Association of Early Steroid Administration With Outcomes of Children Hospitalized for COVID-19 Without Multisystem Inflammatory Syndrome in Children.

Importance: There is limited evidence for therapeutic options for pediatric COVID-19 outside of multisystem inflammatory syndrome in children (MIS-C). Objective: To determine whether the use of steroids within 2 days of admission for non-MIS-C COVID-19 in children is associated with hospital length of stay (LOS). The secondary objective was to determine their association with intensive care unit (ICU) LOS, inflammation, and fever defervescence. Design, Setting, and Participants: This cohort study analyzed data retrospectively for children (<18 years) who required hospitalization for non-MIS-C COVID-19. Data from March 2020 through September 2021 were provided by 58 hospitals in 7 countries who participate in the Society of Critical Care Medicine Discovery Viral Infection and Respiratory Illness Universal Study (VIRUS) COVID-19 registry. Exposure: Administration of steroids within 2 days of admission. Main Outcomes and Measures: Length of stay in the hospital and ICU. Adjustment for confounders was done by mixed linear regression and propensity score matching. Results: A total of 1163 patients met inclusion criteria and had a median (IQR) age of 7 years (0.9-14.3). Almost half of all patients (601/1163, 51.7%) were male, 33.8% (392/1163) were non-Hispanic White, and 27.9% (324/1163) were Hispanic. Of the study population, 184 patients (15.8%) received steroids within 2 days of admission, and 979 (84.2%) did not receive steroids within the first 2 days. Among 1163 patients, 658 (56.5%) required respiratory support during hospitalization. Overall, patients in the steroids group were older and had greater severity of illness, and a larger proportion required respiratory and vasoactive support. On multivariable linear regression, after controlling for treatment with remdesivir within 2 days, country, race and ethnicity, obesity and comorbidity, number of abnormal inflammatory mediators, age, bacterial or viral coinfection, and disease severity according to ICU admission within first 2 days or World Health Organization ordinal scale of 4 or higher on admission, with a random intercept for the site, early steroid treatment was not significantly associated with hospital LOS (exponentiated coefficient, 0.94; 95% CI, 0.81-1.09; P = .42). Separate analyses for patients with an LOS of 2 days or longer (n = 729), those receiving respiratory support at admission (n = 286), and propensity score-matched patients also showed no significant association between steroids and LOS. Early steroid treatment was not associated with ICU LOS, fever defervescence by day 3, or normalization of inflammatory mediators. Conclusions and Relevance: Steroid treatment within 2 days of hospital admission in a heterogeneous cohort of pediatric patients hospitalized for COVID-19 without MIS-C did not have a statistically significant association with hospital LOS.

Comprehensiveness of Testing Among Herpes Simplex Virus Infected Infants: A Multicenter Cohort Study.

Despite clear testing recommendations for herpes simplex virus (HSV) infection in infants, few data exist on the comprehensiveness of HSV testing in practice. In a 23-center study of 112 infants with confirmed HSV disease, less than one-fifth had all recommended testing performed, highlighting the need for increased awareness of and adherence to testing recommendations for this vulnerable population.

Early combination therapy with immunoglobulin and steroids is associated with shorter ICU length of stay in Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19: A retrospective cohort analysis from 28 U.S. Hospitals.

