Meningitis In Humans Research Articles

CALCIUM CHANGES IN Bulimulus tenuissimus (MOLLUSCA) EXPERIMENTALLY INFECTED WITH LARVAL Angiostrongylus cantonensis (NEMATODA)

Only in 2018 Bulimulus tenuissimus was incriminated as an intermediate host of Angiostrongylus cantonensis, the causative agent of infections in domestic, wild animals and humans, causing neural angiostrongyliasis or eosinophilic meningitis in humans. The present study aimed to infect the mollusk B. tenuissimus, using L1 larvae of A. cantonensis, under laboratory conditions, and to analyse the changes that occurred in the calcium metabolism of the host mollusk. The infection caused hypercalcification in the shell of B. tenuissimus infected with A. cantonensis, with a 242% increase in the concentration of CaCO3, at the end of the prepatent period, in the shells of infected mollusks and hypercalcemia, especially at the end of the third week of infection (+18.51%) in relation to the control group. The interference of the parasitic nematode in the calcium metabolism of the host mollusk was evidenced, with reductions in tissue deposits and elevation of calcemia and calcium content in the shell.

Reaching new lands: Updating the distribution of Angiostrongylus cantonensis in South America with the first record in Argentina.

Angiostrongylus cantonensis, commonly known as the rat lungworm, is a metastrongyloid nematode found primarily not only in tropical and subtropical regions but also in temperate areas and considered the leading cause of eosinophilic meningitis in humans. Synanthropic rodents such as Rattus norvegicus and Rattus rattus are the most frequent definitive hosts of this parasite. The presence of this parasite was detected in the pulmonary arteries of three specimens of R. norvegicus in the city of Buenos Aires representing the species' southernmost known record in natural hosts. Species confirmation was achieved through partial sequences of 18S and COI genes. By comparing the COI gene sequences with those available in GenBank through the construction of a haplotype network, we obtained that the analysed specimen presents high similarity with those reported in Japan and Southeast Asia. All infected rats were captured in an area surrounding a port with significant import and export activity, suggesting that A. cantonensis may have been introduced through commercial ships. Specifically, the parasite was detected in a neighbourhood with vulnerable socio-economic conditions and in a nature reserve, which exhibit biotic and abiotic characteristics conducive to sustaining high-density rat populations, scattered waste, areas of spontaneous vegetation, debris accumulation and flooded areas or lagoons offering suitable habitats for intermediate hosts such as snails. Thus, the close proximity of the port to these sites creates a favourable ecological context for the establishment of A. cantonensis. This study shows the need to conduct research to detect A. cantonensis in non-endemic areas but with the characteristics that promote its arrival and development of its life cycle in order to implement control measures to prevent expansion of this parasite and its transmission to humans and other animals.

Nematodes of concern to public and animal health associated with the invasive snail Achatina fulica, in Sergipe, Northeastern Brazil

The giant African land snail Achatina fulica is known to be the intermediate host of a number of nematode species that are prejudicial to human and veterinary health, being also an agricultural and urban pest. The present study investigated the presence of nematodes in A. fulica and other terrestrial mollusks in 24 municipalities of Sergipe State, northeastern of Brazil, in the dry and rainy seasons. In 2019 and 2020, the specimens were collected in standard 20 m × 10 m plots (10 min/01 collector), while in 2021, they were collected by convenience sampling. The nematode species were identified based on sequencing of the mitochondrial Cytochrome C Oxidase Subunit I (COI) gene and the ribosomal nuclear Internal Transcribed Spacer (ITS 2). Specimens of A. fulica infected with Angiostrongylus cantonensis, a nematode that causes eosinophilic meningitis (EM) in humans and animals, were collected in four municipalities. Two nematodes of veterinary importance were also identified, Aelurostrongylus abstrusus and Cruzia tentaculata in seven municipalities. This is the first record of Cruzia tentaculata in Sergipe. Free-living nematodes were also found in 18 municipalities. One of these species – Caenorhabditis briggsae – was identified in the municipality of Itabaiana. Specimens of other mollusks species were also collected. Only one of these mollusks, C. fasciata from Japaratuba, was found infected with free-living nematodes. A. fulica showed greater positivity for nematodes in the dry season, especially in the regions of Lower São Francisco River, Greater Aracaju and South Central Sergipe. The probability of infection increased when mollusks were larger and more recurrent in plots. Given that A. cantonensis is the etiological agent of EM and was found associated to A. fulica in four municipalities, it will be important to implement programs of continuous monitoring of the mollusk's fauna, combined with educational programs that provide information necessary to control A. fulica.

Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Molecular Identification of Kocuria Species Isolated from Pediatric Teaching Hospital in Sulaymaniyah, Iraq

Abstract Background: Kocuria species is now increasingly being recognized as an emerging human pathogen, suggesting that this genus has mostly been neglected or misidentified. Objective: This study attempts to use molecular techniques to correctly identify Kocuria species isolated from inpatient children, and to compare between the identification results obtained from two methods: VITEK 2 and 16S rRNA gene sequencing. Materials and Methods: VITEK 2, an automated system, was used to identify clinical isolates. Isolates identified as Kocuria spp. were submitted to DNA extraction and polymerase chain reaction testing. A partial sequence of the 16S rRNA gene was constructed and compared to GenBank sequences of the 16S rRNA gene. To confirm the strains’ identity, a phylogenetic tree including all Kocuria species was constructed. Results: Three isolates of Kocuria spp. were identified; the first two isolates were confirmed as K. kristinae by VITEK 2, pairwise 16S rRNA sequencing, and phylogenetic tree. However, the third isolate was identified as K. rosea, contradicting the pairwise 16S rRNA sequencing and phylogenetic tree identification as K. turfanensis. Conclusion: It is crucial to utilize molecular techniques for correctly identifying Kocuria species. In this study, it is found that K. turfanensis emerges as a pathogenic species and the first known cause of meningitis in humans and K. kristinae is identified as the first known cause of pediatric urinary tract infections.

Hydrogen Peroxide Is Responsible for the Cytotoxic Effects of Streptococcus pneumoniae on Primary Microglia in the Absence of Pneumolysin

Introduction: Streptococcus pneumoniae is the most common cause of bacterial meningitis and meningoencephalitis in humans. The bacterium produces numerous virulence determinants; among them, hydrogen peroxide (H2O2) and pneumolysin (Ply) contribute to bacterial cytotoxicity. Microglia, the resident phagocytes in the brain, are distinct from other macrophages, and we thus compared their susceptibility to pneumococcal toxicity and their ability to phagocytose pneumococci with those of bone-marrow-derived macrophages (BMDMs). Methods: Microglia and BMDMs were co-incubated with S. pneumoniae D39 to analyze the survival of phagocytes by fluorescence microscopy, bacterial growth by quantitative plating, and phagocytosis by an antibiotic protection assay. Ply was detected by hemolysis assay and Western blot analysis. Results: We found that microglia were killed during pneumococcal infection with a wild-type and an isogenic ply-deficient mutant, whereas the viability of BMDMs was not affected by pneumococci. Treatment with recombinant Ply showed a dose-dependent cytotoxic effect on microglia and BMDMs. However, high concentrations of recombinant Ply were required, and under the chosen experimental conditions, Ply was not detectable in the supernatant during infection of microglia. Inactivation of H2O2 by exogenously added catalase abolished its cytotoxic effect. Consequently, infection of microglia with pneumococci deficient for the pyruvate oxidase SpxB, primarily producing H2O2, resulted in reduced killing of microglia. Conclusion: Taken together, in the absence of Ply, H2O2 caused cell death in primary phagocytes in concentrations produced by pneumococci.

Efficacy of X-Ray Phytosanitary Irradiation on the Infectivity and Reproduction of Angiostrongylus cantonensis in Experimentally Infected Rats.

