Rapid Identification of Bacterial Antibiotic Resistance by qPCR in Infants with Gram-Negative Septicaemia: A Proof-of-Concept Study.

BACKGROUND: Neonatal sepsis remains an important cause of neonatal morbidity and mortality. Tools to rapidly predict antibiotic resistance in neonatal sepsis would be extremely valuable. OBJECTIVES: To develop quantitative polymerase chain reaction (qPCR) primer/probe sets that can rapidly detect antibiotic resistance genes common to a neonatal unit, and to investigate the feasibility of direct detection of antibiotic resistance genes in whole blood of infants with Gram-negative septicaemia without first isolating the organism. METHODS: Primer/probe sets were designed to detect genes that produce aminoglycoside-modifying enzymes or extended-spectrum ß-lactamase. In phase 1, Gram-negative organisms isolated from neonatal clinical specimens within a 12-month period were analysed by qPCR to detect preselected genes. In phase 2, blood specimens of infants with Gram-negative septicaemia were subjected to qPCR analysis to detect antibiotic resistance genes for comparison against conventional antibiotic resistance profile results. RESULTS: Two primer/probe sets showed promising diagnostic utilities for the prediction of antibiotic resistance; the diagnostic utilities (sensitivity, specificity, positive predictive value and negative predictive value) were 90.9, 96.4, 92.6 and 95.5%, respectively, for AAC3-2 [aac(3')-IIa/aacC3/aacC2, aac(3')-IIc/aacC2] to detect gentamicin resistance, and 59.3, 99.3, 94.1 and 92.6%, respectively, for BLA-C1 (blaCTX-M-9, blaCTX-M-14, blaCTX-M-24, blaCTX-M-27) to detect cephalosporin resistance. Twenty-six infants were tested in phase 2, and both gentamicin and cephalosporin resistance patterns were predicted with 100% sensitivity and 100% specificity by AAC3-2 and BLA-C1, respectively. CONCLUSIONS: qPCR with appropriately designed primer/probe sets can predict antibiotic resistance directly from neonatal blood, and it can substantially reduce the turnaround time for antibiotic resistance results.

Gut-associated biomarkers L-FABP, I-FABP, and TFF3 and LIT score for diagnosis of surgical necrotizing enterocolitis in preterm infants.

OBJECTIVES: To evaluate the use of gut barrier proteins, liver-fatty acid binding protein (L-FABP), intestinal-fatty acid binding protein (I-FABP), and trefoil factor 3 (TFF3), as biomarkers for differentiating necrotizing enterocolitis (NEC) from septicemic/control infants and to identify the most severely affected surgical NEC from nonsurgical NEC infants. BACKGROUND: Clinical features and routine radiologic investigations have low diagnostic utilities in identifying surgical NEC patients. METHODS: The diagnostic utilities of individual biomarkers and the combination of biomarkers, the LIT score, were assessed among the NEC (n = 20), septicemia (n = 40), and control groups (n = 40) in a case-control study for the identification of proven NEC and surgical NEC infants. RESULTS: Plasma concentrations of all gut barrier biomarkers and the LIT score were significantly higher in the NEC than in the septicemia or control group (P < 0.01). Using median values of biomarkers and the LIT score in the NEC group as cutoff values for identifying NEC from septicemic/control cases, all had specificities of 95% or more and sensitivities of 50%. Significantly higher levels of biomarkers and the LIT score were found in infants with surgical NEC than in nonsurgical NEC cases (P ≤ 0.02). The median LIT score of 4.5 identified surgical NEC cases with sensitivity and specificity of 83% and 100%%, respectively. A high LIT score of 6 identified nonsurvivors of NEC with sensitivity and specificity of 78% and 91%, respectively. CONCLUSIONS: The LIT score can effectively differentiate surgical NEC from nonsurgical NEC infants and nonsurvivors of NEC from survivors at the onset of clinical presentation. Frontline neonatologists and surgeons may, therefore, target NEC infants who are most in need of close monitoring and those who may benefit from early surgical intervention.

IP-10 is an early diagnostic marker for identification of late-onset bacterial infection in preterm infants.

