Mixed Species Infections Research Articles

Non-random distribution of Plasmodium Species infections and associated clinical features in children in the lake Victoria region, Kenya, 2012–2018

BackgroundWhile Plasmodium falciparum (Pf) stands out as the most lethal malaria parasite species in humans, the impact of other species should not be dismissed. Moreover, there is a notable lack of understanding of mixed-species infections and their clinical implications.MethodsWe conducted eight school-based cross-sectional malariometric surveys in the Lake Victoria region of western Kenya between January–February 2012 and September–October 2018. In each survey, a minimum of 100 children aged 3 to 15 years were randomly chosen from a school in Ungoye village on the mainland and as well as from each school selected in every catchment area on Mfangano island. Plasmodium infection was determined by microscopy and nested polymerase chain reaction (PCR). The multiple-kind lottery (MKL) model calculated the expected distribution of Plasmodium infections in the population and compared it to observed values using a chi-squared test (χ2).ResultsThe Plasmodium prevalence was 25.9% (2521/9724) by microscopy and 51.1% (4969/9724) by PCR. Among all infections detected by PCR, Pf, P. malariae (Pm), and P. ovale (Po) mono-infections were 58.6%, 3.1%, and 1.8%, respectively. Pf/Pm, Pf/Po, Pm/Po, and Pf/Pm/Po co-infections were 23.5%, 4.3%, 0.1%, and 8.6%, respectively. MKL modelling revealed non-random distributions, with frequencies of Pf/Pm and Pf/Pm/Po co-infections being significantly higher than expected (χ2 = 3385.60, p < 0.001). Pf co-infections with Pm and Po were associated with a decreased risk of fever (aOR 0.64, 95% CI 0.46–0.83; p = 0.01) and increased risks of splenomegaly (aOR 12.79, 95% CI 9.69–16.9; p < 0.001) and anaemia (aOR 2.57, 95% CI 2.09–3.15; p < 0.001), compared to single-species infections.ConclusionThis study sheds light on the potential interaction between Pf and Pm and/or Po. Given the clinical significance of mixed-species infections, improved diagnostics, and case management of Pm and Po are urgently needed.

Massive branchial henneguyosis of catfish: A distinct, myxozoan-induced gill disease caused by severe interlamellar Henneguya exilis infection in catfish aquaculture.

Proliferative gill disease (PGD), caused by the myxozoan Henneguya ictaluri, has been the most notorious parasitic gill disease in the US catfish aquaculture industry. In 2019, an unusual gill disease caused by massive burdens of another myxozoan, Henneguya exilis, was described in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish. Targeted metagenomic sequencing and in situ hybridization (ISH) were used to differentiate these conditions by comparing myxozoan communities involved in lesion development and disease pathogenesis between massive H. exilis infections and PGD cases. Thirty ethanol-fixed gill holobranchs from 7 cases of massive H. exilis infection in hybrid catfish were subjected to targeted amplicon sequencing of the 18S rRNA gene and compared to a targeted metagenomic data set previously generated from clinical PGD case submissions. Furthermore, serial sections of 14 formalin-fixed gill holobranchs (2 per case) were analyzed by RNAscope duplex chromogenic ISH assays targeting 8 different myxozoan species. Targeted metagenomic and ISH data were concordant, indicating myxozoan community compositions significantly differ between PGD and massive branchial henneguyosis. Although PGD cases often consist of mixed species infections, massive branchial henneguyosis consisted of nearly pure H. exilis infections. Still, H. ictaluri was identified by ISH in association with infrequent PGD lesions, suggesting coinfections occur, and some cases of massive branchial henneguyosis may contain concurrent PGD lesions contributing to morbidity. These findings establish a case definition for a putative emerging, myxozoan-induced gill disease of farm-raised catfish with a proposed condition name of massive branchial henneguyosis of catfish (MBHC).

