Detection of Mpox Virus Using Microbial Cell-Free DNA: The Potential of Pathogen-Agnostic Sequencing for Rapid Identification of Emerging Pathogens.

BACKGROUND: The 2022 global outbreak of Monkeypox virus (MPXV) highlighted challenges with polymerase chain reaction detection as divergent strains emerged and atypical presentations limited the applicability of swab sampling. Recommended testing in the United States requires a swab of lesions, which arise late in infection and may be unrecognized. We present MPXV detections using plasma microbial cell-free DNA (mcfDNA) sequencing. METHODS: Fifteen plasma samples from 12 case-patients were characterized through mcfDNA sequencing. Assay performance was confirmed through in silico inclusivity and exclusivity assessments. MPXV isolates were genotyped using mcfDNA, and phylodynamic information was imputed using publicly available sequences. RESULTS: MPXV mcfDNA was detected in 12 case-patients. Mpox was not suspected in 5, with 1 having documented resolution of mpox >6 months previously. Six had moderate to severe mpox, supported by high MPXV mcfDNA concentrations; 4 died. In 7 case-patients, mcfDNA sequencing detected coinfections. Genotyping by mcfDNA sequencing identified 22 MPXV mutations at 10 genomic loci in 9 case-patients. Consistent with variation observed in the 2022 outbreak, 21 of 22 variants were G > A/C > T. Phylogenetic analyses imputed isolates to sublineages arising at different time points and from different geographic locations. CONCLUSIONS: We demonstrate the potential of plasma mcfDNA sequencing to detect, quantify, and, for acute infections with high sequencing coverage, subtype MPXV using a single noninvasive test. Sequencing plasma mcfDNA may augment existing mpox testing in vulnerable patient populations or in patients with atypical symptoms or unrecognized mpox. Strain type information may supplement disease surveillance and facilitate tracking emerging pathogens.

Plasma Microbial Cell-Free DNA Sequencing in Immunocompromised Patients With Pneumonia: A Prospective Observational Study.

BACKGROUND: Pneumonia is a common cause of morbidity and mortality, yet a causative pathogen is identified in a minority of cases. Plasma microbial cell-free DNA sequencing may improve diagnostic yield in immunocompromised patients with pneumonia. METHODS: In this prospective, multicenter, observational study of immunocompromised adults undergoing bronchoscopy to establish a pneumonia etiology, plasma microbial cell-free DNA sequencing was compared to standardized usual care testing. Pneumonia etiology was adjudicated by a blinded independent committee. The primary outcome, additive diagnostic value, was assessed in the Per Protocol population (patients with complete testing results and no major protocol deviations) and defined as the percent of patients with an etiology of pneumonia exclusively identified by plasma microbial cell-free DNA sequencing. Clinical additive diagnostic value was assessed in the Per Protocol subgroup with negative usual care testing. RESULTS: Of 257 patients, 173 met Per Protocol criteria. A pneumonia etiology was identified by usual care in 52/173 (30.1%), plasma microbial cell-free DNA sequencing in 49/173 (28.3%) and the combination of both in 73/173 (42.2%) patients. Plasma microbial cell-free DNA sequencing exclusively identified an etiology of pneumonia in 21/173 patients (additive diagnostic value 12.1%, 95% confidence interval [CI], 7.7% to 18.0%, P < .001). In the Per Protocol subgroup with negative usual care testing, plasma microbial cell-free DNA sequencing identified a pneumonia etiology in 21/121 patients (clinical additive diagnostic value 17.4%, 95% CI, 11.1% to 25.3%). CONCLUSIONS: Non-invasive plasma microbial cell-free DNA sequencing significantly increased diagnostic yield in immunocompromised patients with pneumonia undergoing bronchoscopy and extensive microbiologic and molecular testing. CLINICAL TRIALS REGISTRATION: NCT04047719.

Plasma Microbial Cell-Free DNA Sequencing from over 15,000 Patients Identified a Broad Spectrum of Pathogens.

