Neutralization of the Staphylococcus aureus Panton-Valentine leukocidin by African and Caucasian sera.

BACKGROUND: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). METHODS: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). RESULTS: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC50) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC50 = 3.51 and 3.59% respectively). CONCLUSION: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.

The expression and clinical significance of CD59 and FLAER in Chinese adult AML patients.

BACKGROUND: The role of CD59 and fluorescently labeled aerolysin (FLAER) in acute myeloid leukemia (AML) remains unclear and requires further investigation. To explore the relationship between CD59, FLAER, and AML, we investigated CD59 and FLAER expression in AML and analyzed their relationship with clinical characteristics of AML patients. METHODS: We employed flow cytometry (FCM) to analyze CD59 and FLAER expression in 161 AML patients at Tianjin Medical University General Hospital and evaluated its association with sex, white blood cell (WBC) count, platelet (PLT) count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-Dimer(D-D), and lactate dehydrogenase (LDH), followed by analyzing its connection with disease progression and complete remission (CR). RESULTS: CD59 and FLAER deficiencies were identified in AML patients. Compared with CR group, non-CR group patients revealed more CD59 and FLAER deficiency. Compared with non-acute promyelocytic leukemia (M3) group, M3 group patients had more CD59 and FLAER deficiency. CD59- level in primordial cells of M3 patients was positively correlated with primordial cell ratio (r = 0.660, p = 0.003). Additionally, we discovered that the decline in CD59 and FLAER levels might be linked to higher D-D and LDH in AML patients. The difference was statistically significant (p < 0.05). CONCLUSIONS: We demonstrated that the decline in CD59 and FLAER levels was associated with leukemia cell proliferation and abnormal coagulation function in AML, suggesting that they could serve as a predictor of AML coagulation dysfunction, particularly in M3.

Increased Risk of Thrombocytopenia and Death in Patients with Bacteremia Caused by High Alpha Toxin-Producing Methicillin-Resistant Staphylococcus aureus.

Alpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited. We examined the link between in vitro Hla activity and outcome. Study isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production by Western immunoblotting in a subset of isolates and Hla activity by hemolysis assay in all isolates. Relevant demographics, laboratory and clinical data were extracted from patients' medical records to correlate Hla activity of the infecting isolates with outcome. Hla production strongly correlated with hemolytic activity (rs = 0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs. 16.55 HU/mL in normal platelet count, p = 0.012) and from non survivors (median 30.96 vs. 14.87 HU/mL in survivors, p = 0.014) but hemolytic activity of MSSA strains did not differ between patient groups. In vitro Hla activity of MRSA strains obtained from patients with bacteremia is significantly associated with increased risk for thrombocytopenia and death which supports future studies to evaluate feasibility of bedside phenotyping and therapeutic targeting.

Markers of Intestinal Permeability Are Rapidly Improved by Alcohol Withdrawal in Patients with Alcohol-Related Liver Disease.

Changes in intestinal microbiome and barrier function are critical in the development of alcohol-related liver disease (ALD). Here, we determined the effects of a one-week alcohol withdrawal on parameters of intestinal barrier function in heavy drinkers with ALD in comparison to healthy non-drinkers (controls). In serum samples of 17 controls (m = 10/f = 7) and 37 age-matched ALD patients (m = 26/f = 11) undergoing a one-week alcohol withdrawal, markers of liver health and intestinal barrier function were assessed. Liver damage, e.g., fibrosis and hepatic steatosis, were assessed using FibroScan. Before alcohol withdrawal, markers of liver damage, lipopolysaccharide binding protein (LBP) and overall TLR4/TLR2 ligands in serum were significantly higher in ALD patients than in controls, whereas intestinal fatty acid binding protein (I-FABP) and zonulin protein concentrations in serum were lower. All parameters, with the exception of LBP, were significantly improved after alcohol withdrawal; however, not to the level of controls. Our data suggest that one-week of abstinence improves markers of intestinal barrier function and liver health in ALD patients.

Evaluation of the CARDS toxin and its fragment for the serodiagnosis of Mycoplasma pneumoniae infections.

Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen in community-acquired pneumonia. The community-acquired respiratory distress syndrome (CARDS) toxin is the only known virulence factor of M. pneumoniae. It is worth exploring whether this toxin can be used as a candidate antigen for the serodiagnosis of M. pneumoniae. In this study, the full-length, N-terminal, and C-terminal regions of the CARDS toxin were expressed and purified, and serological reactions were evaluated using ELISA. A total of 184 serum samples were collected and tested using a commercialized test kit. Eighty-seven samples were positive, and 97 samples were negative for infection. The purified recombinant proteins were used as antigens to test the serum via indirect ELISA. The sensitivity of the CARDS toxin, the N-terminal region, and the C-terminal region were 90.8%, 90.8%, and 92.0%, respectively. The specificity of the CARDS toxin, the N-terminal region, and the C-terminal region were 85.6%, 73.2%, and 93.8%, respectively. All three CARDS toxin proteins exhibited good reactivity, of which the C-terminal region had a good discrimination ability in human sera. This may have a potential diagnostic value for M. pneumoniae infections.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Absence of Toxemia in Clostridioides difficile Infection: Results from Ultrasensitive Toxin Assay of Serum.

