Real-time prediction model of cVDPV2 outbreaks to aid outbreak response vaccination strategies.

BACKGROUND: Circulating vaccine-derived poliovirus outbreaks are spreading more widely than anticipated, which has generated a crisis for the global polio eradication initiative. Effectively responding with vaccination activities requires a rapid risk assessment. This assessment is made difficult by the low case-to-infection ratio of type 2 poliovirus, variable transmissibility, changing population immunity, surveillance delays, and limited vaccine supply from the global stockpile. The geographical extent of responses have been highly variable between countries. METHODS: We develop a statistical spatio-temporal model of short-term, district-level poliovirus spread that incorporates known risk factors, including historical wild poliovirus transmission risk, routine immunization coverage, population immunity, and exposure to the outbreak virus. RESULTS: We find that proximity to recent cVDPV2 cases is the strongest risk factor for spread of an outbreak, and find significant associations between population immunity, historical risk, routine immunization, and environmental surveillance (p < 0.05). We examine the fit of the model to type 2 vaccine derived poliovirus spread since 2016 and find that our model predicts the location of cVDPV2 cases well (AUC = 0.96). We demonstrate use of the model to estimate appropriate scope of outbreak response activities to current outbreaks. CONCLUSION: As type 2 immunity continues to decline following the cessation of tOPV in 2016, outbreak responses to new cVDPV2 detections will need to be faster and larger in scope. We provide a framework that can be used to support decisions on the appropriate size of a vaccination response when new detections are identified. While the model does not account for all relevant local factors that must be considered in the overall vaccination response, it enables a quantitative basis for outbreak response size.

A recombinase polymerase amplification lateral flow assay for rapid detection of Burkholderia pseudomallei, the causative agent of melioidosis.

Burkholderia pseudomallei causes a fatal and infectious disease, melioidosis or Whitmore's disease in humans and animals. Melioidosis is present in different parts of the world and is endemic in Southeast Asia and Northern Australia. Accurate diagnosis of melioidosis is difficult due to its common flu-like symptoms, potentially long incubation period and erroneous identification as culture contaminant. Early diagnosis of the disease is essentially required for administration of suitable antibiotics and disease containment. The present study reports a rapid, specific and sensitive recombinase polymerase amplification lateral flow assay for detection of B. pseudomallei. Specific primers and probe were designed and the assay was performed at 41 °C for 20 min in a portable incubator. End products were detected using ready-to-use lateral flow strips. RPA lateral flow assay could detect ≥ 250 fg genomic DNA of B. pseudomallei and ≥ 50 copies of recombinant plasmid harbouring the target DNA sequence. The assay was found to be highly specific and did not cross-react with other bacterial strains. In artificially spiked human blood and urine samples, the detection limit of the assay was 4.8 × 104 and 4.95 × 104 CFU/mL of B. pseudomallei, respectively. The detection limit of assay after 6 h of enrichment of artificially spiked urine samples was found to be 4.95 × 103 CFU/mL of B. pseudomallei. Detection limit in artificially spiked tap water and soil samples was determined to be 7.5 × 102 CFU/mL and 3.3 × 104 CFU per 5 g of B. pseudomallei, respectively.

Irreducible Overlapping Pubic Symphysis Dislocation Managed with Distraction Osteotomy of the Contralateral Superior Pubic Ramus: A Rare Case Report.

INTRODUCTION: Overlapping pubic symphysis dislocation (OPSD) or a locked pubic symphysis is a compression of the pelvic ring with the intact pubis trapped into the contralateral obturator foramen. Reduction can be difficult and contralateral suprapubic osteotomy is a good way to address the irreducible OPSD. The technique has only been discussed thrice in the available literature. CASE REPORT: We report the case of a 26-year-old man who had his right pubic ramus entrapped within the contralateral obturator foramen, having an overlap of >4 cm with associated ipsilateral sacroiliac joint (SI joint) disruption and urethral injury. When all the maneuvers of closed and instrumented open reduction failed, we performed a superior pubic ramus osteotomy on the left side and unlocked the incarcerated right pubic ramus. The osteotomy site was stabilized with a 6-hole recon plate and SI joint was stabilized with a 6.5mm percutaneous sacroiliac screw. The patient underwent delayed urethral repair at 10 weeks after the index surgery. At 3-year follow-up, the patient did not report any pubic discomfort, urinary and sexual problems. CONCLUSION: Locked OPSD is a rare injury and is frequently associated with sacroiliac and urethral injuries. Distraction osteotomy of the contralateral superior pubic ramus is a viable option for irreducible cases.