Microbial cell-free DNA (mcfDNA) sequencing is an emerging infectious disease diagnostic tool which enables unbiased pathogen detection and quantification from plasma. The Karius Test, a commercial mcfDNA sequencing assay developed by and available since 2017 from Karius, Inc. (Redwood City, CA), detects and quantifies mcfDNA as molecules/µL in plasma. The commercial sample data and results for all tests conducted from April 2018 through mid-September 2021 were evaluated for laboratory quality metrics, reported pathogens, and data from test requisition forms. A total of 18,690 reports were generated from 15,165 patients in a hospital setting among 39 states and the District of Columbia. The median time from sample receipt to reported result was 26 h (interquartile range [IQR] 25 to 28), and 96% of samples had valid test results. Almost two-thirds (65%) of patients were adults, and 29% at the time of diagnostic testing had ICD-10 codes representing a diverse array of clinical scenarios. There were 10,752 (58%) reports that yielded at least one taxon for a total of 22,792 detections spanning 701 unique microbial taxa. The 50 most common taxa detected included 36 bacteria, 9 viruses, and 5 fungi. Opportunistic fungi (374 Aspergillus spp., 258 Pneumocystis jirovecii, 196 Mucorales, and 33 dematiaceous fungi) comprised 861 (4%) of all detections. Additional diagnostically challenging pathogens (247 zoonotic and vector-borne pathogens, 144 Mycobacterium spp., 80 Legionella spp., 78 systemic dimorphic fungi, 69 Nocardia spp., and 57 protozoan parasites) comprised 675 (3%) of all detections. This is the largest reported cohort of patients tested using plasma mcfDNA sequencing and represents the first report of a clinical grade metagenomic test performed at scale. Data reveal new insights into the breadth and complexity of potential pathogens identified.

Precision medicine integrating whole-genome sequencing, comprehensive metabolomics, and advanced imaging.

Genome sequencing has established clinical utility for rare disease diagnosis. While increasing numbers of individuals have undergone elective genome sequencing, a comprehensive study surveying genome-wide disease-associated genes in adults with deep phenotyping has not been reported. Here we report the results of a 3-y precision medicine study with a goal to integrate whole-genome sequencing with deep phenotyping. A cohort of 1,190 adult participants (402 female [33.8%]; mean age, 54 y [range 20 to 89+]; 70.6% European) had whole-genome sequencing, and were deeply phenotyped using metabolomics, advanced imaging, and clinical laboratory tests in addition to family/medical history. Of 1,190 adults, 206 (17.3%) had at least 1 genetic variant with pathogenic (P) or likely pathogenic (LP) assessment that suggests a predisposition of genetic risk. A multidisciplinary clinical team reviewed all reportable findings for the assessment of genotype and phenotype associations, and 137 (11.5%) had genotype and phenotype associations. A high percentage of genotype and phenotype associations (>75%) was observed for dyslipidemia (n = 24), cardiomyopathy, arrhythmia, and other cardiac diseases (n = 42), and diabetes and endocrine diseases (n = 17). A lack of genotype and phenotype associations, a potential burden for patient care, was observed in 69 (5.8%) individuals with P/LP variants. Genomics and metabolomics associations identified 61 (5.1%) heterozygotes with phenotype manifestations affecting serum metabolite levels in amino acid, lipid and cofactor, and vitamin pathways. Our descriptive analysis provides results on the integration of whole-genome sequencing and deep phenotyping for clinical assessments in adults.

Precision medicine screening using whole-genome sequencing and advanced imaging to identify disease risk in adults.

Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority. We report early results using genomics in combination with advanced imaging and other clinical testing to proactively screen for age-related chronic disease risk among adults. We enrolled active, symptom-free adults in a study of screening for age-related chronic diseases associated with premature mortality. In addition to personal and family medical history and other clinical testing, we obtained whole-genome sequencing (WGS), noncontrast whole-body MRI, dual-energy X-ray absorptiometry (DXA), global metabolomics, a new blood test for prediabetes (Quantose IR), echocardiography (ECHO), ECG, and cardiac rhythm monitoring to identify age-related chronic disease risks. Precision medicine screening using WGS and advanced imaging along with other testing among active, symptom-free adults identified a broad set of complementary age-related chronic disease risks associated with premature mortality and strengthened WGS variant interpretation. This and other similarly designed screening approaches anchored by WGS and advanced imaging may have the potential to extend healthy life among active adults through improved prevention and early detection of age-related chronic diseases (and their risk factors) associated with premature mortality.

Cost-Effectiveness of Plasma Microbial Cell-Free DNA Sequencing When Added to Usual Care Diagnostic Testing for Immunocompromised Host Pneumonia.