Clostridioides difficile infection (CDI) is caused by Toxins A and B, secreted from pathogenic strains of C. difficle. This infection can vary greatly in symptom severity and in clinical presentation. Current assays used to diagnose CDI may lack the required sensitivity to detect the exotoxins circulating in blood. The ultrasensitive single molecule array (Simoa) assay was modified to separately detect toxin A and toxin B in serum with a limit of detection at the low picogram level. When applied to a diverse cohort, Simoa was unable to detect toxins A or B in serum from patients with CDI, including many classified as having severe disease. The detection of toxin may be limited by the inference of antitoxin antibodies circulating in serum. This result does not support the hypothesis that toxemia occurs in C. difficile infection, conflicting with the findings of other published reports.

Osteomyelitis, Venous Thrombosis, and Septic Emboli in a Pediatric Patient: A Case Report.

New virulence factors, such as the Panton-Valentine leukocidin (PVL), are appearing during Staphylococcus aureus infections occurring in the pediatric population. Such factors increase the aggressiveness and risk of dissemination of the bacteria, causing infections to be life-threatening. An early diagnosis is thus especially important. We present a case of osteomyelitis, venous thrombosis, and septic emboli occurring in a pediatric patient that should trigger suspicion of a PVL-positive strain. A multidisciplinary approach is necessary to enable rapid diagnosis and early treatment, which is essential for successful management of these infections. Management is based on broad-spectrum antibiotics, in combination with aggressive surgical treatment and antithrombotic therapy. In patients infected with S. aureus whose condition worsens quickly, PVL gene sequencing should be considered.

The Release of Norepinephrine in C57BL/6J Mice Treated with 6-Hydroxydopamine (6-OHDA) is Associated with Translocations in Enteric Escherichia coli via the QseC Histidine Kinase Receptor.

BACKGROUND We aimed to investigate the effects of norepinephrine (NE) released from endogenous stores on bacterial translocation of Escherichia coli in mice by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. MATERIAL AND METHODS E. coli strain BW25113 and its derivatives (BW25113ΔqseC and BW25113ΔqseC pQseC) were used in this study. The serum concentrations of endotoxin were analyzed. The strains BW25113, BW25113ΔqseC, and BW25113ΔqseC pQseC were detected respectively in tissue specimens harvested from mice treated with 6-OHDA. RESULTS Mice treated with BW25113ΔqseC showed reduced levels of bacterial translocation following administration of 6-OHDA compared with mice treated with BW25113. The defect of E. coli QseC receptor caused the norepinephrine-QseC signal chain to be interrupted, and the invasiveness and penetrating power of the bacteria on the intestinal mucosa was weakened, eventually leading to a significant decrease in the incidence of bacterial translocation. CONCLUSIONS NE modulates the interaction of enteric bacterial pathogens with their hosts via QseC. The blockade of the QseC receptor-mediated effects may be useful to attenuate bacterial translocation.

Clinical Manifestations in Children with Staphylococcal Bacteremia Positive for Panton-Valentine Leucocidin: A Nationwide Survey in a Low Methicillin-Resistance Setting.

A total of 714 pediatric cases of Staphylococcus aureus bacteremia were identified from 2008 to 2015 in Denmark; 98% were methicillin-susceptible S. aureus (MSSA). Fifteen isolates (2,1%) were Panton-Valentine leucocidin positive (0.17/100,000 children/year) and 87% MSSA. Eight cases (53%) were severe, including all pneumonia cases. Panton-Valentine leucocidin positive Staphylococcus aureus bacteremia is rare in our setting with high MSSA-prevalence. Half of the cases were uncomplicated.

Staphylococcus aureus Pneumonia in the Community.

Staphylococcus aureus is an emergent etiology of community-acquired pneumonia (CAP) over the past 2 decades, with severe community-acquired pneumonia (SCAP) caused by methicillin-resistant S. aureus (MRSA) leading to critical illness and death. S. aureus colonization is associated with a high incidence of pneumonia. Panton-Valentine leukocidin (PVL) is one of the most important virulence factors of S. aureus associated with serious complications. In recent years, community-associated MRSA (CA-MRSA) clones that caused infections in young adults and healthy individuals with no exposure to health care settings and no classical risk factors have emerged. Clinical features at admission including concurrent influenza infection, hemoptysis, multilobar infiltrates, and neutropenia should suggest S. aureus CAP. Sputum Gram stains, cultures (or tracheobronchial aspirates or bronchoalveolar lavage in mechanically ventilated patients), polymerase chain reaction (nasopharyngeal or oropharyngeal or lower respiratory tract specimens), and two sets of blood cultures should be obtained from patients presenting with severe S. aureus CAP. For CAP due to methicillin-susceptible S. aureus, first-line therapy is usually cefazolin, oxacillin, or ceftaroline. For CA-MRSA pneumonia, linezolid is recommended. If vancomycin or teicoplanin are used, combination with clindamycin or rifampicin should be considered in cases of PVL-positive MRSA CAP.