Cutaneous-Urinary Schistosomiasis in a Child: A Case Report at the Dermatology Hospital of Bamako

Schistosomiasis is a parasitic disease of human origin caused by flatworms. This condition affects millions of people in developing countries where it is endemic. In 2017, the WHO classified schistosomiasis as a neglected tropical disease (NTD), deserving particular attention due to its potential complications, including infertility. Several initiatives have been undertaken to eradicate this condition, among which regular administration of praziquantel (PZQ), an anthelminthic medication, to school-age children (and other high-risk groups) is the cornerstone of schistosomiasis control. Although the efficacy of PZQ against infections of a single schistosome species is well known, its efficacy against mixed-species infections is less understood, as are the reinfection patterns after treatment. We report a case in a child in Bamako. Patient was a 9-year-old student who presented with pruritic solid lesions on the abdomen that had been continuously evolving, gradually increasing in number and size. The child used water from the marigot for group amusements. Since schistosomes can survive for decades in the host’s bloodstream, they are clearly capable of evading host immune responses, and their ability depends on the properties of the tegument surface. This hypothesis is plausible in our child due to his previous baths in the marigot. This highlights the need for proper management of stagnant water in endemic countries. This observation serves as a warning to clinicians that any hematuria warrants a systematic dermatological examination to search for skin lesions.

A digital microscope for the diagnosis of Plasmodium falciparum and Plasmodium vivax, including P. falciparum with hrp2/hrp3 deletion.

Sensitive and accurate malaria diagnosis is required for case management to accelerate control efforts. Diagnosis is particularly challenging where multiple Plasmodium species are endemic, and where P. falciparum hrp2/3 deletions are frequent. The Noul miLab is a fully automated portable digital microscope that prepares a blood film from a droplet of blood, followed by staining and detection of parasites by an algorithm. Infected red blood cells are displayed on the screen of the instrument. Time-to-result is approximately 20 minutes, with less than two minutes hands-on time. We evaluated the miLab among 659 suspected malaria patients in Gondar, Ethiopia, where P. falciparum and P. vivax are endemic, and the frequency of hrp2/3 deletions is high, and 991 patients in Ghana, where P. falciparum transmission is intense. Across both countries combined, the sensitivity of the miLab for P. falciparum was 94.3% at densities >200 parasites/μL by qPCR, and 83% at densities >20 parasites/μL. The miLab was more sensitive than local microscopy, and comparable to RDT. In Ethiopia, the miLab diagnosed 51/52 (98.1%) of P. falciparum infections with hrp2 deletion at densities >20 parasites/μL. Specificity of the miLab was 94.0%. For P. vivax diagnosis in Ethiopia, the sensitivity of the miLab was 97.0% at densities >200 parasites/μL (RDT: 76.8%, microscopy: 67.0%), 93.9% at densities >20 parasites/μL, and specificity was 97.6%. In Ethiopia, where P. falciparum and P. vivax were frequent, the miLab assigned the wrong species to 15/195 mono-infections at densities >20 parasites/μL by qPCR, and identified only 5/18 mixed-species infections correctly. In conclusion, the miLab was more sensitive than microscopy and thus is a valuable addition to the toolkit for malaria diagnosis, particularly for areas with high frequencies of hrp2/3 deletions.

Glycyrrhiza glabra hydroethanolic extract and manuka honey alone and in combination inhibit bacterial and fungal planktonic cells and biofilms

BackgroundThe natural tendency of polymicrobial infections and co-habitation of bacteria and Candida are currently well-recognized and reported as highly fatal. This kind of interaction is usually associated with drug tolerance, infection relapse, persistence and high mortality. The interest in natural products has emerged and their potential applicability as therapeutic strategies is being constantly explored. PurposeThe main goal of this work is to evaluate the effect of the hydroethanolic extract of Glycyrrhiza glabra, and Manuka honey, in Staphylococcus aureus and Candida tropicalis planktonic cells and single and mixed biofilms. MethodsThe effect of both the G. glabra extract and the honey was evaluated by determination of the minimum inhibitory/bactericidal/fungicidal concentrations and by determination of their antibiofilm activity (alone and in combination) on single and mixed-species biofilms. In addition, phenolic compounds were characterized in the hydroethanolic extract with high performance liquid chromatography and mass spectrometry. ResultsAmong the phenolic compounds identified in the G. glabra extract, flavonoids were the most abundant (≈75 % of the total phenolic compounds), with liquiritigenin being the most representative. S. aureus and C. tropicalis were susceptible to both antimicrobials acting alone. When tested in combination, a significative synergism was observed against C. tropicalis single biofilm formation. Instead, the combination showed an additive effect against mixed biofilms of S. aureus and C. tropicalis. In general the combination G. glabra+honey showed a broader antibiofilm effect than each partner alone. ConclusionThe results showed the good anti S. aureus and C. tropicalis effect of G. glabra and manuka honey against both pathogens tested and highlighted their potential use in combination as therapeutic strategy to combat S. aureus, C. tropicalis and mixed species infections particularly when growing as biofilms.