OBJECTIVES: Suggested therapeutic options for Multisystem Inflammatory Syndrome in Children (MIS-C) include intravenous immunoglobulins (IVIG) and steroids. Prior studies have shown the benefit of combination therapy with both agents on fever control or the resolution of organ dysfunction. The primary objective of this study was to analyze the impact of IVIG and steroids on hospital and ICU length of stay (LOS) in patients with MIS-C associated with Coronavirus Disease 2019 (COVID-19). STUDY DESIGN: This was a retrospective study on 356 hospitalized patients with MIS-C from March 2020 to September 2021 (28 sites in the United States) in the Society of Critical Care Medicine (SCCM) Discovery Viral Infection and Respiratory Illness Universal Study (VIRUS) COVID-19 Registry. The effect of IVIG and steroids initiated in the first 2 days of admission, alone or in combination, on LOS was analyzed. Adjustment for confounders was made by multivariable mixed regression with a random intercept for the site. RESULTS: The median age of the study population was 8.8 (Interquartile range (IQR) 4.0, 13) years. 247/356 (69%) patients required intensive care unit (ICU) admission during hospitalization. Overall hospital mortality was 2% (7/356). Of the total patients, 153 (43%) received IVIG and steroids, 33 (9%) received IVIG only, 43 (12%) received steroids only, and 127 (36%) received neither within 2 days of admission. After adjustment of confounders, only combination therapy showed a significant decrease of ICU LOS by 1.6 days compared to no therapy (exponentiated coefficient 0.71 [95% confidence interval 0.51, 0.97, p = 0.03]). No significant difference was observed in hospital LOS or the secondary outcome variable of the normalization of inflammatory mediators by Day 3. CONCLUSIONS: Combination therapy with IVIG and steroids initiated in the first 2 days of admission favorably impacts ICU but not the overall hospital LOS in children with MIS-C.

Characterization and Outcomes of Hospitalized Children With Coronavirus Disease 2019: A Report From a Multicenter, Viral Infection and Respiratory Illness Universal Study (Coronavirus Disease 2019) Registry.

OBJECTIVES: Multicenter data on the characteristics and outcomes of children hospitalized with coronavirus disease 2019 are limited. Our objective was to describe the characteristics, ICU admissions, and outcomes among children hospitalized with coronavirus disease 2019 using Society of Critical Care Medicine Discovery Viral Infection and Respiratory Illness Universal Study: Coronavirus Disease 2019 registry. DESIGN: Retrospective study. SETTING: Society of Critical Care Medicine Viral Infection and Respiratory Illness Universal Study (Coronavirus Disease 2019) registry. PATIENTS: Children (< 18 yr) hospitalized with coronavirus disease 2019 at participating hospitals from February 2020 to January 2021. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was ICU admission. Secondary outcomes included hospital and ICU duration of stay and ICU, hospital, and 28-day mortality. A total of 874 children with coronavirus disease 2019 were reported to Viral Infection and Respiratory Illness Universal Study registry from 51 participating centers, majority in the United States. Median age was 8 years (interquartile range, 1.25-14 yr) with a male:female ratio of 1:2. A majority were non-Hispanic (492/874; 62.9%). Median body mass index (n = 817) was 19.4 kg/m2 (16-25.8 kg/m2), with 110 (13.4%) overweight and 300 (36.6%) obese. A majority (67%) presented with fever, and 43.2% had comorbidities. A total of 238 of 838 (28.2%) met the Centers for Disease Control and Prevention criteria for multisystem inflammatory syndrome in children, and 404 of 874 (46.2%) were admitted to the ICU. In multivariate logistic regression, age, fever, multisystem inflammatory syndrome in children, and pre-existing seizure disorder were independently associated with a greater odds of ICU admission. Hospital mortality was 16 of 874 (1.8%). Median (interquartile range) duration of ICU (n = 379) and hospital (n = 857) stay were 3.9 days (2-7.7 d) and 4 days (1.9-7.5 d), respectively. For patients with 28-day data, survival was 679 of 787, 86.3% with 13.4% lost to follow-up, and 0.3% deceased. CONCLUSIONS: In this observational, multicenter registry of children with coronavirus disease 2019, ICU admission was common. Older age, fever, multisystem inflammatory syndrome in children, and seizure disorder were independently associated with ICU admission, and mortality was lower among children than mortality reported in adults.

Predictors of Invasive Herpes Simplex Virus Infection in Young Infants.