INTRODUCTION: Immunocompromised host pneumonia (ICHP) is an important cause of morbidity and mortality, yet usual care (UC) diagnostic tests often fail to identify an infectious etiology. A US-based, multicenter study (PICKUP) among ICHP patients with hematological malignancies, including hematological cell transplant recipients, showed that plasma microbial cell-free DNA (mcfDNA) sequencing provided significant additive diagnostic value. AIM: The objective of this study was to perform a cost-effectiveness analysis (CEA) of adding mcfDNA sequencing to UC diagnostic testing for hospitalized ICHP patients. METHODS: A semi-Markov model was utilized from the US third-party payer's perspective such that only direct costs were included, using a lifetime time horizon with discount rates of 3% for costs and benefits. Three comparators were considered: (1) All UC, which included non-invasive (NI) and invasive testing and early bronchoscopy; (2) All UC & mcfDNA; and (3) NI UC & mcfDNA & conditional UC Bronch (later bronchoscopy if the initial tests are negative). The model considered whether a probable causative infectious etiology was identified and if the patient received appropriate antimicrobial treatment through expert adjudication, and if the patient died in-hospital. The primary endpoints were total costs, life-years (LYs), equal value life-years (evLYs), quality-adjusted life-years (QALYs), and the incremental cost-effectiveness ratio per QALY. Extensive scenario and probabilistic sensitivity analyses (PSA) were conducted. RESULTS: At a price of $2000 (2023 USD) for the plasma mcfDNA, All UC & mcfDNA was more costly ($165,247 vs $153,642) but more effective (13.39 vs 12.47 LYs gained; 10.20 vs 9.42 evLYs gained; 10.11 vs 9.42 QALYs gained) compared to All UC alone, giving a cost/QALY of $16,761. NI UC & mcfDNA & conditional UC Bronch was also more costly ($162,655 vs $153,642) and more effective (13.19 vs 12.47 LYs gained; 9.96 vs 9.42 evLYs gained; 9.96 vs 9.42 QALYs gained) compared to All UC alone, with a cost/QALY of $16,729. The PSA showed that above a willingness-to-pay threshold of $50,000/QALY, All UC & mcfDNA was the preferred scenario on cost-effectiveness grounds (as it provides the most QALYs gained). Further scenario analyses found that All UC & mcfDNA always improved patient outcomes but was not cost saving, even when the price of mcfDNA was set to $0. CONCLUSIONS: Based on the evidence available at the time of this analysis, this CEA suggests that mcfDNA may be cost-effective when added to All UC, as well as in a scenario using conditional bronchoscopy when NI testing fails to identify a probable infectious etiology for ICHP. Adding mcfDNA testing to UC diagnostic testing should allow more patients to receive appropriate therapy earlier and improve patient outcomes.

Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial.

CONTEXT: In 1999, the US Congress directed the Centers for Disease Control and Prevention to conduct a pivotal safety and efficacy study of anthrax vaccine adsorbed (AVA). OBJECTIVE: To determine the effects on serological responses and injection site adverse events (AEs) resulting from changing the route of administration of AVA from subcutaneous (s.q.) to intramuscular (i.m.) and omitting the week 2 dose from the licensed schedule. DESIGN, SETTING, AND PARTICIPANTS: Assessment of the first 1005 enrollees in a multisite, randomized, double-blind, noninferiority, phase 4 human clinical trial (ongoing from May 2002). INTERVENTION: Healthy adults received AVA by the s.q. (reference group) or i.m. route at 0, 2, and 4 weeks and 6 months (4-SQ or 4-IM; n = 165-170 per group) or at a reduced 3-dose schedule (3-IM; n = 501). A control group (n = 169) received saline injections at the same time intervals. MAIN OUTCOME MEASURES: Noninferiority at week 8 and month 7 of anti-protective antigen IgG geometric mean concentration (GMC), geometric mean titer (GMT), and proportion of responders with a 4-fold rise in titer (%4 x R). Reactogenicity outcomes were proportions of injection site and systemic AEs. RESULTS: At week 8, the 4-IM group (GMC, 90.8 microg/mL; GMT, 1114.8; %4 x R, 97.7) was noninferior to the 4-SQ group (GMC, 105.1 microg/mL; GMT, 1315.4; %4 x R, 98.8) for all 3 primary end points. The 3-IM group was noninferior for only the %4 x R (GMC, 52.2 microg/mL; GMT, 650.6; %4 x R, 94.4). At month 7, all groups were noninferior to the licensed regimen for all end points. Solicited injection site AEs assessed during examinations occurred at lower proportions in the 4-IM group compared with 4-SQ. The odds ratio for ordinal end point pain reported immediately after injection was reduced by 50% for the 4-IM vs 4-SQ groups (P < .001). Route of administration did not significantly influence the occurrence of systemic AEs. CONCLUSIONS: The 4-IM and 3-IM regimens of AVA provided noninferior immunological priming by month 7 when compared with the 4-SQ licensed regimen. Intramuscular administration significantly reduced the occurrence of injection site AEs. Trial Registration clinicaltrials.gov Identifier: NCT00119067.

Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois, 1998.

We investigated an outbreak of leptospirosis among athletes and community residents after a triathlon was held in Springfield, Illinois. A telephone survey was conducted to collect clinical information and data on possible risk factors, community surveillance was established, and animal specimens and lake water samples were collected to determine the source of the leptospiral contamination. A total of 834 of 876 triathletes were contacted; 98 (12%) reported being ill. Serum samples obtained from 474 athletes were tested; 52 of these samples (11%) tested positive for leptospirosis. Fourteen (6%) of 248 symptomatic community residents tested positive for leptospirosis. Heavy rains that preceded the triathlon are likely to have increased leptospiral contamination of Lake Springfield. Among athletes, ingestion of 1 or more swallows of lake water was a predominant risk factor for illness. This is the largest outbreak of leptospirosis that has been reported in the United States. Health care providers and occupational and recreational users of bodies of freshwater in the United States should be aware of the risk of contracting leptospirosis, particularly after heavy rains.

One-year health assessment of adult survivors of Bacillus anthracis infection.

CONTEXT: Little is known about potential long-term health effects of bioterrorism-related Bacillus anthracis infection. OBJECTIVE: To describe the relationship between anthrax infection and persistent somatic symptoms among adults surviving bioterrorism-related anthrax disease approximately 1 year after illness onset in 2001. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study of 15 of 16 adult survivors from September through December 2002 using a clinical interview, a medical review-of-system questionnaire, 2 standardized self-administered questionnaires, and a review of available medical records. MAIN OUTCOME MEASURES: Health complaints summarized by the body system affected and by symptom categories; psychological distress measured by the Revised 90-Item Symptom Checklist; and health-related quality-of-life indices by the Medical Outcomes Study 36-Item Short-Form Health Survey (version 2). RESULTS: The anthrax survivors reported symptoms affecting multiple body systems, significantly greater overall psychological distress (P<.001), and significantly reduced health-related quality-of-life indices compared with US referent populations. Eight survivors (53%) had not returned to work since their infection. Comparing disease manifestations, inhalational survivors reported significantly lower overall physical health than cutaneous survivors (mean scores, 30 vs 41; P =.02). Available medical records could not explain the persisting health complaints. CONCLUSION: The anthrax survivors continued to report significant health problems and poor life adjustment 1 year after onset of bioterrorism-related anthrax disease.

Assessing the risk of laboratory-acquired meningococcal disease.

Neisseria meningitidis is infrequently reported as a laboratory-acquired infection. Prompted by two cases in the United States in 2000, we assessed this risk among laboratorians. We identified cases of meningococcal disease that were possibly acquired or suspected of being acquired in a laboratory by placing an information request on e-mail discussion groups of infectious disease, microbiology, and infection control professional organizations. A probable case of laboratory-acquired meningococcal disease was defined as illness meeting the case definition for meningococcal disease in a laboratorian who had occupational exposure to an N. meningitidis isolate of the same serogroup within 14 days of illness onset. Sixteen cases of probable laboratory-acquired meningococcal disease occurring worldwide between 1985 and 2001 were identified, including six U.S. cases between 1996 and 2000. Nine cases (56%) were serogroup B; seven (44%) were serogroup C. Eight cases (50%) were fatal. All cases occurred among clinical microbiologists. In 15 cases (94%), isolate manipulation was performed without respiratory protection. We estimated that an average of three microbiologists are exposed to the 3,000 meningococcal isolates seen in U.S. laboratories yearly and calculated an attack rate of 13/100,000 microbiologists between 1996 and 2001, compared to 0.2/100,000 among U.S. adults in general. The rate and case/fatality ratio of meningococcal disease among microbiologists are higher than those in the general U.S. population. Specific risk factors for laboratory-acquired infection are likely associated with exposure to droplets or aerosols containing N. meningitidis. Prevention should focus on the implementation of class II biological safety cabinets or additional respiratory protection during manipulation of suspected meningococcal isolates.

Planning against biological terrorism: lessons from outbreak investigations.