Epidemiological investigation of coccidiosis and associated risk factors in broiler chickens immunized with live anticoccidial vaccines in China.

Coccidiosis is a costly intestinal disease of chickens caused by Eimeria species. This infection is associated with high mortality, reduced feed efficiency, and slowed body weight gain. The diagnosis and control of coccidiosis becomes challenging due to the fact that chickens can be infected by seven different Eimeria species and often occur mixed-species co-infections. Grasping the epidemiology of Eimeria species is crucial to estimate the efficiency of poultry management. This study aimed to explore the distribution of Eimeria species in broiler chickens in China after administering live anticoccidial vaccines. A total of 634 samples were obtained, and the survey results showed that the prevalence of Eimeria was 86.12% (546/634), and the most common species were E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), E. tenella (48.42%), and E. praecox (41.80%). Most samples indicated mixed-species infections (an average of 3.29 species per positive sample). Notably, 63.98% of samples contain 3 to 5 Eimeria species within a single fecal sample. The most prevalent combinations were E. acervulina-E. tenella (38.96%) and E. acervulina-E. necatrix (37.22%). Statistical analysis showed that flocks vaccinated with trivalent vaccines were significantly positive for E. necatrix in grower chickens (OR = 3.30, p < 0.05) compared with starter chickens, and tetravalent vaccinated flocks showed that starter chickens demonstrated a higher susceptibility to E. tenella-E. brunetti (OR = 2.03, p < 0.05) and E. acervulina-E. maxima (OR = 2.05, p < 0.05) compared with adult chickens. Geographically, in the case of tetravalent vaccine-immunized flocks, a substantial positive association was observed between E. necatrix infection rates and flocks from eastern (OR = 3.88, p < 0.001), central (OR = 2.65, p = 0.001), and southern China (OR = 3.17, p < 0.001) compared with southwestern China. This study also found a positive association between E. necatrix (OR = 1.64, p < 0.05), E. acervulina (OR = 1.59, p < 0.05), and E. praecox (OR = 1.81, p < 0.05) infection and coccidiosis occurrence compared with non-infected flocks in tetravalent vaccinated flocks. This molecular epidemiological investigation showed a high prevalence of Eimeria species in the field. The emergent species, E. brunetti and E. praecox, might be incorporated into the widely-used live vaccines in the future. These insights could be useful in refining coccidiosis control strategies in the poultry industry.

Dynamics of asymptomatic Plasmodium falciparum and Plasmodium vivax infections and their infectiousness to mosquitoes in a low transmission setting of Ethiopia: a longitudinal observational study

ObjectiveA 15-month longitudinal study was conducted to determine the duration and infectivity of asymptomatic qPCR-detected Plasmodium falciparum and Plasmodium vivax infections in Ethiopia. MethodTotal parasite and gametocyte kinetics were determined by molecular methods; infectivity to Anopheles arabiensis mosquitoes by repeated membrane feeding assays. Infectivity results were contrasted with passively recruited symptomatic malaria cases. ResultsFor P. falciparum and P. vivax infections detected at enrolment, median durations of infection were 37 days (95% confidence interval [CI], 15-93) and 60 days (95% CI, 18-213), respectively. P. falciparum and P. vivax parasite densities declined over the course of infections. From 47 feeding assays on 22 asymptomatic P. falciparum infections, 6.4% (3/47) were infectious and these infected 1.8% (29/1579) of mosquitoes. No transmission was observed in feeding assays on asymptomatic P. vivax mono-infections (0/56); one mixed-species infection was highly infectious. Among the symptomatic cases, 4.3% (2/47) of P. falciparum and 73.3% (53/86) of P. vivax patients were infectious to mosquitoes. ConclusionThe majority of asymptomatic infections were of short duration and low parasite density. Only a minority of asymptomatic individuals were infectious to mosquitoes. This contrasts with earlier findings and is plausibly due to the low parasite densities in this population.

