Field and laboratory comparative evaluation of a LAMP assay for the diagnosis of urogenital schistosomiasis in Cubal, Central Angola.

OBJECTIVE: To evaluate the performance of Rapid-Heat LAMPellet assay in field conditions for diagnosis of urogenital schistosomiasis in an endemic area in Cubal, Angola, and to assess the reproducibility in a reference laboratory. METHODS: A total of 172 urine samples from school-age children were tested for microhaematuria, microscopic detection of Schistosoma haematobium eggs and LAMP for DNA detection. Urine samples were stored in a basic equipped laboratory. Field-LAMP tests were performed with and without prior DNA extraction from urine samples, and the results were read by turbidity and by colour change. When field procedures were finished, samples were sent to a reference laboratory to be reanalysed by LAMP. RESULTS: A total of 83 of 172 (48.3%) were positive for microhaematuria, 87/172 (50.6%) were microscopy-positive for S. haematobium eggs detection, and 127/172 (73.8%) showed LAMP-positive results for detecting S. haematobium using purified DNA and 109/172 (63.4%) without prior DNA extraction. MacNemar's test showed a statistical significant relation between LAMP results and microscopy-detected S. haematobium infections and microhaematuria (P < 0.001 in both cases), respectively. When samples of purified DNA were reanalysed in a reference laboratory in Spain using the same LAMP methodology, the overall reproducibility achieved 72.1%. CONCLUSIONS: The ease of use, simplicity and feasibility demonstrated by LAMP assay in field conditions together with the acceptable level of reproducibility achieved in a reference laboratory support the use of LAMP assay as an effective test for molecular diagnosis of urogenital schistosomiasis in endemic remote areas.

Detection and molecular characterization of Acanthamoeba spp. in stray cats from Madrid, Spain.

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns.

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents.

We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

Prevalence and genotypes of Giardia duodenalis from dogs in Spain: possible zoonotic transmission and public health importance.

The prevalence of Giardia duodenalis was determinate in faecal samples from dogs and cats in Madrid, Spain and molecular characterisation of isolates. A total of 604 and 144 faecal samples from dogs and cats, respectively, were analysed by routine coprological methods. The prevalence of G. duodenalis was 16.4 % (99/604) in dogs and 4.2 % (6/144) in cats. Sixty-four G. duodenalis isolates (63 from dogs and 1 from a cat) were characterised using glutamate dehydrogenase and ß-giardin genes by PCR-RFLP. The single cat sample showed a mixed infection by assemblages A + F. The assemblages found in the dog samples were A, B, C, D and E, both as single and as mixed infections. The zoonotic assemblages A and B were found in 56 (88.8 %) G. duodenalis-positive samples with 15.9 % of samples having assemblage A (10/63) and 73 % of samples with assemblage B (46/63), indicating high potential zoonotic risk and public health significance.

Evolution of antibodies against SARS-CoV-2 over seven months: Experience of the nationwide seroprevalence ENE-COVID study in Spain.

BACKGROUND: The main aims of this study were to analyze trends of SARS-CoV-2 anti-nucleocapsid IgG throughout the four rounds of the seroepidemiologic study ENE-COVID, and compare the fourth-round results of two immunoassays detecting anti-nucleocapsid and anti-RBD IgG. METHODS: ENE-COVID was developed in 2020 (two phases). Phase one included three rounds carried out in April 27-May 11, May 18-June 1, and June 8-June 22. Phase two included a fourth round in the same cohort (November 16-29). A chemiluminescent microparticle immunoassay was offered to participants in the first three rounds (Abbott; anti-nucleocapsid IgG). In the fourth round, we offered this test and a chemiluminescence immunoassay (Beckman; anti-RBD IgG) to i) a randomly selected sub-cohort, ii) participants who were IgG-positive in any of the three first rounds; and iii) participants who were IgG-positive in the fourth round by point-of-care immunochromatography. RESULTS: 10,153 individuals (82.2% of people invited) participated in the fourth round. Of them, 2595 (35.1% of participants with results in the four rounds) were positive for anti-nucleocapsid IgG in at least one round. Anti-nucleocapsid IgG became undetectable in 43.3% of participants with positive first-round results. In fourth round, anti-nucleocapsid and anti-RBD IgG were detected in 5.5% (321/5827) and 5.4% (315/5827) participants of the randomly selected sub-cohort, and in 26.6% (867/3261) and 25.9% (846/3261) participants with at least one previous positive result, respectively. CONCLUSIONS: The IgG response is heterogeneous and conditioned by infection severity. A proportion of SARS-CoV-2 infected population may have negative serologic results in the post-infection months.

