Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

Highly sensitive detection of Staphylococcus aureus directly from patient blood.

BACKGROUND: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing-polymerase chain reaction (PCR) platform as a model diagnostic system. METHODOLOGY/PRINCIPAL FINDINGS: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90% positive) as opposed to cell-associated bacteria (in WBCs) (71% samples positive) or free bacterial DNA in plasma (62.5% samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95% CI 0.75-0.96) and 0/59 negative controls with the sodA assay (specificity 100%, 95% CI 0.92-1). CONCLUSIONS: We have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.

Rapid universal identification of bacterial pathogens from clinical cultures by using a novel sloppy molecular beacon melting temperature signature technique.

A real-time PCR assay with the ability to rapidly identify all pathogenic bacteria would have widespread medical utility. Current real-time PCR technologies cannot accomplish this task due to severe limitations in multiplexing ability. To this end, we developed a new assay system which supports very high degrees of multiplexing. We developed a new class of mismatch-tolerant "sloppy" molecular beacons, modified them to provide an extended hybridization range, and developed a multiprobe, multimelting temperature (T(m)) signature approach to bacterial species identification. Sloppy molecular beacons were exceptionally versatile, and they were able to generate specific T(m) values for DNA sequences that differed by as little as one nucleotide to as many as 23 polymorphisms. Combining the T(m) values generated by several probe-target hybrids resulted in T(m) signatures that served as highly accurate sequence identifiers. Using this method, PCR assays with as few as six sloppy molecular beacons targeting bacterial 16S rRNA gene segments could reproducibly classify 119 different sequence types of pathogenic and commensal bacteria, representing 64 genera, into 111 T(m) signature types. Blinded studies using the assay to identify the bacteria present in 270 patient-derived clinical cultures including 106 patient blood cultures showed a 95 to 97% concordance with conventional methods. Importantly, no bacteria were misidentified; rather, the few species that could not be identified were classified as "indeterminate," resulting in an assay specificity of 100%. This approach enables highly multiplexed target detection using a simple PCR format that can transform infectious disease diagnostics and improve patient outcomes.

Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology.

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.

Disappearance of vaccine-type invasive pneumococcal disease and emergence of serotype 19A in a minority population with a high prevalence of human immunodeficiency virus and low childhood immunization rates.

We analyzed the epidemiology of invasive pneumococcal disease (IPD) following introduction of pneumococcal conjugated vaccine in an urban population with a 2% human immunodeficiency virus (HIV) prevalence and history of low childhood immunization rates. We observed near-elimination of vaccine-type IPD. Substantial disease remains due to non-vaccine-type pneumococci, highlighting the need to increase pneumococcal immunization among HIV-infected adults.

Invasive pneumococcal disease in an underimmunized, high HIV prevalence population.

OBJECTIVE: To describe the epidemiology of invasive pneumococcal disease (IPD) in a predominantly minority population with low childhood immunization rates and high HIV prevalence, during the early childhood pneumococcal vaccine (PCV7) era. METHODS: A retrospective cases series analysis of 131 patients diagnosed with IPD at University Hospital in Newark, NJ from 2000 through 2005, and who had their pneumococcal isolates serotyped, was conducted. Changes in IPD over time were analyzed with the Cochran-Armitage test and linear regression. Multivariate logistic regression was conducted to determine risk factors for non-vaccine type IPD. RESULTS: Ninety-two percent of cases occurred in older children (>or=5 years) and adults, with 53.4% occurring in the 34-49 year-old age group. 90% of cases were black and 48% were HIV-infected. Among cases five years or older, there was a significant decrease in the proportion of IPD caused by vaccine serotypes (2000: 45.5%, 2001: 50.0%, 2002: 31.8%, 2003: 30.0%, 2004: 0.0%, 2005: 0.0%; p<0.0005). Concomitantly, PCV7 immunization rates among Newark infants increased (2002: 30.5%, 2003: 58.1%, 2004: 70.9%, 2005: 75.6%). Risk factors for non-vaccine type IPD included year of diagnosis and older male. CONCLUSION: At-risk populations, with high HIV prevalence and relatively low infant PCV7 immunization rates, may still be benefiting from PCV7-related herd protection effects.

An outbreak of Fusarium keratitis associated with contact lens use in the northeastern United States.

PURPOSE: To report an outbreak of Fusarium keratitis in contact lens (CL) wearers in the northeastern United States. METHODS: Over a 41-month period, all cases with culture-proven corneal ulceration secondary to Fusarium at 2 tertiary care eye centers were identified through the microbiology departments of each institution, and a retrospective review of charts was performed. Statistical analyses were performed to evaluate a possible association of Fusarium keratitis with specific CL and CL solution brands. RESULTS: Fifteen cases of Fusarium keratitis were reported at the 2 tertiary centers between July 2005 and May 2006 (16.4 cases/yr) compared with 6 cases over the previous 30 months from January 2003 to June 2005 (2.4 cases/yr). All 15 of the more recent cases were CL users, and none had a history of trauma. All 15 patients claimed use of ReNu brand contact lens solution when they developed keratitis. Twelve (80.0%) of 15 patients were Acuvue soft contact lens users. Ten (66.7%) of 15 patients used tap water to rinse their contact lens cases. Six (40.0%) of 15 cases have thus far required corneal transplantation. CONCLUSIONS: The incidence of corneal ulceration secondary to Fusarium has increased sevenfold over the reported 11-month period at 2 tertiary eye care centers in the northeastern United States compared with the previous 30 months. There seems to be an association between the recent outbreak of Fusarium keratitis among CL users and the use of ReNu contact lens solution. Medical treatment of Fusarium keratitis may be ineffective, and emergent penetrating keratoplasty (PKP) may be required in some patients. CL users and their physicians should reconsider the risks of CL use and discuss proper lens care techniques.

A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria.

Bacterial 16S ribosomal RNA (rRNA) genes contain nine "hypervariable regions" (V1-V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. No single region can differentiate among all bacteria; therefore, systematic studies that compare the relative advantage of each region for specific diagnostic goals are needed. We characterized V1-V8 in 110 different bacterial species including common blood borne pathogens, CDC-defined select agents and environmental microflora. Sequence similarity dendrograms were created for hypervariable regions V1-V8, and for selected combinations of regions or short segments within individual hypervariable regions that might be appropriate for DNA probing and real-time PCR. We determined that V1 best differentiated among Staphylococcus aureus and coagulase negative Staphylococcus sp. V2 and V3 were most suitable for distinguishing all bacterial species to the genus level except for closely related enterobacteriaceae. V2 best distinguished among Mycobacterium species and V3 among Haemophilus species. The 58 nucleotides-long V6 could distinguish among most bacterial species except enterobacteriaceae. V6 was also noteworthy for being able to differentiate among all CDC-defined select agents including Bacillus anthracis, which differed from B. cereus by a single polymorphism. V4, V5, V7 and V8 were less useful targets for genus or species-specific probes. The hypervariable sequence-specific dendrograms and the "MEGALIGN" files provided online will be highly useful tools for designing specific probes and primers for molecular assays to detect pathogenic bacteria, including select agents.