Prevalence of polymorphisms in marker genes associated with antimalarial drug resistance in Plasmodium falciparum following 10 years of artemisinin-based combination therapy implementation in urban Kolkata.

Context: Resistance to antimalarial drugs is one of the major challenges for malaria elimination. In India, artemisinin combination therapy (artesunate-sulfadoxin pyrimethamine) was introduced in place of chloroquine (CQ) for the treatment of uncomplicated falciparum malaria in 2010. Periodical monitoring of polymorphisms in antimalarial drug resistance marker genes will be useful for assessing drug pressure, mapping and monitoring of drug resistance status; and will be helpful for searching alternative treatments. Objectives: This study was conducted to study the polymorphisms in antimalarial drug resistance marker genes among clinical Plasmodium falciparum isolates collected from Kolkata after 10 years of artemisinin-based combination therapie (ACT) implementation. Materials and Methods: Blood samples were collected from P. falciparum mono-infected patients and polymorphisms in P. falciparum CQ resistance transporter (pfcrt), P. falciparum multidrug resistance (pfmdr-1), P. falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthetase (pfdhps), pfATPase6 and pfK-13 propeller genes were analysed by polymerase chain reaction and DNA sequencing. Results: In pfcrt gene, C72S, and K76T mutation was recorded in 100% isolates and no mutations was detected in any of the targeted codons of pfmdr-1 gene. A double mutant pfcrt haplotype SVMNT and wildtype haplotype NYD in pfmdr-1 were prevalent in 100% of study isolates. Triple mutant pfdhfr-pfdhps haplotype ANRNI-SGKAA was recorded. No polymorphism in pfK13 gene was documented in any of the isolates. Conclusions: Observed wild codon N86 along with Y184 and D1246 of pfmdr-1 gene might be an indication of the reappearance of CQ sensitivity. The absence of quadruple and quintuple haplotypes in pfdhfr-pfdhps gene along with the wild haplotype of pfK13 is evidence of ACT effectivity. Hence, similar studies with large sample size are highly suggested for monitoring the drug resistance status of P. falciparum.

In vitro activity of the newly approved antimicrobial agent Cefiderocol against Carbapenem resistant Gram negative clinical isolates.

INTRODUCTION: Carbapenem resistant Gram negative bacteria have emerged as priority pathogens in recent years. Cefiderocol is a siderophore cephalosporin licensed in 2019 with claimed activity against ESBL producing and carbapenem resistant bacteria with much better safety margin compared to colistin. The present study was undertaken to assess the in vitro activity of cefiderocol against carbapenem resistant clinical isolates, compared to some select antimicrobial agents including colistin. MATERIALS AND METHODS: Seventy-seven isolates of Gram negative bacteria belonging to the three commonly encountered groups of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp were included. Susceptibility testing for Cefiderocol was determined by Kirby-Bauer's disk diffusion technique as per CLSI guidelines using Cefiderocol disc (30 µg). Sensitivity for the other agents were determined using automated system. RESULTS: Of the 77 isolates, 58.4% belonged to Enterobacterales, followed by P.aeruginosa (27.3%) and Acinetobacter spp (14.3%). Three out of 45 Enterobacterales isolates, one out of 21 P.aeruginosa and none in the Acinetobacter group were found resistant to cefiderocol. All the isolates were intermediate sensitive (I) for colistin since the "susceptible" interpretive category has been eliminated. Tigecycline showed good activity (80.0% sensitive) against Enterobacterales followed by aztreonam (71.1% sensitive). CONCLUSION: Cefiderocol is not yet available in India and our study is possibly the second one from this country demonstrating in vitro resistance to this important antimicrobial agent. However, with a relatively better safety profile compared to colistin, cefiderocol can be an important agent to combat these highly resistant pathogens.

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

INTRODUCTION: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. METHODOLOGY: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. RESULTS: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. CONCLUSIONS: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

Prevalence of histidine-rich protein 2 deletion among the Plasmodium falciparum isolates from Kolkata.

Context: Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. HRP2 has advantages over other biomarkers because of its abundance in the bloodstream, repetitive binding epitopes, and falciparum-specificity. Most HRP2-based RDTs also exhibit some cross-reactivity to a closely related protein (HRP3). Plasmodium falciparum parasites lacking HRP2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs. Objectives: The objective of the study was to study the sensitivity and specificity of hrp2-based RDT for diagnosis of falciparum, to compare the RDT results with microscopy and polymerase chain reaction (PCR), and to determine the prevalence of HRP2 gene deletion among the RDT-negative, microscopy-positive falciparum strains. Materials and Methods: Blood samples were collected and diagnosis was done by microscopic examination, RDTs, and PCR. Results: Out of 1000 patients examined, 138 were positive for P. falciparum. Fever was the most common symptom followed by chills with rigor and headache were recorded among more than >95% of the study patients. Three microscopy-confirmed P. falciparum cases were negative by HRP2-based RDT and were found to have deletion of HRP2 and HRP3 exon 2. Conclusions: Rapid and accurate diagnosis and prompt deployment of effective antimalarial medication are essential components of appropriate case management. P. falciparum strains that evade diagnosis by RDTs represent a major threat to malaria control and elimination efforts.