OBJECTIVES: To identify independent predictors of and derive a risk score for invasive herpes simplex virus (HSV) infection. METHODS: In this 23-center nested case-control study, we matched 149 infants with HSV to 1340 controls; all were ≤60 days old and had cerebrospinal fluid obtained within 24 hours of presentation or had HSV detected. The primary and secondary outcomes were invasive (disseminated or central nervous system) or any HSV infection, respectively. RESULTS: Of all infants included, 90 (60.4%) had invasive and 59 (39.6%) had skin, eyes, and mouth disease. Predictors independently associated with invasive HSV included younger age (adjusted odds ratio [aOR]: 9.1 [95% confidence interval (CI): 3.4-24.5] <14 and 6.4 [95% CI: 2.3 to 17.8] 14-28 days, respectively, compared with >28 days), prematurity (aOR: 2.3, 95% CI: 1.1 to 5.1), seizure at home (aOR: 6.1, 95% CI: 2.3 to 16.4), ill appearance (aOR: 4.2, 95% CI: 2.0 to 8.4), abnormal triage temperature (aOR: 2.9, 95% CI: 1.6 to 5.3), vesicular rash (aOR: 54.8, (95% CI: 16.6 to 180.9), thrombocytopenia (aOR: 4.4, 95% CI: 1.6 to 12.4), and cerebrospinal fluid pleocytosis (aOR: 3.5, 95% CI: 1.2 to 10.0). These variables were transformed to derive the HSV risk score (point range 0-17). Infants with invasive HSV had a higher median score (6, interquartile range: 4-8) than those without invasive HSV (3, interquartile range: 1.5-4), with an area under the curve for invasive HSV disease of 0.85 (95% CI: 0.80-0.91). When using a cut-point of ≥3, the HSV risk score had a sensitivity of 95.6% (95% CI: 84.9% to 99.5%), specificity of 40.1% (95% CI: 36.8% to 43.6%), and positive likelihood ratio 1.60 (95% CI: 1.5 to 1.7) and negative likelihood ratio 0.11 (95% CI: 0.03 to 0.43). CONCLUSIONS: A novel HSV risk score identified infants at extremely low risk for invasive HSV who may not require routine testing or empirical treatment.

Test Characteristics of Cerebrospinal Fluid Gram Stain to Identify Bacterial Meningitis in Infants Younger Than 60 Days.

ABSTRACT: In our cohort of 20,947 infants aged 60 days or younger, cerebrospinal fluid Gram stain had a sensitivity of 34.3% (95% confidence interval, 28.1%-41.1%) and a positive predictive value of 61.4% (95% confidence interval, 52.2%-69.8%) for positive cerebrospinal fluid culture, suggesting that Gram stain alone may lead to both underdiagnosis and overdiagnosis of bacterial meningitis.

Herpes Simplex Virus Infection in Infants Undergoing Meningitis Evaluation.

BACKGROUND: Although neonatal herpes simplex virus (HSV) is a potentially devastating infection requiring prompt evaluation and treatment, large-scale assessments of the frequency in potentially infected infants have not been performed. METHODS: We performed a retrospective cross-sectional study of infants ≤60 days old who had cerebrospinal fluid culture testing performed in 1 of 23 participating North American emergency departments. HSV infection was defined by a positive HSV polymerase chain reaction or viral culture. The primary outcome was the proportion of encounters in which HSV infection was identified. Secondary outcomes included frequency of central nervous system (CNS) and disseminated HSV, and HSV testing and treatment patterns. RESULTS: Of 26 533 eligible encounters, 112 infants had HSV identified (0.42%, 95% confidence interval [CI]: 0.35%-0.51%). Of these, 90 (80.4%) occurred in weeks 1 to 4, 10 (8.9%) in weeks 5 to 6, and 12 (10.7%) in weeks 7 to 9. The median age of HSV-infected infants was 14 days (interquartile range: 9-24 days). HSV infection was more common in 0 to 28-day-old infants compared with 29- to 60-day-old infants (odds ratio 3.9; 95% CI: 2.4-6.2). Sixty-eight (0.26%, 95% CI: 0.21%-0.33%) had CNS or disseminated HSV. The proportion of infants tested for HSV (35%; range 14%-72%) and to whom acyclovir was administered (23%; range 4%-53%) varied widely across sites. CONCLUSIONS: An HSV infection was uncommon in young infants evaluated for CNS infection, particularly in the second month of life. Evidence-based approaches to the evaluation for HSV in young infants are needed.