Seasonal pattern of questing ticks and prevalence of pathogenic Rickettsia and Anaplasmataceae in Khao Yai national park, Thailand

BackgroundTick-borne diseases (TBD) are considered neglected diseases in Thailand with disease burden likely underestimated. To assess risk for emerging TBD in Thailand, the seasonality of questing tick and pathogen prevalence were studied in Khao Yai National Park, a top tourist destination. MethodsDuring 2019, questing ticks around tourist attractions were systematically collected bimonthly and analyzed for Rickettsia and Anaplasmataceae bacterial species by polymerase chain reaction and DNA sequencing. ResultsLarvae and nymphs of questing ticks peaked in Khao Yai National Park during the late rainy-winter season, though no specific trends were observed in adult ticks. Winter (November to February) was the highest risk for human tick-bites due to higher numbers of both ticks and visitors. Of the total 5916 ticks analyzed (651 pools), Anaplasma phagocytophilum, Neoehrlichia mikurensis, Ehrlichia ewingii, and Ehrlichia chaffeensis were detected at low rates (≤0.05%). There was a higher prevalence of human rickettsioses (0.2–7%) in ticks surveyed with Rickettsia tamurae, Rickettsia raoultii, and Rickettsia montana the major species. Amblyomma ticks had the highest prevalence of Rickettsia (85%, 35/44 Amblyomma adults), in which only R. tamurae and R. raoultii were found in Amblyomma with mixed species infections common. We report the first detection of R. africae-like and N. mikurensis in Ixodes granulatus adults in Thailand, suggesting I. granulatus as a potential vector for these pathogens. ConclusionThis study demonstrated the risk of emerging TBD in Thailand and underscores the need for tick-bite prevention among tourists in Thailand.

A Taq-Man-based multiplex quantitative PCR for the simultaneous detection and quantification of Angiostrongylus vasorum, Crenosoma vulpis, and species of respiratory capillarids in canids

In recent years, Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi), commonly referred to as canine lungworms, have gained a growing interest worldwide as the result of their geographical expansion. Each of these nematode species differs considerably in its biology and pathogenicity. Despite their impact on dogs’ health, these parasites are often underdiagnosed owing to diagnostic challenges. Here, we describe the development and validation of a Taq-Man-based multiplex quantitative PCR (qPCR) for the simultaneous detection of the main species of canine lungworms in faeces of infected dogs. Using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each lungworm species, the analytical sensitivity of the assay ascertained was 1.84 ng/μl for A. vasorum, 3.08 ng/μl for C. vulpis and 0.79 ng/μl for Eucoleus spp. The sensitivity of the assays and their ability to detect mixed species infections were compared with microscopy-based techniques (faecal floatation and Baermann technique) applied to faecal samples submitted for lungworm testing through an accredited diagnostic laboratory at the Institute of Parasitology, University of Zurich, Switzerland, and from community dogs as part of a research project on canine endoparasites in Cambodia. The multiplex qPCR displayed high diagnostic sensitivity (42/46, 91.3%; 95% Confidence Interval (CI): 79.1–97.1%) and a diagnostic specificity of 100% (45/45, 95% CI: 90.6–100%), and was able to detect 42.9% additional mixed lungworm species infections compared with microscopy-based methods. Kappa statistics showed substantial agreement between the qPCRs and microscopy for mixed infections (κ = 0.72, 95% CI: 0.4–1) and Eucoleus spp. (κ = 0.65, 95% CI: 0.45–0.85) and almost perfect agreement for C. vulpis (κ = 0.85, 95% CI: 0.63–1) and A. vasorum (κ = 0.92, 95% CI: 0.84–1).This multiplex qPCR enables timely, accurate, and sensitive diagnosis of canine lungworm species in faecal samples and can be used to monitor the geographical distribution and emergence of these parasitic species, globally.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy and beef cattle in Shanxi, China.

Cryptosporidium spp. are key gastrointestinal protists in humans and animals worldwide. Infected cattle are considered the main source of cryptosporidiosis outbreaks in humans. However, little is known about the genetic makeup of Cryptosporidium populations in Shanxi province, China. We analyzed 858 fecal samples collected from farms in Shanxi. The presence of Cryptosporidium spp. was determined via polymerase chain reaction and subsequent sequence analysis of the small subunit rRNA gene as well as restriction fragment length polymorphism analysis. Cryptosporidium parvum was subtyped following sequence analysis of the 60 kDa glycoprotein gene (gp60). The overall prevalence of Cryptosporidium in cattle was 11.19%, with a prevalence of 13.30% and 8.67% in Lingqiu and Yingxian, respectively. The overall prevalence of Cryptosporidium in dairy and beef cattle was 10.78% and 11.50%, respectively. Cryptosporidium infection was detected across all analyzed age groups. The overall prevalence of Cryptosporidium in diarrhea and nondiarrhea samples was 18.24% and 9.72%, respectively, whereas that in intensively farmed and free-range cattle was 17.40% and 3.41%, respectively. We identified five Cryptosporidium species, with C. andersoni being the dominant species. Further, two cases of mixed infections of Cryptosporidium species were detected. All identified C. parvum isolates belonged to the subtype IIdA17G1.

