Non-falciparum malaria infections in Uganda, does it matter? A review of the published literature.

BACKGROUND: Plasmodium falciparum is the dominant malaria species in the sub-Saharan Africa and the main cause of severe disease and death. Notwithstanding, severe malaria and death due to non-falciparum infections have been reported, but at much lower rates than P. falciparum infections. Following increasing use of molecular detection techniques in epidemiological studies, a higher prevalence of non-falciparum species has been reported in the region than previously thought. This article reviews the literature on the prevalence of non-falciparum malaria species in Uganda and the clinical figures of their severe diseases. It aims to elucidate the extent to which mono non-falciparum malaria infections in a highly malaria-endemic country contribute to malaria mortality and outline its policy implications on malaria case management. METHODS: The available English-language published peer-reviewed literature up to March 2024 was sought via PubMed and Google Scholar. The keywords used were severe malaria, AND P. falciparum, P. malariae, P. vivax, P. ovale spp., mixed infections AND Uganda. The review encompassed 53 articles. Articles using molecular diagnosis methods were accounted for analysis. RESULTS: The literature reported a substantial prevalence of non-falciparum infections in Uganda. Plasmodium malariae and Plasmodium ovale spp. were the second and third most prevalent reported malaria species respectively after P. falciparum as dominant species. Non-falciparum malaria infections often occur as mixed infections rather than mono-infections. Besides, molecular diagnostics revealed that 21% of initially reported mono-infections of P. falciparum were, in fact, mixed infections. No article was found on the prevalence of severe malaria or case fatality rate due to mixed or non-falciparum infections. CONCLUSION: A critical knowledge gap exists regarding the impact of mixed and non-falciparum species on severe malaria and death in Uganda. Robust evidence on prevalence, recurrent parasitaemia, and severe clinical manifestations of mixed and non-falciparum malaria infections is crucial for evidence-based and effective policymaking regarding malaria case management.

Coinfection of channel catfish (Ictalurus punctatus) with virulent Aeromonas hydrophila and Flavobacterium covae exacerbates mortality.

Flavobacterium covae and virulent Aeromonas hydrophila are prevalent bacterial pathogens within the US catfish industry that can cause high mortality in production ponds. An assessment of in vivo bacterial coinfection with virulent A. hydrophila (ML09-119) and F. covae (ALG-00-530) was conducted in juvenile channel catfish (Ictalurus punctatus). Catfish were divided into seven treatments: (1) mock control; (2) and (3) high and low doses of virulent A. hydrophila; (4) and (5) high and low doses of F. covae; (6) and (7) simultaneous challenge with high and low doses of virulent A. hydrophila and F. covae. In addition to the mortality assessment, anterior kidney and spleen were collected to evaluate immune gene expression, as well as quantify bacterial load by qPCR. At 96 h post-challenge (hpc), the high dose of virulent A. hydrophila infection (immersed in 2.3 × 107 CFU mL-1 ) resulted in cumulative percent mortality (CPM) of 28.3 ± 9.5%, while the high dose of F. covae (immersed in 5.2 × 106 CFU mL-1 ) yielded CPM of 23.3 ± 12.9%. When these pathogens were delivered in combination, CPM significantly increased for both the high- (98.3 ± 1.36%) and low-dose combinations (76.7 ± 17.05%) (p < .001). Lysozyme activity was found to be different at 24 and 48 hpc, with the high-dose vAh group demonstrating greater levels than unexposed control fish at each time point. Three proinflammatory cytokines (tnfα, il8, il1b) demonstrated increased expression levels at 48 hpc. These results demonstrate the additive effects on mortality when these two pathogens are combined. The synthesis of these mortality and health metrics advances our understanding of coinfections of these two important catfish pathogens and will aid fish health diagnosticians and channel catfish producers in developing therapeutants and prevention methods to control bacterial coinfections.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018-2020.