Effectiveness and Safety of a Single-Dose Ivermectin Treatment for Uncomplicated Strongyloidiasis in Immunosuppressed Patients (ImmunoStrong Study): The Study Protocol.

Strongyloidiasis affects an estimated 600 million people worldwide, especially in tropical and subtropical areas. Single-dose ivermectin treatment has shown to be effective among immunocompetent patients with uncomplicated strongyloidiasis. Here, we present the protocol of the ImmunoStrong study, a prospective observational study aiming to evaluate the effectiveness and safety of a single-dose ivermectin for treatment of uncomplicated strongyloidiasis in immunosuppressed patients. The secondary objectives are to assess accuracy of molecular techniques for the follow-up of these patients and to determine the population pharmacokinetics of ivermectin. The information retrieved by this study will cover relevant information gaps in the strongyloidiasis management among immunosuppressed patients.

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.

Strongyloidiasis in Southern Alicante (Spain): Comparative Retrospective Study of Autochthonous and Imported Cases.

BACKGROUND: Strongyloidiasis is a parasitic disease with global prevalence. In Spain, autochthonous cases are concentrated in the Mediterranean basin. We aimed to analyze clinical and epidemiological characteristics of Strongyloides stercoralis infection in Vega Baja del Segura (Spain), comparing autochthonous versus imported cases. METHODS: Observational retrospective study of all strongyloidiasis cases from January 2009 to January 2019. Cases were diagnosed by stool larvae visualization, positive culture, PCR, Strongyloides serology, and/or compatible histology. RESULTS: We included 36 patients (21 men) with a mean age of 60.8 years ±17.6; 15 cases were autochthonous and 21 imported 80.9% from Latin America. Autochthonous cases were associated with older age (mean 71.3 vs. 53.3 years; p = 0.002), male sex (odds ratio (OR) 5.33; 95% confidence interval (CI) 1.15-24.68; p = 0.041), and agricultural activity (OR 13.5; 95% CI 2.4-73.7; p = 0.002). Fourteen were asymptomatic, three autochthonous cases presented with hyperinfection syndrome, and two patients died. There was no difference between autochthonous versus imported origin in eosinophilia at diagnosis (93.3% vs. 75%; p = 0.207), treatment received, or clinical response (85.7% vs. 88.9% cured; p = 1). CONCLUSION: In our region, imported strongyloidiasis coexists with autochthonous cases, which are mainly in older male farmers who are diagnosed at more advanced stages. Systematic screening programs are needed.

Asymptomatic Strongyloidiasis among Latin American Migrants in Spain: A Community-Based Approach.