Molecular Characterization and Identification of Potential Inhibitors for 'E' Protein of Dengue Virus.

Dengue is an arthropod-borne acute febrile illness caused by Dengue Virus (DENV), a member of Flaviviridae. Severity of the infection ranges from mild self-limiting illness to severe life-threatening hemorrhagic fever (DHF) and dengue shock syndrome (DSS). To date, there is no specific antiviral therapy established to treat the infection. The current study reports the epidemiology of DENV infections and potential inhibitors of DENV 'E' protein. Among the various serotypes, DENV-2 serotype was observed more frequently, followed by DENV-4, DENV-1, and DENV-3. New variants of existing genotypes were observed in DENV-1, 2, and 4 serotypes. Predominantly, the severe form of dengue was attributable to DENV-2 infections, and the incidence was more common in males and pediatric populations. Both the incidence and the disease severity were more common among the residents of non-urban environments. Due to the predominantly self-limiting nature of primary dengue infection and folk medicine practices of non-urban populations, we observed a greater number of secondary dengue cases than primary dengue cases. Hemorrhagic manifestations were more in secondary dengue in particularly in the pediatric group. Through different computational methods, ligands RGBLD1, RGBLD2, RGBLD3, and RGBLD4 are proposed as potential inhibitors in silico against DENV-1, -2, -3, and -4 serotypes.

Sir U.N. Brahmachari and his battle against Kala-Azar.

Kala-azar or visceral leishmaniasis was at one time a scourge in the Bengal Presidency of British India comprising the present Indian states of Bengal, Bihar, Assam, and Odisha. The disease was rampant along the Ganga and Brahmaputra River adjoining areas. In the early 1900s, the treatment initiated was by the intravenous injection of tartar emetic, which had a narrow safety level and long-term use was marked with multiple side effects. In 1920, Upendranath Brahmachari discovered urea stibamine, which is the urea salt of para-amino phenyl stibnic acid and it revolutionized the treatment of Kala-azar with >90% cure rate and with minimal side effects. He is also credited with the description of post-kala-azar dermal leishmaniasis. He was conferred the knighthood of the British Empire as recognition of his important contribution. Although his name was twice nominated for Nobel Prize, unfortunately, he never received it.

The forgotten malariologist: Giovanni Battista Grassi (1854-1925).

The discovery of the mosquito as a vector for malaria parasite was an important discovery at the turn of the 19th century for which Sir Ronald Ross received the Nobel Prize in 1902. Battista Grassi, an Italian physician and a zoologist is also credited with this discovery and he described the species of the mosquito and proved the transmission in healthy human volunteer. Although we remember his name only in this context, he also made numerous other discoveries spanning the fields of protozoology, helminthology, entomology, and zoology.

Staphylococcus aureus grown in anaerobic conditions exhibits elevated glutamine biosynthesis and biofilm units.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L-1 for glutamate and 2.4 ± 0.03 mmol·L-1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Lophomonas blattarum: A new flagellate causing respiratory tract infections.

Lophomonas blattarum is a flagellate protozoan parasite which was originally described as a commensal in the gut of cockroaches. From the 1990s, reports started coming out of peoples Republic of China about its possible role in bronchopulmonary infections, and this was followed by reports from some other parts of the world as well. There had been some skepticism regarding the misidentification of bronchial ciliated epithelial cells as L. blattarum, but recent use of molecular diagnosis has come as an aid in clearing the controversy. This review focuses on the various aspects of the parasite including its biology, epidemiology, clinical manifestations, laboratory diagnosis, and the treatment aspects. Molecular diagnosis has recently been employed and more reports concerning its validation is needed. More basic research concerning the genomic and proteomic analysis is necessary to develop reliable molecular and serological tests for this parasite in future.

Serum procalcitonin as a biomarker of bloodstream infection & focal bacterial infection in febrile patients.