We examined outbreak investigations conducted around the world from 1988 to 1999 by the Centers for Disease Control and Prevention's Epidemic Intelligence Service. In 44 (4.0%) of 1,099 investigations, identified causative agents had bioterrorism potential. In six investigations, intentional use of infectious agents was considered. Healthcare providers reported 270 (24.6%) outbreaks and infection control practitioners reported 129 (11.7%); together they reported 399 (36.3%) of the outbreaks. Health departments reported 335 (30.5%) outbreaks. For six outbreaks in which bioterrorism or intentional contamination was possible, reporting was delayed for up to 26 days. We confirmed that the most critical component for bioterrorism outbreak detection and reporting is the frontline healthcare profession and the local health departments. Bioterrorism preparedness should emphasize education and support of this frontline as well as methods to shorten the time between outbreak and reporting.

Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.

Four rapid tests for the serologic diagnosis of leptospirosis were evaluated, and the performance of each was compared with that of the current standard, the microscopic agglutination test (MAT). The four rapid tests were a microplate immunoglobulin M (IgM)-enzyme-linked immunosorbent assay (ELISA), an indirect hemagglutination assay (IHA), an IgM dipstick assay (LDS), and an IgM dot-ELISA dipstick test (DST). A panel of 276 sera from 133 cases of leptospirosis from four different geographic locations was tested as well as 642 sera from normal individuals or individuals with other infectious or autoimmune diseases. Acute-phase sera from cases (n = 148) were collected or=15 days after onset (median = 29.1). By a traditional method (two-by-two contingency table), the sensitivities for detection of leptospirosis cases were 93.2% by LDS, 92.5% by DST, 86.5% by ELISA, and 79.0% by IHA. Specificity was 98.8% by DST, 97% by ELISA and MAT, 95.8% by IHA, and 89.6% by LDS. With a latent class analysis (LCA) model that included all the rapid tests and the clinical case definition, sensitivity was 95.5% by DST, 94.5% by LDS, 89.9% by ELISA, and 81.1% by IHA. The sensitivity and specificity estimated by the traditional methods were quite close to the LCA estimates. However, LCA allowed estimation of the sensitivity of the MAT (98.2%), which traditional methods do not allow. For acute-phase sera, sensitivity was 52.7% by LDS, 50.0% by DST, 48.7% by MAT and ELISA, and 38.5% by IHA. The sensitivity for convalescent-phase sera was 93.8% by MAT, 84.4% by DST, 83.6% by LDS, 75.0% by ELISA, and 67.2% by IHA. A good overall correlation with the MAT was obtained for each of the assays, with the highest concordance being with the DST (kappa value, 0.85; 95% confidence interval [CI], 0.8 to 0.90). The best correlation was between ELISA and DST (kappa value, 0.86; 95% CI, 0.81 to 0.91). False-positive LDS results were frequent (>or=20%) in sera from individuals with Epstein-Barr virus, human immunodeficiency virus, and periodontal disease and from healthy volunteers. The ease of use and significantly high sensitivity and specificity of DST and ELISA make these good choices for diagnostic testing.

Adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine RB51.

Brucella abortus strain RB51 vaccine, is an attenuated live bacterial vaccine that was licensed conditionally by the Center for Veterinary Biologics, Veterinary Services, Animal and Plant Health Inspection Service, USDA, on 23 February 1996, for vaccination of cattle in the United States. Accidental human inoculations can occur during vaccination of cattle, and previous live Brucella vaccines designed for cattle have been known to cause brucellosis in humans. The Centers for Disease Control and Prevention (CDC) established passive surveillance for accidental inoculation with the RB51 vaccine in the United States to determine if this veterinary vaccine is associated with human disease, to describe the circumstances of accidental inoculation, to evaluate the potential efficacy of post-exposure chemoprophylaxis, and to develop recommendations for post-exposure management following exposure to RB51. Reports were received from 26 individuals. Accidental exposure to RB51 occurred by needle stick injury in 21 people (81%), conjunctival spray exposure in four (15%), and spray exposure of an open wound in one (4%) individual. At least one systemic symptom was reported in 19 (73%) people, including three (12%) who reported persistent local reactions with systemic involvement. One case required surgery, and B. abortus strain RB51 was isolated from the wound of that individual. Seven cases reported no adverse event associated with accidental exposure. Nine cases reported previous exposure to Brucella vaccines, including one case who also reported a previous diagnosis of brucellosis following exposure to S19 vaccine. Accidental needle stick injuries and conjunctival or open wound exposures of humans with the RB51 vaccine are associated with both local and systemic adverse events in the United States that are consistent with brucellosis; however, it remains undetermined if strain RB51 vaccine can cause systemic brucellosis in humans. Early culture attempts on those exposed and developing disease in the future and serologic diagnostic assays for anti-RB-51 antibodies are needed to define if these adverse events are due to RB51 and to define appropriate prophylaxis regimens.