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

Long-Term Monitoring of Aphid-Transmitted Viruses in Melon and Zucchini Crops: Genetic Diversity and Population Structure of Cucurbit Aphid-Borne Yellows Virus and Watermelon Mosaic Virus.

Understanding the emergence and prevalence of viral diseases in crops requires the systematic epidemiological monitoring of viruses, as well as the analysis of how ecological and evolutionary processes combine to shape viral population dynamics. Here, we extensively monitored the occurrence of six aphid-transmitted viruses in melon and zucchini crops in Spain for 10 consecutive cropping seasons between 2011 and 2020. The most prevalent viruses were cucurbit aphid-borne yellows virus (CABYV) and watermelon mosaic virus (WMV), found in 31 and 26% of samples with yellowing and mosaic symptoms. Other viruses, such as zucchini yellow mosaic virus, cucumber mosaic virus, Moroccan watermelon mosaic virus, and papaya ring spot virus, were detected less frequently (<3%) and mostly in mixed infections. Notably, our statistical analysis showed a significant association between CABYV and WMV in melon and zucchini hosts, suggesting that mixed infections might be influencing the evolutionary epidemiology of these viral diseases. We then carried out a comprehensive genetic characterization of the full-length genome sequences from CABYV and WMV isolates by using the Pacific Biosciences single-molecule real-time (PacBio) high-throughput technology to assess the genetic variation and structure of their populations. Our results showed that the CABYV population displayed seven codons under positive selection, and although most isolates clustered in the Mediterranean clade, a subsequent analysis of molecular variance revealed a significant, fine-scale temporal structure, which was in part explained by the level of the variance between isolates from single and mixed infections. In contrast, the WMV population genetic analysis showed that most of the isolates grouped into the Emergent clade, with no genetic differentiation and under purifying selection. These results underlie the epidemiological relevance of mixed infections for CABYV and provide a link between genetic diversity and CABYV dynamics at the whole-genome level.

Mixed infection of three nontuberculous mycobacteria species identified by metagenomic next-generation sequencing in a patient with peritoneal dialysis-associated peritonitis: a rare case report and literature review.

BACKGROUND: Peritonitis caused by nontuberculous mycobacteria (NTM) is an infrequent but important complication in patients undergoing peritoneal dialysis (PD). There has been no report of mixed infections with multiple NTM. Peritoneal dialysis-associated peritonitis (PDAP) caused by Mycobacterium abscessus is more common than that caused by M. smegmatis and M. goodii. CASE PRESENTATION: This case concerns a patient with PDAP caused by gram-positive bacilli, which could not be identified at the species level in successive detections of initial peritoneal effluent. Later, M. smegmatis was detected with no sensitivity results in bacterial culture. However, metagenomic next-generation sequencing (mNGS) and first whole-genome sequences indicated that there were three species coexisting in the culture, including M. smegmatis (24,708 reads), M. abscessus (9224 reads), and M. goodii (8305 reads). This is the first case of PDAP with specific evidence that conventional detection methods isolated a poorly pathogenic NTM, whereas mNGS and first whole-genome sequences identified multiple NTM. Pathogenic bacteria might not be detected using conventional methods due to their lower abundance. This case report is the first description of mixed infections with more than two species of NTM during PDAP. CONCLUSIONS: PDAP caused by multiple NTM is rare, and the diagnosis is difficult. When NTM are isolated by conventional tests in patients who are suspected of infection, clinicians should be vigilant, and further tests should be performed to determine the presence of rare or even previously unknown bacteria, for which the quantity is relatively low, but the pathogenicity is high. The rare pathogen may be a primary agent in causing such complications.

Molecular detection of mixed infection with peste des petits ruminants and retroviruses in Egyptian sheep and goats.

