Mark S. Pasternack

Neonatal Serologic Screening and Early Treatment for Congenital Toxoplasma gondii Infection

BACKGROUND Most infants with congenital Toxoplasma gondii infection have no symptoms at birth, but many will have retinal disease or neurologic abnormalities later in life. Early detection and treatment of congenital toxoplasmosis may reduce these sequelae. METHODS In Massachusetts since January 1986, and in New Hampshire since July 1988, newborns have been screened for intrauterine infection with T. gondii by means of an IgM capture immunoassay of blood specimens routinely collected for screening for metabolic disorders. Congenital infection is confirmed by assays for specific IgG and IgM antibodies in serum from infants and their mothers. For this study, infants with serologic evidence of infection underwent extensive clinical evaluation and received one year of treatment. RESULTS Through June 1992, 100 of 635,000 infants tested had positive screening tests. Congenital infection was confirmed in 52 infants, 50 of whom were identified only through neonatal screening and not through initial clinical examination. However, after the serologic results became available, more detailed examinations revealed abnormalities of either the central nervous system or the retina in 19 of 48 infants evaluated (40 percent). After treatment, only 1 of 46 children had a neurologic deficit (hemiplegia attributable to a cerebral lesion present at birth). Thirty-nine treated children had follow-up ophthalmologic examinations when one to six years old; four (10 percent) had eye lesions that may have developed postnatally (a macular lesion in one child and minor retinal scars in three). CONCLUSIONS Routine neonatal screening for toxoplasmosis identifies congenital infections that are subclinical, and early treatment may reduce the severe long-term sequelae.

β-Chemokines are released from HIV-1-specific cytolytic T-cell granules complexed to proteoglycans

CD8+ lymphocytes are believed to be important in host defence against the human immunodeficiency virus (HIV)-1, inhibiting HIV-1 replication through both cytolytic and non-cytolytic pathways. The cytolytic pathway involves calcium-dependent exocytosis of perforin and granzyme proteases, as well as Fas-mediated programmed cell death, whereas the noncytolytic pathway involves the release of chemokines that prevent viral entry. Using granzyme A as a marker of cytolytic granule proteins, and macrophage inflammatory protein (MIP)-1α and RANTES as markers of HIV-1 inhibitory chemokines, we show that these two very different mediators of viral inhibition are both localized in the cytolytic granules of HIV-1-specific CD8+ cytotoxic T lymphocytes (CTL). Following antigen-specific activation, these mediators are secreted together, facilitating both lysis of virion-producing cells and the inhibition of free virus. In addition, RANTES, MIP-1α and MIP-1β are secreted by CTL as a macromolecular complex containing sulphated proteoglycans. This association appears to have a functional significance, because heparan sulphate facilitates RANTES inhibition of HIV-1 infection of monocytes.

Activation of Caspase-2 in Apoptosis

Members of the CED-3/interleukin-1β-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32–33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 μm N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 μm) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nm), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 μmDEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Cloning and Expression of Secretagogin, a Novel Neuroendocrine- and Pancreatic Islet of Langerhans-specific Ca2+-binding Protein

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.

