Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility.

The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

Pharmacological interventions for prevention and treatment of upper gastrointestinal bleeding in newborn infants.

BACKGROUND: Upper gastrointestinal bleeding is typically a mild, self-limiting condition that can affect both preterm and term neonates, although it can be severe particularly when associated with co-morbidities. Pharmacological interventions with a proton pump inhibitor (PPI), H2 receptor antagonist (H2RA), antacid, bismuth and sucralfate may have effects on both the prevention and treatment of upper gastrointestinal bleeding in infants. OBJECTIVES: To assess how different pharmacological interventions (PPIs, H2RAs, antacids, sucralfate or bismuth salts) administered to preterm and term neonates for the prevention or treatment of upper gastrointestinal bleeding to reduce morbidity and mortality compare with placebo or no treatment, supportive care, or each other. SEARCH METHODS: We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials (CENTRAL 2018, Issue 6), MEDLINE via PubMed (1966 to 12 July 2018), Embase (1980 to 12 July 2018), and CINAHL (1982 to 12 July 2018). We also searched clinical trial databases, conference proceedings, the reference lists of retrieved articles for randomised controlled trials and quasi-randomised trials, and online for Chinese literature articles. SELECTION CRITERIA: We selected randomised, quasi-randomised and cluster-randomised trials involving preterm and term neonates. Trials were included if they used a proton pump inhibitor, H2 receptor antagonist, antacid, sucralfate or bismuth either for the prevention or treatment of upper gastrointestinal bleeding. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed the eligibility of studies for inclusion, extracted data and assessed methodological quality. We conducted meta-analysis using a fixed-effect model. We used the GRADE approach to assess quality of evidence. MAIN RESULTS: Eleven studies with 818 infants met the criteria for inclusion in this review.Four trials with 329 infants assessed the use of an H2 receptor antagonist for prevention of upper gastrointestinal bleeding in high-risk newborn infants. Meta-analysis of these four trials identified a reduction in any upper gastrointestinal bleeding when using an H2 receptor antagonist (typical risk ratio (RR) 0.36, 95% confidence interval (CI) 0.22 to 0.58; typical risk difference (RD) -0.20, 95% CI -0.28 to -0.11; number needed to treat for an additional beneficial outcome (NNTB) 5, 95% CI 4 to 9). The quality of evidence was moderate. A single trial with 53 infants assessing prevention of upper gastrointestinal bleeding reported no difference in mortality in infants assigned H2 receptor antagonist versus no treatment; however the quality of evidence was very low.Seven trials with 489 infants assessed an inhibitor of gastric acid (H2 receptor antagonist or proton pump inhibitor) for treatment of gastrointestinal bleeding in newborn infants. Meta-analysis of two trials (131 infants) showed no difference in mortality from use of a H2 receptor antagonist compared to no treatment. The quality of evidence was low. Meta-analysis of two trials (104 infants) showed a reduction in duration of upper gastrointestinal bleeding from use of an inhibitor of gastric acid compared to no treatment (mean difference -1.06 days, 95% CI -1.28 to -0.84). The quality of evidence was very low. Meta-analysis of six trials (451 infants) showed a reduction in continued upper gastrointestinal bleeding from use of any inhibitor of gastric acid compared to no treatment (typical RR 0.36, 95% CI 0.26 to 0.49; typical RD -0.26, 95% CI -0.33, -0.19; NNTB 4, 95% CI 3 to 5). The quality of evidence was low. There were no significant subgroup differences in duration of upper gastrointestinal bleeding or of continued upper gastrointestinal bleeding according to type of inhibitor of gastric acid. A single trial (38 infants) reported no difference in anaemia requiring blood transfusion from use of a H2 receptor antagonist compared to no treatment.Although no serious adverse events were reported from the use of a H2 receptor antagonist or proton pump inhibitor, some neonatal morbidities - including necrotising enterocolitis, ventilator-associated pneumonia, duration of ventilation and respiratory support, and duration of hospital stay - were not reported. Long-term outcome was not reported. AUTHORS' CONCLUSIONS: There is moderate-quality evidence that use of an H2 receptor antagonist reduces the risk of gastrointestinal bleeding in newborn infants at high risk of gastrointestinal bleeding. There is low-quality evidence that use of an inhibitor of gastric acid (H2 receptor antagonist or proton pump inhibitor) reduces the duration of upper gastrointestinal bleeding and the incidence of continued gastric bleeding in newborn infants with gastrointestinal bleeding. However, there is no evidence that use of an inhibitor of gastric acid in newborn infants affects mortality or the need for blood transfusion. As no study reported the incidence of necrotising enterocolitis, ventilator- or hospital-associated pneumonia, sepsis, or long-term outcome, the safety of inhibitors of gastric acid secretion is unclear.

Chromosomal localization of Wolbachia inserts in the genomes of two subspecies of Chorthippus parallelus forming a Pyrenean hybrid zone.

Wolbachia are endosymbiotic bacteria of arthropods and nematodes that can manipulate the reproduction of various host organisms to facilitate their own maternal transmission. Moreover, Wolbachia's presence in host germ cells may contribute to the many cases of lateral gene transfer from Wolbachia to host genomes that have been described. A previous study in Chorthippus parallelus, a well-known orthopteroid forming a hybrid zone in the Pyrenees, identified Wolbachia sequences from two major supergroups in the genomes of infected and uninfected Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe) subspecies. In this study, we map the Wolbachia genomic inserts to specific regions on the chromosomes of Cpp and Cpe by fluorescent in situ hybridization (FISH) using tyramides to increase the accuracy and detection of these insertions. Additionally, we consider some of the possible roles that these bacterial inserts play in the organization and function of the grasshopper genome, as well as how they can serve as markers for phylogenetic relationships of these organisms.