Background & objectives: Bacteraemia is a serious form of infection in patients presenting with fever, thus, there is a necessity for a biomarker for rapid diagnosis of bacteraemia in such patients to make better therapeutic decisions. This study was conducted to measure the serum procalcitonin (PCT) levels at the time of initial presentation as a biomarker for identifying bacteraemia and as a predictor of mortality in patients admitted with acute fever. Methods: Four hundred and eighty patients, who presented with acute fever requiring admission to a tertiary care teaching hospital in south India, were prospectively studied. All patients were evaluated with a detailed history, physical examination, laboratory and imaging studies. Baseline serum PCT was measured for each patient within six hours of admission. Results: Among patients with single infectious cause (n=275), significantly higher median serum PCT levels were evident in bacteraemia compared to leptospirosis (P=0.002), dengue (P <0.001), scrub typhus (P <0.001) and evident focus of infection without bacteraemia (P=0.036). By receiver-operator characteristic curve analysis, at a cut-off value of >3.2 ng/ml, the sensitivity and specificity of serum PCT levels in predicting bacteraemia were 81.1 and 63.3 per cent, respectively. As per the worst-case scenario analysis, 91 (18.9%) patients had a poor outcome and these had significantly higher median serum PCT levels compared to survivors (n=389) [9.46 (2.03-44.4) vs. 1.23 (0.34-7.645); P <0.001]. At a cut-off value of >3.74 ng/ml, serum PCT levels at initial presentation predicted in-hospital mortality with a sensitivity and specificity of 67 and 67.5 per cent, respectively. Interpretation & conclusions: Our observations suggest that serum PCT level may be a useful biomarker for identifying bacteraemia as well as predicting mortality in patients with acute fever requiring admission to hospital.

Schizophrenia and bipolar disorders: The Toxoplasma connection.

The infectious etiology of psychiatric illnesses has remained an unexplored area till recently. During the past two decades, numerous studies from multiple angles have tried to link chronic toxoplasmosis with schizophrenia and bipolar disorders, among others. Most of the evidence has come from serological studies in the patient population, but other facets have also been explored. This review examines the various areas from which a causal link has been deduced and includes: (a) serological studies, (b) effect of maternal toxoplasmosis on children, (c) neurotransmitter studies, (d) parasite localization in the brain, (e) role of cytokines, and (f) psychotherapy and its effect on Toxoplasma. However, multiple factors may play a role in the etiopathogenesis of psychiatric illnesses, and chronic Toxoplasma infection may be considered an important risk factor for the genesis and symptomatology of schizophrenia and bipolar disorders.

Iron enhances the peptidyl deformylase activity and biofilm formation in Staphylococcus aureus.

Staphylococcus aureus plays a major role in persistent infections and many of these species form structured biofilms on different surfaces which is accompanied by changes in gene expression profiles. Further, iron supplementation plays a critical role in the regulation of several protein(s)/enzyme function, which all aid in the development of active bacterial biofilms. It is well known that for each protein, deformylation is the most crucial step in biosynthesis and is catalyzed by peptidyl deformylase (PDF). Thus, the aim of the current study is to understand the role of iron in biofilm formation and deformylase activity of PDF. Hence, the PDF gene of S. aureus ATCC12600 was PCR amplified using specific primers and sequenced, followed by cloning and expression in Escherichia coli DH5α. The deformylase activity of the purified recombinant PDF was measured in culture supplemented with/without iron where the purified rPDF showed Km of 1.3 mM and Vmax of 0.035 mM/mg/min, which was close to the native PDF (Km = 1.4 mM, Vmax = 0.030 mM/mg/min). Interestingly, the Km decreased and PDF activity increased when the culture was supplemented with iron, corroborating with qPCR results showing 100- to 150-fold more expression compared to control in S. aureus and its drug-resistant strains. Further biofilm-forming units (BU) showed an incredible increase (0.42 ± 0.005 to 6.3 ± 0.05 BU), i.e., almost 15-fold elevation in anaerobic conditions, indicating the significance of iron in S. aureus biofilms.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Antibiotic resistance & pathogen profile in ventilator-associated pneumonia in a tertiary care hospital in India.

