A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization.

Vibrio cholerae, the causative agent of the disease cholera, is responsible for multiple pandemics. V. cholerae binds to and colonizes the gastrointestinal tract within the human host, as well as various surfaces in the marine environment (e.g., zooplankton) during interepidemic periods. A large adhesin, the Flagellar Regulated Hemagglutinin A (FrhA), enhances binding to erythrocytes and epithelial cells and enhances intestinal colonization. We identified a peptide-binding domain (PBD) within FrhA that mediates hemagglutination, binding to epithelial cells, intestinal colonization, and facilitates biofilm formation. Intriguingly, this domain is also found in the ice-binding protein of the Antarctic bacterium Marinomonas primoryensis, where it mediates binding to diatoms. Peptide inhibitors of the M. primoryensis PBD inhibit V. cholerae binding to human cells as well as to diatoms and inhibit biofilm formation. Moreover, the M. primoryensis PBD inserted into FrhA allows V. cholerae to bind human cells and colonize the intestine and also enhances biofilm formation, demonstrating the interchangeability of the PBD from these bacteria. Importantly, peptide inhibitors of PBD reduce V. cholerae intestinal colonization in infant mice. These studies demonstrate how V. cholerae uses a PBD shared with a diatom-binding Antarctic bacterium to facilitate intestinal colonization in humans and biofilm formation in the environment.

A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Bangladesh.

BACKGROUND: The World Health Organization (WHO) recommends the use of oral cholera vaccines (OCVs) as part of an integrated control program, both in highly endemic settings and during cholera epidemics. The available and internationally recommended WHO-prequalified OCVs (Dukoral, Shanchol, Euvichol) contain multiple heat and formalin-killed V. cholerae strains of Inaba and Ogawa serotypes. MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd. in technical collaboration with University of Gothenburg, Sweden has developed a new single strain OCV, Hillchol. This vaccine consists of formaldehyde-inactivated whole cell El Tor V. cholerae O1 bacteria engineered into the Hikojima serotype for stable expression of both the Ogawa (AB) and Inaba (AC) LPS antigens on the bacterial surface. We evaluated the safety and immunogenicity of this novel and potentially much less expensive OCV in comparison with Shanchol. METHODS: We conducted a randomized, non-inferiority, age-descending clinical trial of OCV (Hillchol vs. Shanchol) in the Mirpur area of Dhaka city from July 2016 to May 2017. This study was carried out in three different age cohorts (1-<5, 5-17 and ≥18 years old). Two doses of vaccine were given at 14 days intervals to 560 healthy participants. FINDINGS: No serious adverse events were reported. There were no significant differences in the rates of adverse events between the test vaccine (Hillchol) and the comparator (Shanchol) group. Serum vibriocidal antibody responses in all age groups combined were comparable for all the O1 Ogawa (59% vs. 67%; 90% CI of difference: -14.55, -0.84) and Inaba (70% vs. 71%; 90% CI of difference: -7.24, 5.77) serotypes, showing that the Hillchol vaccine was non-inferior to Shanchol. This new vaccine was also non-inferior to Shanchol in the different age strata. CONCLUSION: The safety and immunogenicity profile of the new OCV Hillchol is comparable to Shanchol in persons residing in a cholera-endemic setting. ClinicalTrials.gov number: NCT02823899.

A randomized Phase I/II study to evaluate safety and reactogenicity of a heat-stable rotavirus vaccine in healthy adults followed by evaluation of the safety, reactogenicity, and immunogenicity in infants.

