Randomized Trial of Metformin With Anti-Tuberculosis Drugs for Early Sputum Conversion in Adults With Pulmonary Tuberculosis.

BACKGROUND: Metformin, by reducing intracellular Mycobacterium tuberculosis growth, can be considered an adjunctive therapy to anti-tuberculosis treatment (ATT). We determined whether metformin with standard ATT reduces time to sputum culture conversion and tissue inflammation in adults with pulmonary tuberculosis (PTB). METHODS: In a randomized, 8-week, clinical trial, newly diagnosed, culture-positive PTB patients were randomized to standard ATT (HREZ = control arm) or standard ATT plus daily 1000 mg metformin (MET-HREZ = Metformin with Rifampicin [METRIF] arm) for 8 weeks during 2018-2020 at 5 sites in India. The primary end point was time to sputum culture conversion by liquid culture during 8 weeks of ATT. Plasma inflammatory markers were estimated in a subset. A Cox proportional hazard model was used to estimate time and predictors of culture conversion. RESULTS: Of the 322 patients randomized, 239 (74%) were male, and 212 (66%) had bilateral disease on chest radiograph with 54 (18%) showing cavitation. The median time to sputum culture conversion by liquid culture was 42 days in the METRIF arm and 41 days in the control arm (hazard ratio, 0.8; 95% confidence interval [CI], .624-1.019). After 8 weeks of ATT, cavitary lesions on X-ray (7, 5.3% vs 18, 12.9%; relative risk, 0.42; 95% CI, .18-.96; P = .041) and inflammatory markers were significantly lower in the METRIF arm. Higher body mass index and lower sputum smear grading were associated with faster sputum culture conversion. CONCLUSIONS: The addition of metformin to standard ATT did not hasten sputum culture conversion but diminished excess inflammation, thus reducing lung tissue damage as seen by faster clearance on X-ray and reduced inflammatory markers. CLINICAL TRIALS REGISTRATION: Clinical Trial Registry of India (CTRI/2018/01/011176).

Vaginosis: Advances in new therapeutic development and microbiome restoration.

Vaginosis is a condition experienced by most women at least once in their lifetime. This condition arises due to the imbalance in the microbiome of the vaginal ecosystem. Most of the pathogens of this disease are organisms which are commonly found in a normal healthy vagina. The vaginal microbiome is important as they act as a primary defence against secondary infections and Sexually transmitted diseases and infections (STDs and STIs). The vagina is mostly dominated by Lactobacillus along with other microbes including Gardnerella vaginalis, Atopobium vaginae., Prevotella spp., Mobiluncus spp., etc. Vaginal microbiome also includes Candida albicans and other species of the genus. The ratio in which these species are present varies from person to person and the dominant species decides the whether a vagina is "normal" or not. Lactobacillus dominated vagina is considered normal and if dominated by Gardnerella and such it is considered to be Bacterial vaginosis (BV) and similarly for Vulvovaginal Candidiasis (VVC). The microbiome also undergoes changes during menstrual cycles and menopausal stages. Due to the dynamic nature of this microbiome, it is tough to perfectly restore the balance. But several treatments are currently available with antibiotics like Clindamycin and derivatives of 5-nitroimidazole drugs like Metronidazole. The extensive use and the non-adherence to the treatment regimen has led to drug resistance through biofilm formation, efflux pumps, single nucleotide polymorphisms and resulting recurrent episode of vaginosis in women. Alternative medicines, preparations from plant sources, anti-microbial peptides and nano formulations are also being explored. Most of these medicines tend to focus on reducing the pathogen load rather than restoring the balance of the ecosystem. Vaginal microbiome transplant, an effort to restore the normalcy in the vaginal environment is becoming a popular treatment. In this review we discuss about the types of vaginosis, available treatments, challenges in treating the condition and the new drugs that are under investigation.

Effect of Metformin on systemic chemokine responses during anti-tuberculosis chemotherapy.