Infant formulas containing hydrolysed protein for prevention of allergic disease.

BACKGROUND: Infant formulas containing hydrolysed proteins have been widely advocated for preventing allergic disease in infants, in place of standard cow's milk formula (CMF). However, it is unclear whether the clinical trial evidence supports this. OBJECTIVES: To compare effects on allergic disease when infants are fed a hydrolysed formula versus CMF or human breast milk. If hydrolysed formulas are effective, to determine what type of hydrolysed formula is most effective, including extensively or partially hydrolysed formula (EHF/PHF). To determine whether infants at low or high risk of allergic disease, and whether infants receiving early short-term (first few days after birth) or prolonged formula feeding benefit from hydrolysed formulas. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL 2017, Issue 11), MEDLINE (1948 to 3 November 2017), and Embase (1974 to 3 November 2017). We also searched clinical trials databases, conference proceedings, and the reference lists of retrieved articles and previous reviews for randomised controlled trials and quasi-randomised trials. SELECTION CRITERIA: We searched for randomised and quasi-randomised trials that compared use of a hydrolysed formula versus human milk or CMF. Outcomes with ≥ 80% follow-up of participants from eligible trials were eligible for inclusion. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, assessed trial quality and extracted data from the included studies. Fixed-effect analyses were performed. The treatment effects were expressed as risk ratio (RR) and risk difference (RD) with 95% confidence intervals and quality of evidence using the GRADE quality of evidence approach. The primary outcome was all allergic disease (including asthma, atopic dermatitis, allergic rhinitis and food allergy). MAIN RESULTS: A total of 16 studies were included.Two studies assessed the effect of three to four days infant supplementation with an EHF while in hospital after birth versus pasteurised human milk feed. A single study enrolling 90 infants reported no difference in all allergic disease (RR 1.43, 95% CI 0.38 to 5.37) or any specific allergic disease up to childhood including cow's milk allergy (CMA) (RR 7.11, 95% CI 0.35 to 143.84). A single study reported no difference in infant CMA (RR 0.87, 95% CI 0.52 to 1.46; participants = 3559). Quality of evidence was assessed as very low for all outcomes.No eligible trials compared prolonged hydrolysed formula versus human milk feeding.Two studies assessed the effect of three to four days infant supplementation with an EHF versus a CMF. A single study enrolling 90 infants reported no difference in all allergic disease (RR 1.37, 95% CI 0.33 to 5.71; participants = 77) or any specific allergic disease including CMA up to childhood. A single study reported a reduction in infant CMA of borderline significance (RR 0.62, 95% CI 0.38 to 1.00; participants = 3473). Quality of evidence was assessed as very low for all outcomes.Twelve studies assessed the effect of prolonged infant feeding with a hydrolysed formula compared with a CMF. The data showed no difference in all allergic disease in infants (typical RR 0.88, 95% CI 0.76 to 1.01; participants = 2852; studies = 8) and children (typical RR 0.85, 95% CI 0.69 to 1.05; participants = 950; studies = 2), and no difference in any specific allergic disease including infant asthma (typical RR 0.57, 95% CI 0.31 to 1.04; participants = 318; studies = 4), eczema (typical RR 0.93, 95% CI 0.79 to 1.09; participants = 2896; studies = 9), rhinitis (typical RR 0.52, 95% CI 0.14 to 1.85; participants = 256; studies = 3), food allergy (typical RR 1.42, 95% CI 0.87 to 2.33; participants = 479; studies = 2), and CMA (RR 2.31, 95% CI 0.24 to 21.97; participants = 338; studies = 1). Quality of evidence was assessed as very low for all outcomes. AUTHORS' CONCLUSIONS: We found no evidence to support short-term or prolonged feeding with a hydrolysed formula compared with exclusive breast feeding for prevention of allergic disease. Very low-quality evidence indicates that short-term use of an EHF compared with a CMF may prevent infant CMA. Further trials are recommended before implementation of this practice.We found no evidence to support prolonged feeding with a hydrolysed formula compared with a CMF for prevention of allergic disease in infants unable to be exclusively breast fed.

Higher versus lower amino acid intake in parenteral nutrition for newborn infants.