Nosocomial infections in pediatric patients with burns

BACKGROUND Nosocomial infections (NI) are believed to occur more commonly in patients with burns than in patients undergoing surgery, but benchmark rates have not been well described, and widely accepted definitions of NI in patients with burns are not available. We present a clinically useful set of definitions for NI for the pediatric burn population and provide benchmark infection rates for NI at selected sites. METHODS Centers for Disease Control and Prevention definitions were modified to more accurately describe nosocomial burn infection and secondary bloodstream infections (BSI) in the burn population. A surveillance system was developed and included calculation of NI rates by 1000 patient or device days, stratified into one of three risk groups (< 30% burn injury, 30% to 60% burn injury, and > 60% burn injury). All patients with acute burns admitted from January 1990 to December 1991 were included, and NI rates were calculated for burn infection, primary and secondary BSI, ventilator-related pneumonia and urinary catheter-related urinary tract infection (UTI). RESULTS Overall 12.5% of patients with central venous catheters had development of primary BSI for a rate of 4.9/1000 central venous catheter-days. Incidence of secondary BSI was 5.8% of patients for a rate of 5.3/1000 patient-days. Incidence of burn infection was 10.1% of patients for a rate of 5.6/1000 patient-days. Incidence of ventilator-related pneumonia was 17.5% of patients for a rate of 11.4/1000 ventilator-days. Incidence of urinary catheter-related UTI was 17.9% of patients, for a rate of 13.2/1000 urinary catheter-days. When rates were stratified by risk groups, incidence increased with increasing burn size for secondary BSI (p < or = 0.0001) and urinary catheter-related UTI (p = 0.08), although rates based on number of patient-days or device-days more accurately reflected risk of infection over time. CONCLUSIONS Infection remains a cause of significant morbidity and death for patients with burns. The definitions and benchmark rates reported here may be useful in evaluation of NI surveillance strategies and calculation of infection rates, which could then be used to evaluate current treatment modalities and improve outcomes for the burn population.

Guidelines for implementation of population-based newborn screening for severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is a Primary Immune Deficiency that is under consideration for population-based newborn screening (NBS) by many NBS programs, and has recently been recommended for inclusion in the US uniform panel of newborn screening conditions. A marker of SCID, the T cell receptor excision circle (TREC), is detectable in the newborn dried blood spot using a unique molecular assay as a primary screen. The New England Newborn Screening Program developed and validated a multiplex TREC assay in which both the TREC analyte and an internal control are acquired from a single punch and run in the same reaction. Massachusetts then implemented a statewide pilot SCID NBS program. The authors describe the rationale for a pilot SCID NBS program, a comprehensive strategy for successful implementation, the screening test algorithm, the screening follow-up algorithm and preliminary experience based on statewide screening in the first year. The Massachusetts experience demonstrates that SCID NBS is a program that can be implemented on a population basis with reasonable rates of false positives.

Inhibitory effect of haptoglobin on granulocyte chemotaxis, phagocytosis and bactericidal activity.

Human haptoglobin (Hp) is synthesized at hepatic and extrahepatic sites as an acute‐phase reactant protein (APP). We investigated the effects of Hp on granulocyte function. The chemotaxis of freshly isolated human granulocytes and differentiated HL‐60 cells in response to the bacterial tripeptide, f‐met‐leu‐phe, was inhibited in the presence of a physiological concentration of Hp, but chemotaxis in the presence of the proinflammatory cytokine interleukin‐8 (IL‐8) was not inhibited. Phagocytosis of viable Escherichia coli, as well as fluoresceinated nonviable E. coli, was inhibited. Hp also reduced granulocyte intracellular bactericidal activity against E. coli. The observed inhibitory effects of Hp on granulocyte function are similar to those reported for C‐reactive protein and suggest that APPs dampen the acute inflammatory response.

Control of naeethicillin-resistant Staphylococcus aureus in a pediatric burn unit

BACKGROUND Control of methicillin-resistant Staphylococcus aureus (MRSA) is particularly difficult in burn units, which are often cited as sources of hospital-wide MRSA outbreaks. We developed a successful MRSA control program and document here its apparent effectiveness in controlling MRSA transmission in a pediatric burn unit. METHODS An MRSA control program that included surveillance culturing, clinician feedback, flexible, site-specific isolation, and a list of known carriers was consistently applied in a pediatric burn unit through a 7-year period. Microbiology reports of MRSA isolates from patients and environmental surfaces and records of all patients from whom MRSA was isolated were reviewed. RESULTS During calendar years 1985 through 1991, a total of 991 acutely burned children were admitted to the Boston unit of the Shriners Burns Institute. Forty MRSA cases (4%) were identified. One patient both had MRSA at admission and met our criteria for nosocomial MRSA. Of the remaining 39 patients, 11 had MRSA at admission and 28 had nosocomial MRSA. There were 17 wound infections, two cases of pneumonia, and two bloodstream infections. No deaths were attributed to MRSA sepsis. CONCLUSION An MRSA control program including surveillance culturing, clinician feedback, flexible, site-specific isolation, and a list of known carriers is associated with a low rate of nosocomial MRSA in a pediatric burn unit.