Concomitant Bacterial Meningitis in Infants With Urinary Tract Infection.

To determine age-stratified prevalence of concomitant bacterial meningitis in infants ≤60 days with a urinary tract infection, we performed a 23-center, retrospective study of 1737 infants with urinary tract infection. Concomitant bacterial meningitis was rare, but more common in infants 0-28 days of age [0.9%; 95% confidence interval (CI): 0.4%-1.9%) compared with infants 29-60 days of age (0.2%; 95% CI: 0%-0.8%).

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors.

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

Afebrile Infants Evaluated in the Emergency Department for Serious Bacterial Infection.

OBJECTIVES: Afebrile infants 0 to 60 days of age are sometimes evaluated for serious bacterial infection (SBI). Our objective was to describe the clinical and laboratory findings in this population and compare them to their febrile counterparts. METHODS: We performed a retrospective observational study comparing afebrile infants undergoing an SBI evaluation to those evaluated for fever. RESULTS: We included infants who were admitted to the hospital and had at least 2 of 3 following bacterial cultures: blood, urine, or cerebrospinal fluid. Of the 1184 infants presenting to the emergency department with chief complaints that may prompt an SBI evaluation, 579 patients met our inclusion criteria with 362 in the fever group and 217 in the afebrile group. The most common chief complaints in the afebrile group were respiratory symptoms (27%), seizure (22%), vomiting/diarrhea (21%), and apparent life-threatening event (11%). Rates of true-positive blood, urine, and cerebrospinal fluid cultures were 2%, 2.4%, and 0.9% respectively. All cases of bacterial meningitis were in the fever group antibiotics (P = 0.16). Infants with fever were more likely to receive antibiotics (P < 0.001), although there were no statistical differences between the 2 groups in the rates of positive blood or urine cultures. CONCLUSIONS: Afebrile infants make up a significant percentage of SBI evaluations in the emergency department. Respiratory symptoms, vomiting, and seizure-like activity are common presentations. Although rates of bacteremia and urinary tract infection are higher in the febrile group, this did not reach statistical significance, and therefore afebrile infants should still be considered at risk for SBI.

Impact of an Institutional Guideline on the Care of Neonates at Risk for Herpes Simplex Virus in the Emergency Department.

BACKGROUND AND OBJECTIVES: Herpes simplex virus (HSV) is rare in neonates but carries significant morbidity and mortality in that group. Emergency department (ED) clinicians have little guidance to decide when to test for HSV and give acyclovir. We created an institutional guideline to provide guidance in patients younger than 6 weeks. Our objective was to evaluate whether guideline implementation affected the ED's decision to test for HSV, and ED use of HSV polymerase chain reactions (PCRs) and acyclovir. METHODS: We reviewed charts for patients 1 year before implementation and 1 year after implementation of our guideline. Inclusion criteria were younger than 60 days, admitted through the ED, symptom onset younger than 6 weeks, and any one of the following criteria: (1) ED blood culture obtained, (2) ED or inpatient HSV PCR obtained, and (3) ED or inpatient acyclovir treatment. Premature patients and transfer patients were excluded. We compared whether the decision to initiate HSV testing, ED use of HSV PCRs, serum alanine aminotransferase, and acyclovir use changed post-guideline implementation. RESULTS: We reviewed 173 charts pre-implementation and 129 post-implementation. We found a significant decrease in ED testing for HSV among patients who did not meet guideline criteria (P < 0.01). We saw an improvement in the use of alanine aminotransferase among patients who met criteria for testing (P = 0.02), but no change in the use of HSV PCRs or acyclovir use among tested patients. CONCLUSIONS: Guideline implementation reduced HSV evaluations in low-risk patients, but did not improve test utilization or acyclovir administration among those tested. Additional work is needed to improve guideline utilization.