BACKGROUND: Sick newborn and preterm infants frequently are not able to be fed enterally, necessitating parenteral fluid and nutrition. Potential benefits of higher parenteral amino acid (AA) intake for improved nitrogen balance, growth, and infant health may be outweighed by the infant's ability to utilise high intake of parenteral AA, especially in the days after birth. OBJECTIVES: The primary objective is to determine whether higher versus lower intake of parenteral AA is associated with improved growth and disability-free survival in newborn infants receiving parenteral nutrition.Secondary objectives include determining whether:• higher versus lower starting or initial intake of amino acids is associated with improved growth and disability-free survival without side effects;• higher versus lower intake of amino acids at maximal intake is associated with improved growth and disability-free survival without side effects; and• increased amino acid intake should replace non-protein energy intake (glucose and lipid), should be added to non-protein energy intake, or should be provided simultaneously with non-protein energy intake.We conducted subgroup analyses to look for any differences in the effects of higher versus lower intake of amino acids according to gestational age, birth weight, age at commencement, and condition of the infant, or concomitant increases in fluid intake. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Review Group to search the Cochrane Central Register of Controlled Trials (2 June 2017), MEDLINE (1966 to 2 June 2017), Embase (1980 to 2 June 2017), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1982 to 2 June 2017). We also searched clinical trials databases, conference proceedings, and citations of articles. SELECTION CRITERIA: Randomised controlled trials of higher versus lower intake of AAs as parenteral nutrition in newborn infants. Comparisons of higher intake at commencement, at maximal intake, and at both commencement and maximal intake were performed. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, assessed trial quality, and extracted data from included studies. We performed fixed-effect analyses and expressed treatment effects as mean difference (MD), risk ratio (RR), and risk difference (RD) with 95% confidence intervals (CIs) and assessed the quality of evidence using the GRADE approach. MAIN RESULTS: Thirty-two studies were eligible for inclusion. Six were short-term biochemical tolerance studies, one was in infants at > 35 weeks' gestation, one in term surgical newborns, and three yielding no usable data. The 21 remaining studies reported clinical outcomes in very preterm or low birth weight infants for inclusion in meta-analysis for this review.Higher AA intake had no effect on mortality before hospital discharge (typical RR 0.90, 95% CI 0.69 to 1.17; participants = 1407; studies = 14; I2 = 0%; quality of evidence: low). Evidence was insufficient to show an effect on neurodevelopment and suggest no reported benefit (quality of evidence: very low). Higher AA intake was associated with a reduction in postnatal growth failure (< 10th centile) at discharge (typical RR 0.74, 95% CI 0.56 to 0.97; participants = 203; studies = 3; I2 = 22%; typical RD -0.15, 95% CI -0.27 to -0.02; number needed to treat for an additional beneficial outcome (NNTB) 7, 95% CI 4 to 50; quality of evidence: very low). Subgroup analyses found reduced postnatal growth failure in infants that commenced on high amino acid intake (> 2 to ≤ 3 g/kg/day); that occurred with increased amino acid and non-protein caloric intake; that commenced on intake at < 24 hours' age; and that occurred with early lipid infusion.Higher AA intake was associated with a reduction in days needed to regain birth weight (MD -1.14, 95% CI -1.73 to -0.56; participants = 950; studies = 13; I2 = 77%). Data show varying effects on growth parameters and no consistent effects on anthropometric z-scores at any time point, as well as increased growth in head circumference at discharge (MD 0.09 cm/week, 95% CI 0.06 to 0.13; participants = 315; studies = 4; I2 = 90%; quality of evidence: very low).Higher AA intake was not associated with effects on days to full enteral feeds, late-onset sepsis, necrotising enterocolitis, chronic lung disease, any or severe intraventricular haemorrhage, or periventricular leukomalacia. Data show a reduction in retinopathy of prematurity (typical RR 0.44, 95% CI 0.21 to 0.93; participants = 269; studies = 4; I2 = 31%; quality of evidence: very low) but no difference in severe retinopathy of prematurity.Higher AA intake was associated with an increase in positive protein balance and nitrogen balance. Potential biochemical intolerances were reported, including risk of abnormal blood urea nitrogen (typical RR 2.77, 95% CI 2.13 to 3.61; participants = 688; studies = 7; I2 = 6%; typical RD 0.26, 95% CI 0.20 to 0.32; number needed to treat for an additional harmful outcome (NNTH) 4; 95% CI 3 to 5; quality of evidence: high). Higher amino acid intake in parenteral nutrition was associated with a reduction in hyperglycaemia (> 8.3 mmol/L) (typical RR 0.69, 95% CI 0.49 to 0.96; participants = 505; studies = 5; I2 = 68%), although the incidence of hyperglycaemia treated with insulin was not different. AUTHORS' CONCLUSIONS: Low-quality evidence suggests that higher AA intake in parenteral nutrition does not affect mortality. Very low-quality evidence suggests that higher AA intake reduces the incidence of postnatal growth failure. Evidence was insufficient to show an effect on neurodevelopment. Very low-quality evidence suggests that higher AA intake reduces retinopathy of prematurity but not severe retinopathy of prematurity. Higher AA intake was associated with potentially adverse biochemical effects resulting from excess amino acid load, including azotaemia. Adequately powered trials in very preterm infants are required to determine the optimal intake of AA and effects of caloric balance in parenteral nutrition on the brain and on neurodevelopment.

Lower versus higher oxygen concentrations titrated to target oxygen saturations during resuscitation of preterm infants at birth.