Peste des petits ruminants (PPR) is a contagious viral disease causing massive economic loss to animal industries in endemic countries including Egypt. Although a vaccine is available, coinfections can overwhelm the animal immune system and interfere with vaccine protection. Small ruminant retrovirus (SRR), including enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV), is responsible for coinfections with PPR. Investigation of clinical cases in this study confirmed the presence of PPR virus by RT-PCR among four flocks. Sequence of five PPR amplicons revealed that all strains had 100% aa similarity and belonged to lineage IV. In addition, these strains had 98-99% nt similarity with all previous Egyptian and African strains from Sudan (MK371449) and Ethiopia (MK371449). Illumina sequencing of a representative sample showed a genome of 5753 nt compatible with ENT-2 virus with 98.42% similarity with the Chinese strain (MN564750.1). Four ORFs representing gag, pro, pol, and env genes were identified and annotated. Pro gene was highly stable while gag, pol, and env showed eight, two, and three aa differences with the reference strains. Sanger sequencing revealed that two amplicons were ENT-2 virus, and one was JSRV. ENT-2 sequences had 100% similarity with KU258870 and KU258871 reference strains while JSRV was 100% similar to the EF68031 reference strain. The phylogenetic tree showed a close relationship between the ENT of goats and the JSRV of sheep. This study highlights the complexity of PPR molecular epidemiology, with SRR that was not molecularly characterized previously in Egypt.

Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections.

BACKGROUND: Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. RESULTS: Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. CONCLUSIONS: Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Mixed genotypes of Orientia tsutsugamushi in conserved genes and a single immune-dominant tsa56 genotype discovered from a patient with scrub typhus in Hainan Island, China: a case report.

BACKGROUND: Orientia tsutsugamushi (O. tsutsugamushi), an obligate intracellular bacterium, is transmitted to humans through infected larval trombiculid mite bites, causing scrub typhus. Mixed genotypes of O. tsutsugamushi in canonical conserved genes were reported in 8-25% of blood samples from patients. Yet, there are few clinical descriptions of these mixed O. tsutsugamushi-infected patients. CASE PRESENTATION: We report a patient with scrub typhus complicated with pulmonary involvement and hepatic dysfunction, who carried mixed genotypes of the conserved genes but had a single immune-dominant 56-kDa type-specific antigen (tsa56) genotype. The patient was successfully recovered by doxycycline treatment. CONCLUSIONS: In this reported case, both patient's eschar and blood samples have repeatedly shown the same results, i.e., no variants were discovered in tsa56 gene that bears multiple hypervariable regions. Whereas the selected highly conserved genes were identified with up to 32 variants in a 2700 base-pair concatenated sequence. The prevalence, disease severity and mechanism of these single-tsa56-genotype mixed infections remain to be investigated on a large scale with more cases.

Double trouble: untangling mixed sequence signals in bird samples with avian haemosporidian co-infections.

Blood parasites comprise some of the most prevalent pathogens in nature, and their detection and identification are major objectives in varied fields such as ecology and biomedicine. Two approaches were compared, one based on Sanger sequencing and the other next-generation sequencing (NGS) based, in terms of their performance in detecting avian blood parasites across tropical Southeast Asian birds. Across a panel of 528 bird individuals, 43 birds were ascertained to be infected with avian haemosporidians using a polymerase chain reaction-based detection method. Among these samples, NGS-based barcoding confirmed co-infections by multiple blood parasites in all eight cases where Sanger sequencing produced double peaks. Importantly however, the NGS-based method produced another five diagnoses of co-infections (62.5%) in which Sanger-based barcoding remained equivocal. In contrast to Sanger sequencing, the NGS-based method was able to identify co-infecting haemosporidian lineages via their barcodes. The accuracy of avian haemosporidian lineage identification was not compromised by the shorter length of NGS sequences, with ~94% of NGS barcodes producing matches identical to those of the Sanger barcodes. The application of NGS-based barcoding methods promises to enhance parasite identification and reduce erroneous inferences based on artefacts.

The diagnosis and molecular epidemiology investigation of avian hepatitis E in Shandong province, China.