The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax.

Cutaneous anthrax is a rare zoonotic disease in the United States. The clinical diagnosis traditionally has been established by conventional microbiological methods, such as culture and gram staining. However, these methods often yield negative results when patients have received antibiotics. During the bioterrorism event of 2001, we applied two novel immunohistochemical assays that can detect Bacillus anthracis antigens in skin biopsy samples even after prolonged antibiotic treatment. These assays provided a highly sensitive and specific method for the diagnosis of cutaneous anthrax, and were critical in the early and rapid diagnosis of 8 of 11 cases of cutaneous anthrax during the outbreak investigation. Skin biopsies were obtained from 10 of these 11 cases, and histopathological findings included various degrees of ulceration, hemorrhage, edema, coagulative necrosis, perivascular inflammation, and vasculitis. Serology was also an important investigation tool, but the results required several weeks because of the need to test paired serum specimens. Other tests, including culture, special stains, and polymerase chain reaction assay, were less valuable in the diagnosis and epidemiological investigation of these cutaneous anthrax cases. This report underscores the critical role of pathology in investigating potential bioterrorism events and in guiding epidemiological studies, a role that was clearly demonstrated in 2001 when B. anthracis spores were intentionally released through the United States postal system.

Pathology and pathogenesis of bioterrorism-related inhalational anthrax.

During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Gram's staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with gram's stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.

Surveillance for unexplained deaths and critical illnesses due to possibly infectious causes, United States, 1995-1998.

Population-based surveillance for unexplained death and critical illness possibly due to infectious causes (UNEX) was conducted in four U.S. Emerging Infections Program sites (population 7.7 million) from May 1, 1995, to December 31, 1998, to define the incidence, epidemiologic features, and etiology of this syndrome. A case was defined as death or critical illness in a hospitalized, previously healthy person, 1 to 49 years of age, with infection hallmarks but no cause identified after routine testing. A total of 137 cases were identified (incidence rate 0.5 per 100,000 per year). Patients' median age was 20 years, 72 (53%) were female, 112 (82%) were white, and 41 (30%) died. The most common clinical presentations were neurologic (29%), respiratory (27%), and cardiac (21%). Infectious causes were identified for 34 cases (28% of the 122 cases with clinical specimens); 23 (68%) were diagnosed by reference serologic tests, and 11 (32%) by polymerase chain reaction-based methods. The UNEX network model would improve U.S. diagnostic capacities and preparedness for emerging infections.

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Serogroup W-135 meningococcal disease during the Hajj, 2000.

An outbreak of serogroup W-135 meningococcal disease occurred during the 2000 Hajj in Saudi Arabia. Disease was reported worldwide in Hajj pilgrims and their close contacts; however, most cases were identified in Saudi Arabia. Trends in Saudi meningococcal disease were evaluated and the epidemiology of Saudi cases from this outbreak described. Saudi national meningococcal disease incidence data for 1990 to 2000 were reviewed; cases from January 24 to June 5, 2000, were retrospectively reviewed. The 2000 Hajj outbreak consisted of distinct serogroup A and serogroup W-135 outbreaks. Of 253 identified cases in Saudi Arabia, 161 (64%) had serogroup identification; serogroups W-135 and A caused 93 (37%) and 60 (24%) cases with attack rates of 9 and 6 cases per 100,000 population, respectively. The 2000 Hajj outbreak was the first large serogroup W-135 meningococcal disease outbreak identified worldwide. Enhanced surveillance for serogroup W-135, especially in Africa, is essential to control this emerging epidemic disease.

First case of bioterrorism-related inhalational anthrax in the United States, Palm Beach County, Florida, 2001.

On October 4, 2001, we confirmed the first bioterrorism-related anthrax case identified in the United States in a resident of Palm Beach County, Florida. Epidemiologic investigation indicated that exposure occurred at the workplace through intentionally contaminated mail. One additional case of inhalational anthrax was identified from the index patient's workplace. Among 1,076 nasal cultures performed to assess exposure, Bacillus anthracis was isolated from a co-worker later confirmed as being infected, as well as from an asymptomatic mail-handler in the same workplace. Environmental cultures for B. anthracis showed contamination at the workplace and six county postal facilities. Environmental and nasal swab cultures were useful epidemiologic tools that helped direct the investigation towards the infection source and transmission vehicle. We identified 1,114 persons at risk and offered antimicrobial prophylaxis.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.