Species-specific in situ hybridization confirms arrested development of Henneguya ictaluri in hybrid catfish (Channel Catfish × Blue Catfish) under experimental conditions, with notes on mixed-species infections in clinical cases of proliferative gill disease from Mississippi catfish aquaculture.

Proliferative gill disease (PGD) in Channel Catfish Ictalurus punctatus and hybrid catfish (Channel Catfish × Blue Catfish I. furcatus) is attributed to the myxozoan Henneguya ictaluri. Despite evidence of decreased H. ictaluri transmission and impaired parasite development in hybrid catfish, PGD still occurs in hybrid production systems. Previous metagenomic assessments of clinical PGD cases revealed numerous myxozoans within affected gill tissues in addition to H. ictaluri. The objective of this study was to investigate the development and pathologic contributions of H. ictaluri and other myxozoans in naturally and experimentally induced PGD. Henneguya species-specific in situ hybridization (ISH) assays were developed using RNAscope technology. Natural infections were sourced from diagnostic case submissions in 2019. Experimental challenges involved Channel Catfish and hybrid catfish exposed to pond water from an active PGD outbreak, and the fish were sampled at 1, 7, 10, 12, 14, 16, 18, and 20 weeks postchallenge. Nine unique ISH probes were designed, targeting a diagnostic variable region of the 18S ribosomal RNA gene of select myxozoan taxa identified in clinical PGD cases. Partial validation from pure H. ictaluri, H. adiposa, H. postexilis, and H. exilis infections illustrated species-specific labeling and no cross-reactivity between different myxozoan species or the catfish hosts. After experimental challenge, mature plasmodia of H. ictaluri and H. postexilis formed in Channel Catfish but were not observed in hybrids, suggesting impaired or delayed sporogenesis in the hybridized host. These investigations also confirmed the presence of mixed infections in clinical PGD cases. Although H. ictaluri appears to be the primary cause of PGD, presporogonic stages of other myxozoans were also present, which may contribute to disease pathology and exacerbate respiratory compromise by further altering normal gill morphology. This work provides molecular confirmation and more resolute developmental timelines of H. ictaluri and H. postexilis in Channel Catfish and supports previous research indicating impaired or precluded H. ictaluri sporogony in hybrid catfish.

A novel sensitive hexaplex high-resolution melt assay for identification of five human Plasmodium species plus internal control

BackgroundThe diagnosis of malaria infection in humans remains challenging, further complicated by mixed Plasmodium species infections, potentially altering disease severity and morbidity. To facilitate appropriate control measures and treatment, rapid, sensitive, and specific detection assays, including those for the second minor species, would be required. This study aimed to develop a multiplex high-resolution melting (hexaplex PCR-HRM) assay with seven distinct peaks corresponding to five Plasmodium species of the Plasmodium genus, and an internal control to limit false negatives providing quality assurance testing results. MethodsFive species-specific primers for human malaria species were designed targeting on the Plasmodium 18 small subunit ribosomal RNA (18S rRNA) and mitochondrial genes. The hexaplex PCR-HRM was developed for the simultaneous and rapid detection and differentiation of five human Plasmodium spp. The limit of detection (LoD), sensitivity, and specificity of the assay were evaluated. Artificial mixing was used to assess the ability to determine the second minor species. Furthermore, a hexaplex PCR-HRM assay was used to identify 120 Plasmodium-infected clinical isolates from Kanchanaburi, Western Thailand, where malaria is endemic. ResultsThe hexaplex PCR-HRM assay detected the targeted genome of five Plasmodium species at levels as low as 2.354–3.316 copies/uL with 91.76 % sensitivity and 98.04 % specificity. In artificial mixing, the assay could detect minority parasite species at 0.001 % of the predominant parasite population. Plasmodium vivax infections (99 %) accounted for the majority of malaria cases in Kanchanaburi, Thailand. ConclusionsThe developed hexaplex PCR-HRM assay we present in this study is a novel approach for multiplexing the Plasmodium genus and detecting five Plasmodium species with the advantage of detecting second minority parasite species. The developed one-step assay without any nesting protocols would reduce the risks of cross-contamination. Moreover, it also provides a simple, sensitive, specific, and low-cost approach for optional molecular detection of malaria.

