Ryan T. Novak

Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion System of Burkholderia pseudomallei

ABSTRACT Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 × 103 genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 × 103 CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.

Serogroup A meningococcal conjugate vaccination in Burkina Faso: analysis of national surveillance data

BACKGROUND An affordable, highly immunogenic Neisseria meningitidis serogroup A meningococcal conjugate vaccine (PsA-TT) was licensed for use in sub-Saharan Africa in 2009. In 2010, Burkina Faso became the first country to implement a national prevention campaign, vaccinating 11·4 million people aged 1-29 years. We analysed national surveillance data around PsA-TT introduction to investigate the early effect of the vaccine on meningitis incidence and epidemics. METHODS We examined national population-based meningitis surveillance data from Burkina Faso using two sources, one with cases and deaths aggregated at the district level from 1997 to 2011, and the other enhanced with results of cerebrospinal fluid examination and laboratory testing from 2007 to 2011. We compared mortality rates and incidence of suspected meningitis, probable meningococcal meningitis by age, and serogroup-specific meningococcal disease before and during the first year after PsA-TT implementation. We assessed the risk of meningitis disease and death between years. FINDINGS During the 14 year period before PsA-TT introduction, Burkina Faso had 148 603 cases of suspected meningitis with 17 965 deaths, and 174 district-level epidemics. After vaccine introduction, there was a 71% decline in risk of meningitis (hazard ratio 0·29, 95% CI 0·28-0·30, p<0·0001) and a 64% decline in risk of fatal meningitis (0·36, 0·33-0·40, p<0·0001). We identified a statistically significant decline in risk of probable meningococcal meningitis across the age group targeted for vaccination (62%, cumulative incidence ratio [CIR] 0·38, 95% CI 0·31-0·45, p<0·0001), and among children aged less than 1 year (54%, 0·46, 0·24-0·86, p=0·02) and people aged 30 years and older (55%, 0·45, 0·22-0·91, p=0·003) who were ineligible for vaccination. No cases of serogroup A meningococcal meningitis occurred among vaccinated individuals, and epidemics were eliminated. The incidence of laboratory-confirmed serogroup A N meningitidis dropped significantly to 0·01 per 100 000 individuals per year, representing a 99·8% reduction in the risk of meningococcal A meningitis (CIR 0·002, 95% CI 0·0004-0·02, p<0·0001). INTERPRETATION Early evidence suggests the conjugate vaccine has substantially reduced the rate of meningitis in people in the target age group, and in the general population because of high coverage and herd immunity. These data suggest that fully implementing the PsA-TT vaccine could end epidemic meningitis of serogroup A in sub-Saharan Africa. FUNDING None.

Persistence of Protection Against Hepatitis B Virus Infection Among Adolescents Vaccinated With Recombinant Hepatitis B Vaccine Beginning at Birth : A 15-Year Follow-Up Study

Background: Long-term follow-up studies of populations that received recombinant hepatitis B (HB) vaccination beginning at birth are limited. Methods: Micronesian adolescents who had received 3 doses of recombinant HB vaccine (Recombivax 5 μg at birth, 2.5 μg at 2 months, 2.5 μg at 6 months) and tested negative for antibody to HB core antigen (anti-HBc) 2 years after primary vaccination (baseline testing) were followed up 15 years after primary vaccination. After testing for anti-HBc, HB surface antigen (HBsAg), and antibody to HBsAg (anti-HBs), participants received a booster dose of HB vaccine. An anamnestic response was defined as an increase in anti-HBs concentrations to a level ≥10 mIU/mL 14 days postbooster. Results: Of the 105 participants, 42 (40.0%) had anti-HBs concentrations ≥10 mIU/mL on baseline testing. At 15 years, 8 (7.6%) were anti-HBc positive; none were HBsAg positive. Of the remaining 97, 7 (7.3%) had anti-HBs concentrations ≥10 mIU/mL. Of the 96 who received a booster dose, 46 (47.9%) had an anamnestic response; final antibody concentrations were 10-99 mIU/mL for 17 (17.7%) and >100 mIU/mL for 29 (30.2%). Participants with anti-HBs concentrations ≥10 mIU/mL on baseline testing were more likely to have an anamnestic response at 15 years [26/39 (66.7%) versus 20/57 (35.1%); P = 0.003]. Conclusions: Fifteen years after primary vaccination starting at birth, 8% of participants had evidence of past HB virus infection, but none had chronic infection. Absence of an anamnestic response to an additional vaccine dose, seen in half of participants, might indicate waning immunity.

Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America.