BACKGROUND: Initial resuscitation with air is well tolerated by most infants born at term. However, the optimal fractional inspired oxygen concentration (FiO2 - proportion of the breathed air that is oxygen) targeted to oxygen saturation (SpO2 - an estimate of the amount of oxygen in the blood) for infants born preterm is unclear. OBJECTIVES: To determine whether lower or higher initial oxygen concentrations, when titrated according to oxygen saturation targets during the resuscitation of preterm infants at birth, lead to improved short- and long-term mortality and morbidity. SEARCH METHODS: We conducted electronic searches of the Cochrane Central Register of Controlled Trials (13 October 2017), Ovid MEDLINE (1946 to 13 October 2017), Embase (1974 to 13 October 2017) and CINAHL (1982 to 13 October 2017); we also searched previous reviews (including cross-references), contacted expert informants, and handsearched journals. SELECTION CRITERIA: We included randomised controlled trials (including cluster- and quasi-randomised trials) which enrolled preterm infants requiring resuscitation following birth and allocated them to receive either lower (FiO2 < 0.4) or higher (FiO2 ≥ 0.4) initial oxygen concentrations titrated to target oxygen saturation. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed the eligibility of studies for inclusion, extracted data and assessed methodological quality. Primary outcomes included mortality near term or at discharge (latest reported) and neurodevelopmental disability. We conducted meta-analysis using a fixed-effect model. We assessed the quality of the evidence using GRADE. MAIN RESULTS: The search identified 10 eligible trials. Meta-analysis of the 10 included studies (914 infants) showed no difference in mortality to discharge between lower (FiO2 < 0.4) and higher (FiO2 ≥ 0.4) initial oxygen concentrations targeted to oxygen saturation (risk ratio (RR) 1.05, 95% confidence interval (CI) 0.68 to 1.63). We identified no heterogeneity in this analysis. We graded the quality of the evidence as low due to risk of bias and imprecision. There were no significant subgroup effects according to inspired oxygen concentration strata (FiO2 0.21 versus ≥ 0.4 to < 0.6; FiO2 0.21 versus ≥ 0.6 to 1.0; and FiO2 ≥ 0.3 to < 0.4 versus ≥ 0.6 to 1.0). Subgroup analysis identified a single trial that reported increased mortality from use of lower (FiO2 0.21) versus higher (FiO2 1.0) initial oxygen concentration targeted to a lowest SpO2 of less than 85%, whereas meta-analysis of nine trials targeting a lowest SpO2 of 85% to 90% found no difference in mortality.Meta-analysis of two trials (208 infants) showed no difference in neurodevelopmental disability at 24 months between infants receiving lower (FiO2 < 0.4) versus higher (FiO2 > 0.4) initial oxygen concentrations targeted to oxygen saturation. Other outcomes were incompletely reported by studies. Overall, we found no difference in use of intermittent positive pressure ventilation or intubation in the delivery room; retinopathy (damage to the retina of the eyes, measured as any retinopathy and severe retinopathy); intraventricular haemorrhage (any and severe); periventricular leukomalacia (a type of white-matter brain injury); necrotising enterocolitis (a condition where a portion of the bowel dies); chronic lung disease at 36 weeks' gestation; mortality to follow up; postnatal growth failure; and patent ductus arteriosus. We graded the quality of the evidence for these outcomes as low or very low. AUTHORS' CONCLUSIONS: There is uncertainty as to whether initiating post birth resuscitation in preterm infants using lower (FiO2 < 0.4) or higher (FiO2 ≥ 0.4) oxygen concentrations, targeted to oxygen saturations in the first 10 minutes, has an important effect on mortality or major morbidity, intubation during post birth resuscitation, other resuscitation outcomes, and long-term outcomes including neurodevelopmental disability. We assessed the quality of the evidence for all outcomes as low to very low. Further large, well designed trials are needed to assess the effect of using different initial oxygen concentrations and the effect of targeting different oxygen saturations.

WITHDRAWN: Infant formulas containing hydrolysed protein for prevention of allergic disease and food allergy.

BACKGROUND: Allergy is common and may be associated with foods, including cow's milk formula (CMF). Formulas containing hydrolysed proteins have been used to treat infants with allergy. However, it is unclear whether hydrolysed formulas can be advocated for prevention of allergy in infants. OBJECTIVES: To compare effects on allergy and food allergy when infants are fed a hydrolysed formula versus CMF or human breast milk. If hydrolysed formulas are effective, to determine what type of hydrolysed formula is most effective, including extensively or partially hydrolysed formula (EHF/PHF). To determine which infants at low or high risk of allergy and which infants receiving early, short-term or prolonged formula feeding may benefit from hydrolysed formulas. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Review Group supplemented by cross referencing of previous reviews and publications (updated August 2016). SELECTION CRITERIA: We searched for randomised and quasi-randomised trials that compared use of a hydrolysed formula versus human milk or CMF. Trials with ≥ 80% follow-up of participants were eligible for inclusion. DATA COLLECTION AND ANALYSIS: We independently assessed eligibility of studies for inclusion, methodological quality and data extraction. Primary outcomes included clinical allergy, specific allergy and food allergy. We conducted meta-analysis using a fixed-effect (FE) model. MAIN RESULTS: Two studies assessed the effect of three to four days' infant supplementation with an EHF whilst in hospital after birth versus pasteurised human milk feed. Results showed no difference in infant allergy or childhood cow's milk allergy (CMA). No eligible trials compared prolonged hydrolysed formula versus human milk feeding.Two studies assessed the effect of three to four days infant supplementation with an EHF versus a CMF. One large quasi-random study reported a reduction in infant CMA of borderline significance among low-risk infants (risk ratio (RR) 0.62, 95% confidence interval (CI) 0.38 to 1.00).Prolonged infant feeding with a hydrolysed formula compared with a CMF was associated with a reduction in infant allergy (eight studies, 2852 infants; FE RR 0.82, 95% CI 0.72 to 0.95; risk difference (RD) -0.04, 95% CI -0.08 to -0.01; number needed to treat for an additional beneficial outcome (NNTB) 25, 95% CI 12.5 to 100) and infant CMA (two studies, 405 infants; FE RR 0.38, 95% CI 0.16 to 0.86). We had substantial methodological concerns regarding studies and concerns regarding publication bias, as substantial numbers of studies including those in high-risk infants have not comprehensively reported allergy outcomes (GRADE quality of evidence 'very low').Prolonged infant feeding with a hydrolysed formula compared with a CMF was not associated with a difference in childhood allergy and led to no differences in specific allergy, including infant and childhood asthma, eczema and rhinitis and infant food allergy. Many of the analyses assessing specific allergy are underpowered.Subroup analyses showed that infant allergy was reduced in studies that enrolled infants at high risk of allergy who used a hydrolysed formula compared with a CMF; used a PHF compared with a CMF; used prolonged and exclusive feeding of a hydrolysed formula compared with a CMF; and used a partially hydrolysed whey formula compared with a CMF. Studies that enrolled infants at high risk of allergy; used a PHF compared with a CMF; used prolonged and exclusive feeding of a hydrolysed formula compared with a CMF; and used a partially hydrolysed whey formula compared with a CMF found a reduction in infant CMA. AUTHORS' CONCLUSIONS: We found no evidence to support short-term or prolonged feeding with a hydrolysed formula compared with exclusive breast feeding for prevention of allergy. Very low-quality evidence indicates that short-term use of an EHF compared with a CMF may prevent infant CMA.In infants at high risk of allergy not exclusively breast fed, very low-quality evidence suggests that prolonged hydrolysed formula feeding compared with CMF feeding reduces infant allergy and infant CMA. Studies have found no difference in childhood allergy and no difference in specific allergy, including infant and childhood asthma, eczema and rhinitis and infant food allergy.Very low-quality evidence shows that prolonged use of a partially hydrolysed formula compared with a CMF for partial or exclusive feeding was associated with a reduction in infant allergy incidence and CMA incidence, and that prolonged use of an EHF versus a PHF reduces infant food allergy.