Two cases of urinary schistosomiasis with unusual egg presentations: Dra 1 repeat sequence not detected

Introduction: Schistosoma haematobium is the primary cause of urinary schistosomiasis in man. It is rare to find other human schistosome species in urine because they are located in the intestines, or those of animal origin. Mixed infections of human and animal species of schistosomes may occur in cattle breeding areas like Nigeria. Case Report: During a prevalence study on urinary schistosomiasis, two teenage boys from different local government areas (LGAs) of Kaduna State, Nigeria had mixed urinary Schistosoma infections. Their urine samples were centrifuged at 3000 rpm (revolutions per minute) for 5 minutes. Microscopic examination of the urine sediments revealed highly polymorphic eggs (or morphotypes). After subjecting the genomic DNA for detection of S. haematobium Dra 1 tandem repeat sequence by polymerase chain reaction (PCR), it was not amplified. However, there was amplification in a classical urinary schistosomiasis caused by S. haematobium (which served as positive control). Conclusion: Unusual egg presentations in urinary schistosomiasis may present a dilemma in making diagnostic conclusion. Hence, these two cases suggest the possibility of human–animal Schistosoma hybrids circulating in the area, especially S. haematobium–S. bovis hybrids.

First report of Fusarium equiseti causing root and crown rot in tomato in Mexico.

Tomato (Solanum lycopersicum L.) is one of the most important crops in Mexico due to its economic and nutritional value. Among the main diseases in tomato production is Fusarium wilt, which can cause 60% production losses (Ascencio et al, 2008). Mixed infections of Fusarium species or other fungi genera, would increase disease severity. During April to May of 2021, tomato plants with more than 60 days old, were collected from the main production areas of Aguascalientes (22°03'46.5"N 102°05'17.4"W and 22°04'53.64"N 101°58'55.81"W) and Zacatecas (23°05'59.2"N 102°41'07.3"W and 22°16'52.1"N 102°00'11.8"W) Mexico states. Plants showed main root rot, vascular bundles necrosis with corky appearance, stem crown rot, and ascending yellowing. The main root and stem crown were cut in 0.25 cm2 pieces and disinfested in 2% NaClO for one minute, rinsed with distilled water two times, placed on acidified potato dextrose agar (PDA) medium, and incubated at 25 ± 2°C for 7 days. Characteristic Fusarium growths were purified by hyphal tip on PDA, subsequently pure strains were obtained by single-spore isolation method. Several fungi colonies were obtained, but we focused on the colonies that showed abundant aerial mycelium of white color and irregular growth, which turned yellowish to golden and brown color as it ages. Carnation leaf agar (CLA) medium were used for conidia and sporodochium development. Chains of terminal, intercalary and agglomerated chlamydospores with thick, rough brown walls of 18.9 (7.46) µm in diameter (n=120) were observed in the mycelium. Macroconidia with 5 to 7 septa were 30 to 75 (28.32) µm in long and 1.2 to 4.8 (3.2) µm in wide (n=72). Basal cell developed in foot-shape, apical cell was elongated and slightly curved, and some macroconidia had swollen midd-cell. Sporodochium was orange to brown in color and microconidia were absent (Figure 1). Two representative strains from each state, LCA-3.1 and EMA-1 from Aguascalientes and ECZ-4 and LRZ-6 from Zacatecas, were selected for DNA amplification of ITS, TEF-1α and RPB2 regions, with universal primers ITS1/ITS4, EF1/EF2 and 2-5F2/7cR (White et al.1990; O'Donnell et al. 1998, 2013). PCR products were sequenced by Psomagen, Inc. (USA). The sequences obtained showed 100% of similarity among themselves and within species of the Fusarium incarnatum-equiseti species complex (FIESC) with nucleotide NCBI accessions NR_121457 (Type material) for ITS and MW362069 for TEF-1α; and 99.28% with MN170399 for RPB2 in FUSARIOID-ID database. According to morphological (Leslie and Summerell, 2006) and molecular characteristics, isolates were identified as Fusarium equiseti (FIESC 14). The LCA-3.1 sequences were selected to be deposited in GenBank with accession numbers OM812801 (ITS), OM937108 (TEF-1α) and ON653596 (RPB2). Pathogenicity tests were performed twice, under greenhouse conditions in tomato seedlings of cv. Rio Grande. Five tomato seedlings were inoculated by root immersion method (Lopez et al, 2018) in a 1x106 spores/mL solution for 8 min, and transplanted to 1L pots with sterile peat. Five controls plants were immersed in sterile water. At 14 days after inoculation, a general plant decline and slower growth compared to the control plants were observed. Subsequently, plants showed root rot, vascular necrosis, and a brown ring in stem crown. Controls were symptomless. The fungi were re-isolated from symptomatic plants and were morphologically similar to the inoculated strains. Patel et al. (2017) described the pathogenic and toxic effects of F. equiseti on tomato, causing low seed germination, and low root and shoot growth. This is the first report of F. equiseti causing root and stem rot in tomato plants in Mexico.