Protein biomarker elucidation for the verification of biological agents in the taxonomic group of Gammaproteobacteria using tandem mass spectrometry.

Some pathogenic microbes can be used for nefarious applications and instigate population-based fear. In a bio-threat scenario, rapid and accurate methods to detect biological agents in a wide range of complex environmental and clinical matrices, is of paramount importance for the implementation of mitigation protocols and medical countermeasures. This study describes targeted and shot-gun tandem MS based approaches for the verification of biological agents from the environmental samples. The marker proteins and peptides were elucidated by an exhaustive literature mining, in silico analysis of prioritized proteins, and MS/MS analysis of abundant proteins from selected bacterial species. For the shot-gun methodology, tandem MS analysis of abundant peptides was carried from spiked samples. The validation experiments employing a combination of shot-gun tandem MS analysis and a targeted search reported here is a proof of concept to show the applicability of the methodology for the unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples.

A real-time loop mediated isothermal amplification assay for molecular detection of Burkholderia mallei, the aetiological agent of a zoonotic and re-emerging disease glanders.

Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings.

Development of a rapid and sensitive recombinase polymerase amplification-lateral flow assay for detection of Burkholderia mallei.

Burkholderia mallei, a potential biothreat agent is the aetiological agent of glanders, a zoonotic disease primarily affecting equines. B. mallei shares close genetic proximity with B. pseudomallei, the aetiological agent of melioidosis. Hence, molecular detection of B. mallei and its differentiation from B. pseudomallei has always been challenging. Early diagnosis of glanders is critical for timely treatment in humans and disease containment in animals. In this study a recombinase polymerase amplification-lateral flow (RPA-LF) assay has been developed for early and accurate detection of B. mallei. RPA-LF assay was found to be highly sensitive and detected as low as 10 fg genomic DNA of B. mallei. The assay was highly specific and could differentiate B. mallei and B. pseudomallei. The assay also detected B. mallei in artificially spiked blood, tap water and garden soil. The established assay is simple, rapid and does not require complex instrumentation. The field deployable assay can have better implications in rapid glanders diagnosis and environmental detection of B. mallei over PCR-based detection tools in glanders endemic areas with limited laboratory resources.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis, which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli. The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis. Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis.

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices.

Anthrax: A disease of biowarfare and public health importance.

Bioterrorism has received a lot of attention in the first decade of this century. Biological agents are considered attractive weapons for bioterrorism as these are easy to obtain, comparatively inexpensive to produce and exhibit widespread fear and panic than the actual potential of physical damage. Bacillus anthracis (B. anthracis), the etiologic agent of anthrax is a Gram positive, spore forming, non-motile bacterium. This is supposed to be one of the most potent BW agents because its spores are extremely resistant to natural conditions and can survive for several decades in the environment. B. anthracis spores enter the body through skin lesion (cutaneous anthrax), lungs (pulmonary anthrax), or gastrointestinal route (gastrointestinal anthrax) and germinate, giving rise to the vegetative form. Anthrax is a concern of public health also in many countries where agriculture is the main source of income including India. Anthrax has been associated with human history for a very long time and regained its popularity after Sept 2001 incidence in United States. The present review article describes the history, biology, life cycle, pathogenicity, virulence, epidemiology and potential of B. anthracis as biological weapon.

Distribution of airborne microbes and antibiotic susceptibility pattern of bacteria during Gwalior trade fair, Central India.