Angiostrongylus cantonensis is a globally distributed nematode and the leading cause of eosinophilic meningitis in humans. As a global hotspot for this disease, Hawaii's agricultural exports may be contributing to the spread of A. cantonensis. Phytosanitary irradiation doses of 150 or 400 Gy provide quarantine security against multiple insect pests. We evaluated the invitro and invivo effects of phytosanitary irradiation on infectious, third-stage, A. cantonensis larvae. In vitro experiments directly exposed larvae to irradiation doses ranging from 200 to 1,000 Gy. Results showed low mortality and no dose response across all treatments 27 days post-irradiation. In vivo studies isolated larvae from wild-caught Parmarion martensi after exposure to x-ray irradiation at doses of 0, 150, and 400 Gy and infected them into laboratory rats. Fourteen rats were assigned to each treatment and infected with 50 larvae from their assigned irradiation dose. Results at 3 and 6 weeks post-infection demonstrated a significant negative dose response in regard to the number of larvae that migrated to the brain and adults found in the pulmonary artery. No irradiated larvae that grew into adults were able to produce eggs. These findings indicate that x-ray irradiation does not result in the direct mortality of A. cantonensis larvae; however, it does affect the infectivity and reproduction of A. cantonensis within its definitive host, the rat. Phytosanitary irradiation at doses ≥150 Gy appears to be an effective means of preventing the establishment of viable populations of A. cantonensis, thus reducing the potential for global spread due to agricultural exports from Hawaii.

Bacterial and host factors involved in zoonotic Streptococcal meningitis

Zoonotic streptococci cause several invasive diseases with high mortality rates, especially meningitis. Numerous studies elucidated the meningitis pathogenesis of zoonotic streptococci, some specific to certain bacterial species. In contrast, others are shared among different bacterial species, involving colonization and invasion of mucosal barriers, survival in the bloodstream, breaching the blood–brain and/or blood–cerebrospinal fluid barrier to access the central nervous system, and triggering inflammation of the meninges. This review focuses on the recent advancements in comprehending the molecular and cellular events of five major zoonotic streptococci responsible for causing meningitis in humans or animals, including Streptococcus agalactiae, Streptococcus equi subspecies zooepidemicus, Streptococcus suis, Streptococcus dysgalactiae, and Streptococcus iniae. The underlying mechanism was summarized into four themes, including 1) bacterial survival in blood, 2) brain microvascular endothelial cell adhesion and invasion, 3) penetration of the blood–brain barrier, and 4) activation of the immune system and inflammatory reaction within the brain. This review may contribute to developing therapeutics to prevent or mitigate injury of streptococcal meningitis and improve risk stratification.

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

Engagement of AKT and ERK signaling pathways facilitates infection of human neuronal cells with West Nile virus

West Nile virus (WNV) is an important neurotropic virus that accounts for the emergence of human arboviral encephalitis and meningitis. The interaction of WNV with signaling pathways plays a key role in controlling WNV infection. We have investigated the roles of the AKT and ERK pathways in supporting WNV propagation and modulating the inflammatory response following WNV infection. WNV established a productive infection in neuronal cell lines originated from human and mouse. Expression of IL-11 and TNF-α was markedly up-regulated in the infected human neuronal cells, indicating elicitation of inflammation response upon WNV infection. WNV incubation rapidly activated signaling cascades of AKT (AKT-S6-4E-BP1) and ERK (MEK-ERK-p90RSK) pathways. Treatment with AKT inhibitor MK-2206 or MEK inhibitor U0126 abrogated WNV-induced AKT or ERK activation. Strong activation of AKT and ERK signaling pathways could be detectable at 24 h after WNV infection, while such activation was abolished at 48 h post infection. U0126 treatment or knockdown of ERK expression significantly increased WNV RNA levels and viral titers and efficiently decreased IL-11 production induced by WNV, suggesting the involvement of ERK pathway in WNV propagation and IL-11 induction. MK-2206 treatment enhanced WNV RNA replication accompanied with a moderate decrease in IL-11 production. These results demonstrate that engagement of AKT and ERK signaling pathways facilitates viral infection and may be implicated in WNV pathogenesis.

