The effects of differences in pspA alleles and capsular types on the resistance of Streptococcus pneumoniae to killing by apolactoferrin.

Pneumococcal surface protein A (PspA) is the only pneumococcal surface protein known to strongly bind lactoferrin on the bacterial surface. In the absence of PspA Streptococcus pneumoniae becomes more susceptible to killing by human apolactoferrin (apo-hLf), the iron-free form of lactoferrin. In the present study we examined diverse strains of S. pneumoniae that differed by 2 logs in their susceptibility to apo-hLf. Among these strains, the amount of apo-hLf that bound to cell surface PspA correlated directly with the resistance of the strain to killing by apo-hLf. Moreover examination of different pspA alleles on shared genetic backgrounds revealed that those PspAs that bound more lactoferrin conferred greater resistance to killing by apo-hLf. The effects of capsule on killing of pneumococci by apo-hLf were generally small, but on one genetic background, however, the lack of capsule was associated with 4-times as much apo-hLf binding and 30-times more resistance to killing by apo-hLf. Overall these finding strongly support the hypothesis that most of the variation in the ability of apo-hLf is dependent on the variation in the binding of apo-hLf to surface PspA and this binding is dependent on variation in PspA as well as variation in capsule which may enhance killing by reducing the binding of apo-hLf to PspA.

Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production.

There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

Colonization potential to reconstitute a microbe community in patients detected early after fecal microbe transplant for recurrent C. difficile.

BACKGROUND: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient's gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice. RESULTS: A total of nine samples from three human donors, recipient's pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01%). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8%, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55%, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2-4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7% of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10%. CONCLUSION: We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.

Loss of Vancomycin-Resistant Enterococcus Fecal Dominance in an Organ Transplant Patient With Clostridium difficile Colitis After Fecal Microbiota Transplant.

We report the use of fecal microbiota transplantation in a single heart-kidney transplant recipient with recurrent Clostridium difficile, vancomycin-resistant Enterococcus (VRE) fecal dominance, and recurrent VRE infections. Fecal microbiota transplantation resulted in the reconstruction of a diverse microbiota with (1) reduced relative abundance of C difficile and VRE and (2) positive clinical outcome.

Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.

Asymptomatic bacteriuria: prevalence rates of causal microorganisms, etiology of infection in different patient populations, and recent advances in molecular detection.

Bacteriuria, or the presence of bacteria in urine, is associated with both asymptomatic and symptomatic urinary tract infection and underpins much of the dynamic of microbial colonization of the urinary tract. The prevalence of bacteriuria in dissimilar patient groups such as healthy adults, institutionalized elderly, pregnant women, and immune-compromised patients varies widely. In addition, assessing the importance of 'significant bacteriuria' in infected individuals represents a diagnostic challenge, partly due to various causal microorganisms, and requires careful consideration of the distinct etiologies of bacteriuria in different populations and circumstances. Recent molecular discoveries have revealed how some bacterial traits can enable organisms to grow in human urine, which, as a fitness adaptation, is likely to influence the progression of bacteriuria in some individuals. In this review, we comprehensively analyze currently available data on the prevalence of causal organisms with a focus on asymptomatic bacteriuria in dissimilar populations. We evaluate recent advances in the molecular detection of bacteriuria from a diagnostic viewpoint and briefly discuss the potential benefits and some of the challenges of these approaches. Overall, this review provides an update on the comparative prevalence and etiology of bacteriuria from both microbiological and clinical perspectives.

Prognostic value of semi-quantitative bacteruria counts in the diagnosis of group B streptococcus urinary tract infection: a 4-year retrospective study in adult patients.

BACKGROUND: Semi-quantitative bacteruria counts (s-QBC) are important in the diagnosis of urinary tract infection (UTI) due to most uropathogens. The prognostic value of s-QBC for diagnosis of UTI due to group B streptococcus (GBS) is unknown. In this study, we assessed the value of s-QBC for differentiating acute GBS UTI from asymptomatic bacteruria (ABU), independent of other potential prognostic indicators. METHODS: Medical record review and urinalysis (UA) values for 1593 patients who had urinary GBS isolated (103 to ≥105 CFU/ml) during a four-year period were analyzed using binary logistic regression to determine the predictive values of s-QBC, age, and gender for infection category (acute UTI, ABU) based on the clinical diagnosis. RESULTS: s-QBC alone had a strong predictive value for infection category but only for ABU. Multivariate logistic regression showed similar predictive power of s-QBC for infection category using age as a co-predictor, which was also independently associated with infection category. Typical s-QBC cut-off values that are commonly used in diagnostic settings had no significant power in predicting infection category. Among other UA measures, proteinuria and hematuria were significantly associated with acute infection. CONCLUSIONS: Together, these data show that s-QBC is not useful in the differential diagnosis of GBS UTI. Among the patients in this study, age was an equally effective prognostic indicator compared to s-QBC for identifying high- and low-risk patients for acute GBS UTI. Collectively, these findings indicate that age-based associations may be equally as useful as s-QBC for predicting infection category in the setting of adult patients with GBS-positive urine cultures.

Group B streptococcus cystitis presenting in a diabetic patient with a massive abdominopelvic abscess: a case report.

