Differences in SpeB protease activity among group A streptococci associated with superficial, invasive, and autoimmune disease.

The secreted cysteine proteinase SpeB is an important virulence factor of group A streptococci (GAS), whereby SpeB activity varies widely among strains. To establish the degree to which SpeB activity correlates with disease, GAS organisms were recovered from patients with pharyngitis, impetigo, invasive disease or acute rheumatic fever (ARF), and selected for analysis using rigorous sampling criteria; >300 GAS isolates were tested for SpeB activity by casein digestion assays, and each GAS isolate was scored as a SpeB-producer or non-producer. Highly significant statistical differences (p < 0.01) in SpeB production are observed between GAS recovered from patients with ARF (41.5% SpeB-non-producers) compared to pharyngitis (20.5%), invasive disease (16.7%), and impetigo (5.5%). SpeB activity differences between pharyngitis and impetigo isolates are also significant, whereas pharyngitis versus invasive isolates show no significant difference. The disproportionately greater number of SpeB-non-producers among ARF-associated isolates may indicate an altered transcriptional program for many rheumatogenic strains and/or a protective role for SpeB in GAS-triggered autoimmunity.

Long-term highly suppressed HIV-infected children and adolescents with negative rapid HIV tests due to significant antibody loss.

BACKGROUND: Rapid HIV test devices are widely used throughout the world and are important as diagnostic tools with relatively high sensitivity and specificity. Loss of HIV specific antibodies in late-stage AIDS patients has previously been reported in patients with advanced disease (i.e., AIDS). OBJECTIVE: To study rate of antibody loss that may lead to false negative HIV-antibodies results in children and adolescents who received long term antiretroviral (ARV) treatment with persistently undetectable viral loads. STUDY DESIGN: Five FDA approved rapid HIV test kits including Trinity Uni-Gold Recombigen HIV-1, OraQuick Advance HIV-1/2, Reveal G3 HIV-1, Clearview STAT-PAK HIV-1/2, and Clearview COMPLETE HIV-1/2 were used to test 98 stored samples from 27 patients. Samples were tested at baseline and at least twice in 6-14 years post initiation of ARV treatment and full viral load suppression. RESULTS: Of the 403 tests, 43 (10.7%) were found to be false-negative using rapid HIV kits. Loss of positivity was correlated with decrease of HIV antibody titer. CONCLUSIONS: There is a slow but persistent loss of HIV specific antibodies in highly suppressed HIV infected children and adolescents that may lead to false-negative results in rapid HIV antibody tests. The temporal loss of signal is dependent on the baseline level of antibodies and the type of HIV rapid test kit used.

Higher incidence of perineal community acquired MRSA infections among toddlers.

BACKGROUND: A six-fold increase in pediatric MRSA infections, prompted us to examine the clinical profile of children with MRSA infections seen at Mercy Children's Hospital, Toledo, Ohio and to characterize the responsible strains. METHODS: Records were reviewed of pediatric patients who cultured positive for MRSA from June 1 to December 31, 2007. Strain typing by pulsed field gel electrophoresis (PFT) and DiversiLab, SCCmec typing, and PCR-based lukSF-PV gene (encodes Panton-Valentine leukocidin), arginine catabolic mobile element (ACME) and cap5 gene detection was performed. RESULTS: Chart review of 63 patients with MRSA infections revealed that 58(92%) were community acquired MRSA (CAMRSA). All CAMRSA were skin and soft tissue infections (SSTI). Twenty five (43%) patients were aged < 3 yrs, 19(33%) aged 4-12 and 14(24%) aged 13-18. Nineteen (76%) of those aged < 3 yrs had higher incidence of perineal infections compared to only 2(11%) of the 4-12 yrs and none of the 13-18 yrs of age. Infections in the extremities were more common in the older youth compared to the youngest children. Overall, there was a significant association between site of the infection and age group (Fisher's Exact p-value < 0.001). All CAMRSA were USA300 PFT, clindamycin susceptible, SCCmec type IVa and lukSF-PV gene positive. Nearly all contained ACME and about 80% were cap5 positive. Of the 58 USA300 strains by PFT, 55(95%) were also identified as USA300 via the automated repetitive sequence-based PCR method from DiversiLab. CONCLUSIONS: CAMRSA SSTI of the perineum was significantly more common among toddlers and that of the extremities in older children. The infecting strains were all USA300 PFT. Further studies are needed to identify the unique virulence and colonization characteristics of USA300 strains in these infections.