Therapeutic response to artemisinin combination therapies among individuals with Plasmodium falciparum single infection vs mixed Plasmodium species infections: a retrospective posthoc analysis in Kisumu County, western Kenya

ObjectivesThis study examined the treatment response of mixed vs single-species Plasmodium falciparum infections to artemisinin-based combination therapies (ACTs). MethodsA total of 1211 blood samples collected on days 0, 7, 14, 21, 28, 35, and 42 from 173 individuals enrolled in two randomized ACT efficacy studies were tested for malaria using 18s ribosomal RNA-based real-time polymerase chain reaction. All recurrent parasitemia were characterized for Plasmodium species composition and time to reinfection during 42-day follow-up compared across ACTs. ResultsDay 0 samples had 71.1% (116/163) single P. falciparum infections and 28.2% (46/163) coinfections. A total of 54.0% (88/163) of individuals tested positive for Plasmodium at least once between days 7-42. A total of 19.3% (17/88) of individuals with recurrent infections were infected with a different Plasmodium species than observed at day 0, with 76.5% (13/17) of these “hidden” infections appearing after clearing P. falciparum present at day 0. Artesunate-mefloquine (16.4 hours) and dihydroartemisinin-piperaquine (17.6 hours) had increased clearance rates over artemether-lumefantrine (21.0 hours). Dihydroartemisinin-piperaquine exhibited the longest duration of reinfection prophylaxis. Cure rates were comparable across each species composition. ConclusionNo differences in clearance rates were found depending on whether the infection contained species other than P. falciparum. Significantly longer durations of protection were observed for individuals treated with dihydroartemisinin-piperaquine.

In Vivo Activity of Hydrogen-Peroxide Generating Electrochemical Bandage Against Murine Wound Infections.

Biofilms formed by antibiotic-resistant bacteria in wound beds present unique challenges in terms of treating wound infections. In this work, the in vivo activity of a novel electrochemical bandage (e-bandage) composed of carbon fabric and controlled by a wearable potentiostat, designed to continuously deliver low amounts of hydrogen peroxide (H2O2) was evaluated against methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MDR-PA) and mixed-species (MRSA and MDR-PA) wound infections. Wounds created on Swiss Webster mice were infected with the above-named bacteria and biofilms allowed to establish on wound beds for 3 days. e-Bandages, which electrochemically reduce dissolved oxygen to H2O2 when polarized at -0.6 VAg/AgCl, were placed atop the infected wound bed and polarized continuously for 48 hours. Polarized e-bandage treatment resulted in significant reductions (p <0.001) of both mono-species and mixed-species wound infections. After e-bandage treatment, electron microscopy showed degradation of bacterial cells, and histopathology showed no obvious alteration to the inflammatory host response. Blood biochemistries showed no abnormalities. Taken all together, results of this work suggest that the described H2O2-producing e-bandage can effectively reduce in vivo MRSA, MDR-PA and mixed-species wound biofilms, and should be further developed as a potential antibiotic-free strategy for treatment of wound infections.

