis and inadequate therapy.

Crusted scabies is an infrequent disease caused by Sarcoptes scabiei that usually affects patients with underlying medical conditions leading to immunosuppression. Here, we present the case of an 81 years old man, diagnosed with crusted scabies who came to our attention after multiple misdiagnosis and incorrect and potentially detrimental treatment with steroids. He was admitted to our inpatients ward and treated with oral ivermectin plus local permethrin. The hospitalization was complicated by a secondary bacterial skin infection caused by methicillin-sensitive Staphylococcus aureus. Crusted scabies is commonly misdiagnosed in elderly and immunosuppressed people due to its unusual occurrence and atypical clinical presentation. It should be considered in the differential diagnosis of skin lesions associated with pruritus in patients with underling medical conditions leading to immunosuppression. A prompt diagnosis and treatment are warranted due to the potential secondary infections and subsequent related morbidity and mortality.

Occurrence of Lyme disease infection in a non-endemic area in Northern Italy.

BACKGROUND: In Italy, the incidence of human tick-borne disease has increased over the last decades. Since 2015 a multidisciplinary group has been established in Sacco Hospital for the management of the patients affected by Lyme disease (LD). A retrospective evaluation (2015-2017) was performed for LD in non-endemic areas. METHODS: Retrospective analysis of all 1000 samples for 800 patients screened for LD antibodies at the Sacco Hospital in 3 years (2015-2017). Clinical and epidemiological data were collected and compared with the serological results. RESULTS: Among the 800 patients screened, 134 of them were diagnosed with borreliosis during 2015 (37 cases), 2016 (31 cases) and 2017 (66 cases). Localized LD was diagnosed 100 out of 134 cases (69%): in most of them (N.=63) erythema migrans has been documented; in 37 out of 100 it was not possible to detect it. In only three cases, patients complained of different clinical symptoms such as headache, arm and facial pain respectively. 23 out of 134 cases (16%) showed a persistence of serological positivity and symptoms with osteomuscular involvement and fatigue, despite the therapy (late LD). In that same study 11 out of 134 patients (7%) received a diagnosis of neuroborreliosis. CONCLUSIONS: Our data reported a high percentage of LD infection (19%) in a non-endemic area. The definition of a Multidisciplinary Working Group and a clinical care pathway allowed a better clinical management of LD cases treated in Sacco Hospital, Milan.

Lyme borreliosis incidence in Lombardy, Italy (2000-2015): Spatiotemporal analysis and environmental risk factors.

Lyme borreliosis cases have been reported from Lombardy in northern Italy, where Ixodes ricinus is the main vector of Borrelia burgdorferi sensu lato. However, spatial and temporal variation in the incidence of Lyme borreliosis is not well understood. In the present study, based on new notified cases of Lyme borreliosis from 2000 to 2015, an average of 1.24 new cases per million residents per year was documented. New cases, georeferenced at the municipal level, were analyzed by retrospective space-time analysis (using SaTScan v. 9.3.1); and land cover, extrapolated from a Corine Land Cover dataset (using QGIS 2.8.1), was used to implement an environmental risk factor analysis. Firstly, a temporal high-risk cluster was detected in Lombardy: the relative risk of Lyme borreliosis was 3.73 times higher during 2008-2015 compared with the entire study period. Moreover, in a spatiotemporal high-risk cluster with a circular base, land cover consisting of wildland-urban interface, meadow, forest and meadow-forest transition were significantly higher compared to low-risk areas. Results of the present study demonstrate that the incidence of Lyme borreliosis is increasing in Lombardy and that environmental conditions are suitable for I. ricinus ticks infected with B. burgdorferi s.l.: citizens and health systems should be aware of Lyme borreliosis to reduce tick bites with personal protective behaviors and to avoid misdiagnosis, particularly within the area including the observed high-risk cluster. Economic resources should be invested to inform about methods to prevent tick bites, how to check people and pets after frequenting risk areas, and ways of removing the biting ticks when they are found.

A case of Plasmodium malariae recurrence: recrudescence or reinfection?

BACKGROUND: Plasmodium malariae is the most neglected of the six human malaria species and it is still unknown which is the mechanism underlying the long latency of this Plasmodium. CASE PRESENTATION: A case of PCR-confirmed P. malariae recurrence in a 52-year old Italian man was observed 5 months after a primary attack. In the interval between the two observed episodes of malaria the patient denied any further stay in endemic areas except for a visit to Libya, a country considered malaria-free. Genomic DNA of the P. malariae strain using five microsatellites (PM2, PM9, PM11, PM25, PM34) and the antigen marker of circumsporozoite (csp) was amplified and sequenced. Analysis of polymorphisms of the P. malariae csp central repeat region showed differences between the strains responsible of the first and second episode of malaria. A difference in the allele size was also observed for the sequence analysis of PM2 microsatellites. CONCLUSIONS: Plasmodium malariae is a challenging human malaria parasite and even with the use of molecular techniques the pathogenesis of recurrent episodes cannot be precisely explained.

Diagnosis of Louse-Borne Relapsing Fever despite Negative Microscopy in Two Asylum Seekers from Eastern Africa.

