Novel Antitubercular Agents: Design, Synthesis, Molecular Dynamic and Biological Studies of Pyrazole - 1,2,4-Triazole Conjugates.

Mycobacterium tuberculosis (Mtb) has numerous cell wall and non-cell wall mediated receptors for drug action, of which cell wall mediated targets were found to be more promising because of their pivotal role in bacterial protection and survival. Herein, we reported the design and synthesis of a series of pyrazole-linked triazoles based on the reported structural features of promising drug candidates that target DprE1 receptors through a Structure-based drug design (SBDD) approach (6a-6j and 7a-7j). The synthesized compounds were evaluated for their in-vitro antitubercular activity against virulent strains of Mtb H37Rv. In-silico studies revealed that most compounds exhibit binding interactions with crucial amino acids like Lys418, Tyr314, Tyr60, and Asp386 at DprE1. Furthermore, the protein-ligand (7j) shows appreciable stability compared to innate protein in a 100 ns molecular dynamic simulation study. In-vitro MAB assay revealed that 14 compounds exhibit significant antitubercular activity with minimum inhibitory concentration (MIC) of the 3.15-4.87 µM of the 20 compounds tested. An in-vitro cytotoxicity study on normal cell lines (MCF10) revealed safe compounds (IC50 values:341.85 to 726.08 µM). Hence, the present study opens the development of new pyrazole-linked triazoles as probable DprE1 inhibitors.

Idiopathic CD4+ T lymphocytopenia: A case report.

Idiopathic CD4+ T lymphocytopenia (ICL) is a very rare immunodeficiency syndrome with an unexplained depletion of CD4+ T lymphocytes and no evidence of Human Immunodeficiency Virus (HIV) infection. Here we report a 29-year-old male patient who had severe ulcerative colitis with low level CD4+ count of 254 cells/mm3, and had no evidence of HIV or Human T cell Lymphotrophic virus type I or II infections. He had recurrent Candidiasis infection and his CD4 count was just 53 cells/mm3 after 3 months. The cause for the decline of CD4 T lymphocytes was unknown.

Retrospective analysis on distribution and antifungal susceptibility profile of Candida in clinical samples: a study from Southern India.

Introduction: Candida is one of the rising primary causes of infections connected with health care. However, their distribution and susceptibility patterns vary widely amongst different regions. Method: The study was carried out to retrospectively analyze the distribution of Candida in various clinical samples, their species types and susceptibility, in a tertiary care hospital, in India for 4 years using the Vitek-2 database. Results: Candida infection was identified in 751 clinical samples, and the major source of infection was found to be urine samples accounting for about 58.32%. A total of 18 different Candida species were recorded. Non-albicans Candida (NAC) 73.64% (n = 553) predominated Candida albicans 26.36% (n = 198). Candida tropicalis was found to be identified at a higher frequency followed by C. albicans, Candida glabrata and Candida parapsilosis. Candida tropicalis was the only species which were recovered from bile; Candida pelliculosa was recorded merely from blood and Candida lipolytica from urine and blood and not in any other samples. In vaginal swabs, C. albicans accounted for 63.64% (n = 14) compared to NAC 36.36% (n = 8). The susceptibility test revealed that 75.44% (n = 559) isolates were susceptible and 24.56% (n = 182) were resistant to one or more drugs tested. Major resistance was exhibited to flucytosine by C. tropicalis 77.46% (n = 55) compared to C. albicans 11.27% (n = 8). Apart from C. albicans, NAC-C. tropicalis, C. glabrata and Candida krusei showed resistance to echinocandins, and Candida haemulonii to amphotericin-B. Conclusion: The knowledge of the incidence, resistance and emergence of different species might guide clinicians to select an appropriate antifungal therapy and plan effective strategies to control invasive and systemic Candida infections.

Exploring the antibiogram of soil isolates from an indian hospital precinct: link to antibiotic usage.