We report on three cases of meningococcal disease caused by ciprofloxacin-resistant Neisseria meningitidis, one in North Dakota and two in Minnesota. The cases were caused by the same serogroup B strain. To assess local carriage of resistant N. meningitidis, we conducted a pharyngeal-carriage survey and isolated the resistant strain from one asymptomatic carrier. Sequencing of the gene encoding subunit A of DNA gyrase (gyrA) revealed a mutation associated with fluoroquinolone resistance and suggests that the resistance was acquired by means of horizontal gene transfer with the commensal N. lactamica. In susceptibility testing of invasive N. meningitidis isolates from the Active Bacterial Core surveillance system between January 2007 and January 2008, an additional ciprofloxacin-resistant isolate was found, in this case from California. Ciprofloxacin-resistant N. meningitidis has emerged in North America.

Use of 16S rRNA Gene Sequencing for Rapid Confirmatory Identification of Brucella Isolates

ABSTRACT Members of the genus Brucella are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65 Brucella strains along with 17 related strains likely to present a differential diagnostic challenge. All Brucella 16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of Brucella spp. and their differentiation from closely related organisms.

Effectively introducing a new meningococcal A conjugate vaccine in Africa: The Burkina Faso experience

A new Group A meningococcal (Men A) conjugate vaccine, MenAfriVac™, was prequalified by the World Health Organization (WHO) in June 2010. Because Burkina Faso has repeatedly suffered meningitis epidemics due to Group A Neisseria meningitidis special efforts were made to conduct a country-wide campaign with the new vaccine in late 2010 and before the onset of the next epidemic meningococcal disease season beginning in January 2011. In the ensuing five months (July-November 2010) the following challenges were successfully managed: (1) doing a large safety study and registering the new vaccine in Burkina Faso; (2) developing a comprehensive communication plan; (3) strengthening the surveillance system with particular attention to improving the capacity for real-time polymerase chain reaction (PCR) testing of spinal fluid specimens; (4) improving cold chain capacity and waste disposal; (5) developing and funding a sound campaign strategy; and (6) ensuring effective collaboration across all partners. Each of these issues required specific strategies that were managed through a WHO-led consortium that included all major partners (Ministry of Health/Burkina Faso, Serum Institute of India Ltd., UNICEF, Global Alliance for Vaccines and Immunization, Meningitis Vaccine Project, CDC/Atlanta, and the Norwegian Institute of Public Health/Oslo). Biweekly teleconferences that were led by WHO ensured that problems were identified in a timely fashion. The new meningococcal A conjugate vaccine was introduced on December 6, 2010, in a national ceremony led by His Excellency Blaise Compaore, the President of Burkina Faso. The ensuing 10-day national campaign was hugely successful, and over 11.4 million Burkinabes between the ages of 1 and 29 years (100% of target population) were vaccinated. African national immunization programs are capable of achieving very high coverage for a vaccine desired by the public, introduced in a well-organized campaign, and supported at the highest political level. The Burkina Faso success augurs well for further rollout of the Men A conjugate vaccine in meningitis belt countries.

Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing

BackgroundBacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.ResultsA retrospective analysis of 55 clinical B. cereus isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27), gastrointestinal (GI) illness (n = 18), and associated isolates from food (n = 10) were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST) in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates.ConclusionSTs of clinical B. cereus isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to B. anthracis and emetic B. cereus isolates.

Clinical Evaluation of a Type III Secretion System Real-Time PCR Assay for Diagnosing Melioidosis

ABSTRACT A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

Neisseria meningitidis serogroup W, Burkina Faso, 2012.

In 2010, Burkina Faso became the first country to introduce meningococcal serogroup A conjugate vaccine (PsA-TT). During 2012, Burkina Faso reported increases in Neisseria meningitidis serogroup W, raising questions about whether these cases were a natural increase in disease or resulted from serogroup replacement after PsA-TT introduction. We analyzed national surveillance data to describe the epidemiology of serogroup W and genotyped 61 serogroup W isolates. In 2012, a total of 5,807 meningitis cases were reported through enhanced surveillance, of which 2,353 (41%) were laboratory confirmed. The predominant organism identified was N. meningitidis serogroup W (62%), and all serogroup W isolates characterized belonged to clonal complex 11. Although additional years of data are needed before we can understand the epidemiology of serogroup W after PsA–TT introduction, these data suggest that serogroup W will remain a major cause of sporadic disease and has epidemic potential, underscoring the need to maintain high-quality case-based meningitis surveillance after PsA–TT introduction.

Hepatitis C virus transmission in hemodialysis units: importance of infection control practices and aseptic technique.

We investigated 4 hepatitis C virus (HCV) infection outbreaks at hemodialysis units to identify practices associated with transmission. Apparent failures to follow recommended infection control precautions resulted in patient-to-patient HCV transmission, through cross-contamination of the environment or intravenous medication vials. Fastidious attention to aseptic technique and infection control precautions are essential to prevent HCV transmission.