Infant formulas containing hydrolysed protein for prevention of allergic disease and food allergy.

BACKGROUND: Allergy is common and may be associated with foods, including cow's milk formula (CMF). Formulas containing hydrolysed proteins have been used to treat infants with allergy. However, it is unclear whether hydrolysed formulas can be advocated for prevention of allergy in infants. OBJECTIVES: To compare effects on allergy and food allergy when infants are fed a hydrolysed formula versus CMF or human breast milk. If hydrolysed formulas are effective, to determine what type of hydrolysed formula is most effective, including extensively or partially hydrolysed formula (EHF/PHF). To determine which infants at low or high risk of allergy and which infants receiving early, short-term or prolonged formula feeding may benefit from hydrolysed formulas. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Review Group supplemented by cross referencing of previous reviews and publications (updated August 2016). SELECTION CRITERIA: We searched for randomised and quasi-randomised trials that compared use of a hydrolysed formula versus human milk or CMF. Trials with ≥ 80% follow-up of participants were eligible for inclusion. DATA COLLECTION AND ANALYSIS: We independently assessed eligibility of studies for inclusion, methodological quality and data extraction. Primary outcomes included clinical allergy, specific allergy and food allergy. We conducted meta-analysis using a fixed-effect (FE) model. MAIN RESULTS: Two studies assessed the effect of three to four days' infant supplementation with an EHF whilst in hospital after birth versus pasteurised human milk feed. Results showed no difference in infant allergy or childhood cow's milk allergy (CMA). No eligible trials compared prolonged hydrolysed formula versus human milk feeding.Two studies assessed the effect of three to four days' infant supplementation with an EHF versus a CMF. One large quasi-random study reported a reduction in infant CMA of borderline significance among low-risk infants (risk ratio (RR) 0.62, 95% confidence interval (CI) 0.38 to 1.00).Prolonged infant feeding with a hydrolysed formula compared with a CMF was associated with a reduction in infant allergy (eight studies, 2852 infants; FE RR 0.82, 95% CI 0.72 to 0.95; risk difference (RD) -0.04, 95% CI -0.08 to -0.01; number needed to treat for an additional beneficial outcome (NNTB) 25, 95% CI 12.5 to 100) and infant CMA (two studies, 405 infants; FE RR 0.38, 95% CI 0.16 to 0.86). We had substantial methodological concerns regarding studies and concerns regarding publication bias, as substantial numbers of studies including those in high-risk infants have not comprehensively reported allergy outcomes (GRADE quality of evidence 'very low').Prolonged infant feeding with a hydrolysed formula compared with a CMF was not associated with a difference in childhood allergy and led to no differences in specific allergy, including infant and childhood asthma, eczema and rhinitis and infant food allergy. Many of the analyses assessing specific allergy are underpowered.Subroup analyses showed that infant allergy was reduced in studies that enrolled infants at high risk of allergy who used a hydrolysed formula compared with a CMF; used a PHF compared with a CMF; used prolonged and exclusive feeding of a hydrolysed formula compared with a CMF; and used a partially hydrolysed whey formula compared with a CMF. Studies that enrolled infants at high risk of allergy; used a PHF compared with a CMF; used prolonged and exclusive feeding of a hydrolysed formula compared with a CMF; and used a partially hydrolysed whey formula compared with a CMF found a reduction in infant CMA. AUTHORS' CONCLUSIONS: We found no evidence to support short-term or prolonged feeding with a hydrolysed formula compared with exclusive breast feeding for prevention of allergy. Very low-quality evidence indicates that short-term use of an EHF compared with a CMF may prevent infant CMA.In infants at high risk of allergy not exclusively breast fed, very low-quality evidence suggests that prolonged hydrolysed formula feeding compared with CMF feeding reduces infant allergy and infant CMA. Studies have found no difference in childhood allergy and no difference in specific allergy, including infant and childhood asthma, eczema and rhinitis and infant food allergy.Very low-quality evidence shows that prolonged use of a partially hydrolysed formula compared with a CMF for partial or exclusive feeding was associated with a reduction in infant allergy incidence and CMA incidence, and that prolonged use of an EHF versus a PHF reduces infant food allergy.

Specialist teams for neonatal transport to neonatal intensive care units for prevention of morbidity and mortality.