BACKGROUND: Metformin (MET), by boosting immunity, has been suggested as a host-adjunctive therapy to anti-tuberculosis treatment (ATT). METHODS: We evaluated whether adding MET to the standard ATT can alter the host chemokine response. We investigated the influence of metformin on the plasma levels of a wide panel of chemokines in a group of active tuberculosis patients before treatment, at 2nd month of ATT and at 6-months of ATT as part of our clinical study to examine the effect of metformin on ATT. RESULTS: Our results demonstrated that addition of metformin resulted in diminished CC (CCL1 and CCL3) and CXC (CXCL-2 and CXCL-10) chemokines in MET arm as compared to non-MET arm at the 2nd month and 6th month of ATT. In addition to this, MET arm showed significantly diminished chemokines in individuals with high bacterial burden and cavitary disease. CONCLUSION: Our current data suggest that metformin alters chemokines responses that could potentially curb excessive inflammation during ATT.

Effect of coadministration of moxifloxacin and rifampin on Mycobacterium tuberculosis in a murine aerosol infection model.

Coadministration of moxifloxacin and rifampin was evaluated in a murine model of Mycobacterium tuberculosis pulmonary infection to determine whether the finding of antagonism documented in a hollow-fiber infection model could be recapitulated in vivo. Colony counts were followed in a no-treatment control group, groups administered moxifloxacin or rifampin monotherapy, and a group administered a combination of the two agents. Following 18 days of once-daily oral administration to mice infected with M. tuberculosis, there was a reduction in the plasma exposure to rifampin that decreased further when rifampin was coadministered with moxifloxacin. Pharmacodynamic analysis demonstrated a mild antagonistic interaction between moxifloxacin and rifampin with respect to cell kill in the mouse model for tuberculosis (TB). No emergence of resistance was noted over 28 days of therapy, even with monotherapy. This was true even though one of the agents in the combination (moxifloxacin) induces error-prone replication. The previously noted antagonism with respect to cell kill shown in the hollow-fiber infection model was recapitulated in the murine TB lung model, although to a lesser extent.

Immunochemical characterisation of a 29-Kda surface-associated molecule of Entamoeba histolytica and its recognition by serum from patients with amoebiasis.

A 29-Kda cytotoxic molecule of axenically-grown pathogenic Entamoeba histolytica (strain HM1) was purified from an amoebic extract by immuno-affinity chromatography with monoclonal antibodies. Immunoreactivity of the purified 29-Kda molecule altered significantly (p less than 0.01) after exposure to heat or trypsin, but remained unaltered after treatment with sodium metaperiodate. The 29-Kda molecule was recognised by serum from each of 13 patients with amoebic liver abscess. In an ELISA system, the molecule produced significantly higher (p less than 0.01) OD readings with these serum samples than with samples from asymptomatic cyst passers. No serum from healthy subjects or from patients with idiopathic ulcerative colitis or giardiasis had antibodies that reacted with the 29-Kda molecule. The immune response to the 29-Kda amoebic protein in man may indicate a specific role for this molecule in invasive amoebiasis.

Uses and limitations in the demonstration of specific circulating immune complexes in patients with amoebiasis.

A micro-enzyme linked immunosorbent assay (micro-ELISA) has been evaluated as a diagnostic test to detect amoebic antigen in polyethylene glycol (PEG) precipitated circulating immune complexes (CIC) in sera from patients with amoebiasis. The immune complexes were captured on rabbit anti-amoebic IgG-coated wells of microtitration plates and the complexed antigen was detected by enzyme linked antihuman immunoglobulins. A titre of greater than 160 for the immune complexes was considered to be of clinical significance. The immunoassay detected amoebic, antigen-specific CIC in 35 (94.5%) of 37 patients with confirmed amoebic liver abscess. Twenty (55.5%) of 36 clinically suspected cases of amoebic liver abscess had amoebic antigen-specific CIC and responded favourably to anti-amoebic chemotherapy. Only two (20%) of 10 cases of non-dysenteric symptomatic intestinal amoebic infection had amoebic antigen-specific CIC. One (10%) of 10 patients with non-amoebic intestinal disorders also had amoebic antigen in CIC. However, none of 15 cases of non-amoebic hepatic disorders that included hydatid disease, metastatic adenocarcinoma, hepatocellular carcinoma, cholecystitis and choledocal cyst, 13 cases of rheumatoid arthritis and 25 apparently healthy subjects had amoebic antigen in CIC. The levels of the amoebic antigen-specific CIC did not correlate (p greater than 0.05) with either the number of abscess(es) or lobe(s) of the liver involved. However, the levels of antigen-specific CIC were higher (p less than 0.01) in patients with a liver size of more than 5 cm below the right costal margin. Antigen-specific CIC levels tended to decline or disappear during 3-6 months following completion of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies which inhibit in vitro cytotoxicity of axenic Entamoeba histolytica.

