Safety and Tolerability of ShigActive™, a Shigella spp. Targeting Bacteriophage Preparation, in a Phase 1 Randomized, Double-Blind, Controlled Clinical Trial.

Bacterial diseases of the gastrointestinal (GI) tract continue to be a major worldwide cause of human morbidity and mortality. Among various enteric pathogens, Shigella spp. are some of the most common and deadly bacterial pathogens. They are responsible for ~125 million worldwide cases of shigellosis, and ~14,000 deaths annually, the majority in children under the age of 5 and occurring in developing countries. Preventing and treating shigellosis with conventional drugs (e.g., vaccines and antibiotics) has proven to be very difficult. Here, we assessed the safety and tolerability of ShigActive™, a lytic bacteriophage preparation targeting Shigella spp., in a randomized, placebo-controlled, double-blind Phase 1 clinical trial. Ten participants randomized 4:1 received ShigActive™ or placebo co-administered with sodium bicarbonate orally three times daily for 7 days. Solicited and unsolicited adverse events (AEs) were observed for 29 days. Fifty percent of the subjects receiving ShigActive™ reported mild GI-related symptoms, while one participant experienced moderate fatigue. No serious or medically attended AEs occurred through day 90. Additionally, no significant differences in GI-associated inflammatory mediators or fecal microbiome changes were observed between placebo- and ShigActive™-treated subjects, or from a participants' baseline value. The results of this first-in-human (FIH) randomized, controlled Phase 1 trial of ShigActive™ demonstrate that it is safe and well tolerated when orally administered with no significant differences compared to placebo controls.

Microbiological Methods Used in the Enterics for Global Health Shigella Surveillance Study.

Background: Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. Conclusions: Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms.

Detecting Residual Chronic Salmonella Typhi Carriers on the Road to Typhoid Elimination in Santiago, Chile, 2017-2019.

BACKGROUND: In Santiago, Chile, where typhoid had been hyperendemic (1977-1991), we investigated whether residual chronic carriers could be detected among household contacts of non-travel-related typhoid cases occurring during 2017-2019. METHODS: Culture-confirmed cases were classified as autochthonous (domestically acquired) versus travel/immigration related. Household contacts of cases had stool cultures and serum Vi antibody measurements to detect chronic Salmonella Typhi carriers. Whole genome sequences of acute cases and their epidemiologically linked chronic carrier isolates were compared. RESULTS: Five of 16 autochthonous typhoid cases (31.3%) were linked to 4 chronic carriers in case households; 2 cases (onsets 23 months apart) were linked to the same carrier. Carriers were women aged 69-79 years with gallbladder dysfunction and Typhi fecal excretion; 3 had highly elevated serum anti-Vi titers. Genomic analyses revealed close identity (≤11 core genome single-nucleotide polymorphism [SNP] differences) between case and epidemiologically linked carrier isolates; all were genotypes prevalent in 1980s Santiago. A cluster of 4 additional autochthonous cases unlinked to a carrier was identified based on genomic identity (0-1 SNPs). Travel/immigration isolate genotypes were typical for the countries of travel/immigration. CONCLUSIONS: Although autochthonous typhoid cases in Santiago are currently rare, 5 of 16 such cases (31.3%) were linked to elderly chronic carriers identified among household contacts of cases.

Detection of Salmonella Typhi in Bile by Quantitative Real-Time PCR.

In countries where the incidence of typhoid fever is high, fecal material from short-term carriers of Salmonella Typhi contaminates inadequately treated water supplies. As treated water supplies and improved sanitation become available, chronic (mainly gallbladder) carriers of S. Typhi become important. The objective of this study was to develop a method for detection of S. Typhi in bile by quantitative real-time PCR (qPCR) in patients undergoing cholecystectomy. We evaluated sensitivity and specificity of probesets that target oriC, viaB, fliC-d, STY0201, and stoD. We optimized DNA extraction from bile and compared the sensitivity of culture and our qPCR method to detect S. Typhi in bile samples containing various cephalosporins. With the use of an optimized DNA extraction technique, our limit of detection of S. Typhi in spiked human bile samples was 7.4 × 102 CFU/mL. We observed that S. Typhi could be detected by qPCR in samples containing cefazolin, cefotaxime, or ceftriaxone whereas culture could only detect Typhi in samples containing cefazolin but not cefotaxime or ceftriaxone. Our qPCR detection method for S. Typhi in bile should be preferred in areas where antibiotic usage is common. IMPORTANCE New Salmonella Typhi conjugate vaccines have been deployed, which will potentially lead to a fall in incidence rates of typhoid fever in endemic areas. Identification of chronic carriers of S. Typhi will be important as these individuals can be a potential source of transmission to susceptible persons. To address this public health concern, we have developed a novel method to detect S. Typhi in bile using real-time PCR. Our method can be used to identify carriers of S. Typhi among patients undergoing cholecystectomy (gallbladder removal surgery). The sensitivity of our molecular-based assay was superior to culture when performed in the presence of antibiotics commonly used during surgery. Our methodology will complement efforts to eliminate typhoid disease.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