BACKGROUND: Maternal antenatal transfers provide better neonatal outcomes. However, there will inevitably be some infants who require acute transport to a neonatal intensive care unit (NICU). Because of this, many institutions develop services to provide neonatal transport by specially trained health personnel. However, few studies report on relevant clinical outcomes in infants requiring transport to NICU. OBJECTIVES: To determine the effects of specialist transport teams compared with non-specialist transport teams on the risk of neonatal mortality and morbidity among high-risk newborn infants requiring transport to neonatal intensive care. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Review Group to search the Cochrane Central Register of Controlled Trials (CENTRAL 2015, Issue 7), MEDLINE (1966 to 31 July 2015), EMBASE (1980 to 31 July 2015), CINAHL (1982 to 31 July 2015), conference proceedings, and the reference lists of retrieved articles for randomised controlled trials and quasi-randomised trials. STUDY DESIGN: randomised, quasi-randomised or cluster randomised controlled trials. POPULATION: neonates requiring transport to a neonatal intensive care unit. INTERVENTION: transport by a specialist team compared to a non-specialist team. OUTCOMES: any of the following outcomes - death; adverse events during transport leading to respiratory compromise; and condition on admission to the neonatal intensive care unit. DATA COLLECTION AND ANALYSIS: The methodological quality of the trials was assessed using the information provided in the studies and by personal communication with the author. Data on relevant outcomes were extracted and the effect size estimated and reported as risk ratio (RR), risk difference (RD), number needed to treat for an additional beneficial outcome (NNTB) or number needed to treat for an additional harmful outcome (NNTH) and mean difference (MD) for continuous outcomes. Data from cluster randomised trials were not combined for analysis. MAIN RESULTS: One trial met the inclusion criteria of this review but was considered ineligible owing to serious bias in the reporting of the results. AUTHORS' CONCLUSIONS: There is no reliable evidence from randomised trials to support or refute the effects of specialist neonatal transport teams for neonatal retrieval on infant morbidity and mortality. Cluster randomised trial study designs may be best suited to provide us with answers on effectiveness and clinical outcomes.

Intravenous in-line filters for preventing morbidity and mortality in neonates.

BACKGROUND: Venous access is an essential part of caring for the sick neonate. However, problems such as contamination of fluids with bacteria, endotoxins and particulates have been associated with intravenous infusion therapy. Intravenous in-line filters claim to be an effective strategy for the removal of bacteria, endotoxins and particulates associated with intravenous therapy in adults and are increasingly being recommended for use in neonates. OBJECTIVES: To determine the effect of intravenous in-line filters on morbidity and mortality in neonates. SEARCH METHODS: We used the standard search strategy of the Cochrane Neonatal Review Group. We searched the electronic databases MEDLINE (from 1966 to May, 2015), EMBASE (from 1980 to May, 2015), CINAHL (from 1982 to May 2015) and the Cochrane Central Register of Controlled Trials (CENTRAL; 2015, Issue 5). We did not impose any language restrictions. Further searching included cross references, abstracts, conferences, symposia proceedings, expert informants and journal handsearching. SELECTION CRITERIA: We included randomised controlled trials (RCTs) or quasi-RCTs that compared the use of intravenous in-line filters with placebo or nothing in neonates. DATA COLLECTION AND ANALYSIS: We followed the procedures of the Cochrane Neonatal Review Group throughout. We checked titles and abstracts identified from the search. We obtained the full text of all studies of possible relevance. We independently assessed the trials for their methodological quality and subsequent inclusion in the review. We contacted authors for further information as needed. Statistical analysis followed the procedures of the Cochrane Neonatal Review Group. MAIN RESULTS: There were four eligible studies that recruited a total of 704 neonates. This review of low to very low quality evidence found that the use of in-line filters compared with unfiltered fluids for intravenous infusion had no statistically significant difference in effectiveness on overall mortality (typical RR 0.87, 95% CI 0.52 to 1.47; typical RD -0.01, 95% CI -0.06 to 0.04; two studies, 530 infants), proven and suspect septicaemia (typical RR 0.86, 95% CI 0.59 to 1.27; typical RD -0.02, 95% CI -0.09 to 0.04; two studies, 530 infants), or other secondary outcomes (including local phlebitis and thrombus, necrotising enterocolitis, duration of cannula patency, length of stay in hospital, number of catheters inserted and financial costs). AUTHORS' CONCLUSIONS: There is insufficient evidence to recommend the use of intravenous in-line filters to prevent morbidity and mortality in neonates.

Regional (spinal, epidural, caudal) versus general anaesthesia in preterm infants undergoing inguinal herniorrhaphy in early infancy.