Objectives: To assess the safety and reactogenicity of single oral dose of heat-stable rotavirus vaccine (HSRV) in healthy adults aged 18-45 years followed by assessment of safety, reactogenicity, and immunogenicity of three doses of HSRV in healthy infants aged 6-8 weeks at enrollment.Trial Design: Single-center randomized controlled, sequential, blinded (adults) and open-label (infants).Setting: Single site at International Center for Diarrheal Disease Research, Bangladesh (icddr,b).Participants: Fifty eligible adults randomized in 1:1 ratio (HSRV: Placebo) followed by 50 eligible infants randomized in 1:1 ratio (HSRV: Comparator (RotaTeq®, pentavalent human-bovine (WC3) reassortant live-attenuated, rotavirus vaccine)).Intervention: Adults received either a single dose of HSRV or placebo and followed for 14 days. Infants received three doses of either HSRV or comparator with a follow-up for 28 days after each dose.Main Outcome Measures: Solicited and unsolicited adverse events (AEs) along with any serious adverse events (SAEs) were part of the safety and reactogenicity assessment in adults and infants whereas serum anti-rotavirus IgA response rates were part of immunogenicity assessment in infants only. Post-vaccination fecal shedding of vaccine-virus rotavirus strains was also determined in adults and infants.Results: In this study, HSRV, when compared with placebo, did not result in increase in solicited adverse events (solicited AEs) in adults. In infants, HSRV had a safety profile similar to comparator vis-à-vis solicited AEs. In infants, fecal shedding of vaccine-virus strains was not detected in HSRV recipients but was observed in two comparator recipients. Percentage of infants exhibiting threefold rise in serum anti-rotavirus IgA titers from baseline to 1-month post-dose 3 in HSRV group was 88% (22/25) and 84% (21/25) in comparator group.Conclusion: HSRV was found to be generally well-tolerated in both adults and infants and immunogenic in infants.

Risk factors for mortality among inpatients with smear positive pulmonary tuberculosis.

OBJECTIVE: To evaluate risk factors having significant effect on mortality of smear positive Pulmonary Tuberculosis (PTB) inpatients. METHODS: A descriptive cross-sectional study was conducted at Ojha Institute of Chest Diseases, Dow University Hospital Ojha Campus, Karachi. One hundred and seventy (170) inpatients of smear positive PTB confirmed by Acid Fast Bacilli (AFB) smear, aged between 13-80 years were enrolled by using consecutive sampling technique while patients with drug resistant Tuberculosis (TB) and extra pulmonary TB were excluded from the study. Selected patients were interviewed for collecting demographic data and risk factor data by using a standard questionnaire. RESULTS: Out of 170 PTB inpatients, mortality was observed in 23 (13.5%) patients among which male patients were 12 (52.2%), and female were 11 (47.8%). Mortality was significantly associated with increasing age (p=0.003), socioeconomic status (p=0.019), anemia (p=0.03), Chronic Liver Disease (CLD) (p=0.005), Diabetes Mellitus (DM) (p=0.001), Human Immunodeficiency Virus (HIV) (p=0.007), Hypertension (HTN) (p=0.006), recurrent TB (p=0.001), and smoking (p=0.001). CONCLUSION: Increasing age, poverty, smoking history, and presence of comorbidities like DM, CLD, HIV, hypertension, and anemia are associated with higher mortality in smear positive PTB cases.

Peripapillary Retinal Nerve Fiber Layer (RNFL) Thickness Measurements by Topcon SD-OCT in Myopic Patients.

OBJECTIVE: To evaluate the effect of low-to-moderate myopia on peripapillary retinal nerve fiber layer (RNFL) thickness measured by Topcon SD optical coherence tomography (OCT). STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Ophthalmology Department, Shifa Foundation Falahee Clinic, over a period of one year starting from June 2015. METHODOLOGY: A total of 43 eyes of 43 patients, having mild to moderate myopic refractive error, were enrolled in the study. Refractive error/spherical equivalent was calculated. RNFL thickness was obtained from all four peripapillary quadrants: temporal, superior, nasal, and inferior; and 12 sub-quadrants using Topcon SD OCT. Pearson correlation coefficients (r) were calculated to evaluate relationships between the RNFL thickness and spherical equivalent (SE) before and after adjustment for ocular magnification. RESULTS: The study included 51.2% females and 48.8% males. Mean age was 30.9 ±6.45 years. Mean axial length was 24.25 ±0.91 mm. Mean SE was -3.25 ±1.93 DS. Mean of average RNFL thickness (with Littmann's correction) was 97.28 ±8.15 µm. Correlation analysis among all subjects showed that the average, mean nasal quadrant, upper nasal, and inferonasal sub-quadrant RNFL thickness had positive correlation with spherical equivalent (r = 0.31, p = 0.045).However, correction of the magnification effect by applying Littmann formula eliminated this effect. CONCLUSION: In low-to-moderate myopia, RNFL measurements vary with refractive error of the eye. Since ocular magnification significantly affects the RNFL measurement, it should be considered in diagnosing glaucoma.