A panel of 12 independent hybridoma cell lines secreting monoclonal antibodies to axenic E. histolytica (HM1) have been developed. A hybridoma cell line P4 C4 P2 F8 C8 (clone C8) produced monoclonal antibodies (MoAb C8) of IgG1 isotype which recognised a 29 KD surface associated antigen of amoebic trophozoites in Western immunoblot. Immunofluorescent probing with MoAb C8 employing live and acetone fixed amoebic trophozoites indicated 29 KD molecule on the surface plasma membrane of E. histolytica trophozoites. The MoAb C8 also agglutinated the live amoebic trophozoites. Pretreatment of amoebic trophozoites with anti 29 KD monoclonal antibody significantly (P less than 0.01) inhibited in vitro cytotoxicity of amoebic trophozoites to the cultured baby hamster kidney (BHK-21) cells. MoAb recognised a 29 KD molecule of E. histolytica trophozoites which mediated cytotoxic potentials of the parasite. The absence or variable degree of expression of cytotoxic 29 KD molecule may possibly serve as a marker to differentiate virulent/avirulent populations or strains of E. histolytica.

Characteristics of Escherichia coli isolates from infantile and childhood diarrhea.

Seventy-five strains of Escherichia coli from cases of infantile and childhood diarrhea were serogrouped and analysed in terms of hydrophobicity, mannose resistant haemagglutination and enterotoxigenicity. The strains were distributed over 21 serogroups of which 54.6% were hydrophobic, 37.3% were haemagglutinating and 66.5% were enterotoxigenic. EPEC strains were less than ETEC strains. LT producers were more than LTST or ST producers. MRHA activity was found to be well correlated with hydrophobicity.

Pharmacokinetics and dose response of anti-TB drugs in rat infection model of tuberculosis.

Robust and physiologically relevant infection models are required to investigate pharmacokinetic-pharmacodynamic (PK/PD) correlations for anti-tuberculosis agents at preclinical discovery. We have validated an inhalation-based rat infection model of tuberculosis harbouring mycobacteria in a replicating state, that is suitable for investigating pharmacokinetics and drug action of anti-tubercular agents. A reproducible and actively replicating lung infection was established in Wistar rats by inhalation of a series of graded inocula of Mycobacterium tuberculosis. Following an initial instillation of ∼10(5) log10 CFU/lung, M. tuberculosis grew logarithmically for the first 3 weeks, and then entered into a chronic phase with no net increase in pulmonary bacterial loads. Dose response of front-line anti-TB drugs was investigated following pharmacokinetic measurements in the plasma of infected rats. Rifampicin, Isoniazid, and Ethambutol dosed per orally exhibited bactericidality and good dose response with maximal effect of 5.66, 4.66, and 4.80 log10 CFU reductions in the lungs, respectively. In contrast, Pyrazinamide was merely bacteriostatic with 1.92 log10 CFU/lung reduction and did not reduce the bacterial burden beyond the initial bacterial loads present at beginning of treatment in spite of high Pyrazinamide blood levels. Rat infection model with actively replicating bacilli provides a physiologically distinct and pharmacologically relevant model that can be exploited to distinguish investigational compounds in to bacteriostatic or bactericidal scaffolds. We propose that this rat infection model though need more drug substance, can be used in early discovery settings to investigate pharmacology of novel anti-tubercular agents for the treatment of active pulmonary tuberculosis.

Complete Genome Sequence of Potato leafroll virus Isolates Infecting Potato in the Different Geographical Areas of India Shows Low Level Genetic Diversity.