BACKGROUND: With improvements in neonatal intensive care, more preterm infants are surviving the neonatal period and presenting for surgery in early infancy. Inguinal hernia is the most common condition requiring early surgery, appearing in 38% of infants whose birth weight is between 751 grams and 1000 grams. Approximately 20% to 30% of otherwise healthy preterm infants having general anaesthesia for inguinal hernia surgery at a postmature age have at least one apnoeic episode within the postoperative period. Research studies have failed to adequately distinguish the effects of apnoeic episodes from other complications of extreme preterm gestation on the risk of brain injury, or to investigate the potential impact of postoperative apnoea upon longer term neurodevelopment. In addition to episodes of apnoea, there are concerns that anaesthetic and sedative agents may have a direct toxic effect on the developing brain of preterm infants even after reaching postmature age. It is proposed that regional anaesthesia may reduce the risk of postoperative apnoea, avoid the risk of anaesthetic-related neurotoxicity and improve neurodevelopmental outcomes in preterm infants requiring surgery for inguinal hernia at a postmature age. OBJECTIVES: To determine if regional anaesthesia reduces postoperative apnoea, bradycardia, the use of assisted ventilation, and neurological impairment, in comparison to general anaesthesia, in preterm infants undergoing inguinal herniorrhaphy at a postmature age. SEARCH METHODS: The following databases and resources were searched: the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, 2015, Issue 2), MEDLINE (December 2002 to 25 February 2015), EMBASE (December 2002 to 25 February 2015), controlled-trials.com and clinicaltrials.gov, reference lists of published trials and abstracts published in Pediatric Research and Pediatric Anesthesia. SELECTION CRITERIA: Randomised and quasi-randomised controlled trials of regional (spinal, epidural, caudal) versus general anaesthesia, or combined regional and general anaesthesia, in former preterm infants undergoing inguinal herniorrhaphy in early infancy. DATA COLLECTION AND ANALYSIS: At least two of three review authors (LJ, JF, AL) independently extracted data and performed analyses. Authors were contacted to obtain missing data. The methodological quality of each study was assessed according to the criteria of the Cochrane Neonatal Review Group. Data were analysed using Review Manager 5. Meta-analyses were performed with calculation of risk ratios (RR) and risk difference (RD), along with their 95% confidence intervals (CI) where appropriate. MAIN RESULTS: Seven small trials comparing spinal with general anaesthesia in the repair of inguinal hernia were identified. Two trial reports are listed as 'Studies awaiting classification' due to insufficient information on which to base an eligibility assessment. There was no statistically significant difference in the risk of postoperative apnoea/bradycardia (typical RR 0.72, 95% CI 0.48 to 1.06; 4 studies, 138 infants), postoperative oxygen desaturation (typical RR 0.82, 95% CI 0.61 to 1.11; 2 studies, 48 infants), the use of postoperative analgesics (RR 0.42, 95% CI 0.15 to 1.18; 1 study, 44 infants), or postoperative respiratory support (typical RR 0.09, 95% CI 0.01 to1.64; 3 studies, 98 infants) between infants receiving spinal or general anaesthesia. When infants who had received preoperative sedatives were excluded, the meta-analysis supported a reduction in the risk of postoperative apnoea in the spinal anaesthesia group (typical RR 0.53, 95% CI 0.34 to 0.82; 4 studies, 129 infants). Infants with no history of apnoea in the preoperative period and receiving spinal anaesthesia (including a subset of infants who had received sedatives) had a reduced risk of postoperative apnoea and this reached statistical significance (typical RR 0.34, 95% CI 0.14 to 0.81; 4 studies, 134 infants). Infants receiving spinal rather than general anaesthesia had a statistically significant increased risk of anaesthetic agent failure (typical RR 7.83, 95% CI 1.51 to 40.58; 3 studies, 92 infants). Infants randomised to receive spinal anaesthesia had an increased risk of anaesthetic placement failure of borderline statistical significance (typical RR 7.38, 95% CI 0.98 to 55.52; typical RD 0.15, 95% CI 0.03 to 0.27; 3 studies, 90 infants). AUTHORS' CONCLUSIONS: There is moderate-quality evidence to suggest that the administration of spinal in preference to general anaesthesia without pre- or intraoperative sedative administration may reduce the risk of postoperative apnoea by up to 47% in preterm infants undergoing inguinal herniorrhaphy at a postmature age. For every four infants treated with spinal anaesthesia, one infant may be prevented from having an episode of postoperative apnoea (NNTB=4). In those infants without preoperative apnoea, there is low-quality evidence that spinal rather than general anaesthesia may reduce the risk of preoperative apnoea by up to 66%. There was no difference in the effect of spinal compared with general anaesthesia on the overall incidence of postoperative apnoea, bradycardia, oxygen desaturation, need for postoperative analgesics or respiratory support. Limitations on these results included varying use of sedative agents, or different anaesthetic agents, or combinations of these factors, in addition to trial quality aspects such as allocation concealment and inadequate blinding of intervention and outcome assessment. The meta-analyses may have inadequate power to detect a difference between groups for some outcomes, with estimates of effect based on a total population of fewer than 140 infants.The effect of newer, rapidly acting, quickly metabolised general anaesthetic agents on safety with regard to the risk of postoperative apnoea and neurotoxic exposure has not so far been established in randomised trials. There is potential for harm from postoperative apnoea and direct brain toxicity from general anaesthetic agents superimposed upon pre-existing altered brain development in infants born at very to extreme preterm gestation. This highlights the clear need for the examination of neurodevelopmental outcomes in the context of large randomised controlled trials of general, compared with spinal, anaesthesia, in former preterm infants undergoing surgery for inguinal hernia.There is a particular need to examine the impact of the choice of spinal over general anaesthesia on respiratory and neurological outcomes in high-risk infant subgroups with severe respiratory disease and previous brain injury.

Current approaches to antiarrhythmic therapy in heart failure.

Atrial fibrillation (AF) is exceedingly common in patients with heart failure (HF), as they share common risk factors. Rate control is the cornerstone of treatment for AF; however, restoration of sinus rhythm should be considered when more than minimal symptoms are present. Life-threatening ventricular arrhythmias are responsible for the primary mode of death in patients with NYHA I, II, or III HF. Although implantable cardioverter defibrillators protect against sudden cardiac arrest, many patients will present with VT or ICD shocks. Antiarrhythmic drug therapy beyond beta-blocker therapy remains fundamental to the termination of acute VT and the prevention of ICD shocks.

Depressive symptoms are associated with clinical outcomes in heart failure with reduced ejection fraction.