Yield of closed pleural biopsy and cytology in exudative pleural effusion.

OBJECTIVE: To determine diagnostic yield of Closed Pleural Biopsy (CPB) and Cytology in Exudative Pleural Effusion (PE). METHODS: This prospective comparative study was conducted at Chest Unit-II & Medical Unit-IV of Dow University of Health Sciences, Karachi Pakistan from January 2011 till December 2014. RESULTS: Ninety-four patients with exudative PE were finally included. The mean age (SD) was 44.0 (13.8) years. Overall Specific Diagnosis was reached in 76/94 patients; 46 Tuberculosis PE (TPE) & 30 Malignant PE (MPE). CPB diagnosed all TPE patients alone and 28/30 of MPE. Cytology diagnosed only 10/30 patients of MPE with 8 patients having both CPB & Cytology positive for malignancy whereas in the remaining two cases only Cytology positive. The sensitivity of CPB in detecting TPE and MPE was 93.9% and 82.4% respectively whereas specificity for both was 100%. The diagnostic yield of cytology in detecting MPE is only (33.3%). The diagnostic yield of CPB for TPE and MPE is 100% and 93.3% respectively. The overall specific diagnostic yield of CPB is 78.7%. CONCLUSION: CPB is better than pleural fluid cytology alone with the later adding little to diagnostic yield when both combined in distinguishing TPE from MPE, the two main differential of exudative PE in a TB-Endemic country.

A bacterial hemerythrin domain regulates the activity of a Vibrio cholerae diguanylate cyclase.

The first demonstrated example of a regulatory function for a bacterial hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O(2) and undergo autoxidation. The amino acid sequence encoded by the VC1216 gene from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial diguanylate cyclases (DGCs) that catalyze formation of cyclic di-(3',5')-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon being exposed to air. Vc Bhr-DGC showed approximately 10 times higher DGC activity in the diferrous than in the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in V. cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low-dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity.

Mannose-containing oligosaccharides of non-specific human secretory immunoglobulin A mediate inhibition of Vibrio cholerae biofilm formation.

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA(-/-) mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA(+/+) pups. Importantly, cross-fostering of IgA(-/-) pups with IgA(+/+) nursing dams reversed the greater susceptibility to MO10 infection, suggesting a role for non-specific SIgA in protection against the infection. Since biofilm formation is associated with virulence of MO10, we further examined the role of human non-specific SIgA on this virulence phenotype of the pathogen. Human non-specific SIgA, in a dose-dependent fashion, significantly reduced the biofilm formation by MO10 without affecting the viability of the bacterium. Such an inhibitory effect was not induced by human serum IgA, IgG, or IgM, suggesting a role for the oligosaccharide-rich secretory component (SC) of SIgA. This was supported by the demonstration that SIgA treated with endoglycosidase H, to cleave the high-mannose containing terminal chitobiose residues, did not induce a reduction in biofilm formation by MO10. Furthermore, the addition of free mannose per se, across a wide dose range, induced significant reduction in MO10 biofilm formation. Collectively, these results suggest that mannose containing oligosaccharides within human non-specific secretory IgA can alter important virulence phenotypes of Vibrio cholerae such as biofilm formation, without affecting viability of the microorganism. Such effects may contribute significantly to innate immune defenses against invading pathogens in vivo in the gastrointestinal tract.

The ToxT-dependent methyl-accepting chemoreceptors AcfB and TcpI contribute to Vibrio cholerae intestinal colonization.

Vibrio cholerae colonizes the human intestine and causes the acute diarrheal disease cholera. Flagellar-mediated chemotaxis contributes to intestinal colonization as well as infectivity. The virulence-regulatory protein ToxT activates transcription of the genes encoding the major virulence factors cholera toxin and toxin coregulated pilus. ToxT additionally activates transcription of two genes, tcpI and acfB, located within the Vibrio Pathogenicity Island predicted to encode methyl-accepting chemoreceptors. We show that disruption of either tcpI or acfB individually does not noticeably affect V. cholerae intestinal colonization within the infant mouse, but disruption of both tcpI and acfB leads to a decrease in intestinal colonization. These results suggest that TcpI and AcfB may have overlapping or redundant chemotactic functions that contribute to V. cholerae intestinal colonization.