Exploring inactivation of SARS-CoV-2, MERS-CoV, Ebola, Lassa, and Nipah viruses on N95 and KN95 respirator material using photoactivated methylene blue to enable reuse.

BACKGROUND: The COVID-19 pandemic resulted in a worldwide shortage of N95 respirators, prompting the development of decontamination methods to enable limited reuse. Countries lacking reliable supply chains would also benefit from the ability to safely reuse PPE. Methylene blue (MB) is a light-activated dye with demonstrated antimicrobial activity used to sterilize blood plasma. Decontamination of respirators using photoactivated MB requires no specialized equipment, making it attractive for use in the field during outbreaks. METHODS: We examined decontamination of N95 and KN95 respirators using photoactivated MB and 3 variants of SARS-CoV-2, the virus that causes COVID-19; and 4 World Health Organization priority pathogens: Ebola virus, Middle East respiratory syndrome coronavirus, Nipah virus, and Lassa virus. Virus inactivation by pretreating respirator material was also tested. RESULTS: Photoactivated MB inactivated all tested viruses on respirator material, albeit with varying efficiency. Virus applied to respirator material pre-treated with MB was also inactivated, thus MB pretreatment may potentially protect respirator wearers from virus exposure in real-time. CONCLUSIONS: These results demonstrate that photoactivated MB represents a cost-effective, rapid, and widely deployable method to decontaminate N95 respirators for reuse during supply shortages.

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus.

BACKGROUND: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure). METHODS: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate. RESULTS: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces. DISCUSSION AND CONCLUSIONS: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction.

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

Use of Ebola Vaccine: Expansion of Recommendations of the Advisory Committee on Immunization Practices To Include Two Additional Populations - United States, 2021.

On December 19, 2019, the Food and Drug Administration (FDA) approved rVSVΔG-ZEBOV-GP Ebola vaccine (ERVEBO, Merck) for the prevention of Ebola virus disease (EVD) caused by infection with Ebola virus, species Zaire ebolavirus, in adults aged ≥18 years. In February 2020, the Advisory Committee on Immunization Practices (ACIP) recommended preexposure vaccination with ERVEBO for adults aged ≥18 years in the United States who are at highest risk for potential occupational exposure to Ebola virus because they are responding to an outbreak of EVD, work as health care personnel at federally designated Ebola treatment centers in the United States, or work as laboratorians or other staff members at biosafety level 4 facilities in the United States (1).

Addressing personal protective equipment (PPE) decontamination: Methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus. DESIGN: The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance. METHODS: MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. RESULTS: Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3. CONCLUSIONS: MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.

Lessons Learned From a COVID-19 Biohazard Spill During Swabbing at a Quarantine Facility.

The need for increased testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has resulted in an increase of testing facilities outside of traditional clinical settings and sample handling by individuals without appropriate biohazard and biocontainment training. During the repatriation and quarantine of passengers from the Grand Princess cruise ship at a US military base, biocontainment of a potentially infectious sample from a passenger was compromised. This study describes the steps taken to contain the spill, decontaminate the area, and discusses the needs for adequate training in a biohazard response.

Use of Ebola Vaccine: Recommendations of the Advisory Committee on Immunization Practices, United States, 2020.

This report summarizes the recommendations of the Advisory Committee on Immunization Practices (ACIP) for use of the rVSVΔG-ZEBOV-GP Ebola vaccine (Ervebo) in the United States. The vaccine contains rice-derived recombinant human serum albumin and live attenuated recombinant vesicular stomatitis virus (VSV) in which the gene encoding the glycoprotein of VSV was replaced with the gene encoding the glycoprotein of Ebola virus species Zaire ebolavirus. Persons with a history of severe allergic reaction (e.g., anaphylaxis) to rice protein should not receive Ervebo. This is the first and only vaccine currently licensed by the Food and Drug Administration for the prevention of Ebola virus disease (EVD). These guidelines will be updated based on availability of new data or as new vaccines are licensed to protect against EVD.ACIP recommends preexposure vaccination with Ervebo for adults aged ≥18 years in the U.S. population who are at highest risk for potential occupational exposure to Ebola virus species Zaire ebolavirus because they are responding to an outbreak of EVD, work as health care personnel at federally designated Ebola treatment centers in the United States, or work as laboratorians or other staff at biosafety level 4 facilities in the United States. Recommendations for use of Ervebo in additional populations at risk for exposure and other settings will be considered and discussed by ACIP in the future.