Enterococcus Cecorum bacteremia and meningitis in an asplenic patient: A case report

This paper presents a case of Enterococcus cecorum (E. cecorum) meningitis with bacteremia in a middle-aged woman who had previously undergone a splenectomy. Within 24 hours, the bacterium was identified from a blood culture. The patient was clinically diagnosed with meningitis, which was radiologically verified by an MRI brain scan. Fortunately, she had a favorable outcome. To the best of our knowledge, this is the first reported case of E. cecorum-induced meningitis in humans and the second documented incidence of E. cecorum infection in humans in Australia. The purpose of this case report is to emphasize the potential for serious human infections caused by an extremely rare pathogen.

Antimicrobial Activity of Garlic and Honey on Staphylococcus Aureus and Escherichia Coli

The numerous side effects of conventional antibiotics and increasing antibiotics resistance of some strains of bacteria such as Staphylococcus aureus and Escherichia coli which are commonly implicated in skin infection, surgical site infections, urinary tract infections (UTIs), neonatal meningitis, systemic infections and enteric infections in humans makes it is pertinent to source for alternative remedies. Aim: To assess the antimicrobial activity of honey and garlic on Staphylococcus aureus and Escherichia coli Methodology: Standard microbiological procedures were employed to isolate and identify the bacteria species. Antimicrobial activity was determined using the agar well diffusion method. Result: The S. aureus and E. coli were susceptible to the honey and garlic extracts. S. aureus was susceptible to garlic, honey and the mixture of both with zones of inhibition ranging from 6.9-17.2mm, 4.8- 12.9mm and 6.0-17.0mm respectively while those of E. coli ranged from 5.8-16.4mm, 5.0-11.8mm and 5.6-16.0 respectively. There was significant difference (p ≤ 0.05) in the antimicrobial activity of the garlic, honey and the mixture. Conclusion: This study therefore reveals that honey and garlic possess significant antimicrobial activity against the tested organisms with high antibacterial activity occurring at high concentrations. Honey and garlic can therefore, serve as substitute to antibiotics

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance.

Streptococcus suis serotype 2 (SS2) is a Gram-positive bacterium. It is a common and significant pathogen in pigs and a common cause of zoonotic meningitis in humans. It can lead to sepsis, endocarditis, arthritis, and pneumonia. If not diagnosed and treated promptly, it has a high mortality rate. The pan-genome of SS2 is open, and with an increasing number of genes, the core genome and accessory genome may exhibit more pronounced differences. Due to the diversity of SS2, the genes related to its virulence and resistance are still unclear. In this study, a strain of SS2 was isolated from a pig farm in Sichuan Province, China, and subjected to whole-genome sequencing and characterization. Subsequently, we conducted a Pan-Genome-Wide Association Study (Pan-GWAS) on 230 strains of SS2. Our analysis indicates that the core genome is composed of 1,458 genes related to the basic life processes of the bacterium. The accessory genome, consisting of 4,337 genes, is highly variable and a major contributor to the genetic diversity of SS2. Furthermore, we identified important virulence and resistance genes in SS2 through pan-GWAS. The virulence genes of SS2 are mainly associated with bacterial adhesion. In addition, resistance genes in the core genome may confer natural resistance of SS2 to fluoroquinolone and glycopeptide antibiotics. This study lays the foundation for further research on the virulence and resistance of SS2, providing potential new drug and vaccine targets against SS2.

Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis

BackgroundRibosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear.ResultsHere, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis.ConclusionsTogether, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces’ RPSA.

Comparative Genome Analysis of Two Streptococcus suis Serotype 8 Strains Identifies Two New Virulence-Associated Genes.