AIMS: The objective of this study was to examine associations between elevated depressive symptoms and increased risk of adverse clinical events patients with heart failure and reduced ejection fraction (HFrEF), as well as the potential contribution of health behaviours. METHODS AND RESULTS: One hundred forty-two men and women with HFrEF were enrolled through heart failure (HF) clinics and followed over time. At baseline and 6 months, depressive symptoms were assessed by the Beck Depression Inventory-II (BDI-II) and HFrEF disease activity by B-type natriuretic peptide (BNP). The Self-Care of Heart Failure Index (SCHFI) was used to assess HF self-care behaviours. Proportional hazards regression models assessed the contribution of depressive symptoms and HFrEF disease biomarkers on death or cardiovascular hospitalization. Over a median follow-up period of 4 years, 42 patients (30%) died, and 84 (60%) had cardiovascular hospitalizations. A 10-point higher baseline BDI-II score was associated with a 35% greater risk of death or cardiovascular hospitalization. Higher baseline BDI-II scores were associated with poorer HF self-care maintenance behaviours (R = -0.30, P < 0.001) and fewer daily steps (R = -0.19, P = 0.04), suggesting that elevated depressive symptoms may diminish important health behaviours. Increases in plasma BNP over 6 months were associated with worse outcomes. Changes in BDI-II and plasma BNP over 6 months were positively related (R = 0.25, P = 0.004). CONCLUSIONS: This study confirms that elevated depressive symptoms are associated with an increased likelihood of adverse clinical outcomes in patients with HFrEF. Poor health behaviours may contribute to the adverse association of elevated depressive symptoms with the increased hazard of adverse clinical outcomes.

Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B6 precursor, and indoles. Growth restriction of C. parvum by indoles does not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impairs host mitochondrial function and reduces total cellular ATP, as well as directly reducing the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole-producing bacteria, delays life cycle progression of the parasite in vitro and reduces the severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites impair mitochondrial function and contribute to colonization resistance to Cryptosporidium infection.

Microbiota produced indole metabolites disrupt host cell mitochondrial energy production and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. Susceptibility rapidly declines with age, associated with changes in the microbiota. To explore microbial influences on susceptibility, we screened 85 microbiota- associated metabolites enriched in the adult gut for their effects on C. parvum growth in vitro. We identified eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B 6 precursor, and indoles. Growth restriction of C. parvum by indoles did not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impaired host mitochondrial function and reduced total cellular ATP, as well as directly reduced the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole producing bacteria, delayed life cycle progression of the parasite in vitro and reduced severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites contribute to colonization resistance to Cryptosporidium infection.

Association of Depression Symptoms and Biomarkers of Risk on Clinical Outcomes in HFrEF.

BACKGROUND: Prior studies have demonstrated an association of depression with adverse clinical outcomes in patients with HFrEF, but the possible mechanisms responsible for the association are not unserstood. METHODS: 142 men and women with HFrEF were enrolled through HF clinics and followed over time. At baseline and 6-months, depression was assessed by the Beck Depression Inventory (BDI-II) and disease activity by B-type natriuretic peptide (BNP). Proportional Hazards Regression Models assessed the contribution of depressive symptoms and HFrEF disease biomarkers on death or cardiovascular hospitalization. RESULTS: Over a median follow-up period of 4 years, 42 patients (30%) died, and 84 (60%) had cardiovascular hospitalizations. A 10-point higher baseline BDI-II score was associated with a 35% higher hazard of death or cardiovascular hospitalization. Greater baseline BDI-II scores were associated with poorer HF self-care maintenance (R=-0.30, p<0.001) and fewer daily steps (R=-0.19, p=0.04), suggesting that depression may adversely affect important health behaviors. Increases in plasma BNP over 6 months were associated with worse outcomes. Changes in BDI-II score and plasma BNP over 6 months were positively correlated (R=0.25, p=0.004). CONCLUSIONS: This study underscores the importance of elevated depression symptoms and their association with an increased likelihood of adverse clinical outcomes in patients with HFrEF. Health behaviors may play a greater role than direct biobehavioral pathways in the adverse effects of depression on the HF disease trajectory and resultant clinical outcomes.

Unexpected case of chagas disease reactivation in endomyocardial biopsy for evaluation of cardiac allograft rejection.

Acute Chagas disease reactivation (CDR) after cardiac transplantation is a well-known phenomenon in endemic countries of Central and South America and Mexico, but is rare outside of those countries. In this report, we describe a case of a 49-year-old male who presented 25 weeks after heart transplant with clinical features concerning for acute rejection, including malaise, anorexia, weight loss, and fever. His immunosuppression therapy included tacrolimus, mycophenolate, and prednisone. An endomyocardial biopsy revealed lymphocytic and eosinophilic inflammation, myocyte damage, and rare foci of intracellular organisms consistent with Trypanosoma cruzi amastigotes. The patient had no known history of Chagas disease. Upon additional questioning, the patient endorsed bites from reduviid bugs during childhood in El Salvador. Follow-up serum PCR testing was positive for T. cruzi DNA. Tests for other infectious organisms and donor specific antibodies were negative. This case illustrates the striking clinical and histologic similarities between acute cellular rejection and acute CDR with cardiac involvement in heart transplant patients, and thus emphasizes the importance of pre-transplant testing for Chagas in patients with epidemiologic risk factors.

Heart Failure: Advanced Refractory Heart Failure.

End-stage heart failure (HF) is associated with an extremely poor prognosis. Progressive and/or persistent HF signs and symptoms in the setting of optimal therapy is the hallmark of more advanced disease. Physicians must be able to recognize patients with features of refractory HF to aid in timely evaluation for advanced therapy options. Left ventricular assist device implantation and heart transplantation prolong survival in patients with end-stage HF, but are options only for select patients. Timely referral for evaluation is necessary to prevent secondary irreversible end-organ dysfunction and to ascertain whether there are factors that can be addressed and corrected. All patients with end-stage HF should be offered referral for palliative care to aid in symptom management and improve quality of life. In addition, for patients who are not candidates for advanced therapy options, hospice should be discussed. In some cases, palliative home inotrope infusion can be considered for symptom management.

Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro.

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.