Penicillin Use in Meningococcal Disease Management: Active Bacterial Core Surveillance Sites, 2009.

In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines.

Population-Based Surveillance of Neisseria meningitidis Antimicrobial Resistance in the United States.

Background. Antimicrobial treatment and chemoprophylaxis of patients and their close contacts is critical to reduce the morbidity and mortality and prevent secondary cases of meningococcal disease. Through the 1990's, the prevalence of antimicrobial resistance to commonly used antimicrobials among Neisseria meningitidis was low in the United States. Susceptibility testing was performed to ascertain whether the proportions of isolates with reduced susceptibility to antimicrobials commonly used for N meningitidis have increased since 2004 in the United States. Methods. Antimicrobial susceptibility testing was performed by broth microdilution on 466 isolates of N meningitidis collected in 2004, 2008, 2010, and 2011 from an active, population-based surveillance system for susceptibility to ceftriaxone, ciprofloxacin, penicillin G, rifampin, and azithromycin. The molecular mechanism of reduced susceptibility was investigated for isolates with intermediate or resistant phenotypes. Results. All isolates were susceptible to ceftriaxone and azithromycin, 10.3% were penicillin G intermediate (range, 8% in 2008-16.7% in 2010), and <1% were ciprofloxacin, rifampin, or penicillin G resistant. Of the penicillin G intermediate or resistant isolates, 63% contained mutations in the penA gene associated with reduced susceptibility to penicillin G. All ciprofloxacin-resistant isolates contained mutations in the gyrA gene associated with reduced susceptibility. Conclusions. Resistance of N meningitidis to antimicrobials used for empirical treatment of meningitis in the United States has not been detected, and resistance to penicillin G and chemoprophylaxis agents remains uncommon. Therapeutic agent recommendations remain valid. Although periodic surveillance is warranted to monitor trends in susceptibility, routine clinical testing may be of little use.

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis.

BACKGROUND: Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. METHODS AND FINDINGS: CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. RESULTS: Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. CONCLUSION: Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

Genomic basis of a polyagglutinating isolate of Neisseria meningitidis.

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.

Neisseria Base: a comparative genomics database for Neisseria meningitidis.

Neisseria meningitidis is an important pathogen, causing life-threatening diseases including meningitis, septicemia and in some cases pneumonia. Genomic studies hold great promise for N. meningitidis research, but substantial database resources are needed to deal with the wealth of information that comes with completely sequenced and annotated genomes. To address this need, we developed Neisseria Base (NBase), a comparative genomics database and genome browser that houses and displays publicly available N. meningitidis genomes. In addition to existing N. meningitidis genome sequences, we sequenced and annotated 19 new genomes using 454 pyrosequencing and the CG-Pipeline genome analysis tool. In total, NBase hosts 27 complete N. meningitidis genome sequences along with their associated annotations. The NBase platform is designed to be scalable, via the underlying database schema and modular code architecture, such that it can readily incorporate new genomes and their associated annotations. The front page of NBase provides user access to these genomes through searching, browsing and downloading. NBase search utility includes BLAST-based sequence similarity searches along with a variety of semantic search options. All genomes can be browsed using a modified version of the GBrowse platform, and a plethora of information on each gene can be viewed using a customized details page. NBase also has a whole-genome comparison tool that yields single-nucleotide polymorphism differences between two user-defined groups of genomes. Using the virulent ST-11 lineage as an example, we demonstrate how this comparative genomics utility can be used to identify novel genomic markers for molecular profiling of N. meningitidis.

Serologically confirmed household transmission of 2009 pandemic influenza A (H1N1) virus during the first pandemic wave--New York City, April-May 2009.

BACKGROUND: Understanding transmissibility of influenza viruses within households is critical for guiding public health response to pandemics. We studied serologically confirmed infection and disease among household contacts of index case patients with 2009 pandemic influenza A (H1N1) virus (pH1N1) infection in a setting of minimal community pH1N1 transmission. METHODS: We defined index case patients as students and staff of a New York City high school with laboratory-confirmed pH1N1 infection during the earliest phase of the pH1N1 outbreak in April 2009. We visited households of index case patients twice, once in early May and again in June/July 2009. At each visit, household members (both index case patents and household contacts) provided serum samples and completed questionnaires about illness and possible risk factors. Serologic testing was performed using microneutralization and hemagglutination-inhibition assays. RESULTS: Of 79 eligible household contacts in 28 households, 19% had serologically confirmed pH1N1 infection, and 28% of those infected were asymptomatic. Serologically confirmed infection varied by age among household contacts: 36% of contacts younger than 10 years were infected, compared with 46% of contacts age 10-18 years, 8% of contacts aged 19-54 years, and 22% of contacts aged 55 years and older. CONCLUSIONS: Infection rates were high for household contacts of persons with confirmed pH1N1, particularly for contacts aged 10-18 years, and asymptomatic infection was common. Efforts to reduce household transmission during influenza pandemics are important adjuncts to strategies to reduce community illness.