OBJECTIVE: Hospitals serve as hotspots of antibiotic resistance. Despite several studies exploring antibiotic resistance in hospitals, none have explored the resistance profile of soil bacteria from a hospital precinct. This study examined and compared the antibiogram of the soil isolates from a hospital and its affiliated university precinct, to determine if antibiotic resistant bacteria were present closer to the hospital. RESULTS: 120 soil samples were collected from JSS Hospital and JSS University in Mysore, India across three consecutive seasons (monsoon, winter and summer). 366 isolates were randomly selected from culture. Antibiotic susceptibility testing was performed on 128 isolates of Pseudomonas (n = 73), Acinetobacter (n = 30), Klebsiella species (n = 15) and Escherichia coli (n = 10). Pseudomonas species exhibited the highest antibiotic resistance. Ticarcillin-clavulanic acid, an extended-spectrum carboxypenicillin antibiotic used to treat moderate-to-severe infections, ranked highest amongst the antibiotics to whom these isolates were resistant (n = 51 out of 73, 69.9%). Moreover, 56.8% (n = 29) were from the hospital and 43.1% (n = 22) were from the university precinct, indicating antibiotic resistant bacteria were closer to the hospital setting. This study highlights the effect of antibiotic usage in hospitals and the influence of anthropogenic activities in the hospital on the dissemination of antibiotic resistance into hospital precinct soil.

Case Report: Pharyngeal Diphtheria in a Pregnant Woman from South India.

Diphtheria is a vaccine-preventable disease and is caused by toxigenic strains of Corynebacterium diphtheriae. Several case reports have been published in the recent years, and this resurgence of cases has occurred mainly in adolescent and adult populations. Also, several research articles have reported waning immunity against diphtheria in adults who have completed childhood immunization. Thus, it is an important need to conduct larger sero-surveillance studies to understand the cause of rising diphtheria cases. Here, we report a case of a 23-year-old pregnant women of 8 weeks' gestation who presented to the outpatient department with fever, severe throat pain, odynophagia, dysphagia, neck pain, and neck swelling of 3 days' duration. On clinical examination, a gray, leathery membrane was noted on the soft palate. An Albert's stain from the membrane revealed organisms resembling Corynebacterium diphtheriae. Appropriate treatment was initiated immediately, and follow-up examination at 2 weeks from date of discharge was uneventful. The gray membrane had completely resolved. Contact tracing was done and the appropriate antimicrobial agent was administered. This case study indicates the importance of timely clinical and microbiological diagnosis and reinforces the previously reported resurgence of diphtheria infection.

Antifungal Resistance of Candida Species Isolated from HIV Patients in a Tertiary Care Hospital, Mysuru, Karnataka.

CONTEXT: Candidiasis still remains as a common opportunistic infection in patients with human immunodeficiency virus (HIV). Drug resistance has become a serious health concern because of indiscriminate usage and dosage. AIM: To determine the antifungal resistance pattern of Candida albicans and non-albicans Candida (NAC) from HIV patients. SUBJECTS AND METHODS: The study was carried out in the department of microbiology at a tertiary care hospital. Candida isolates obtained from HIV patients were tested for drug susceptibility by Vitek-2 automated system. RESULTS: Antifungal susceptibility pattern (n=109) revealed that 15% of the isolates were resistant to at-least one and 85% were sensitive to all the drugs tested. About 10% and 19% of C. albicans showed resistance to fluconazole and flucytosine respectively. Among non-albicans tested, only C. tropicalis (14%) exhibited resistance to flucytosine. CONCLUSIONS: Knowledge on epidemiology, species prevalence, and drug resistance pattern may guide for effective therapy. This reduces morbidity and also improves the quality of life.

Performance of molecular and serologic tests for the diagnosis of scrub typhus.

Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its non-specific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluates the diagnostic performance of various tests. The present study aims at assessing the accuracy of various rapid diagnostic tests, serologic tests, and nucleic acid amplification methods on well-characterized patient samples. Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood, eschar or tissue were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative real time PCR using 47kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil-Felix test(WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT).Among the 316 participants, 158 had confirmed scrub typhus (cases) and 158 were controls. ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies had excellent discriminative potential with sensitivities and specificities of 92%, 94% and 92%, 92% respectively. The sensitivity and specificity of IFA were found to be 95% and 74% respectively. IgM serology had a false positivity rate of 8% with other acute febrile illnesses such as dengue, leptospirosis and spotted fever due to the nonspecific binding of the pentavalent IgM. LAMP assay had 91.7% sensitivity and 77.2% specificity while qPCR provided excellent sensitivity (97%) and perfect specificity. In conclusion, ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies have excellent sensitivity and specificity while the accuracy of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories particularly for diagnosis of early disease with less than 7 days duration. This study provides a comprehensive evaluation of all currently available diagnostic tests for scrub typhus.