Bilateral cavernous sinus thromboses and intraorbital abscesses secondary to Streptococcus milleri.

PURPOSE To report the first case of bilateral cavernous sinus thromboses and bilateral intraorbital abscesses secondary to Streptococcus milleri. STUDY DESIGN Single interventional case report. INTERVENTION AND TESTING The findings of the ophthalmic evaluation, radiographic imaging, medical and surgical intervention, specimen cultures, and clinical course were analyzed. RESULTS A 17-year-old female had bilateral proptosis, decreased vision in the left eye, and altered mental status at presentation. An orbital compartment syndrome developed in the left eye and purulent material was present after lateral canthotomy, suggestive of an intraorbital abscess. Magnetic resonance imaging (MRI) scans revealed bilateral cavernous sinus thromboses, and subsequent computed tomographic (CT) scans revealed bilateral intraorbital abscesses in the setting of acute ethmoid and sphenoid sinusitis. Antibiotic treatment and surgical drainage of the orbital abscess and sinuses was performed, and specimen cultures revealed S. milleri. After surgery, the patient experienced hearing loss and a right internal capsule infarct, in addition to complete vision loss in the left eye. A second intraorbital abscess developed in the right eye and was drained surgically. The vision remained 20/20. CONCLUSIONS Streptococcus milleri is a virulent organism with a propensity to form abscesses in multiple areas of the body and should be considered as a possible etiologic agent in abscess formation of the orbit and cavernous sinus thrombosis.

Incidence of catheter-associated bloodstream infection after introduction of minocycline and rifampin antimicrobial-coated catheters in a pediatric burn population.

The Centers for Disease Control and Prevention guidelines for prevention of intravascular catheter-related infections suggest that antimicrobial-coated catheters can decrease the risk of developing catheter-related bloodstream infection in a variety of adult patient populations. There are limited data on their efficacy in the pediatric population, particularly among children with burn injuries. A study was conducted at Shriners Hospitals for Children®, Boston, to determine whether minocycline/rifampin (MR)-coated catheters could decrease the incidence of catheter-associated bloodstream infection (CABSI) in a pediatric burn population. A historical control group included all patients with double- or triple-lumen catheters inserted in the 18-month period from January 2006 to June 2007. The study group included all patients with MR antimicrobial double- or triple-lumen catheters inserted in the subsequent 18-month period, July 2007 to December 2008. Data collected included name, age, date of burn/injury, date of admission, percent TBSA area burn injury or other diagnosis, catheter site (subclavian, internal jugular, or femoral), method of insertion (new percutaneous stick or guidewire), type of catheter (double or triple lumen), date inserted, duration of catheter placement (days), and positive blood cultures recovered while the central venous catheter was in place. CABSI was defined using the Centers for Disease Control and Prevention definition of laboratory-confirmed bloodstream infection. There were a total of 66 patients with 252 catheters (1780 catheter days) in the control group and 75 patients with 263 catheters (1633 catheter days) in the study group. Age, percent burn injury, catheter site, and method of insertion were not statistically different between the two groups. The percentage of infected catheters and the rate of infection were significantly different for the two groups, with the MR antimicrobial catheters only half as likely to become infected. In a subset of these patients with catheters in place for more than 4 days, the percentage of infected catheters and rate of infection were also significantly different with results similar to those in the entire group. MR antimicrobial-coated catheters significantly reduced the incidence of CABSI in this pediatric burn population compared with noncoated catheters.