Using single-nucleotide polymorphisms to discriminate disease-associated from carried genomes of Neisseria meningitidis.

Neisseria meningitidis is one of the main agents of bacterial meningitis, causing substantial morbidity and mortality worldwide. However, most of the time N. meningitidis is carried as a commensal not associated with invasive disease. The genomic basis of the difference between disease-associated and carried isolates of N. meningitidis may provide critical insight into mechanisms of virulence, yet it has remained elusive. Here, we have taken a comparative genomics approach to interrogate the difference between disease-associated and carried isolates of N. meningitidis at the level of individual nucleotide variations (i.e., single nucleotide polymorphisms [SNPs]). We aligned complete genome sequences of 8 disease-associated and 4 carried isolates of N. meningitidis to search for SNPs that show mutually exclusive patterns of variation between the two groups. We found 63 SNPs that distinguish the 8 disease-associated genomes from the 4 carried genomes of N. meningitidis, which is far more than can be expected by chance alone given the level of nucleotide variation among the genomes. The putative list of SNPs that discriminate between disease-associated and carriage genomes may be expected to change with increased sampling or changes in the identities of the isolates being compared. Nevertheless, we show that these discriminating SNPs are more likely to reflect phenotypic differences than shared evolutionary history. Discriminating SNPs were mapped to genes, and the functions of the genes were evaluated for possible connections to virulence mechanisms. A number of overrepresented functional categories related to virulence were uncovered among SNP-associated genes, including genes related to the category "symbiosis, encompassing mutualism through parasitism."

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.

A computational genomics pipeline for prokaryotic sequencing projects.

MOTIVATION: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. RESULTS: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. AVAILABILITY AND IMPLEMENTATION: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Population structure and capsular switching of invasive Neisseria meningitidis isolates in the pre-meningococcal conjugate vaccine era--United States, 2000-2005.

BACKGROUND: A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups. METHODS: Meningococcal isolates from 10 Active Bacterial Core surveillance sites from 2000 through 2005 were analyzed to identify changes occurring after MCV4 licensure. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsular switching. RESULTS: Among 1160 isolates, the most common genetic lineages were the sequence type (ST)-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared with 36 (12.9%) for serogroup C (P < .001); most serogroup C switches were from virulent serogroup B and/or serogroup Y lineages. CONCLUSIONS: A limited number of genetic lineages caused the majority of invasive meningococcal infections. A substantial proportion of isolates had evidence of capsular switching. The high prevalence of capsular switching requires surveillance to detect changes in the meningococcal population structure that may affect the effectiveness of meningococcal vaccines.

Development of a neutralization assay for Nipah virus using pseudotype particles.

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

Meningococcus genome informatics platform: a system for analyzing multilocus sequence typing data.

The Meningococcus Genome Informatics Platform (MGIP) is a suite of computational tools for the analysis of multilocus sequence typing (MLST) data, at http://mgip.biology.gatech.edu. MLST is used to generate allelic profiles to characterize strains of Neisseria meningitidis, a major cause of bacterial meningitis worldwide. Neisseria meningitidis strains are characterized with MLST as specific sequence types (ST) and clonal complexes (CC) based on the DNA sequences at defined loci. These data are vital to molecular epidemiology studies of N. meningitidis, including outbreak investigations and population biology. MGIP analyzes DNA sequence trace files, returns individual allele calls and characterizes the STs and CCs. MGIP represents a substantial advance over existing software in several respects: (i) ease of use-MGIP is user friendly, intuitive and thoroughly documented; (ii) flexibility--because MGIP is a website, it is compatible with any computer with an internet connection, can be used from any geographic location, and there is no installation; (iii) speed--MGIP takes just over one minute to process a set of 96 trace files; and (iv) expandability--MGIP has the potential to expand to more loci than those used in MLST and even to other bacterial species.