Differential gene expression of soluble CD8+ T-cell mediated suppression of HIV replication in three older children.

Suppression of human immunodeficiency virus (HIV) replication by CD8+ T-cells (CD8 suppression) contributes to survival in adults and children <1 year. Soluble CD8 suppression can also be seen in some older children with AIDS. The factor responsible, CD8-derived antiviral factor (CAF), acts at the level of HIV RNA transcription. Differential gene expression techniques have been used to define the gene(s) mediating this phenomenon in adults. Recently, CAF has been linked to exosomes secreted by CD8+ T-cells. To compare the gene expression profiles from pediatric patients with each other, with those reported in 2 previous studies in adults and in those reportedly related to exosomes, we used differential gene expression to study three older children with HIV infection, one who did demonstrate soluble CD-8 suppression and two who did not. Eighteen differentially expressed genes were also seen in one adult study (P = 0.002, χ(2) test), and 38 such genes (P < 0.0001, χ(2) test) in a second adult study. In addition, two exosome components and some RNA's related to exosomal proteins were also differentially expressed. In children with HIV infection, we found significant differentially expressed genes that correlated to those previously reported in two studies in adults. Our data also lends some support to the recent identification of CAF with exosomes secreted by CD8+ T-cells.

Phase I/II, open-label trial of safety and immunogenicity of meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccine in human immunodeficiency virus-infected adolescents.

BACKGROUND: Quadrivalent meningococcal polysaccharide conjugate vaccine (MCV4) is routinely recommended for healthy youth in the United States, but there are no data about its use in HIV-infected people. METHODS: P1065 is a Phase I/II trial of MCV4 safety and immunogenicity in HIV-infected children and youth performed at 27 US sites of the IMPAACT network. All youth (11-24 years old) received 1 dose of open-label MCV4 at entry. Standardized questionnaires were used to evaluate safety. Baseline protective immunity was defined as rabbit serum bactericidal antibody (rSBA) titer > or = 1:128. Immunogenic response was defined as a > or = 4-fold rise in rSBA against each meningococcal serogroup. Multivariable logistic regression analysis was used to evaluate the association of demographic and clinical characteristics on immunogenic response to serogroup C. RESULTS: Among 319 subjects who received MCV4, 10 (3.1%) reported immediate adverse events which were local and mild, and 7 (2.2%) experienced Grade > or = 3 adverse events, unrelated to vaccine. The 305 subjects with serologic data had a median age of 17 years and were 59% male, 50% Black, and 38% Latino. Subjects were stratified by entry CD4%: 12%, CD4 <15%; 40%, 15% to 24%; and 48%, > or = 25%. Baseline protective immunity varied by serogroup: A, 41%; C, 11%; W-135, 15%; Y, 35% The immunogenic response rates to serogroups A, C, W-135, and Y were 68%, 52%, 73%, and 63%, respectively. In multivariable logistic regression models, lower entry CD4%, higher entry viral load, and CDC Class B/C diagnosis were associated with significantly lower odds of response to serogroup C. CONCLUSION: Many HIV-infected youth naturally acquire meningococcal immunity. MCV4 is safe and immunogenic in HIV-infected youth, but response rates are lower than in healthy youth, particularly for those with more advanced HIV clinical, immunologic, and virologic status.

Seven-year surveillance of north american pediatric group a streptococcal pharyngitis isolates.

