Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru.

E. coli that produce extended-spectrum ß-lactamases (ESBLs) are major multidrug-resistant bacteria. In Peru, only a few reports have characterised the whole genome of ESBL enterobacteria. We aimed to confirm the identity and antimicrobial resistance (AMR) profile of two ESBL isolates from dog faeces and drinking water of rural Andean households and determine serotype, phylogroup, sequence type (ST)/clonal complex (CC), pathogenicity, virulence genes, ESBL genes, and their plasmids. To confirm the identity and AMR profiles, we used the VITEK®2 system. Whole-genome sequencing (WGS) and bioinformatics analysis were performed subsequently. Both isolates were identified as E. coli, with serotypes -:H46 and O9:H10, phylogroups E and A, and ST/CC 5259/- and 227/10, respectively. The isolates were ESBL-producing, carbapenem-resistant, and not harbouring carbapenemase-encoding genes. Isolate 1143 ST5259 harboured the astA gene, encoding the EAST1 heat-stable toxin. Both genomes carried ESBL genes (blaEC-15, blaCTX-M-8, and blaCTX-M-55). Nine plasmids were detected, namely IncR, IncFIC(FII), IncI, IncFIB(AP001918), Col(pHAD28), IncFII, IncFII(pHN7A8), IncI1, and IncFIB(AP001918). Finding these potentially pathogenic bacteria is worrisome given their sources and highlights the importance of One-Health research efforts in remote Andean communities.

Protein Arrays for the Identification of Seroreactive Protein Markers for Infectious Diseases.

The protein array is a powerful platform to study humoral responses to infectious agents using small sample volumes [<3 µL]. Its success can be largely attributed to the development of new strategies for high-throughput cloning and expression, and improved manufacturing techniques for the construction of arrays. Here, we describe a method to hybridize protein arrays with malaria patients' sera in order to identify seroreactive antigens, some of which may have a high potential of conferring protection from severe forms of malaria.

Comprehensive proteomics investigation of P. vivax-infected human plasma and parasite isolates.

BACKGROUND: In recent times, Plasmodium vivax (P. vivax) has become a serious threat to public health due to its ability to cause severe infection with fatal outcomes. Its unique biology makes it resilient to control measures that are otherwise effective against P. falciparum. A deeper understanding of P. vivax biology and pathogenesis is, therefore, essential for developing the right control strategies. Proteomics of P. falciparum has been helpful in studying disease biology and elucidating molecular mechanisms involved in the development of disease. However, unlike P. falciparum, proteomics data for P. vivax infection is minimal due to the absence of a continuous culture system. The dependence on clinical samples and animal models has drastically limited P. vivax research, creating critical knowledge gaps in our understanding of the disease. This study describes an in-depth proteomics analysis of P. vivax-infected human plasma and parasite isolates, to understand parasite biology, pathogenesis, and to identify new diagnostic targets for P. vivax malaria. METHODS: A mass-spectrometry- (MS) based proteomics approach (Q Exactive) was applied to analyze human plasma and parasite isolates from vivax malaria patients visiting a primary health centre in India. Additionally, a targeted proteomics assay was standardized for validating unique peptides of most recurring parasite proteins. RESULTS: Thirty-eight P. vivax proteins were detected in human plasma with high confidence. Several glycolytic enzymes were found along with hypothetical, cytoskeletal, ribosomal, and nuclear proteins. Additionally, 103 highly abundant P. vivax proteins were detected in parasite isolates. This represents the highest number of parasite proteins to be reported from clinical samples so far. Interestingly, five of these; three Plasmodium exported proteins (PVX_003545, PVX_003555 and PVX_121935), a hypothetical protein (PVX_083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX_094303) were found in both plasma and parasite isolates. CONCLUSIONS: A parasite proteomics investigation is essential to understand disease pathobiology and design novel interventions. Control strategies against P. vivax also depend on early diagnosis. This work provides deeper insights into the biology of P. vivax by identifying proteins expressed by the parasite during its complex life-cycle within the human host. The study also reports antigens that may be explored as diagnostic candidates.

Hospital-derived antibody profiles of malaria patients in Southwest India.