BACKGROUND/PURPOSE: Research into the distribution of bioaerosols during events associated with huge groups of people is lacking, especially in developing countries. The purpose of this study was to understand the distribution pattern of bioaerosols during an annual trade fair in the historical city of Gwalior, central India, a very important historical fair that was started by the King of Gwalior Maharaja Madho Rao in 1905. METHODS: Air samples were collected from six different sites at the fair ground and three different sites in a residential area before/during/after the fair using an impactor sampler on microbial content test agar and rose bengal agar for total bacteria and fungi, respectively. The representative strains of bacteria and fungi were further identified and selected bacterial strains were subjected to antibiotic susceptibility testing according to US Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: The bacterial bioaerosol count [colony-forming units (CFU)/m(3)] at fair sites was found to be 9.0 × 10(3), 4.0 × 10(4), and 1.0 × 10(4) before the start of the fair, during the fair, and after the fair, respectively. The fungal bioaerosol count at fair sites was 2.6 × 10(3) CFU/m(3), 6.3 × 10(3) CFU/m(3), and 1.7 × 10(3) CFU/m(3) before the fair, during the fair, and after the fair, respectively. Bacterial/fungal bioaerosols during-fair were increased significantly from the bacterial/fungal bioaerosols of the before-fair period (p < 0.05); they were also significantly higher than the bacterial/fungal bioaerosols at non-fair sites during the event (p < 0.0001). The proportion of antibiotic-resistant bacteria over the fair ground was significantly increased during-fair and was still higher in the after-fair period. Methicillin-resistant staphylococci (MRS) were also reported at the fair ground. CONCLUSION: The study indicates significantly higher bacterial and fungal bioaerosols during the fair event. Therefore, further research is needed to explore the health aspects and guidelines to control microbial load during such types of events.

Exoproteome analysis of a novel strain of Bacillus cereus implicated in disease resembling cutaneous anthrax.

Bacillus cereus belongs to B. cereus sensu lato group, shared by six other related species including Bacillus anthracis. B. anthracis is the causative agent for serious illness affecting a wide range of animals as well as humans and is a category A Biological and Toxin Warfare (BTW) agent. Recent studies indicate that a Bacillus species other than B. anthracis can cause anthrax-like disease and role of anthrax virulence plasmids (pXO1 and pXO2) on the pathogenicity of B. cereus has been documented. B. cereus strain TF5 was isolated from the tissue fluid of cutaneous anthrax-like skin lesions of a human patient from an anthrax endemic area in India. The strain harboured a PA gene, however, presence of pXO1 or pXO2-like plasmids could not be ascertained using reported primers. Abundant exoproteome of the strain in the early stationary phase was elucidated using a 2-DE MS approach and compared with that from a reference B. cereus strain. Analysis of proteins showing qualitative and quantitative differences between the two strains indicated an altered regulatory mechanism and putative role of S-layer protein and sphingomyelinase in the pathogenesis of strain TF5. Phylogenetic analysis of the S-layer protein indicated close affiliation of the strain with anthracis-like B. cereus strains such as B. cereus var. anthracis strain CI; whereas sphingomyelinase exhibited specific relationship with all the strains of B. anthracis apart from that with anthracis-like B. cereus strains.

Detection of protective antigen, an anthrax specific toxin in human serum by using surface plasmon resonance.

In this study, surface plasmon resonance (SPR) technology was used for the sensitive detection of protective antigen (PA), an anthrax specific toxin in spiked human serum samples. A monoclonal antibody raised against Bacillus anthracis PA was immobilized on carboxymethyldextran-modified gold chip, and its interaction with PA was characterized in situ by SPR. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74 m°, respectively. The change in Gibb's free energy (∆G= -78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 1 pg/mL purified PA. In PA-spiked human serum samples, 10 pg/mL of PA could be detected. Presence of PA in blood samples serves as an important early diagnostic marker for B. anthracis infections. Thus, SPR test can be a sensitive assay for detection of anthrax at early stages of infection.

A field usable qualitative anti-protective antigen enzyme-linked immunosorbent assay for serodiagnosis of human anthrax.

Although all mammals, including humans, are vulnerable when they come into direct contact with infected animals, anthrax is primarily a disease of herbivorous animals. In countries like India, cutaneous anthrax is a public health problem in several regions. Hence, a simple and efficacious serodiagnostic assay for large scale surveillance of endemic populations is required. In the present study, a field-usable, qualitative ELISA was developed for serodiagnosis of human anthrax. Results are assessed on a visual basis and no sophisticated instruments are required. Anti-protective antigen (PA) IgG was determined by visual examination of ELISA results of 225 human serum samples (160 from healthy humans, 5 from PA vaccinated individuals and 60 from confirmed anthrax cases). Comparison of the ELISA results with the results obtained from optical density values showed compatible sensitivity and specificity. Assay sensitivity, specificity, and positive and negative predictive values were found to be 100%. The developed assay could be a very useful tool for serological diagnosis of anthrax infection in humans.

Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System.

Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning throughout the world. Staphylococcal enterotoxin B (SEB) is one of the most potent and a listed biological warfare agent. Therefore, its quick, accurate and sensitive detection is of paramount importance. But availability of sensitive and specific antibodies against SEB is the major bottleneck in the development of an immunodetection system. Therefore, in the present study seb gene was cloned and expressed in a heterologous host resulting in a yield of 92 mg pure toxin per litre of culture broth after Ni-NTA affinity purification. Antibodies raised against the recombinant toxin did not cross react with related enterotoxins and organisms that can gain access in the food. Further, a sandwich ELISA was developed to detect SEB after extraction from artificially spiked food samples like milk, orange juice, skim milk and khoya. The sandwich ELISA was able to detect SEB in the range of 0.25 to 0.49 ng/ml or g of food. The detection system developed in the present study is at least as specific and sensitive as other commercially available kits which use monoclonal antibodies.

Molecular characterization reveals involvement of altered El Tor biotype Vibrio cholerae O1 strains in cholera outbreak at Hyderabad, India.

Thirty-four Vibrio cholerae isolates collected from a cholera outbreak in Hyderabad, South India were found to belong to serogroup Ol biotype El Tor serotype Ogawa. The genotype of all the isolates was confirmed by PCR assays. All the isolates were found PCR positive for ctxAB, ompW, rflOl, rtxC, and tcpA genes. All the isolates but one harboured rstR ( El Tor ) allele. However, one isolate carried both rstR ( EL Tor ) as well as rstR ( Classical ) alleles. Cholera toxin (ctxB) genotyping of the isolates confirmed the presence of altered cholera toxin B of classical biotype in all the isolates. All the isolates except VCH35 harboured an RS1-CTX prophage array on the large chromosome. The isolate VCH35 contained a tandem repeat of classical CTX prophage on the small chromosome. The clonal relationship among the V. cholerae isolates as carried out by enterobacterial repetitive intergenic consensus sequences PCR, BOX PCR and randomly amplified polymorphic DNA, uniformly showed a genetic relationship among the outbreak isolates. The results of this study suggest that altered El Tor biotype V. cholerae with the classical cholera toxin gene are involved in cholera outbreaks in India.

Characterization of an environmental strain of Bacillus thuringiensis from a hot spring in Western Himalayas.

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B.

Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

Nucleolar proteins suppress Caenorhabditis elegans innate immunity by inhibiting p53/CEP-1.

The tumor suppressor p53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, and cell cycle regulation. A genetic screen for negative regulators of innate immunity in Caenorhabditis elegans led to the identification of a mutation in NOL-6, a nucleolar RNA-associated protein (NRAP), which is involved in ribosome biogenesis and conserved across eukaryotic organisms. Mutation or silencing of NOL-6 and other nucleolar proteins results in an enhanced resistance to bacterial infections. A full-genome microarray analysis on animals with altered immune function due to mutation in nol-6 shows increased transcriptional levels of genes regulated by a p53 homologue, CEP-1. Further studies indicate that the activation of innate immunity by inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1. Since nucleoli and p53/CEP-1 are conserved, our results reveal an ancient immune mechanism by which the nucleolus may regulate immune responses against bacterial pathogens.

Isolation and characterization of heat resistant enterotoxigenic Staphylococcus aureus from a food poisoning outbreak in Indian subcontinent.

Outbreaks of staphylococcal food poisoning (SFP) are very common across the world; however, there is hardly any report of SFP from the Indian subcontinent. An outbreak occurred in the state of Madhya Pradesh (India) after the consumption of a snack called "Bhalla" made up of potato balls fried in vegetable oil. More than 100 children and adults who ate the snack suffered from the typical symptoms of SFP and required hospitalization. Food and clinical samples were found to contain a large number of enterotoxigenic Staphylococcus aureus. All enterotoxigenic isolates produced a combination of SEB and SED enterotoxins and were sensitive to oxacillin and vancomycin. Isolates were characterized by molecular biology tools, viz., SDS-PAGE, amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD) and nucleotide sequencing of seb, sed, and 16S rDNA genes. Results of these studies suggested that the isolates, irrespective of their isolation from food or clinical samples, were clonal in origin. Further, seb gene sequence of isolates showed nucleotide variations at multiple sites when compared with other sequences available in the database. Representative isolates, one each from food and clinical samples, were found to be highly heat resistant (D(60) approximately 15-16 min). Isolates obtained in the current outbreak need to be further studied to find out the impact on food safety guidelines with respect to thermal processing.

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples.

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10(3) CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10(4) CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.