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and septicemia in swine and humans. Among numerous pathogenic serotypes, S. suis serotype 8 has distinctive characteristics such as a high detection rate and causing multi-host infection. There is no complete genome of serotype 8 strains so far. In this study, the complete genome of two S. suis serotype 8 strains, virulent strain 2018WUSS151 and non-virulent strain WUSS030, were sequenced. Comparative genomic analysis showed that the homology of the two genomes reaches 99.68%, and the main difference is the distinctive prophages. There are 83 genes unique to virulent strain 2018WUSS151, including three putative virulence-associated genes (PVGs). Two PVGs, padR and marR, are passenger genes in ISSsu2 family transposons that are able to form circular DNA intermediates during transposition, indicating the possibility of horizontal transmission among S. suis strains. The deletion mutant of PVGs marR or atpase attenuated the virulence of serotype 2 virulent SC070731 in a mouse infection model, confirming their role in S. suis virulence. These findings contribute to clarifying the genomic characterization of S. suis serotype 8 and S. suis pathogenesis.

Characterization of pig tonsils as niches for the generation of Streptococcus suis diversity

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.

In silico discovery of diagnostic/vaccine candidate antigenic epitopes and a multi-epitope peptide vaccine (NaeVac) design for the brain-eating amoeba Naegleria fowleri causing human meningitis

Naegleria fowleri, the brain-eating amoeba, is a free-living amoeboflagellate with three different life cycles (trophozoite, flagellated, and cyst) that lives in a variety of habitats around the world including warm freshwater and soil. It causes a disease called naegleriasis leading meningitis and primary amoebic meningoencephalitis (PAM) in humans. N. fowleri is transmitted through contaminated water sources such as insufficiently chlorinated swimming pool water or contaminated tap water, and swimmers are at risk. N. fowleri is found all over the world, and most infections were reported in both developed and developing countries with high mortality rates and serious clinical findings. Until now, there is no FDA approved vaccine and early diagnosis is urgent against this pathogen. In this study, by analyzing the N. fowleri vaccine candidate proteins (Mp2CL5, Nfa1, Nf314, proNP-A and proNP-B), it was aimed to discover diagnostic/vaccine candidate epitopes and to design a multi-epitope peptide vaccine against this pathogen. After the in silico evaluation, three prominent diagnostic/vaccine candidate epitopes (EAKDSK, LLPHIRILVY, and FYAKLLPHIRILVYS) with the highest antigenicities were discovered and a potentially highly immunogenic/antigenic multi-epitope peptide vaccine (NaeVac) was designed against the brain-eating amoeba N. fowleri causing human meningitis.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Molecular study of the status of Angiostrongylus cantonensis in rats in Haiti

Angiostrongylus cantonensis, commonly known as the rat lungworm, causes Eosinophilic meningitis in humans. Our study aimed to investigate the prevalence and distribution of this parasite in rats in Haiti. Rats were trapped at 8 sites, 7 in Artibonite (rural region) and one in an urban area of Port-au-Prince. After euthanasia, hearts and lungs were sampled and preserved in 70% ethanol. Subsequently, the organs were dissected to detect adult worms. Parasite DNA was amplified using PCR targeting either the nematode ITS2 gene for rodent lung tissue or cox1 for isolated worms. Subsequent sequencing allowed parasite identification. A total of 70 rats were captured, i.e. 23 Rattus norvegicus and 47 Rattus rattus. Adult nematodes morphologically compatible with A. cantonensis were isolated from 5/70 rats (7%) and identification was confirmed by sequencing. Molecular analysis of lung tissue revealed a parasite prevalence of 31.4% (22/70), and its presence at 4 of the 8 sites investigated, including Port-au-Prince. The molecular approach on lung tissue targeting the ITS2 gene enabled us to detect a prevalence 4 times higher than the visual search for adult worms alone. Only one COX1 haplotype was identified, belonging to genotype II-G, widely distributed in Brazil, the French Antilles (Guadeloupe), French Polynesia, Hawaii, and Japan. These results confirm that A. cantonensis is an endemic parasite in Haiti not only in the capital Port-au-Prince, but also in several rural areas. Direct molecular screening for Angiostrongylus DNA in rat lung tissue showed higher sensitivity than visual detection of worms during dissection and could be useful for further prevalence studies.