BACKGROUND: Pharyngeal group A streptococcal (GAS) emm type surveillance enhances understanding of the epidemiology of pharyngitis and invasive GAS disease and formulation of multivalent type-specific vaccines. In addition, such surveillance provides pre-GAS vaccine baseline data. We assessed geographic and temporal trends in GAS emm-type distribution among pediatric pharyngeal isolates collected systematically in the United States and Canada from 2000 to 2007. METHODS: We collected approximately 100 acute GAS pharyngitis isolates from each of 13 widely scattered sites (10 in the United States and 3 in Canada) annually for 7 seasons (2000-2007) from 3- to 18-year-old children. We assessed emm type and subtype by DNA sequencing and analyzed temporal and geographic trends. RESULTS: A total of 7040 US and 1434 Canadian GAS isolates were studied. The 6 most prevalent emm types (in descending order) were 1, 12, 28, 4, 3, and 2 in the United States and 12, 1, 28, 4, 3, 2, and 77 in Canada, constituting 70%-71% of isolates in each country; 10 emm types constituted 87%-89% total. Fifty-six emm types were identified in the United States, including 8 new types, and 33 types in Canada. Although a few types predominated nationally, marked variability among individual sites and at individual sites from year to year was observed. US-Canadian differences in type distribution were apparent. Twenty percent of isolates represented emm subtypes that differed slightly from reference types; 110 new subtypes were identified. An experimental 26-valent M protein vaccine covers 85% of pharyngitis isolates. CONCLUSIONS: Although overall US and Canadian emm type distribution was consistent and relatively few types dominated nationally, striking intersite and temporal variations within individual sites in prevalent emm types of GAS occurred. These results have important implications for the development and formulation of type-specific GAS vaccines.

Performance of a rapid antigen-detection test and throat culture in community pediatric offices: implications for management of pharyngitis.

OBJECTIVES: The goals were to establish performance characteristics of a rapid antigen-detection test and blood agar plate culture performed and interpreted in community pediatric offices and to assess the effect of the pretest likelihood of group A streptococcus pharyngitis on test performance (spectrum bias). METHODS: Two throat swabs were collected from 1848 children 3 to 18 years of age who were evaluated for acute pharyngitis between November 15, 2004, and May 15, 2005, in 6 community pediatric offices. One swab was used to perform the rapid antigen-detection test and a blood agar plate culture in the office and the other was sent to our laboratory for blood agar plate culture. Clinical findings were used to calculate the McIsaac score for each patient. The sensitivities of the office tests were calculated, with the hospital laboratory culture results as the criterion standard. RESULTS: Thirty percent of laboratory blood agar plate cultures yielded group A streptococcus (range among sites: 21%-36%). Rapid antigen-detection test sensitivity was 70% (range: 61%-80%). Office culture sensitivity was significantly greater, 81% (range: 71%-91%). Rapid antigen-detection test specificity was 98% (range: 98%-99.5%), and office culture specificity was 97% (range: 94%-99%), a difference that was not statistically significant. The sensitivity of a combined approach using the rapid antigen-detection test and back-up office culture was 85%. Among patients with McIsaac scores of >2, rapid antigen-detection test sensitivity was 78%, office culture sensitivity was 87%, and combined approach sensitivity was 91%. Positive diagnostic test results were significantly associated with McIsaac scores of >2. CONCLUSIONS: The sensitivity of the office culture was significantly greater than the sensitivity of the rapid antigen-detection test, but neither test was highly sensitive. The sensitivities of each diagnostic modality and the recommended combined approach were best among patients with greater pretest likelihood of group A streptococcus pharyngitis.

Age-associated differences in prevalence of group A streptococcal type-specific M antibodies in children.

Our prior studies of the molecular epidemiology of group A streptococcus (GAS) pharyngitis indicated that the most common emm types associated with pediatric pharyngitis in North America were 12, 1, 28, and 4. We previously reported that the proportions of pediatric pharyngitis due to emm types 12 and 4 decreased with increasing age throughout childhood. We hypothesized that this is due to age-associated acquisition of antibodies to the amino-terminal type-specific region of common GAS M proteins during childhood. We sought to demonstrate this in sera from healthy children by using ELISAs for M 12, 1, 28, and 4. Enzyme-linked immunosorbent assays (ELISA) using chemically synthesized peptides copying amino-terminal type-specific regions of the M proteins were performed on sera from four age groups of healthy children (group I: 3-6 years, group II: 7-10 years, group III: 11-14 years, group IV: 15-18 years). ELISA data were correlated with opsonophagocytic assays for a subset of sera and M 1 GAS. Sera from healthy 12-20-month-old children were used as negative controls. Our results showed that the highest percentage of positivity was for M12, which also showed progressive seropositivity in older children. For the other serotypes, the highest seroprevalence rates were in the 11-14-year-old age group. The presence of ELISA antibodies against M1 correlated with opsonophagocytic activity, a previously studied indicator of immunologic protection.

Update on the laboratory diagnosis and monitoring of HIV infection.