INTRODUCTION: Streptococcus agalactiae or group B streptococcus is a Gram-positive pathogen that is typically associated with neonatal disease and infection in pregnant women. Group B streptococcus also causes invasive infections in non-pregnant adults including urinary tract infections. The spectrum of urinary tract infections caused by group B streptococcus includes cystitis, pyelonephritis, urosepsis and asymptomatic bacteriuria, which is particularly common among elderly individuals. A rare form of invasive group B streptococcus infection in adults is secondary abscess. Here, we present the first reported case of a patient who developed an unusual, massive abdominopelvic abscess secondary to acute group B streptococcus urinary tract infection. CASE PRESENTATION: A 46-year-old African-American woman presented to the University Emergency Department complaining of urinary tract infection symptoms and severe abdominal pain. Diagnostic imaging by transvaginal ultrasound and computed tomography revealed a massive peripherally-enhancing, low-attenuating fluid collection within her pelvis. The patient's abdominopelvic abscess was drained by ultrasound-guided drainage and this yielded a septic aspirate that was culture positive for abundant S. agalactiae. A recent history of urinary tract infection symptoms in the patient suggested that her abscess developed secondary to cystitis. Complete resolution of the abscess as a favorable outcome was achieved in this case following surgical drainage and appropriate antimicrobial therapy. CONCLUSION: Acute bacterial urinary tract infection leading to an abdominopelvic abscess has not previously been reported in the literature. This case report defines a new disease etiology associated with acute streptococcal cystitis and it will be of interest in cases of urinary tract infections where there is an association with abdominal and/or pelvic pain. A brief review of the literature on unusual secondary abscesses due to group B streptococcus is provided alongside this case to highlight the clinical significance and prognoses of these rare infections. Finally, this case emphasizes the requirement to distinguish unusual etiologies of pyogenic abscesses in order to guide successful clinical management and to treat patients with antibiotics active against the causal organism.

Device for carrying blood samples at 37°C for cryoglobulin test.

To reliably transport blood samples for cryoglobulin analysis, we have created a sample transport device containing a mixture of two waxes that solidifies at 38°C and maintains sample temperature at 38°C. Samples arriving at the laboratory at 37 to 38°C increased to 95% from 34% with the use of the device.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection.

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae.

Streptococcus pneumoniae expresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.

Serine protease PrtA from Streptococcus pneumoniae plays a role in the killing of S. pneumoniae by apolactoferrin.

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

Hit-and-run stimulation: a novel concept to reactivate latent HIV-1 infection without cytokine gene induction.

Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4(+) memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-kappaB) activity. While this short NF-kappaB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-kappaB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and gamma interferon (IFN-gamma). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-kappaB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-kappaB activity profile could be used to selectively trigger HIV-1 reactivation.

Group B Streptococcus (GBS) urinary tract infection involves binding of GBS to bladder uroepithelium and potent but GBS-specific induction of interleukin 1alpha.

Group B Streptococcus (GBS) causes urinary tract infections, but the pathogenic mechanisms underlying GBS urinary tract infections are unknown. We investigated whether uropathogenic GBS can bind to bladder uroepithelium to initiate urinary tract infection. Uropathogenic GBS isolated from a patient with acute cystitis bound to human T24 bladder uroepithelial cells in close association with F-actin in statistically significantly higher numbers compared with nonuropathogenic GBS. In vivo modeling using transurethrally infected mice revealed superior fitness of uropathogenic GBS for bladder colonization and potent uropathogenic GBS-specific up-regulation of interleukin 1alpha during infection. Thus, binding of uropathogenic GBS to uroepithelium and vigorous induction of interleukin 1alpha represents the initial stages of GBS urinary tract infection.

Diversity of group B streptococcus serotypes causing urinary tract infection in adults.

Serotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCR-reverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >10(7) CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS.

PIR-B-deficient mice are susceptible to Salmonella infection.

Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B(-/-)) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B(-/-) mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7-14 days postinfection: diffuse spreading along the sinusoids in PIR-B(-/-) mice vs nodular restricted localization in WT mice. PIR-B(-/-) mice have more inflammatory cells in the liver but fewer B cells and CD8(+) T cells in the spleen than WT mice at 14 days postinfection. PIR-B(-/-) bone marrow-derived macrophages (BMMphi) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMMphi. PIR-B(-/-) BMMphi also produced more nitrite and TNF-alpha upon exposure to Salmonella than WT BMMphi did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.

Risk of infection and tracking of work-related infectious diseases in the funeral industry.

This review demonstrates that funeral service professionals (FSPs) have a risk of exposure to bacterial and viral pathogens as well as to prion-mediated diseases. It reveals a lack of published studies focusing on the implementation and effectiveness of infection control policies for this occupational group as well as the difficulty involved in determining actual infection rates related to workplace exposure events. Possible reasons for this lack of data include the categorization of these workers by the Bureau of Labor Statistics Standard Occupational Classification System as service providers rather than health care professionals.

Severe hemolytic disease of the newborn in a group B African-American infant delivered by a group O mother.

Maternal-fetal ABO incompatibility is a common hematological problem affecting the newborn. In general, hemolysis is minimal and the clinical course is relatively benign, rarely causing the escalating levels of hyperbilirubinemia and significant anemia commonly associated with Rh hemolytic disease of the newborn (HDN). The incidence of HDN ranges from one in 150 births to 1:3000 births, depending on the degree of anemia and level of serum bilirubin. The etiology of ABO hemolytic disease of the newborn (ABO-HDN) is complex because anti-A and anti-B antibodies are composed mainly of IgM. Since only IgG antibodies cross the placenta, those pregnant women with high levels of IgG anti-A,B, anti-A, or anti-B with an ABO incompatible fetus will be the ones to give birth to an infant with ABO-HDN. We describe a case of a B/Rh positive term newborn born to an O/Rh negative African-American mother demonstrating aggressive hemolysis and a robust response of the bone marrow. This case was successfully managed with phototherapy and simple RBC transfusion without the need for exchange transfusion.

Validation of a multiplex pneumococcal serotyping assay with clinical samples.

We have recently developed a rapid pneumococcal serotyping method called "multibead assay" (J. Yu et al., J. Clin. Microbiol. 43:156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis. 148:1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.