Strongyloides stercoralis infection is frequently underdiagnosed since many infections remain asymptomatic. AIM: To estimate the prevalence and characteristics of asymptomatic S. stercoralis infection in Latin American migrants attending a community-based screening program for Chagas disease in Spain. METHODOLOGY: Three community-based Chagas disease screening campaigns were performed in Alicante (Spain) in 2016, 2017, and 2018. Serological testing for S. stercoralis infection was performed using a non-automatized IVD-ELISA detecting IgG (DRG Instruments GmbH, Marburg, Germany). RESULTS: Of the 616 migrants from Central and South America who were screened, 601 were included in the study: 100 children and adolescents (<18 years of age) and 501 adults. Among the younger group, 6 participants tested positive (prevalence 6%, 95% confidence interval [CI] 2.5% to 13.1%), while 60 adults did so (prevalence 12%, 95% CI 9.3% to 15.3%). S. stercoralis infection was more common in men than in women (odds ratio adjusted [ORa] 2.28, 95% CI 1.289 to 4.03) and in those from Bolivia (ORa 2.03, 95% CI 1.15 to 3.59). Prevalence increased with age (ORa 1.02, 95% CI 0.99 to 1.05). In contrast, a university education had a protective effect (ORa 0.29, 95% CI 0.31 to 0.88). Forty-one (41/66; 62.1%) of the total cases of S. stercoralis infection were treated at the health care center. Positive stool samples were observed in 19.5% of the followed-up positive cases. CONCLUSION: Incorporating serological screening for S. stercoralis into community-based screening for Chagas disease is a useful intervention to detect asymptomatic S. stercoralis infection in Central and South American migrants and an opportunity to tackle neglected tropical diseases in a transversal way. The remaining challenge is to achieve patients' adherence to the medical follow-up.

Comparative performance evaluation of four commercial multiplex real-time PCR assays for the detection of the diarrhoea-causing protozoa Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica.

BACKGROUND: Multiplex molecular panels are relentlessly replacing conventional methods for the detection of enteric pathogens from stool samples in clinical and research laboratories. Here we evaluated four commercial multiplex real-time PCR assays for the detection of Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica. METHODS: The diagnostic performance of the Gastroenteritis/Parasite Panel I (Diagenode), the RIDAGENE Parasitic Stool Panel (R-Biopharm), the Allplex Gastrointestinal Parasite Panel 4 (Seegene) and the FTD Stool Parasites (Fast Track) real-time PCR methods was assessed against a reference panel of 126 well-characterized DNA samples including Cryptosporidium hominis (n = 29), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 47), Entamoeba histolytica (n = 3), other parasite species (n = 20), and apparently healthy subjects (n = 24). PRINCIPAL FINDINGS: Obtained diagnostic sensitivities ranged from 53-88% for Cryptosporidium hominis/parvum, and from 68-100% for G. duodenalis. The R-Biopharm method achieved the best performance for the detection of Cryptosporidium hominis/parvum both in terms of diagnostic sensitivity (87.5%) and detection limit (a 100-fold increase compared to other tests). The Fast Track method was particularly suited for the detection of G. duodenalis, achieving a 100% sensitivity and a detection limit at least 10-fold superior. Detection of E. histolytica was similarly achieved by all compared methods except Diagenode. CONCLUSIONS: Diagnostic performance varied largely depending on the method used and the targeted pathogen species. Factors including test sensitivity/specificity, cost, patient population surveyed, laboratory workflow, and diagnostic algorithm should be carefully considered when choosing the most appropriate multiplex PCR platform.

High Prevalence and Diversity of Zoonotic and Other Intestinal Parasites in Dogs from Eastern Spain.

The diversity and frequency of enteric parasites in dog populations in the Castellón province (Eastern Spain) were assessed using a prospective cross-sectional epidemiological survey. A total of 263 canine fecal samples were collected between July 2014 and July 2016. Detection of intestinal parasites was conducted by routine coprological methods. In addition, identification of Giardia duodenalis and Cryptosporidium spp. was carried out by direct immunofluorescence microscopy, whereas the presence of Strongyloides spp. was assessed by real-time PCR in a selected number of specimens. Based on conventional and/or immunofluorescence microscopy examination, 65.8% (95% confidence interval: 59.7-71.5) of the investigated dogs were found infected by at least one gastrointestinal parasite. G. duodenalis (35.4%) and members of the family Ancylostomatidae (27.0%) were the most prevalent protozoan and helminth parasites found, respectively. Other pathogens potentially infective to humans included Toxocara canis (8.0%), Cryptosporidium spp. (6.8%), and Strongyloides spp. (1.1%). Frequency of occurrence of helminthic, but not protozoan, enteroparasites was geographical origin dependent (p = 0.02), with dogs living in coastal areas presenting higher infection rates than those living in inland regions. Similarly, rural dogs were significantly more infected than urban dogs (p < 0.001). Our results revealed that zoonotic agents were common in dogs from the Castellón province. Animals from rural areas and sheltered dogs were particularly at risk of these infections.