Very low birth weight (VLBW) infants with suspected late-onset infection requiring sepsis screening were enrolled in a prospective study to evaluate the diagnostic utilities of a comprehensive panel of key chemokines and cytokines, both individually and in combination, to identify diagnostic markers for early recognition of bacterial sepsis and necrotizing enterocolitis (NEC). Plasma chemokines interleukin (IL)-8, interferon-gamma-inducible protein 10 (IP-10), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein 1 (MCP-1), growth-related oncogene-alpha (GRO-alpha), and regulated upon activation of normal T cell expressed and secreted (RANTES) and cytokines IL-1beta, IL-6, IL-10, IL-12p70, and tumor necrosis factor alpha (TNF-alpha) were measured at the onset of sepsis (0 h) and 24 h later. Of 155 suspected infection episodes, 44 were classified as infected. Concentrations of all studied inflammatory mediators (except IL-1beta and RANTES) were significantly higher in the infected than in the noninfected group at 0 h, but the levels decreased precipitously by 24 h. IP-10 with a plasma cutoff concentration > or = 1250 pg/mL could identify all septicemic and NEC cases and had the highest overall sensitivity (93%) and specificity (89%) at 0 h. We conclude that preterm infants have the ability to induce a robust chemokine and cytokine response during sepsis, and IP-10 is a sensitive early marker of infection.

Early prediction of sepsis-induced disseminated intravascular coagulation with interleukin-10, interleukin-6, and RANTES in preterm infants.

BACKGROUND: The progression to disseminated intravascular coagulation (DIC) in infected very low birth weight (VLBW; <1500 g) infants is difficult to predict with precision at the onset of sepsis. We investigated the immunologic profiles of preterm infants with sepsis, using chemokine and cytokine measurements to predict the development of sepsis-induced DIC at the onset of infection. METHODS: We measured a panel of chemokines and cytokines at 0 and 24 h after clinical presentation in VLBW infants with suspected infection requiring full sepsis screening. The chemokines measured were interleukin (IL)-8, interferon-gamma-inducible protein-10 (IP-10), monokine induced by interferon-gamma, monocyte chemoattractant protein-1, and regulated upon activation normal T-cell expressed and secreted (RANTES), and the cytokines were IL-6, IL-10, and tumor necrosis factor-alpha. RESULTS: Of 195 episodes of suspected clinical sepsis investigated, 62 were culture-confirmed septicemia or necrotizing enterocolitis (28 of these infants developed DIC), 22 were culture-negative clinical infections, and 111 involved noninfected episodes. All studied inflammatory mediators except RANTES showed significantly greater up-regulation in culture-positive infected infants than in noninfected infants at 0 and 24 h, whereas RANTES showed significant down-regulation. The model that used plasma IL-10 (>208 ng/L), IL-6 (>168 ng/L), and RANTES (<3110 ng/L) at 0 h had sensitivity, specificity, and positive and negative predictive values of 100%, 97%, 85%, and 100%, respectively, for identifying infected patients who subsequently developed DIC. CONCLUSIONS: IL-10, IL-6, and RANTES measured at clinical presentation sensitively and accurately predicted the development of DIC in severely infected infants. This information could be vital for early and effective treatment of neonatal sepsis.

Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection.

This prospective study aimed to evaluate the diagnostic utilities of neutrophil CD64 expression for the identification of early-onset clinical infection and pneumonia in term infants and to define the optimal cutoff value so that it may act as a reference with which future studies can be compared. Term newborns in whom infection was suspected when they were <72 h of age were recruited into the study. C-reactive protein (CRP) and expression of CD64 on neutrophils were measured at 0 h (at the time of sepsis evaluation) and 24 h. The sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of CRP, CD64, and the combination of these two markers for predicting neonatal sepsis were determined. A total of 338 infants with suspected clinical sepsis were investigated, 115 of whom were found to be clinically infected. CRP and CD64 in infected infants were both significantly elevated at 0 and 24 h compared with noninfected infants (p < 0.001). The calculated optimal cutoff value for CD64 was 6136 antibody-phycoerythrin molecules bound/cell. CD64 has a very high sensitivity (96%) and NPV (97%) at 24 h. The addition of CRP only marginally enhanced the sensitivity and NPV (97 and 98%, respectively). In conclusion, neutrophil CD64 is a very sensitive diagnostic marker for the identification of early-onset clinical infection and pneumonia in term newborns. The results strongly suggest that measurement of neutrophil CD64 may allow neonatal clinicians to discontinue antibiotic treatment at 24 h in infants who are clinically stable and whose CD64 expressions are below the optimal cutoff level.