Development of a biofilm inhibitor molecule against multidrug resistant Staphylococcus aureus associated with gestational urinary tract infections.

Urinary Tract Infection (UTI) is a globally widespread human infection caused by an infestation of uropathogens. Eventhough, Escherichia coli is often quoted as being the chief among them, Staphylococcus aureus involvement in UTI especially in gestational UTI is often understated. Staphylococcal accessory regulator A (SarA) is a quorum regulator of S. aureus that controls the expression of various virulence and biofilm phenotypes. Since SarA had been a focussed target for antibiofilm agent development, the study aims to develop a potential drug molecule targeting the SarA of S. aureus to combat biofilm associated infections in which it is involved. In our previous studies, we have reported the antibiofilm activity of SarA based biofilm inhibitor, (SarABI) with a 50% minimum biofilm inhibitory concentration (MBIC50) value of 200 µg/mL against S. aureus associated with vascular graft infections and also the antibiofilm activity of the root ethanolic extracts of Melia dubia against uropathogenic E. coli. In the present study, in silico design of a hybrid molecule composed of a molecule screened from M. dubia root ethanolic extracts and a modified SarA based inhibitor (SarABI(M)) was undertaken. SarABI(M) is a modified form of SarABI where the fluorine groups are absent in SarABI(M). Chemical synthesis of the hybrid molecule, 4-(Benzylamino)cyclohexyl 2-hydroxycinnamate (henceforth referred to as UTI Quorum-Quencher, UTI(QQ)) was then performed, followed by in vitro and in vivo validation. The MBIC50 and MBIC90 of UTI(QQ) were found to be 15 and 65 µg/mL, respectively. Confocal laser scanning microscopy (CLSM) images witnessed biofilm reduction and bacterial killing in either UTI(QQ) or in combined use of antibiotic gentamicin and UTI (QQ) . Similar results were observed with in vivo studies of experimental UTI in rat model. So, we propose that the drug UTI(QQ) would be a promising candidate when used alone or, in combination with an antibiotic for staphylococcal associated UTI.

A study of dermatophytes and their in-vitro antifungal sensitivity.

Of the 150 clinically suspected cases of Dermatophytosis studied, majority of the cases were from age group 11-20 and 21-30 (51.4%), Tinea corporis (48.7%) and Tinea capitis (18%) were the commonest clinical types. The isolation rate was 24% (36) of which 19 (52.7%) were Trichophyton rubrum, 11 (30.55%) were Trichophyton mentagrophytes and 4 (11.1%) were Trichophyton violaceum. One isolate each of Microsporum gypseum & Epidermophyton floccosum were obtained. Griseofulvin proved to be the best drug with a sensitivity of 94.4% followed by Miconazole (75% sensitive). Tolnaftate showed a sensitivity of 47.22%. For Clotrimazole only 30.55% of the isolates were sensitive.

Subperiodic, asymptomatic microfilaremia in an adult male from Mysore: a nonendemic area.

Wuchereria bancrofti is found throughout tropics and subtropics like Asia, Pacific islands, Africa, areas of South America and Caribbean basin. In all these areas, except Pacific islands, microfilaria occurs in the periodic form, in which case the microfilaria are found in large numbers in the peripheral blood during night. In the Pacific islands, they occur in the subperiodic form, i.e., microfilaria are present in the peripheral blood at all times and reach the maximum level of parasitemia in the afternoon. Microfilaria of Wuchereria bancrofti and Brugia malayi occurring in India displays a nocturnal periodicity, appearing in large numbers at night. This is the biological adaptation to the nocturnal biting habits of the vector mosquitoes. The maximum density in blood is reported between 10 PM and 2 AM. Here is a case report of asymptomatic microfilaremia showing subperiodicity, which is very unusual in India.