Validation of a measurement instrument for parental child feeding in a low and middle-income country

Background

Parental child feeding practices (PCFP) are a key factor influencing children’s dietary intake, especially in the preschool years when eating behavior is being established. Instruments to measure PCFP have been developed and validated in high-income countries with a high prevalence of childhood obesity. The aim of this study was to test the appropriateness, content, and construct validity of selected measures of PCFP in a low and middle-income country (LMIC) in which there is both undernutrition and obesity in children.

Methods

An expert panel selected subscales and items from measures of PCFP that have been well-tested in high-income countries to measure both “coercive” and “structural” behaviors. Two sequential cross-sectional studies (Study 1, n =?154; Study 2, n =?238) were conducted in two provinces in Indonesia. Findings of the first study were used to refine subscales used in Study 2. An additional qualitative study tested content validity from the perspective of mothers (the intended respondents). Factorial validation and reliability were also tested. Convergent validity was tested with child nutritional status.

Results

In Study 1, a confirmatory factor analysis (CFA) model with 11 factors provided good fit (RMSEA?=?0.045; CFI?=?0.95 and TLI?=?0.95) after two subscales were removed. Reliability was good among seven of the subscales. Following a decision to take out an additional subscale, the instrument was tested for factorial validity (Study 2). A CFA model with 10 subscales provided good fit (RMSEA?=?0.03; CFI?=?0.92 and TLI?=?0.90). The reliability of subscales was lower than in Study 1. Convergent validity with nutrition status was found with two subscales.

Conclusions

The two studies provide evidence of acceptable psychometric properties for 10 subscales from tested instruments to measure PCFP in Indonesia. This provides the first evidence of the validity of these measures in a LMIC setting. Some shortcomings, such in the reliability of some subscales and further tests of predictive validity, require further investigation.

     

Perforin is a critical physiologic regulator of T-cell activation

Individuals with impaired perforin-dependent cytotoxic function (Ctx(-)) develop a fatal inflammatory disorder called hemophagocytic lymphohistiocytosis (HLH). It has been hypothesized that immune hyperactivation during HLH is caused by heightened infection, defective apoptosis/responsiveness of Ctx(-) lymphocytes, or enhanced antigen presentation. Whereas clinical and experimental data suggest that increased T-cell activation drives HLH, potential abnormalities of T-cell activation have not been well characterized in Ctx(-) hosts. To define such abnormalities and to test these hypotheses, we assessed in vivo T-cell activation kinetics and viral loads after lymphocytic choriomeningitis virus (LCMV) infection of Ctx(-) mice. We found that increased T-cell activation occurred early during infection of Ctx(-) mice, while they had viral burdens that were identical to those of WT animals, demonstrating that T-cell hyperactivation was independent of viral load. Furthermore, cell transfer and signaling studies indicated that increased antigenic stimulation, not a cell-intrinsic defect of responsiveness, underlay heightened T-cell activation in vivo. Finally, direct measurement of viral antigen presentation demonstrated an increase in Ctx(-) mice that was proportional to abnormal T-cell activation. We conclude that perforin-dependent cytotoxicity has an immunoregulatory role that is distinguishable from its pathogen clearance function and limits T-cell activation in the physiologic context by suppressing antigen presentation.     

Evaluation of the DiversiLab System for Detection of Hospital Outbreaks of Infections by Different Bacterial Species

Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMérieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpson''s index of diversity and adjusted Rand''s and Wallace''s coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.Pulsed-field gel electrophoresis (PFGE) is generally considered the “gold standard” method for the typing of many bacterial species. Other commonly used typing procedures include multilocus sequence typing (MLST), Multiple-locus variable number of tandem repeats analysis (MLVA), and amplified fragment length polymorphism analysis (6, 8, 11, 14, 21). But all of these methods suffer from different drawbacks. They are specific for one species, time-consuming, or poorly reproducible. The recently introduced DiversiLab (DL) system (bioMérieux) potentially overcomes these limitations. This typing technique is based on the repetitive-sequence-based PCR (rep-PCR) for typing (3, 4, 23, 24). This method was developed in the 1990s and, though still used today, suffers from reproducibility problems. The DL system is a semiautomated rep-PCR with a high level of standardization, in particular for the electrophoresis step by using a Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). This reduces reproducibility problems due to variation in assay conditions. The analysis software allows the comparison of individual amplification product patterns (peak patterns), which enables easier interpretation of the patterns, but a virtual gel image is also generated. The patterns can be stored in a database and used for comparison. An important advantage of DL is that a result can be obtained in 1 day starting from a pure culture. A number of studies investigating DL have been published (5, 7, 16, 18, 19), but these were limited to one species and sometimes used collections or made comparisons at the level of MLST, a method which is not discriminatory enough for hospital outbreak analysis.In this study, we evaluated the DL system for the identification of established hospital outbreaks of a number of bacterial species, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Escherichia coli, the Enterobacter cloacae complex, Acinetobacter species, Klebsiella species, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa, using typed collections.     

Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis

Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.     

Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility.     

Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.     

Gene expression profiling of peripheral blood mononuclear cells from children with active hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetically heterogeneous autosomal recessive immune disorder that results when the critical regulatory pathways that mediate immune defense mechanisms and the natural termination of immune/inflammatory responses are disrupted or overwhelmed. To advance the understanding of FHL, we performed gene expression profiling of peripheral blood mononuclear cells from 11 children with untreated FHL. Total RNA was isolated and gene expression levels were determined using microarray analysis. Comparisons between patients with FHL and normal pediatric controls (n = 30) identified 915 down-regulated and 550 up-regulated genes with more than or equal to 2.5-fold difference in expression (P ≤ .05). The expression of genes associated with natural killer cell functions, innate and adaptive immune responses, proapoptotic proteins, and B- and T-cell differentiation were down-regulated in patients with FHL. Genes associated with the canonical pathways of interleukin-6 (IL-6), IL-10 IL-1, IL-8, TREM1, LXR/RXR activation, and PPAR signaling and genes encoding of antiapoptotic proteins were overexpressed in patients with FHL. This first study of genome-wide expression profiling in children with FHL demonstrates the complexity of gene expression patterns, which underlie the immunobiology of FHL.