Immunogenicity of a two-dose regime of varicella vaccine in children with cancers.

OBJECTIVE: Live-attenuated varicella vaccine is effective and safe in immunocompetent children. In this study, we assess the immunogenicity and adverse events following varicella vaccination in immunosuppressed cancer children. METHODS: Varicella-zoster virus (VZV)-seronegative cancer children received two doses of live-attenuated VZV vaccine (Varilrix) in a span of 3 months. Patients with acute lymphoblastic leukaemia (ALL) were in the maintenance phase of chemotherapy, whereas those with solid tumours joined the study around 3-6 months from treatment discontinuation. VZV-specific cellular and humoral immune responses were measured before and after VZV vaccination. RESULTS: The median (range) age of the 17 patients was 4.4 yr (2.0-14.5). Thirteen had ALL, one had myelodysplastic syndrome and three had solid tumours. Following vaccination, the VZV-specific stimulation index (SI) increased from 1.7 (0.9-2.9) to 17.9 (5.9-36.0) (P < 0.001). Similarly, SI to phytohaemagglutinin mitogen increased from 1136 (499-1930) to 1714 (848-2518) (P = 0.028). There were also significant increases in CD4+ cells and CD4:CD8 ratio as well as a reduction in CD16/56+ cells in peripheral blood lymphocytes. Seroconversion rate to VZV was 19% after one dose and increased to 94% after the second dose of VZV vaccine. Serum VZV-specific IgG concentrations also increased significantly following two doses when compared with one dose of VZV vaccine (P = 0.0004). One subject developed possibly vaccine-related chickenpox with self-limiting hepatitis at 5 wk following vaccination. None of the patients developed herpes-zoster at a median (range) follow-up of 27.5 months (24.0-30.0). CONCLUSIONS: Non-immune cancer children can be effectively vaccinated against chickenpox at the defined period. However, the safety of chickenpox vaccine in these immunosuppressed children needs to be further studied.

Neutrophil CD64 expression: a sensitive diagnostic marker for late-onset nosocomial infection in very low birthweight infants.

This study aims to evaluate the diagnostic utilities of four leukocyte surface antigens-two lymphocyte antigens (CD25 and CD45RO) and two neutrophil antigens (CD11b and CD64)-for identification of late-onset nosocomial bacterial infection in preterm, very low birthweight infants, and to define the optimal cutoff value for each marker so that it may act as a reference with which future studies can be compared. Very low birthweight infants in whom infection was suspected when they were >72 h of age were eligible for the study. A full sepsis screen was performed in each episode. IL-6, C-reactive protein, and leukocyte surface antigens (CD25, CD45RO, CD11b, and CD64) were measured at 0 (at the time of sepsis evaluation), 24, and 48 h by standard biochemical methods and quantitative flow cytometric analysis. The diagnostic utilities including sensitivity, specificity, and positive and negative predictive values of each marker and combination of markers for predicting late-onset neonatal infection were determined. One hundred twenty-seven episodes of suspected clinical sepsis were investigated in 80 infants. Thirty-seven episodes were proven infection. The calculated optimal cutoff values for CD25, CD45RO, CD11b, and CD64 were 3,100, 2,900, 10,450, and 4,000 phycoerythrin-molecules bound per cell, respectively. An interim analysis of data after 68 episodes suggested that CD25 and CD45RO were poor predictors of neonatal infection with sensitivity or specificity <75% during a single measurement. Thus, these two markers were excluded from further investigation. In the final analysis, CD64 has the highest sensitivity (95-97%) and negative predictive value (97-99%) at 0 and 24 h after the onset. The addition of IL-6 or C-reactive protein (0 h) to CD64 (24 h) further enhanced the sensitivity and negative predictive value to 100%, and has the specificity and positive predictive value exceeding 88% and 80%, respectively. Neutrophil CD64 expression is a very sensitive marker for diagnosing late-onset nosocomial infection in very low birthweight infants. If further validated, the use of CD64 as an infection marker should allow early discontinuation of antibiotic treatment at 24 h without waiting for the definitive microbiologic culture results. The quantitative flow cytometric analysis applied in this study could be developed into a routine clinical test with high comparability and reproducibility across different laboratories.