Sevelamer Use in End-Stage Kidney Disease (ESKD) Patients Associates with Poor Vitamin K Status and High Levels of Gut-Derived Uremic Toxins: A Drug-Bug Interaction?

Gut microbial metabolism is not only an important source of uremic toxins but may also help to maintain the vitamin K stores of the host. We hypothesized that sevelamer therapy, a commonly used phosphate binder in patients with end-stage kidney disease (ESKD), associates with a disturbed gut microbial metabolism. Important representatives of gut-derived uremic toxins, including indoxyl sulfate (IndS), p-Cresyl sulfate (pCS), trimethylamine N-oxide (TMAO), phenylacetylglutamine (PAG) and non-phosphorylated, uncarboxylated matrix-Gla protein (dp-ucMGP; a marker of vitamin K status), were analyzed in blood samples from 423 patients (65% males, median age 54 years) with ESKD. Demographics and laboratory data were extracted from electronic files. Sevelamer users (n = 172, 41%) were characterized by higher phosphate, IndS, TMAO, PAG and dp-ucMGP levels compared to non-users. Sevelamer was significantly associated with increased IndS, PAG and dp-ucMGP levels, independent of age, sex, calcium-containing phosphate binder, cohort, phosphate, creatinine and dialysis vintage. High dp-ucMGP levels, reflecting vitamin K deficiency, were independently and positively associated with PAG and TMAO levels. Sevelamer therapy associates with an unfavorable gut microbial metabolism pattern. Although the observational design precludes causal inference, present findings implicate a disturbed microbial metabolism and vitamin K deficiency as potential trade-offs of sevelamer therapy.

Gut-Derived Metabolites and Their Role in Immune Dysfunction in Chronic Kidney Disease.

Several of the uremic toxins, which are difficult to remove by dialysis, originate from the gut bacterial metabolism. This opens opportunities for novel targets trying to decrease circulating levels of these toxins and their pathophysiological effects. The current review focuses on immunomodulatory effects of these toxins both at their side of origin and in the circulation. In the gut end products of the bacterial metabolism such as p-cresol, trimethylamine and H2S affect the intestinal barrier structure and function while in the circulation the related uremic toxins stimulate cells of the immune system. Both conditions contribute to the pro-inflammatory status of patients with chronic kidney disease (CKD). Generation and/or absorption of these toxin precursors could be targeted to decrease plasma levels of their respective uremic toxins and to reduce micro-inflammation in CKD.

Prevalence, clinical expression, invasiveness and outcome of Staphylococcus aureus containing Panton-Valentine leukocidin in children treated in a university hospital of Lithuania.

Background: There is a high prevalence of Staphylococcus aureus virulence factor Panton-Valentine leukocidin (PVL) in North-East parts of Europe. The aim was to evaluate data regarding the PVL occurrences in Lithuania, determine the relationship with Methicillin resistant Staphylococcus aureus (MRSA), association with demographic and clinical conditions, invasiveness and severity of the disease in children treated in hospital Kauno klinikos (KK).Methods: We performed a prospective case-cohort single-center study on paediatric patients hospitalized from 2012 to 2015 to KK. We compared characteristics in PVL positive [SA-PVL(+)] and PVL negative [SA-PVL(-)] groups among non-invasive and invasive infections. Logistic regression was performed to detect PVL predicting factors and Cox regression was presented to define factors associated with admission to intensive care unit (ICU).Results: PVL was detected in 51.5%, MRSA in 7.0% and MRSA-PVL(+) in 4.8% of cases. In general, PVL was associated with older age comparing with SA-PVL(-) (median 8.5 vs. 4.0 years, p < .001). Skin and soft tissue infections were presented in 87.9% of all SA-PVL(+) cases. Invasive infections (44.7% vs. 12.1%, p < .001) and co-morbidities (20.5% vs. 2.9%, p < .001) were associated with SA-PVL(-) infections compared to SA-PVL(+), but ICU admission number was higher in invasive SA-PVL(+) cases comparing to invasive SA-PVL(-) cases (41.2% vs. 10.2%, p = .007).Conclusions: There was a high prevalence of pvl gene in patients treated in KK. SA-PVL(+) infections were associated with SSTI and were not common in invasive infections, but the invasive infections caused by SA-PVL(+) were related to severe disease progression and admission to ICU.

Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection.

BACKGROUND: Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. METHODS: To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. RESULTS: One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. CONCLUSIONS: In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.

Circulating Anti-cytolethal Distending Toxin B and Anti-vinculin Antibodies as Biomarkers in Community and Healthcare Populations With Functional Dyspepsia and Irritable Bowel Syndrome.

OBJECTIVES: Anti-cytolethal distending toxin B (CdtB) and anti-vinculin antibodies have been proposed as biomarkers that discriminate irritable bowel syndrome (IBS) diarrhea from inflammatory bowel disease; however, it is unknown whether they can also discriminate patients with IBS and IBS subtypes and functional dyspepsia (FD) from healthy individuals in the general population. We aimed to determine whether anti-CdtB and anti-vinculin can discriminate IBS and FD from health and from organic gastrointestinal (GI) disease. METHODS: Adults were enrolled from 2 Australian studies: (i) a random, population-based study (n = 331) with subjects diagnosed with IBS (n = 63) or FD (n = 61) by modified Rome III criteria or healthy control subjects (n = 246) who did not meet criteria for IBS and/or FD and (ii) an outpatient-based study with subjects diagnosed with IBS (n = 256) and/or FD (n = 55) or organic GI disease (n = 182) by an independent clinician. Serum levels of anti-CdtB/anti-vinculin antibodies were determined by enzyme-linked immunosorbent assay. RESULTS: There was a significantly higher mean value of anti-CdtB in FD vs healthy controls (mean = 2.46 [SD = 0.72] vs mean = 2.14 [SD = 0.77]; P = 0.005) and IBS/FD overlap vs healthy controls (mean = 2.47 [SD = 0.78] vs mean = 2.14 [SD = 0.77]; P = 0.02). There were no significant differences in anti-CdtB in IBS and FD outpatients or IBS/FD subgroups compared with patients with organic GI disease. In terms of anti-vinculin, there were no significant differences between IBS and FD and healthy controls or between IBS and FD and organic GI disease controls. DISCUSSION: We did not confirm that anti-CdtB/anti-vinculin discriminated IBS diarrhea from organic GI disease in Australian subjects. However, we did find higher anti-CdtB in FD and IBS/FD overlap vs healthy controls. Postinfectious FD may be more common than currently recognized.

Second-Generation Biomarker Testing for Irritable Bowel Syndrome Using Plasma Anti-CdtB and Anti-Vinculin Levels.

BACKGROUND: ELISA testing for anti-CdtB and anti-vinculin can discriminate patients with irritable bowel syndrome with diarrhea (IBS-D) from those with inflammatory bowel disease (IBD). However, recent findings suggest the antigens can suffer from epitope instability. AIM: This study aimed to assess effects of incorporating epitope stabilization on test characteristics for distinguishing IBS-D from IBD subjects. METHODS: Plasma samples from IBS-D subjects from a large-scale clinical trial and subjects with endoscopically active IBD without concurrent immunomodulator therapy were used. After epitope stabilization, CdtB and vinculin were used in ELISA testing. Optical density readings were compared between IBS-D and IBD subjects. RESULTS: Samples from 100 IBS-D and 31 IBD (22 UC and 9 CD) subjects were tested. IBS-D subjects had higher anti-CdtB titers (P = 0.0001) and higher anti-vinculin titers (P = 0.004) than IBD subjects. The specificities of anti-CdtB and anti-vinculin to differentiate IBS-D from IBD were 93.5% and 90.9%, respectively, with sensitivities of 43.0% and 52.2%, respectively. The positive likelihood ratios of identifying IBS-D with anti-CdtB and anti-vinculin were 6.7 and 5.7, respectively. Assuming a pretest probability of 57% for diagnosis of IBS-D in patients with abdominal pain and change in bowel habits, testing positive for both antibodies resulted in a posttest probability of > 98%. CONCLUSIONS: Performing epitope stabilization for CdtB and vinculin enhances the test characteristics of ELISAs for anti-CdtB and anti-vinculin in discriminating IBS-D from IBD. Measurement of anti-CdtB and anti-vinculin with this second-generation methodology may further advance our understanding of the role of immunity in functional bowel diseases.

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Accurate and selective quantification of anthrax protective antigen in plasma by immunocapture and isotope dilution mass spectrometry.

Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 µL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF and EF did not interfere with accuracy. Applied to a study of inhalation anthrax in rhesus macaques, total-PA suggested triphasic kinetics, similar to that previously observed for LF and EF. This study is the first to report circulating PA83 in inhalation anthrax, typically at less than 4% of the levels of PA63, providing the first evidence that activated PA63 is the primary form of PA throughout infection.