Real-Time Polymerase Chain Reaction in Stool Detects Transmission of Strongyloides stercoralis from an Infected Donor to Solid Organ Transplant Recipients.

Solid organ transplant recipients can acquire Strongyloides stercoralis from an infected donor. The diagnosis of S. stercoralis in immunocompromised individuals may be challenging due to a lower sensitivity of available parasitological and serological methods, compared with immunocompetent individuals. Recently, a real-time polymerase chain reaction (RT-PCR) in stool has been developed for S. stercoralis diagnosis. We report two cases of S. stercoralis infection transmitted by a donor to two solid organ transplant recipients, who were diagnosed with RT-PCR in stool. This test could play an important role inS. stercoralis diagnosis in immunosuppressed patients, facilitating rapid treatment initiation and reducing the risk of severe strongyloidiasis. Adherence to current recommendations of screening among donors and recipients from endemic areas is also urgently needed.

Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model.

BACKGROUND: Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. METHODOLOGY/PRINCIPAL FINDINGS: Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. CONCLUSIONS/SIGNIFICANCE: Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.

Usefulness of Strongyloides stercoralis serology in the management of patients with eosinophilia.

Strongyloides stercoralis infection is being increasingly diagnosed out of endemic areas. The aim of this study is to evaluate the usefulness of S. stercoralis serology for the management of probable strongyloidiasis in patients presenting with eosinophilia. Overall, 147 patients were included, 89 (60.5%) patients had a positive S. stercoralis serology. Strongyloides stercoralis larvae were detected only in 15 (10.2%) patients. Twenty-eight patients had human immunodeficiency virus infection. Eighty patients received ivermectin 200 mcg/Kg/day for 2 days, and follow-up 6 months after treatment could be performed in 32 patients: 26 (81.3%) patients reached the response to treatment criteria (negative serology 6 months after treatment or when by enzyme-linked immunosorbent assay the optical density ratio of post-treatment to pre-treatment decreased to 0.6), and 11 (34.4%) patients fulfilled the cure criteria (negative serology 6 months after treatment). Strongyloides stercoralis serology is a useful diagnostic tool both in the diagnosis of probable strongyloidiasis and follow-up after treatment.

Congenital toxoplasmosis in wild boar (Sus scrofa) and identification of the Toxoplasma gondii types involved.

Congenital toxoplasmosis has been little described in wild animals. We report a case of vertical transmission in wild boar (Sus scrofa). Necropsy and histopathologic examination of a pregnant female and her three fetuses revealed all to have lesions compatible with acute toxoplasmosis. Nested polymerase chain reaction B1 gene detected Toxoplasma gondii in maternal (heart and diaphragm) and fetal (central nervous system, retina, optic nerve, heart, lung, tongue, and diaphragm) samples. The mother had a mixed infection of T. gondii types I and III. One fetus with type III infection developed no malformations, but the others-one with type I infection and one infected by types I and III-showed bilateral ocular agenesis, prognathism, and agenesis of the nasal cartilage. These results suggest the pathogenicity of the various T. gondii types may differ in wild boars.

Synthesis of 16-mercaptohexadecylphosphocholine, a miltefosine analog with leishmanicidal activity.