BACKGROUND: Avian hepatitis E virus (HEV) is the pathogenic agent of big liver and spleen disease (BLS) and of hepatitis-splenomegaly syndrome (HSS) in chickens, which have caused economic losses to the poultry industry in China. In this study, 18 samples of BLS chickens were collected to reveal the molecular epidemiological characteristics of avian HEV in the province of Shandong, China. RESULTS: Gross and microscopic lesions of clinical samples were observed; then, virology detection and genetic analysis of avian HEV were performed. The results showed that there was significant swelling and rupture in the liver and that the spleen was enlarged. Microscopic lesions demonstrated obvious hemorrhage in the liver, with infiltration of heterophilic granulocytes, lymphocytes, and macrophages, as well as the reduction of lymphocytes in the spleen. Eleven of the 18 samples were positive for avian HEV, with a positive rate of 61.11%. More importantly, all avian HEV-positive samples were mixed infections: among these, the mixed infections of avian HEV and chicken infectious anemia virus (CIAV) and avian HEV and fowl adenovirus (FAdV) were the most common. Furthermore, the genetic evolution analysis showed that all avian HEV strains obtained here did not belong to the reported 4 genotypes, thus constituting a potential novel genotype. CONCLUSIONS: These results of this study further enrich the epidemiological data on avian HEV in Shandong, prove the genetic diversity of avian HEV in China, and uncover the complex mixed infections of avian HEV clinical samples.

SAG3 Toxoplasma gondii cloning reveals unexpected fivefold infection in the blood of feral cats in the Mexican Caribbean.

BACKGROUND: Currently, more than 300 genotypes of Toxoplasma gondii (T. gondii) have been described throughout the world, demonstrating its wide genetic diversity. The SAG3 locus is one of the genes included in the genotyping panel of this parasite. It is associated with its virulence since it participates during the invasion process of the host cells. Therefore, cloning, sequencing, and bioinformatic analysis were used to deepen the understanding of the SAG3 locus genetic diversity of T. gondii in blood samples from feral cats. RESULTS: Six different SAG3 sequences were detected, five of which were detected in one feline. Three sequences were first reported here; one of them was an intragenic recombinant. In the cladogram, four out of ten SAG3 sequences did not share nodes with others reported worldwide. CONCLUSIONS: Cloning and sequencing of samples with more than one restriction pattern by PCR-RFLP were very helpful tools to demonstrate the presence of more than three genotypes of T. gondii in the blood of feral cats from southeastern Mexico. This suggests a potential mixed infection of multiple T. gondii strains and high genetic diversity of the parasites in felines in this tropical region of Mexico.

First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab, Pakistan.

Theileriosis and anaplasmosis are important tick-borne hemoparasites of bovines. The first surveillance study aimed to assess the suitability of duplex PCR for simultaneous detection of Theileria annulata and Anaplasma marginale field infections in Jhang and Rawalpindi districts of Punjab, Pakistan. Cattle blood samples (n = 480) were collected from selected union councils of all tehsils using a multistage sampling technique. The sampling unit consisted of asymptomatic cattle belonging to either age, sex, and breed. Epidemiological data related to host, area, management, and season were collected using a questionnaire. Based on duplex PCR, the overall prevalence of the two concurrent tick-borne pathogens was 19.79% (95/480). Chi-square analysis indicated that age, breed, tick infestation, history of tick-borne diseases, frequency of acaricidial application, and season were significantly associated with tick-borne pathogens. Phylogenetic analysis of A. marginale and T. annulata isolates based on msp1ß and cytochrome b genes, respectively, revealed that nucleotide sequences acquired from these two pathogens are novel, grouped separately from different countries. All our A. marginale isolates showed 88.2 to 80.5% similarity with isolates from Egypt, Israel, Mexico, and lesser homology with South African isolates. Similarly, the phylogenetic tree based on cytochrome b partial sequences of T. annulata revealed that our sequences are closer to those from India and Iran. Based on this first study on concomitant detection of tick-borne pathogens, it can be concluded that mixed infections are endemic in the study districts and mPCR is suitable for detecting concurrent field infections. Simultaneous infections should be considered while performing surveillance and chemotherapeutic trials for better prevention and control of tick-borne diseases.