Malaria parasite density and detailed qualitative microscopy enhances large-scale profiling of infection endemicity in Nigeria

With global progress towards malaria reduction stalling, further analysis of epidemiology is required, particularly in countries with the highest burden. National surveys have mostly analysed infection prevalence, while large-scale data on parasite density and different developmental forms rarely available. In Nigeria, the country with the largest burden globally, blood slide microscopy of children up to 5 years of age was conducted in the 2018 National Demographic and Health Survey, and parasite prevalence previously reported. In the current study, malaria parasite density measurements are reported and analysed for 7783 of the children sampled across the 36 states within the six geopolitical zones of the country. Asexual and sexual stages, and infections with different malaria parasite species are analysed. Across all states of Nigeria, there was a positive correlation between mean asexual parasite density within infected individuals and prevalence of infection in the community (Spearman’s rho = 0.39, P = 0.02). Asexual parasite densities were highest in the northern geopolitical zones (geometric means > 2000 μL−1), extending the evidence of exceptionally high infection burden in many areas. Sexual parasite prevalence in each state was highly correlated with asexual parasite prevalence (Spearman’s rho = 0.70, P < 0.001), although sexual parasite densities were low (geometric means < 100 μL−1 in all zones). Infants had lower parasite densities than children above 1 year of age, but there were no differences between male and female children. Most infections were of P. falciparum, which had higher asexual densities but lower sexual parasite densities than P. malariae or P. ovale mono-infections. However, mixed species infections had the highest asexual parasite densities. It is recommended that future large surveys in high burden countries measure parasite densities as well as developmental stages and species, to improve the quality of malaria epidemiology and tracking of future changes.

Enterococcus faecalis suppresses Staphylococcus aureus-induced NETosis and promotes bacterial survival in polymicrobial infections.

Enterococcus faecalis is an opportunistic pathogen that is frequently co-isolated with other microbes in wound infections. While E. faecalis can subvert the host immune response and promote the survival of other microbes via interbacterial synergy, little is known about the impact of E. faecalis-mediated immune suppression on co-infecting microbes. We hypothesized that E. faecalis can attenuate neutrophil-mediated responses in mixed-species infection to promote survival of the co-infecting species. We found that neutrophils control E. faecalis infection via phagocytosis, ROS production, and degranulation of azurophilic granules, but it does not trigger neutrophil extracellular trap formation (NETosis). However, E. faecalis attenuates Staphylococcus aureus-induced NETosis in polymicrobial infection by interfering with citrullination of histone, suggesting E. faecalis can actively suppress NETosis in neutrophils. Residual S. aureus-induced NETs that remain during co-infection do not impact E. faecalis, further suggesting that E. faecalis possess mechanisms to evade or survive NET-associated killing mechanisms. E. faecalis-driven reduction of NETosis corresponds with higher S. aureus survival, indicating that this immunomodulating effect could be a risk factor in promoting the virulence polymicrobial infection. These findings highlight the complexity of the immune response to polymicrobial infections and suggest that attenuated pathogen-specific immune responses contribute to pathogenesis in the mammalian host.

Molecular Epidemiology of Plasmodium species in Conflicted Federally Administered Tribal Area (FATA) Pakistan.

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan's war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

First Report of Tomato Spotted Wilt Virus in Oxypetalum coeruleum in Korea

&lt;i&gt;Oxypetalum coeruleum&lt;/i&gt;, commonly known as Tweedia, is a perennial herbaceous plant of the &lt;i&gt;Apocynaceae&lt;/i&gt; family native to southern Brazil and Uruguay. Tweedia plants are grown as one of the most popular ornamental flowers for floral arrangement in Korea. In May 2021, several tweedia plants in a single greenhouse in Gimje, Jeollabuk-do were found to show virus-like symptoms including necrotic rings, vein-clearing, chlorotic mottle, and mosaic on the leaves, and necrosis on the stems. Here, we have identified tomato spotted wilt virus (TSWV) in symptomatic tweedia leaves by applying high-throughput RNA sequencing. In the result, a single infection by TSWV was verified without mixed infections of different virus species. To confirm the presence of TSWV, a reverse transcription polymerase chain reaction was performed with a specific primer set to the N gene of TSWV. The complete genomic sequence of L, M, and S segments of TSWV ’Oxy’ isolate were determined and deposited in GenBank under accession numbers LC671525, LC671638, and LC671639, respectively. In the phylogenetic tree analysis by maximum likelihood method, ’Oxy’ isolate showed a high relationship with TSWV ’Gumi’ isolate from &lt;i&gt;Gerbera jamesonii&lt;/i&gt; in Gyeongsangbuk-do, Korea; for all three RNA segments. To our knowledge, this is the first report of TSWV infection of &lt;i&gt;O. coeruleum&lt;/i&gt; in Korea.