BACKGROUND: Naturally acquired immunity to malaria across the globe varies in intensity and protective powers. Many of the studies on immunity are from hyperendemic regions of Africa. In Asia, particularly in India, there are unique opportunities for exploring and understanding malaria immunity relative to host age, co-occurrence of Plasmodium falciparum and Plasmodium vivax infections, varying travel history, and varying disease severity. Variation in immunity in hospital settings is particularly understudied. METHODS: A US NIH ICEMR (South Asia) team examined the level of immunity in an Indian malaria patient population visiting or admitted to Goa Medical College and Hospital in Goa, India. Sera from 200 patients of different ages, in different seasons, infected with P. falciparum or P. vivax or both species, and with different clinical severity were applied to an established protein array system with over 1000 P. falciparum and P. vivax antigens. Differential binding of patient IgG to different antigens was measured. RESULTS: Even though Goa itself has much more P. vivax than P. falciparum, IgG reactivity towards P. falciparum antigens was very strong and comparable to that seen in regions of the world with high P. falciparum endemicity. Of 248 seropositive P. falciparum antigens, the strongest were VAR, MSP10, HSP70, PTP5, AP2, AMA1, and SYN6. In P. vivax patients, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 were the strongest IgG binders. Both P. falciparum and P. vivax patients also revealed strong binding to new antigens with unknown functions. Seropositives showed antigens unique to the young (HSP40, ACS6, GCVH) or to non-severe malaria (MSP3.8 and PHIST). CONCLUSION: Seroreactivity at a major hospital in Southwest India reveals antibody responses to P. falciparum and P. vivax in a low malaria transmission region with much migration. In addition to markers of transmission, the data points to specific leads for possible protective immunity against severe disease. Several, but not all, key antigens overlap with work from different settings around the globe and from other parts of India. Together, these studies confidently help define antigens with the greatest potential chance of universal application for surveillance and possibly for disease protection, in many different parts of India and the world.

Identification of Highly Expressed Plasmodium Vivax Proteins from Clinical Isolates Using Proteomics.

Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX_090265), Pv-fam-d protein (PVX_101520), Plasmodium exported protein (PVX_003545), Pvstp1 (PVX_094303) and hypothetical protein (PVX_083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts.

A Perspective on Proteomics of Infectious Diseases.

Pneumonia, HIV/AIDS, tuberculosis, malaria and several other diseases caused by pathogens largely contribute to the enormous burden of infectious diseases. Over the last few decades, the impact of infectious diseases on a population has been drastic and remains a major health concern even today. Despite advances in science and technology in this era of health and development, there is a substantial knowledge gap in our understanding of the molecular basis of these infectious diseases. The availability of valuable genomic information for a number of pathogens and their hosts has improved our understanding of disease pathogenesis but has not always been useful in addressing important biological questions. The primary reason lies in the fact that genes do not best reflect the status of a cell. Proteins represent the functional molecules of a cell and are ultimately responsible for controlling most aspects of cellular function. Their existence as different isoforms owing to posttranslational modifications suggests that many proteins can be produced by the same gene. Furthermore, not all mRNAs are translated at all times justifying the need to develop additional tools to study proteins as separate molecular entities. Their presence or absence under disease conditions, varying levels, different forms, and functions need to be carefully studied to understand molecular alterations in response to a disease. Here, we describe the applications of proteomics-based approaches to study infectious diseases with a note on the objectives of the Human Proteome Project (HPP)-Human Infectious Diseases (HID) project under the HUPO's flagship program.

Clinical Proteomics and Cytokine Profiling for Dengue Fever Disease Severity Biomarkers.

Dengue fever (DF) is a major global health burden with a pathophysiology that is still incompletely understood. Biomarkers that predict and explain susceptibility to DF and its progression to its more severe hemorrhagic form are much needed. DF is endemic in tropical and subtropical regions of the world, with a rapidly increasing incidence of disease severity. We conducted a clinical biomarker discovery study using both a case-control and longitudinal study design. Plasma proteome alterations in patients with DF (n = 12) and dengue hemorrhagic fever (DHF, n = 24) were analyzed in comparison to healthy controls (HCs, n = 16), using the isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics methodology (false discovery rate of 1%, ≥2 peptides). Several proteins such as the alpha-2 macroglobulin, angiotensinogen, apolipoprotein B-100, serotransferrin, and ceruloplasmin were upregulated (fold change >1.2) in all DHF cases, and downregulated in DF (fold change <0.83), compared with HCs. Plasma cytokine profiling (8 DF, 8 DHF, and 8 HC) on two consecutive time points, at day 0 (day of admission) and days 5-7, found significant elevation in IL-1RA, IL-7, TNF-α, MCP1-MCAF, and MIP-1ß levels, but only in the DHF cases, which is the severe disease, and not in DF, compared with HCs (p < 0.05). These new observations on changes in the plasma proteome and cytokine profiles in patients with dengue infection identify several putative molecular leads for future biomarker development and precision medicine in relation to forecasting DF disease severity.

Quantitative Proteomics Analysis of Plasmodium vivax Induced Alterations in Human Serum during the Acute and Convalescent Phases of Infection.

The radial distribution of Plasmodium vivax malaria burden has evoked enormous concern among the global research community. In this study, we have investigated the serum proteome alterations in non-severe vivax malaria patients before and during patient recuperation starting from the early febrile to the defervescence and convalescent stages of the infection. We have also performed an extensive quantitative proteomics analysis to compare the serum proteome profiles of vivax malaria patients with low (LPVM) and moderately-high (MPVM) parasitemia with healthy community controls. Interestingly, some of the serum proteins such as Serum amyloid A, Apolipoprotein A1, C-reactive protein, Titin and Haptoglobin, were found to be sequentially altered with respect to increased parasite counts. Analysis of a longitudinal cohort of malaria patients indicated reversible alterations in serum levels of some proteins such as Haptoglobin, Apolipoprotein E, Apolipoprotein A1, Carbonic anhydrase 1, and Hemoglobin subunit alpha upon treatment; however, the levels of a few other proteins did not return to the baseline even during the convalescent phase of the infection. Here we present the first comprehensive serum proteomics analysis of vivax malaria patients with different levels of parasitemia and during the acute and convalescent phases of the infection.

Proteomics of Plasmodium vivax malaria: new insights, progress and potential.

INTRODUCTION: Plasmodium vivax has accounted for an enormous share of the global malaria burden in recent years, along with Plasmodium falciparum. The wide distribution of P. vivax and recent evidences of severe and complicated vivax malaria across several endemic regions of the world suggest that this disease may have been more overlooked than benign. While P. falciparum has been extensively studied, P. vivax has received limited research attention owing to its complex nature and absence of a continuous culture system. AREAS COVERED: This review briefly describes the epidemiology of vivax malaria, analyzes challenges towards effective control and summarizes major insights provided by genomics and transcriptomics research in the area. Subsequently, the review provides a detailed description of the applications of proteomics in vivax malaria research, focusing on both host responses and parasite proteomics studies to understand P. vivax biology. Expert commentary: In recent years, proteomics technologies are being used effectively to understand P. vivax biology and the underlying pathogenesis. Technological advances in mass spectrometry configurations, multiomics investigations and emerging strategies such as targeted proteomics may also immensely aid in studying disease severity, improving existing diagnosis and identifying new drug and vaccine targets.

Clinicopathological Analysis and Multipronged Quantitative Proteomics Reveal Oxidative Stress and Cytoskeletal Proteins as Possible Markers for Severe Vivax Malaria.

In Plasmodium vivax malaria, mechanisms that trigger transition from uncomplicated to fatal severe infections are obscure. In this multi-disciplinary study we have performed a comprehensive analysis of clinicopathological parameters and serum proteome profiles of vivax malaria patients with different severity levels of infection to investigate pathogenesis of severe malaria and identify surrogate markers of severity. Clinicopathological analysis and proteomics profiling has provided evidences for the modulation of diverse physiological pathways including oxidative stress, cytoskeletal regulation, lipid metabolism and complement cascades in severe malaria. Strikingly, unlike severe falciparum malaria the blood coagulation cascade was not found to be affected adversely in acute P. vivax infection. To the best of our knowledge, this is the first comprehensive proteomics study, which identified some possible cues for severe P. vivax infection. Our results suggest that Superoxide dismutase, Vitronectin, Titin, Apolipoprotein E, Serum amyloid A, and Haptoglobin are potential predictive markers for malaria severity.

Challenges and prospects for biomarker research: a current perspective from the developing world.

Majority of deaths due to communicable and non-communicable diseases occur in the low and middle-income nations (LMNs), mainly due to the lack of early diagnoses and timely treatments. In such a scenario, biomarkers serve as an indispensible resource that can be used as indicators of biological processes, specific disease conditions or response to therapeutic interventions. Evaluation, diagnosis and management of diseases in developing world by following/extrapolating the findings obtained on the basis of the research work involving only the populations from the developed countries, could often be highly misleading due to existence of diverse patterns of diseases in developing countries compared to the developed world. Biomarker candidates identified from high-throughput integrated omics technologies have promising potential; however, their actual clinical applications are found to be limited, primarily due to the challenges of disease heterogeneity and pre-analytical variability associated with the biomarker discovery pipeline. Additionally, in the developing world, economic crunches, lack of awareness and education, paucity of biorepositories, enormous diversities in socio-epidemiological background, ethnicity, lifestyle, diet, exposure to various environmental risk factors and infectious agents, and ethical and social issues also cumulatively hinder biomarker discovery ventures. Establishment of standard operating procedures, comprehensive data repositories and exchange of scientific findings are crucial for reducing the variability and fragmentation of data. This review highlights the challenges associated with the discovery, validation and translational phases of biomarker research in LMNs with some of their amenable solutions and future prospects. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.