Molecular insights on potato yellow vein crinivirus infections in the highlands of Colombia.

Potato yellow vein virus (PYVV) was detected in potatoes grown in the Central highlands, north of Bogotá (~3000 m altitude), Colombia. At this altitude viral whitefly vectors are largely absent, but infection persists because of the use of uncertified tubers. Plants with typical PYVV-induced yellowing symptoms, as well as with atypical yellowing or non-symptomatic symptoms were sampled at three separate geographical locations. PYVV presence was assessed by RT-PCR, and several plants were subjected to high-throughput sequencing (HTS) of their small RNA (sRNA) populations. Complete or almost complete sequences of four PYVV isolates were thus reconstructed, all from symptomatic plants. Three viral isolates infected plants singly, while the fourth co-infected the plant together with a potyvirus. Relative proportions of sRNAs to each of the three crinivirus genomic RNAs were found to remain comparable among the four infections. Genomic regions were identified as hotspots of sRNA formation, or as regions that poorly induced sRNAs. Furthermore, PYVV titres in the mixed versus single infections remained comparable, indicating an absence of synergistic/antagonistic effects of the potyvirus on the accumulation of PYVV. Daughter plants raised in the greenhouse from tubers of the infected, field-sampled plants displayed mild PYVV infection symptoms that disappeared with time, demonstrating the occurrence of recovery and asymptomatic infection phenotypes in this pathosystem.

Prevalence and genetic diversity of gastroenteritis viruses in hospitalized children < 5 years of age in Maharashtra state, Western India, 2017-2019.

Four gastroenteritis viruses were responsible for 54% of the acute gastroenteritis (AGE) cases in children hospitalized between May 2017 and December 2019 in Pune city of Maharashtra state, Western India. The majority (79%) of the children were <2 years of age. The prevalence of Rotavirus A (RVA) was 30.5% followed by 14.3% for norovirus, 8.4% for adenovirus, and 5.5% for astrovirus. The severity of the disease was highest in patients with coinfections compared with the patients with a single infection or negative for all (p = 0.024). Genotyping analysis showed that the majority of the RVA-positive samples (66%) could be typed as G3P[8], 63.6% of the norovirus as GII.4 Sydney [P16], 44% of the adenovirus as type 41%, and 56.2% of the astrovirus as astrovirus type 1. The almost equivalent prevalence of rotavirus and nonrotaviruses and acute gastroenteritis (AGE) cases without known etiology in around 46% of the cases was noted in the present study. Our data highlight that after the recent inclusion of rotavirus vaccines as a part of the National Immunization schedule in India, conducting extensive AGE surveillance in children should include nonrotaviruses such as norovirus.

Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs.

Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies.

Severe polymicrobial and fungal periprosthetic osteomyelitis persisting after hip disarticulations treated with caspofungin in risk patients: a case series.

BACKGROUND: Periprosthetic fungal infections are considered rare and opportunistic infections. Treatment is difficult, and established standards do not yet exist. The choice of the appropriate antifungal drug might affect the patient outcome. CASES: All the three cases presented showed polybacterial recurrent infection of the revision hip arthroplasty. All patients were of younger age, had multiple revisions of the endoprosthesis, each had a large partial femoral replacement greater than 40% of the femoral length, gentamycin-loaded cement, and a long anchoring distance of the used intramedullary stem. Due to the severe life-threatening infection with deep osteomyelitis, an amputation had to be performed. However, despite surgical intervention, the fungal dominated infection persisted. Finally, only the use of caspofungin allowed permanent infection control. CONCLUSION: The polybacterial infection is driven by the symbiosis between fungi and bacteria. Therefore, eradication of the fungus is required to achieve elimination of the bacteria. Antimycotics of the echinocandin-class, such as caspofungin, may be considered as initial treatment.

Small RNA and Transcriptome Sequencing of a Symptomatic Peony Plant Reveals Mixed Infections with Novel Viruses.

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus, apple stem grooving virus, lychnis mottle virus, grapevine line pattern virus, and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleotides (nt) in length, comprising three open reading frames (ORFs), and shared 63.8 to 75.9% nt sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57 and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length, with five ORFs encoding the REP (ORF1), triple gene block (ORF2 to 4) and coat protein (CP, ORF5). Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

Therapeutic Potential of Antimicrobial Peptides in Polymicrobial Biofilm-Associated Infections.

It is widely recognized that many chronic infections of the human body have a polymicrobial etiology. These include diabetic foot ulcer infections, lung infections in cystic fibrosis patients, periodontitis, otitis, urinary tract infections and even a proportion of systemic infections. The treatment of mixed infections poses serious challenges in the clinic. First, polymicrobial communities of microorganisms often organize themselves as biofilms that are notoriously recalcitrant to antimicrobial therapy and clearance by the host immune system. Secondly, a plethora of interactions among community members may affect the expression of virulence factors and the susceptibility to antimicrobials of individual species in the community. Therefore, new strategies able to target multiple pathogens in mixed populations need to be urgently developed and evaluated. In this regard, antimicrobial or host defense peptides (AMPs) deserve particular attention as they are endowed with many favorable features that may serve to this end. The aim of the present review is to offer a comprehensive and updated overview of studies addressing the therapeutic potential of AMPs in mixed infections, highlighting the opportunities offered by this class of antimicrobials in the fight against polymicrobial infections, but also the limits that may arise in their use for this type of application.

Application of MIRU-VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients.

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). According to multiple studies, MIRU-VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU-VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU-VNTR on smear slides in detecting mixed infections. Ziehl-Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU-VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU-VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU-VNTR on smear slides can be conveniently used for the detection of mixed infections.

Microbiota in vaginal health and pathogenesis of recurrent vulvovaginal infections: a critical review.

Recurrent vulvovaginal infections (RVVI) has not only become an epidemiological and clinical problem but also include large social and psychological consequences. Understanding the mechanisms of both commensalism and pathogenesis are necessary for the development of efficient diagnosis and treatment strategies for these enigmatic vaginal infections. Through this review, an attempt has been made to analyze vaginal microbiota (VMB) from scratch and to provide an update on its current understanding in relation to health and common RVVI i.e. bacterial vaginosis, vulvovaginal candidiaisis and Trichomoniasis, making the present review first of its kind. For this, potentially relevant studies were retrieved from data sources and critical analysis of the literature was made. Though, culture-independent methods have greatly unfolded the mystery regarding vaginal bacterial microbiome, there are only a few studies regarding the composition and diversity of vaginal mycobiome and different Trichomonas vaginalis strains. This scenario suggests a need of further studies based on comparative genomics of RVVI pathogens to improve our perceptive of RVVI pathogenesis that is still not clear (Fig. 5). Besides this, the review details the rationale for Lactobacilli dominance and changes that occur in healthy VMB throughout a women's life. Moreover, the list of possible agents continues to expand and new species recognised in both health and VVI are updated in this review. The review concludes with the controversies challenging the widely accepted dogma i.e. "VMB dominated with Lactobacilli is healthier than a diverse VMB". These controversies, over the past decade, have complicated the definition of vaginal health and vaginal infections with no definite conclusion. Thus, further studies on newly recognised microbial agents may reveal answers to these controversies. Conversely, VMB of women could be an answer but it is not enough to just look at the microbiology. We have to look at the woman itself, as VMB which is fine for one woman may be troublesome for others. These differences in women's response to the same VMB may be determined by a permutation of behavioural, cultural, genetic and various other anonymous factors, exploration of which may lead to proper definition of vaginal health and disease.