The alkylphosphocholine miltefosine (n-hexadecylphosphocholine, MT) has been introduced recently as a very effective drug for the oral treatment of human leishmaniasis. However, the parasiticidal mechanism of MT at a molecular level is far from being understood. Here we report the synthesis and biological characterization of 16-mercaptohexadecylphosphocholine, a thiol analog of MT which was designed to facilitate the search of MT interacting targets within the parasite by a variety of analytical methods. This analog presents the same leishmanicidal effect as the parent drug against Leishmania donovani promastigotes and Leishmania pifanoi axenic amastigotes, and has been used to develop an affinity chromatography method to attempt the isolation of putative Leishmania proteins that bind to the phosphocholine part of the molecule.

Activity of cecropin A-melittin hybrid peptides against colistin-resistant clinical strains of Acinetobacter baumannii: molecular basis for the differential mechanisms of action.

Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin (polymyxin E), the last universally active drug against this pathogen. The possible widespread distribution of colistin-resistant A. baumannii strains may create an alarming clinical situation. In a previous work, we reported differences in lethal mechanisms between polymyxin B (PXB) and the cecropin A-melittin (CA-M) hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) on colistin-susceptible strains (J. M. Saugar, T. Alarcón, S. López-Hernández, M. López-Brea, D. Andreu, and L. Rivas, Antimicrob. Agents Chemother. 46:875-878, 2002). We now demonstrate that CA(1-8)M(1-18) and three short analogues, namely CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), its Nalpha-octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH2), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH2) are active against two colistin-resistant clinical strains. In vitro, resistance to colistin sulfate was targeted to the outer membrane, as spheroplasts were equally lysed by a given peptide, regardless of their respective level of colistin resistance. The CA-M hybrids were more efficient than colistin in displacing lipopolysaccharide-bound dansyl-polymyxin B from colistin-resistant but not from colistin-susceptible strains. Similar improved performance of the CA-M hybrids in permeation of the inner membrane was observed, regardless of the resistance pattern of the strain. These results argue in favor of a possible use of CA-M peptides, and by extension other antimicrobial peptides with similar features, as alternative chemotherapy in colistin-resistant Acinetobacter infections.

Role of positional hydrophobicity in the leishmanicidal activity of magainin 2.

The emergence of membrane-active antimicrobial peptides as new alternatives against pathogens with multiantibiotic resistance requires the design of better analogues. Among the different physicochemical parameters involved in the optimization of linear antimicrobial peptides, positional hydrophobicity has recently been incorporated. This takes into consideration the concept of the topological distribution of hydrophobic residues throughout the sequence rather than the classical concept of hydrophobicity as a global parameter of the peptide, calculated as the summation of the individual hydrophobicities of the residues. In order to assess the contribution of this parameter to the leishmanicidal mechanisms of magainin 2 analogues, the activities of two of these analogues, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), which have similar charges, amino acid compositions, and hydrophobicities but different positional hydrophobicities, against Leishmania donovani promastigotes were assayed (T. Tachi, R. F. Epand, R. M. Epand, and K. Matsuzaki, Biochemistry 41:10723-10731, 2002). The activities were compared with that of the parental peptide, F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS). The three peptides were active at micromolar concentrations, in the order MG-H2 > MG-H1 > F5W-magainin 2. These activities differ from their hemolytic and bactericidal activities. The results demonstrate that positional hydrophobicity, which reflects the presence of short stretches of sequences rich in hydrophobic amino acids, plays an important role in the activities of leishmanicidal peptides.

Activities of polymyxin B and cecropin A-,melittin peptide CA(1-8)M(1-18) against a multiresistant strain of Acinetobacter baumannii.

Polymyxin B (PXB) and the cecropin A-melittin hybrid CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) were compared for antibiotic activity on reference and multiresistant Acinetobacter baumannii strains. Significant differences for both peptides were observed on their inner membrane interaction and inhibition by environmental factors, supporting the use of CA(1-8)M(1-18) as a potential alternative to PXB against ACINETOBACTER: