Advances in Human Pathogen Control–A 21st Century Challenge

Immunogens mimicking the native-like structure of surface-exposed viral antigens are considered promising vaccine candidates. Influenza viruses are important zoonotic respiratory viruses with high pandemic potential. Recombinant soluble hemagglutinin (HA) glycoprotein-based protein subunit vaccines against Influenza have been shown to induce protective efficacy when administered intramuscularly. Here, we have expressed a recombinant soluble trimeric HA protein in Expi 293F cells and purified the protein derived from the Inf A/Guangdong-Maonan/ SWL1536/2019 virus which was found to be highly virulent in the mouse. The trimeric HA protein was found to be in the oligomeric state, highly stable, and the efficacy study in the BALB/c mouse challenge model through intradermal immunization with the prime-boost regimen conferred complete protection against a high lethal dose of homologous and mouse-adapted InfA/PR8 virus challenge. Furthermore, the immunogen induced high hemagglutinin inhibition (HI) titers and showed cross-protection against other Inf A and Inf B subtypes. The results are promising and warrant trimeric HA as a suitable vaccine candidate. Full article

The World Health Organization (WHO) declared in May 2021 that SARS-CoV-2 is transmitted not only by close contact with infectious respiratory fluids from infected people or contaminated materials but also indirectly through air. Airborne transmission has serious implications for the control measures we can deploy, given the emergence of more transmissible variants. This emphasizes the need to deploy a mechanism to reduce the viral load in the air, especially in closed and crowded places such as hospitals, public transport buses, etc. In this study, we explored ultraviolet C (UVC) radiation for its ability to inactivate the SARS-CoV-2 particles present in aerosols and designed an air disinfection system to eliminate infectious viruses. We studied the virus inactivation kinetics to identify the UVC dosage required to achieve maximum virus inactivation. Based on the experimental data, UVC-based devices were designed for the sanitization of air through HVAC systems in closed spaces. Further, a risk assessment model to estimate the risk reduction was applied which showed that the use of UVC radiation could result in the reduction of the risk of infection in occupied spaces by up to 90%. Full article

Background: D-dimer, generated upon the degradation of fibrin, is extensively used to detect thrombosis in various diseases. It is also explored as a marker for thrombosis in cases with COVID-19 disease. Few studies have confirmed its utility as a marker for assessing disease severity. Objectives: The current research was undertaken to determine the role of D-dimer in patients with COVID-19 and to investigate any association with the progression and severity of the disease in the Saudi population. Methods: Clinical indices in confirmed COVID-19 patients were collected from tertiary care hospitals in Aljouf and Qassim regions. The plasma D-dimer levels were quantified directly in the samples collected from COVID-19 patients (n = 148) using an immunofluorescence assay, and the data were presented in Fibrinogen Equivalent Units (mg/L). The collected data of D-dimer were analyzed based on COVID-19 severity, age, and the gender of patients. Results: The findings show that the plasma D-dimer concentrations were significantly (p = 0.0027) elevated in COVID-19 cases (n = 148), compared to in the normal healthy uninfected controls (n = 309). Moreover, the D-dimer levels were analyzed according to the severity of the disease in the patients. The data revealed that D-dimer concentrations were significantly increased in patients with mild infection to moderate disease, and the levels were the highest in patients with severe COVID-19 disease (p < 0.05). Our analysis demonstrates that the D-dimer levels have no association with the age or gender of COVID-19 patients (p > 0.05) in the study population. Conclusions: D-dimer can serve as a biomarker not only for the detection of COVID-19 infection, but also for determining the severity of infection of COVID-19 disease. Full article

Acute HIV infection (AHI), i.e., the early stage of HIV infection, plays an important role in immune system failure and HIV transmission, but most AHI patients are missed due to their non-specific symptoms. To facilitate the identification of patients with high AHI risk and reduction of missed diagnosis, we characterized 61 AHI patients in a Southwest China hospital with 4300 beds; specifically, we characterized their general clinical characteristics, evolution in results of a novel HIV screening assay called Elecsys^® HIV Duo, and by programming, we analyzed the ability of all routine laboratory tests (e.g., routine blood analysis) to identify AHI patients. Among 61 AHI patients, 85.2% were male and the median age was 42 (interquartile range, 25–62) years. A total of 61.9% of patients visit the emergency department first during AHI. Clinical presentation of AHI patients included fever, fatigue, chills, rash, and various respiratory, digestive, and nervous system symptoms. Two of three results from Elecsys^® HIV Duo show clear evolution trends: HIV P24 antigen decreased while HIV antibody increased in consecutive samples of nearly all patients. High fluorescence lymphocytes have a very high positive likelihood ratio (LR+) of 10.33 and a relatively high “rate of out-of-range tests” of 56.8% (21 in 37 patients who received this test had a result outside the reference range). In addition, we identified more than ten tests with LR+ greater than two. In summary, the emergency department is important for AHI screening. The evolution of HIV P24 Ag and HIV Ab and those laboratory tests with a high “rate of out-of-range tests” or high LR+ may aid the AHI identification and missed diagnosis reduction. Full article

Coronaviruses interact with protein or carbohydrate receptors through their spike proteins to infect cells. Even if the known protein receptors for these viruses have no evolutionary relationships, they do share ontological commonalities that the virus might leverage to exacerbate the pathophysiology. ANPEP/CD13, DPP IV/CD26, and ACE2 are the three protein receptors that are known to be exploited by several human coronaviruses. These receptors are moonlighting enzymes involved in several physiological processes such as digestion, metabolism, and blood pressure regulation; moreover, the three proteins are expressed in kidney, intestine, endothelium, and other tissues/cell types. Here, we spot the commonalities between the three enzymes, the physiological functions of the enzymes are outlined, and how blocking either enzyme results in systemic deregulations and multi-organ failures via viral infection or therapeutic interventions is addressed. It can be difficult to pinpoint any coronavirus as the target when creating a medication to fight them, due to the multiple processes that receptors are linked to and their extensive expression. Full article

Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG). We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells. Full article

Although clothes washing machines remove dirt, microorganisms are not reliably removed by modern cold-water machine-washing practices. Microbial bioburden on clothing originates from the wearer’s skin, the environment (indoor and outdoor), and the washing machine itself. While most clothing microbes are commensals, microbes causing odors and opportunistic pathogens may also be present. Understanding the extent of microbial transfer from washing machines to clothes may inform strategies for odor control and for mitigating the transmission of microbes through the laundering process. This study was designed to quantify and identify bacteria/fungi transferred from laundromat machines to sentinel cotton washcloths under standard cold-water conditions. Bacterial 16S rRNA and fungal ITS sequencing enabled identification of microorganisms in the washcloths following laundering. Total plate-based enumeration of viable microorganisms also was performed, using growth media appropriate for bacteria and fungi. Opportunistic human bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., were recovered. The fungal bioburden was ~two-fold lower than the bacterial bioburden. Most sequences recovered were assigned to non-pathogenic fungi, such as those from genera Malassezia and Ascomycota. These results suggest that public washing machines represent a source of non-pathogenic and pathogenic microbial contamination of laundered garments. Full article

Infectious diseases caused by various nosocomial microorganisms affect worldwide both immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (−)-myrtenol and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and benzalkonium chloride on 64–81% of the clinical isolates of S. aureus and C. albicans, including MRSA and fluconazole-resistant fungi, while (−)-myrtenol increased the properties of amikacin and fluconazole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myrtenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an S. aureus–C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both (−)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by membrane damage since the treatment with both terpenes led to a significant drop in membrane potential similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it seems to be a promising agent to increase the efficiency of the treatment of infections caused by bacteria and be fungi of the genus Candida as well as mixed fungal–bacterial infections, including resistant strains. Full article

Group B Streptococcus (GBS), a commensal in the body, causes a wide range of infectious diseases. This bacterium is dangerous for pregnant women and their babies, in whom it is responsible for early neonatal bacterial sepsis (EOS). The colonisation levels of GBS and its resistance profile to antibiotics provide important information that is useful for orienting prevention strategies. There are few data available on the subject on the determination of resistance phenotypes in Cameroon. We therefore aimed to determine the prevalence of colonisation and antibiotic resistance, including patterns of inducible resistance to clindamycin, of GBS in the city of Yaounde. To achieve this goal, a prospective cross-sectional study with an analytical component was carried out from 28 June to 29 August 2020 at the BIOSANTE laboratory and the Yaounde Gynaeco-Obstetrics and Paediatrics hospital. Vaginal swabs and urine were collected from 163 women. This samples were analysed using 5% defibrinated sheep blood agar and chocolate plus polyvitex agar. The isolates were identified using the morphology of the colony, Gram staining, haemolysis, catalase tests and latex grouping tests. Antibiotic susceptibility testing was carried out by disk diffusion method following the recommendations of the ACFSM 2019. The double disk diffusion method was used to identify isolates with clindamycin-inducible resistance. Our data were analysed with SPSS version 2.1. The results obtained showed that the overall prevalence of colonisation by GBS was 37% (57/163), or 40.3% in non-pregnant women and 59.7% in pregnant women. Pregnancy (p-value = 0.019) and earlier (from the second semester of pregnancy) gestational age (p-value = 0.025) constituted the risk factors of maternal colonisation by GBS. In addition, the strains of GBS were resistant to all 16 antibiotics tested. A D test showed that 64.7% of GBS strains were constitutively resistant to clindamycin. We also note the presence of M phenotypes. As a whole, our results demonstrated that the rate of GBS colonisation in this study was similar to or higher than those in previous reports in Cameroon. All these results indicate that attention should be paid to this bacterium in the monitoring of antimicrobial resistance and in the care of pregnant women and newborns. Full article

Healthcare settings have been utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) since 2010. MALDI-TOF MS has various benefits over the conventional method of biochemical identification, including ease of use, speed, accuracy, and low cost. This approach can solve many of the obstacles to identifying bacteria, fungi and viruses. As technology advanced, more and more databases kept track of spectra, allowing species with similar morphological, genotypic, and biochemical traits to be identified. Using MALDI-TOF MS for identification has become more accurate and quicker due to advances in sample preparation and database enrichment. Rapid sample detection and colony identification using MALDI-TOF MS have produced promising results. A key application of MALDI-TOF MS is quickly identifying highly virulent and drug-resistant diseases. Here, we present a review of the scientific literature assessing the effectiveness of MALDI-TOF MS for locating clinically relevant pathogenic bacteria, fungi, and viruses. MALDI-TOF MS is a useful strategy for locating clinical pathogens, however, it also has some drawbacks. A small number of spectra in the database and inherent similarities among organisms can make it difficult to distinguish between different species, which can result in misidentifications. The majority of the time additional testing may correct these problems, which happen very seldom. In conclusion, infectious illness diagnosis and clinical care are being revolutionized by the use of MALDI-TOF MS in the clinical microbiology laboratory. Full article

Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease, has infected 6 million people, putting 70 million people at risk worldwide. Presently, very limited drugs are available, and these have severe side effects. Hence, there is an urgency to delve into other pathways and targets for novel drugs. Trypanosoma cruzi (T. cruzi) expresses a number of different cyclic AMP (cAMP)-specific phosphodiesterases (PDEs). cAMP is one of the key regulators of mammalian cell proliferation and differentiation, and it also plays an important role in T. cruzi growth. Very few studies have demonstrated the important role of cyclic nucleotide-specific PDEs in T. cruzi’s survival. T. cruzi phosphodiesterase C (TcrPDEC) has been proposed as a potential new drug target for treating Chagas disease. In the current study, we screen several analogs of xanthine for potency against trypomastigote and amastigote growth in vitro using three different strains of T. cruzi (Tulahuen, Y and CA-1/CL72). One of the potent analogs, GVK14, has been shown to inhibit all three strains of amastigotes in host cells as well as axenic cultures. In conclusion, xanthine analogs that inhibit T. cruzi PDE may provide novel alternative therapeutic options for Chagas disease. Full article

Human lymphatic filariae have evolved numerous immune evasion strategies to secure their long-term survival in a host. These strategies include regulation of pattern recognition receptors, mimicry with host glycans and immune molecules, manipulation of innate and adaptive immune cells, induction of apoptosis in effector immune cells, and neutralization of free radicals. This creates an anti-inflammatory and immunoregulatory milieu in the host: a modified Th2 immune response. Therefore, targeting filarial immunomodulators and manipulating the filariae-driven immune system against the filariae can be a potential therapeutic and prophylactic strategy. Filariae-derived immunosuppression can also be exploited to treat other inflammatory diseases and immunopathologic states of parasitic diseases, such as cerebral malaria, and to prevent leishmaniasis. This paper reviews immunomodulatory mechanisms acquired by these filariae for their own survival and their potential application in the development of novel therapeutic approaches against parasitic and inflammatory diseases. Insight into the intricate network of host immune-parasite interactions would aid in the development of effective immune-therapeutic options for both infectious and immune-pathological diseases. Full article

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)₂) and the counter-ion (Ph₄P⁺ or Et₄N⁺). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC₅₀ values ranged from 0.1–8 $\mathsf{\mu }$M (A2780) and 0.8–29 $\mathsf{\mu }$M (OVCAR8). The complexes with the Ph₄P⁺ ([P]) counter-ion are in general more active than their Et₄N⁺ ([N]) analogues, presenting IC₅₀ values in the same order of magnitude or even lower than Auranofin. Studies in the zebrafish embryo model further showed that, despite their marked anticancer effect, the complexes with [P] counter-ion exhibited low in vivo toxicity. In general, the exocyclic exchange of sulfur by oxygen or ylidenemalononitrile (C(CN)₂) enhanced the compounds toxicity. Most complexes containing the [P] counter ion exhibited exceptional antiplasmodial activity against the Plasmodium berghei parasite liver stages, with submicromolar IC₅₀ values ranging from 400–700 nM. In contrast, antibacterial/fungi activities were highest for most complexes with the [N] counter-ion. Auranofin and two selected complexes [P][AuSBu(=S)] and [P][AuSEt(=S)] did not present anti-HIV activity in TZM-bl cells. Mechanistic studies for selected complexes support the idea that thioredoxin reductase, but not DNA, is a possible target for some of these complexes. The complexes [P] [AuSBu(=S)], [P] [AuSEt(=S)], [P] [AuSEt(=Se)] and [P] [AuSeiPr(=S)] displayed a strong quenching of the fluorescence intensity of human serum albumin (HSA), which indicates a strong interaction with this protein. Overall, the results highlight the promising biological activities of these complexes, warranting their further evaluation as future drug candidates with clinical applicability. Full article

Antimicrobials in wastewater promote the emergence of antibiotic resistance, facilitated by selective pressure and transfer of resistant genes. Enteric bacteria belonging to Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Citrobacter species (n = 126) from hospital effluents and proximate wastewater treatment plant were assayed for susceptibility to four antimicrobial classes. The β-lactamase encoding genes harbored in plasmids were genotyped and the plasmids were sequenced. A multidrug resistance phenotype was found in 72% (n = 58) of E. coli isolates, 70% (n = 43) of Klebsiella species isolates, and 40% (n = 25) of Enterobacter and Citrobacter species. Moreover, 86% (n = 50) of E. coli, 77% (n = 33) of Klebsiella species, and 25% (n = 4) of Citrobacter species isolates phenotypically expressed extended spectrum β-lactamase. Regarding ESBL genes, bla_CTX-M-27 and bla_TEM-1 were found in E. coli, while Klebsiella species harbored bla_CTX-M-15, bla_CTX-M-30, or bla_SHV-12. Genes coding for aminoglycoside modifying enzymes, adenylyltransferases (aadA1, aadA5), phosphotransferases (aph(6)-1d, aph(3″)-Ib), acetyltransferases (aac(3)-IIa), (aac(6)-Ib), sulfonamide/trimethoprim resistant dihydropteroate synthase (sul), dihydrofolate reductase (dfrA), and quinolone resistance protein (qnrB1) were also identified. Monitoring wastewater from human sources for acquired resistance in clinically important bacteria may provide a cheaper alternative in regions facing challenges that limit clinical surveillance. Full article

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis. Full article