The Clinical Presentation of Culture-positive and Culture-negative, Quantitative Polymerase Chain Reaction (qPCR)-Attributable Shigellosis in the Global Enteric Multicenter Study and Derivation of a Shigella Severity Score: Implications for Pediatric Shigella Vaccine Trials.

BACKGROUND: Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. METHODS: We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold <27.9). Among Shigella MSD cases, we determined risk factors for death and derived a clinical severity score. RESULTS: Compared to culture-positive Shigella MSD cases (n = 745), culture-negative/qPCR-attributable Shigella cases (n = 852) were more likely to be under 12 months, stunted, have a longer duration of diarrhea, and less likely to have high stool frequency or a fever. There was no difference in dehydration, hospitalization, or severe classification from a modified Vesikari score. Twenty-two (1.8%) Shigella MSD cases died within the 14-days after presentation to health facilities, and 59.1% of these deaths were in culture-negative cases. Age <12 months, diarrhea duration prior to presentation, vomiting, stunting, wasting, and hospitalization were associated with mortality. A model-derived score assigned points for dehydration, hospital admission, and longer diarrhea duration but was not significantly better at predicting 14-day mortality than a modified Vesikari score. CONCLUSIONS: A composite severity score consistent with severe disease or dysentery may be a pragmatic clinical endpoint for severe shigellosis in vaccine trials. Reliance on culture for microbiologic confirmation may miss a substantial number of Shigella cases but is currently required to measure serotype specific immunity.

The Diversity of Lipopolysaccharide (O) and Capsular Polysaccharide (K) Antigens of Invasive Klebsiella pneumoniae in a Multi-Country Collection.

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645 K. pneumoniae isolates were collected from the blood of patients in 13 countries during 2005-2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and wzi capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of K. pneumoniae strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were gmlABC-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and wzi capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent K. pneumoniae invasive infection.

Characterization of Invasive Salmonella Serogroup C1 Infections in Mali.

Nontyphoidal Salmonella (NTS) are the leading cause of foodborne infections worldwide and a major cause of bloodstream infections in infants and HIV-infected adults in sub-Saharan Africa (SSA). Salmonella Typhimurium (serogroup B) and Salmonella Enteritidis (serogroup D) are the most common serovars in this region. However, data describing rarer invasive NTS serovars, particularly those belonging to serogroups C1 and C2, circulating in SSA are lacking. We previously conducted systematic blood culture surveillance on pediatric patients in Bamako, Mali, from 2002 to 2014, and the results showed that serovars Typhimurium and Enteritidis accounted for 32% and 36% of isolates, respectively. Here, we present data on 27 Salmonella serogroup C1 strains that were isolated during this previous study. The strains were typed by serum agglutination and multilocus sequence typing (MLST). Sixteen strains were Salmonella Paratyphi C, four were Salmonella Colindale, and two were Salmonella Virchow. Interestingly, five strains were identified as the very rare Salmonella Brazzaville using a combination of serum agglutination and flagellin gene typing. Phenotypic characterization showed that Salmonella Brazzaville produced biofilm and exhibited catalase activity, which were not statistically different from the gastroenteritis-associated Salmonella Typhimurium sequence type (ST) 19. All tested Salmonella Paratyphi C strains were poor biofilm producers and showed significantly less catalase activity than Salmonella Typhimurium ST19. Overall, our study provides insight into the Salmonella serogroup C1 serovars that cause invasive disease in infants in Mali. In addition, we show that MLST and flagellin gene sequencing, in association with traditional serum agglutination, are invaluable tools to help identify rare Salmonella serovars.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study.

BACKGROUND: Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). METHODS: GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. FINDINGS: We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. INTERPRETATION: A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. FUNDING: Bill & Melinda Gates Foundation.

Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe). RESULTS: Each one of the HIV-1 genes was cloned and expressed individually in fission yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between fission yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in fission yeast and mammalian cells. Consistent with the observation in fission yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress. CONCLUSIONS: Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy.

Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that prohibit PR enzymatic activities through non-competitive binding to PR, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chances of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 resistant strains.