In China, the estimated number of HIV infected cases is approaching one million. Although public education has been initiated for awareness and behavioral modification for this devastating infection, better diagnostic methods are needed to identify infected persons and manage infection. Simple and more accurate diagnostic tools have become available, particularly for early detection and to monitor treatment in those who receive anti-retroviral treatment. In this short review, we summarize some of the common and new methodologies that can be used in clinical laboratories, in the field, or in private laboratories. These range from simple antibody tests to more sophistical methods that are used to monitor disease progression and identify drug resistance. These tools can assist physicians, medical practitioners, and laboratory personnel to select suitable diagnostic tools for the diagnosis, blood screening, monitoring of disease progression, and for detection of drug resistance to anti-retroviral therapies.

Community-based surveillance in the united states of macrolide-resistant pediatric pharyngeal group A streptococci during 3 respiratory disease seasons.

BACKGROUND: In 2001, a total of 48% of pharyngeal group A streptococci (GAS) from Pittsburgh children were macrolide resistant. We assessed macrolide resistance, resistance genes, and emm types among GAS in the United States. METHODS: In prospective, multicenter, community-based surveillance of pharyngeal GAS recovered from children 3-18 years old during 3 respiratory seasons (the 2000-2001 season, the 2001-2002 season, and the 2002-2003 season), GAS were tested for macrolide resistance and underwent emm gene sequencing. Macrolide-resistant GAS were tested for resistance to clindamycin, and resistance genes were determined. RESULTS: Erythromycin resistance was observed in 4.4% of isolates from the 2000-2001 season, 4.3% from the 2001-2002 season, and 3.8% from the 2002-2003 season (P=.80). Clindamycin resistance was found in 1.04% of isolates; annual rates of clindamycin resistance were stable (P=.75). The predominant resistance genotype each year was mef A (65%-76.9%; overall, 70.3%). Resistant isolates included strains representing 8-11 different emm types each year. Heterogeneity of emm subtypes, resistance genes, and clindamycin resistance was evident among resistant isolates within some emm types. Geographic variability in resistance rates was present each year. CONCLUSIONS: The macrolide resistance rate among pharyngeal GAS was <5% and was stable over the 3 seasons. However, rates varied among sites each year. There was no evidence of spread of a specific resistant clone, increasing clindamycin resistance, or escalation in median erythromycin MICs.

Antimicrobial susceptibilities of invasive pediatric Abiotrophia and Granulicatella isolates.

Abiotrophia and Granulicatella species have been associated with various infections. Antimicrobial susceptibility data for these nutritionally variant streptococcus-like organisms, especially for pediatric isolates, are very limited. Little is known about the genetic bases of their resistance mechanisms. We report the results of identification to bacterial species level, antimicrobial susceptibility testing, macrolide resistance testing, and detection of genes encoding that resistance for a collection of 15 pediatric clinical isolates from normally sterile sites. Our results indicate that the prevalence of beta-lactam and macrolide resistance is high and that both erm and mef are found in these isolates.

Anti-Vpr activity of a yeast chaperone protein.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G(2) arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

Group A streptococcal pharyngitis serotype surveillance in North America, 2000-2002.

Geographic and interseasonal heterogeneity of pharyngeal group A streptococcal (GAS) genotypes (emm types) is poorly characterized. We evaluated emm type and subtype distribution among pediatric pharyngitis isolates obtained from 9 sites in the United States during 2000-2001 (year 1) and from 10 sites in the United States and 1 site in Canada during 2001-2002 (year 2). The 7 predominant types were the same in both years, although their order changed. emm 12, 1, and 28 accounted for 49.2% of year 1 isolates, and emm 1, 12, and 4 accounted for 47.1% of year 2 isolates; 6 types accounted for 72.1% in year 1 and 69.4% in year 2. From year 1 to year 2, the proportions of emm 12 and 28 decreased and emm 1 and 6 increased. Striking intersite and interseasonal variations in the distribution of predominant emm types were observed. We conclude that the most-predominant GAS genotypes were similar for each year despite fluctuations, that intersite and intrasite variations in the distribution of emm types were apparent, and that emm type surveillance is needed as M protein vaccine development proceeds.

Quantification of human immunodeficiency virus type 1 proviral DNA by using TaqMan technology.

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.