BACKGROUND & OBJECTIVES: Ventilator-associated pneumonia (VAP) is an important hospital-acquired infection with substantial mortality. Only a few studies are available from India addressing the microbiological aspects of VAP, which have been done with small study populations. This study was carried out in the intensive care units (ICUs) of a tertiary care hospital to assess the profile of pathogens and to determine the pattern of antimicrobial resistance. METHODS: This was a retrospective study of clinically suspected cases of VAP. Over a three year period, a total of 247 cases in 2011, 297 in 2012 and 303 in 2013 admitted in ICUs on mechanical ventilation with clinical evidence of VAP were included in our study. The endotracheal aspirate samples from these suspected cases were subjected to quantitative culture technique, and colony count of ≥10[5] colony forming units/ml was considered significant. Antimicrobial susceptibility test for the isolates was done. RESULTS: VAP rates of 44.1, 43.8 and 26.3 were seen in 2011, 2012 and 2013, respectively. In all the three years, non-fermentative Gram-negative bacilli were the predominant organisms, followed by Pseudomonas spp. and Klebsiella spp. Staphylococcus aureus exhibited a downwards trend in prevalence from 50.0 per cent in 2011 to 34.9 per cent in 2013. An increase in vancomycin-resistant enterococci was seen from 4.3 per cent in 2012 to 8.3 per cent in 2013, while methicillin resistance amongst the S. aureus crossed the 50 per cent mark in 2013. An increasing trend in resistance was shown by Pseudomonas spp. for piperacillin-tazobactam (PTZ), amikacin and imipenem (IPM). For the non-fermenters, resistance frequency remained very high except for IPM (33.1%) and polymyxin-B (2.4%). INTERPRETATION & CONCLUSIONS: Our findings show VAP as an important problem in the ICU setting. The incidence of multidrug-resistant pathogens was on the rise. The resistance pattern of these pathogens can help an institution to formulate effective antimicrobial policy. To have a comprehensive pan-India picture, multicentric studies are needed.

Factor V Leiden mutation is not a predisposing factor for acute coronary syndromes.

BACKGROUND: The prevalence of Coronary artery disease (CAD) in India has increased considerably over the past few years and could become the number one killer disease if interventions are not done. Factor V Leiden (FVL) mutation and FII G20210A polymorphism are two recently described genetic factors with a propensity towards venous thrombosis. This warrants the investigations for thrombophilia in myocardial infarction patients in India. METHODS: The study cohort consisted of 51 patients aged below 50 years presenting with acute coronary syndromes. In both patient group and normal individuals the major risk factors Protein C deficiency, Protein S deficiency, anticardiolipin antibodies, Fibrinogen and Lipoprotein [a] were studied. Factor V Leiden (FVL) G1691A mutation in both control and patient group was looked by using Polymerase chain reaction (PCR) followed by sequencing of the PCR products. RESULTS: Our results indicated significantly higher levels of anticardiolipin antibodies and fibrinogen in the patients and absence of FVL (G1691A) mutation in our study cohort. One of the patients (H5) showed insertion of an extra A nucleotide in exon 10 of the Factor V gene resulting in frame shift mutation in this patient. CONCLUSION: The results of present study showed absence of FVL mutation in our population. However, there is a need to confirm the above findings on patients from different populations from different parts of the country. The insertion of an extra A in exon 10 in the patient needs to be ascertained to confirm that it is one of its kinds or is prevalent in the population.

Catheter lock technique: in vitro efficacy of ethanol for eradication of methicillin-resistant staphylococcal biofilm compared with other agents.

Biofilm formation in central venous catheters (CVC) is a prerequisite for catheter-related bloodstream infection (CRBSI). The catheter lock technique has been used to treat biofilm infection, but the ideal agent, concentration and the minimum exposure time necessary to eradicate the biofilms are not clearly known. In this study, biofilm-producing strains of staphylococci were used to find out the minimum biofilm eradication concentration of ethanol compared with three other conventional antibacterial agents. Eight representative methicillin-resistant staphylococci, from colonized CVCs, were studied. The biofilms were exposed to 1, 5 and 10 mg mL(-1) of gentamicin, ciprofloxacin and vancomycin. The ethanol concentrations used were 20%, 40% and 80%. Biofilms were examined for the presence of live organisms after exposure to these agents from 30 min to 24 h. The three antibiotics were unable to eradicate the biofilms even after 24 h, while ethanol at 40% concentration could do so for all the isolates in 1 h. Our study highlights the efficacy and rationale of using 40% ethanol for a short period as catheter lock solution to eradicate biofilms and thus to prevent CRBSI, instead of using high concentrations of antibiotics for extended periods.

Seroprevalence of IgG antibodies to Chlamydia pneumoniae and Helicobacter pylori among coronary heart disease patients and normal individuals in South Indian population.

In recent years, the role of infectious agents in the aetiology of atherosclerotic disease has come to the forefront. In the present study, seroprevalence (IgG) of chlamydia pneumoniae and helicobacter pylori in patients with atherosclerotic coronary heart disease was compared to normal healthy adults. Out of a total of 117 patients 101 had unstable angina (UA) and 16 had chronic stable angina (CSA). C. pneumoniae seropositivity was found in 66% of patients with UA and 94% of CSA patients. The corresponding figures for H. pylori were 58% and 56% respectively. In comparison, 81% of healthy adults were seropositive for C. pneumoniae and 53% for H.pylori. No significant association was found between CHD and the infectious agents. However, this study has revealed a high infection by C. pneumoniae as well as H.pylori in this part of India.