Health and Social Needs in Three Migrant Worker Communities around La Romana, Dominican Republic, and the Role of Volunteers: A Thematic Analysis and Evaluation.

Objective. For decades, Haitian migrant workers living in bateyes around La Romana, Dominican Republic, have been the focus of short-term volunteer medical groups from North America. To assist these efforts, this study aimed to characterize various health and social needs that could be addressed by volunteer groups. Design. Needs were assessed using semistructured interviews of community and professional informants, using a questionnaire based on a social determinants of health framework, and responses were qualitatively analysed for common themes. Results. Key themes in community responses included significant access limitations to basic necessities and healthcare, including limited access to regular electricity and potable water, lack of health insurance, high out-of-pocket costs, and discrimination. Healthcare providers identified the expansion of a community health promoter program and mobile medical teams as potential solutions. English and French language training, health promotion, and medical skills development were identified as additional strategies by which teams could support community development. Conclusion. Visiting volunteer groups could work in partnership with community organizations to address these barriers by providing short-term access to services, while developing local capacity in education, healthcare, and health promotion in the long-term. Future work should also carefully evaluate the impacts and contributions of such volunteer efforts.

Initial Presentation of Neonatal Herpes Simplex Virus Infection.

OBJECTIVE: To inform the decision to test and empirically treat for herpes simplex virus (HSV) by describing the initial clinical presentation and laboratory findings of infants with a confirmed diagnosis of neonatal HSV. STUDY DESIGN: This is a retrospective case series performed at 2 pediatric tertiary care centers. Infants who developed symptoms prior to 42 days of age with laboratory confirmed HSV from 2002 through 2012 were included. We excluded infants <34 weeks gestation, those who developed illness before discharge from their birth hospital, and those who developed symptoms after 42 days of age. RESULTS: We identified 49 infants with HSV meeting these criteria. Most infants (43/49, 88%) came to medical attention at ≤28 days. Of 49 infants, 22 (45%) had disseminated, 16 (33%) central nervous system, and 10 (20%) skin, eye, mouth HSV disease. Eight infants (16%) had nonspecific presentations without the classic signs of seizure, vesicular rash, or critical illness (intensive care admission). All infants with nonspecific presentation were ≤14 days, had cerebrospinal fluid pleocytosis, or both. CONCLUSIONS: The majority of infants with HSV (84%) presented with seizure, vesicular rash, or critical illness. A subset of patients (16%) lacked classic signs at hospitalization; most manifested signs suggestive of HSV within 24 hours. Further studies are needed to validate the risk factors identified in this study including age <14 days and cerebrospinal fluid pleocytosis at presentation.

A game plan: Gamification design principles in mHealth applications for chronic disease management.

Effective chronic disease management is essential to improve positive health outcomes, and incentive strategies are useful in promoting self-care with longevity. Gamification, applied with mHealth (mobile health) applications, has the potential to better facilitate patient self-management. This review article addresses a knowledge gap around the effective use of gamification design principles, or mechanics, in developing mHealth applications. Badges, leaderboards, points and levels, challenges and quests, social engagement loops, and onboarding are mechanics that comprise gamification. These mechanics are defined and explained from a design and development perspective. Health and fitness applications with gamification mechanics include: bant which uses points, levels, and social engagement, mySugr which uses challenges and quests, RunKeeper which uses leaderboards as well as social engagement loops and onboarding, Fitocracy which uses badges, and Mango Health, which uses points and levels. Specific design considerations are explored, an example of the efficacy of a gamified mHealth implementation in facilitating improved self-management is provided, limitations to this work are discussed, a link between the principles of gaming and gamification in health and wellness technologies is provided, and suggestions for future work are made. We conclude that gamification could be leveraged in developing applications with the potential to better facilitate self-management in persons with chronic conditions.

Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism.

Development of an ELISA based bridging assay as a surrogate measure of ADCC.

Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing. To provide an alternative to the cell lysis based ADCC assay, a non-cell based measure of ADCC activity was developed to determine the ADCC activity of an anti-CD20 mAb, which measures the ability of the mAb to bind to CD20 antigen and FcγRIIIa, simultaneously. The bridging of CD20 and FcγRIIIa is an essential interaction for the initiation of ADCC activity. This ADCC bridging method is simple, offers the ease of use of a standard ELISA, and shows reproducible dose-response curves in the concentration range of 50-1000 ng/mL. With interassay variability of 7-10% and recovery of 89-115%, the assay demonstrates acceptable precision and accuracy. The assay is able to detect degradative changes in anti-CD20 mAb samples subjected to light and acid exposure, suggesting that it is suitable for use as a stability-indicating method. The assay is also sensitive to mAb fucose levels. A linear relationship between the ADCC bridging assay and the cell lysis ADCC assay was demonstrated, strongly suggesting that the ADCC bridging assay can be used as a surrogate measure of ADCC.

Inhibition of antithrombin by Plasmodium falciparum histidine-rich protein II.

Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner. The observed 50% inhibitory concentration (IC(50)) for the HRPII neutralization of AT activity is approximately 30nM for factor Xa inhibition and 90nM for thrombin inhibition. Zn(2+) was required for these reactions with a distribution coefficient (K(d)) of approximately 7µM. Substituting Zn(2+) with Cu(2+), but not with Ca(2+), Mg(2+), or Fe(2+), maintained the HRPII effect. HRPII attenuated the prolongation in plasma clotting time induced by heparin, suggesting that HRPII inhibits AT activity by preventing its stimulation by heparin. In the microvasculature, where erythrocytes infected with P falciparum are sequestered, high levels of released HRPII may bind cellular glycosaminoglycans, prevent their interaction with AT, and thereby contribute to the procoagulant state associated with P falciparum infection.

The PICM chemical scanning method for identifying domain-domain and protein-protein interfaces: applications to the core signaling complex of E. coli chemotaxis.

The number of known protein structures is growing exponentially (Berman et al., 2000), but the structural mapping of essential domain-domain and protein-protein interaction surfaces has advanced more slowly. It is particularly difficult to analyze the interaction surfaces of membrane proteins on a structural level, both because membrane proteins are less accessible to high-resolution structural analysis and because the membrane environment is often required for native complex formation. The Protein-Interactions-by-Cysteine-Modification (PICM) method is a generalizable, in vitro chemical scanning approach that can be applied to many protein complexes, in both membrane-bound and soluble systems. The method begins by engineering Cys residues on the surface of a protein of known structure, then a bulky probe is coupled to each Cys residue. Next, the effects of both Cys substitution and bulky probe attachment are measured on the assembly and the activity of the target complex. Bulky probe coupling at an essential docking site disrupts complex assembly and/or activity, while coupling outside the site typically has little or no effect. PICM has been successfully applied to the core signaling complex of the E. coli and S. typhimurium chemotaxis pathway, where it has mapped out essential docking surfaces on transmembrane chemoreceptor (Tar) and histidine kinase (CheA) components (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The approach shares similarities with other important scanning methods like alanine and tryptophan scanning (Cunningham and Wells, 1989; Sharp et al., 1995a), but has two unique features: (1) functional effects are determined for both small volume (Cys) and large volume (bulky probe) side chain substitutions in the same experiment, and (2) nonperturbing positions are identified at which Cys residues and bulky probes can be introduced for subsequent biochemical and biophysical studies, without significant effects on complex assembly or activity.