We report two cases of louse-borne relapsing fever observed at our Institution in June 2016. Both patients were young asylum seekers from Africa who had recently arrived in Milan, Italy. Notably, direct microscopic examination of peripheral blood smears was repeatedly negative for the presence of spirochetes and the diagnosis, supported by clinical and epidemiologic evidence, required molecular confirmation by polymerase chain reaction amplification of DNA extracted from blood and sequencing of the amplified products.

Chagas disease in Europe: A review for the internist in the globalized world.

Chagas disease (CD) or American trypanosomiasis identified in 1909 by Carlos Chagas, has become over the last 40years a global health concern due to the huge migration flows from Latin America to Europe, United States, Canada and Japan. In Europe, most migrants from CD-endemic areas are concentrated in Spain, Italy, France, United Kingdom and Switzerland. Pooled seroprevalence studies conducted in Europe show an overall 4.2% prevalence, with the highest infection rates observed among individuals from Bolivia (18.1%). However, in most European countries the disease is neglected with absence of screening programmes and low access to diagnosis and treatment. Physicians working in Europe should also be aware of the risk of autochthonous transmission of Trypanosoma cruzi to newborns by their infected mothers and to recipients of blood or transplanted organs from infected donors. Finally, physicians should be able to recognize and treat the most frequent and serious complications of chronic Chagas disease, namely cardiomyopathy, megacolon and megaesophagus. This review aims to highlights the problem of CD in Europe by reviewing papers published by European researchers on this argument, in order to raise the awareness of internists who are bound to increasingly encounter patients with the disease in their routine daily activities.

Severe Plasmodium falciparum malaria in the intensive care unit: A 6-year experience in Milano, Italy.

BACKGROUND: Severe imported Plasmodium falciparum malaria is a potentially life-threatening disease with a reported mortality rate of 5-10% when patients are admitted to the Intensive Care Unit. METHODS: To retrospectively review the clinical aspects, the value of severity predictive scores and the management of patients with severe P. falciparum malaria admitted to an ICU in Milano, Italy between January 2010 and December 2015. RESULTS: Twelve patients were included: seven were male and five female with a median age of 43 years. All were initially treated with intravenous quinine. Median parasitaemia upon admission was 14,5% (range 1-20%). At the time of ICU admission, 3 patients (25%) had 5 or more World Health Organization criteria for severe malaria while another 6 of them developed one or more of the latter during their stay in ICU. Five required mechanical ventilation because of respiratory failure due to ARDS. Four patients required renal replacement therapy. Three patients underwent blood exchange transfusion. All patients survived. CONCLUSIONS: Our retrospective evaluation of adults patients admitted to the ICU with severe imported P. falciparum malaria demonstrated a favourable outcome. Severity predictive scores currently in use probably overestimate the risk of malaria mortality in patients treated in health care systems of high income countries.

Pancreas divisum: correlation between anatomical abnormalities and bile precipitation in the gallbladder in seven patients.

Pancreas divisum is a genetic defect associated with recurrent acute pancreatitis due to insufficient drainage of the accessory pancreatic duct. Seven young patients diagnosed with pancreatic divisum and thickening of the gallbladder bile as shown on magnetic resonance cholangio-pancreatography without pancreatic ductal changes underwent laparoscopic cholecystectomy. During the mean follow-up of 32 months no episode of pancreatitis was reported. There is an association between PD and higher concentration of bile in the gallbladder. Cholecystectomy can be considered curative in patients with PD in the absence of indications for major surgery.

CAND-LO 2014-15 study: changing epidemiology of candidemia in Lombardy (Italy).

PURPOSE: The aim of this study was to monitor recent changes in the epidemiology of candidemia and in the antifungal susceptibility profiles of Candida isolates in one Italian region (Lombardy) in 2014-2015 in comparison with two other studies performed in the same area in 1997-1999 and in 2009. METHODS: A laboratory-based surveillance was conducted in 11 microbiology laboratories. Identification of Candida isolates from 868 episodes and antifungal susceptibility testing (YeastOne) was performed locally. RESULTS: A progressive increase in the rate of candidemia up to 1.27/1000 admissions and 1.59/10,000 patient days was documented. In all the three surveys, Candida albicans remains the most frequently isolated species, ranging from 52 to 59 % of the etiology of BSIs. The epidemiological shift to the more resistant C. glabrata, observed between 1997-1999 and 2009 surveys, was not confirmed by our more recent data. The pattern of etiology of BSIs occurred in 2014-2015 overlaps that of the 90s. Acquired antifungal resistance is a rare event. No isolate had an amphoterin B minimal inhibitory concentration (MIC, mg/L) value higher than the epidemiological cutoff. All the echinocandin MIC distributions are typical for wild-type organisms except for those of two C. glabrata isolates. Fluconazole resistance declined from 24.9 % in the 2009 survey to 5.4 % in the recent one. CONCLUSIONS: Data from regional surveys may highlight the influence of therapeutic practices on the epidemiology of Candida BSIs and may optimize empirical therapies.