Five Potato leafroll virus (PLRV) isolates were collected from five states representing different potato growing parts of India. The ssRNA genome sequences of these isolates were determined. The genome comprised of 5,883 nucleotides and deduced genome organization resembled other PLRV isolates. About 97.6-98.7 % similarities was observed within the Indian isolates and were more close to European, Canadian, African, American and Czech isolates (95.8-98.6 %) than to an Australian isolate (92.9-93.4 %). These isolates were 43.7-53.1 % similar to other poleroviruses and 29.1-29.3 % to Barley yellow dwarf virus, a luteovirus. Out of five isolates, the isolate PBI-6 was recombinant one as detected by RDP3 software. Multiple sequence alignment of nucleotide and amino acid sequences of different ORFs indicated that the ORF 3 and ORF 4, corresponding to coat protein and movement proteins are more conserved than other ORFs. Amino acid changes specific to Indian isolates were observed and it was more in ORF 2 than in ORF 0, ORF 3 and ORF 4. This is the first report of complete genome sequence of PLRV isolates from India, which reveals low level genetic diversity.

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C(6) or C(2) carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the DeltailvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the DeltailvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies.

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.

Cytopathogenicity of Entamoeba histolytica to human intestinal epithelial cells: inhibition by monoclonal antibodies and sera from amoebic patients.

Interactions of pathogenic Entamoeba histolytica (HM 1) with human intestinal epithelial cells (Henle-407) were investigated. The E. histolytica trophozoites adhered and cytolysed 87% of cultured epithelial cell monolayers. A significant (P less than 0.001) inhibition of cytopathic effect of amoebic trophozoites pretreated with monoclonal antibodies to a 29 kDa surface associated protein suggested utilization of the 29 kDa surface protein in recognition and cytolysis of epithelial target cells. The polyclonal sera from treated patients of amoebic liver abscess and anti-amoebic hyperimmune serum inhibited cytopathogenicity to a greater degree (P less than 0.001) than did the monoclonal antibodies. The data thus suggest involvement of several amoebic molecules in exercising cytopathogenicity to epithelial cells.

Immunoreactivity of Entamoeba histolytica antigens with sera from amoebic patients.

Immune sera from 15 patients with cured amoebic liver abscess were used to recognise the antigens of Entamoeba histolytica (HMI) by immunoblotting. The amoebic proteins most frequently recognised by sera from patients with cured amoebic liver abscess had molecular masses of 8, 13, 18, 22, 29, 38, 45, 67 and 94 kDa. Six plasma membrane-associated amoebic proteins of molecular mass 29, 38, 45-67 complex, 85 and 94 kDa were strongly recognised by such sera. Two plasma membrane-associated antigens of 108 and 129 kDa were not recognised by any sera. None of the crude or plasma membrane-associated antigens were recognised by sera from five patients of idiopathic ulcerative colitis, five patients of persistent giardiasis and five normal healthy subjects. Identification of such antigens, especially plasma membrane-associated antigens may pave a way to develop specific diagnostic and immunoprotective agents.

Study of the interaction between bacteriophage T4 asiA and Escherichia coli sigma(70), using the yeast two-hybrid system: neutralization of asiA toxicity to E. coli cells by coexpression of a truncated sigma(70) fragment.

The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma(70) was studied by using the yeast two-hybrid system. Truncation of sigma(70) to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma(70) fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma(70) and asiA could be expressed in large quantities and purified.

Pharmacokinetics-pharmacodynamics of rifampin in an aerosol infection model of tuberculosis.

Limited information exists on the pharmacokinetic (PK)-pharmacodynamic (PD) relationships of drugs against Mycobacterium tuberculosis. Our aim was to identify the PK-PD parameter that best describes the efficacy of rifampin on the basis of in vitro and PK properties. Consistent with 83.8% protein binding by equilibrium dialysis, the rifampin MIC for M. tuberculosis strain H37Rv rose from 0.1 in a serum-free system to 1.0 mg/ml when it was tested in the presence of 50% serum. In time-kill studies, rifampin exhibited area under the concentration-time curve (AUC)-dependent killing in vitro, with maximal killing seen on all days and with the potency increasing steadily over a 9-day exposure period. MIC and time-kill studies performed with intracellular organisms in a macrophage monolayer model yielded similar results. By use of a murine aerosol infection model with dose ranging and dose fractionation over 6 days, the PD parameter that best correlated with a reduction in bacterial counts was found to be AUC/MIC (r(2) = 0.95), whereas the maximum concentration in serum/MIC (r(2) = 0.86) and the time that the concentration remained above the MIC (r(2) = 0.44) showed lesser degrees of correlation.