Multiple sclerosis is a disease characterised by axonal demyelination in the central nervous system (CNS). The atypical antipsychotic drug clozapine attenuates experimental autoimmune encephalomyelitis (EAE), a mouse model used to study multiple sclerosis, but the precise mechanism is unknown and could include both peripheral and CNS–mediated effects. To better understand where clozapine exerts its protective effects, we investigated the tissue distribution and localisation of clozapine using matrix-assisted laser desorption ionization imaging mass spectrometry and liquid chromatography-mass spectrometry. We found that clozapine was detectable in the brain and enriched in specific brain regions , but the distribution was not altered by EAE. Furthermore, although not altered in other organs, clozapine levels were significantly elevated in serum during EAE. Because clozapine antagonises dopamine receptors, we analysed dopamine levels in serum and brain as well as dopamine receptor expression on brain-resident and infiltrating immune cells. While neither clozapine nor EAE significantly affected dopamine levels, we observed a significant downregulation of dopamine receptors 1 and 5 and up-regulation of dopamine receptor 2 on microglia and CD4+-infiltrating T cells during EAE. Together these findings provide insight into how neuroinflammation, as modelled by EAE, alters the distribution and downstream effects of clozapine. Given that neuroinflammation occurs during schizophrenia3, clozapine’s immunomodulatory ability may contribute to its efficacy in reducing disease severity.Clozapine is an atypical antipsychotic agent used for neurological disorders including schizophrenia and Parkinson’s disease. Clozapine reduces psychosis by antagonising both the serotonin 2A and C (5-HT2A and 5-HT2C) and the dopamine receptor 2 but has also been shown to be immunomodulatory in the central nervous system (CNS)5. EAE is characterised by the infiltration of autoreactive CD4 + T cells and other immune cells (e.g. monocytes) into the CNS causing an inflammatory state. It has been shown previously that innate and adaptive immune cells are increased in the CNS and that pro-inflammatory cytokines, like interferon gamma and interleukin-17, associated with a high level of inflammation are upregulated7. We have shown previously that clozapine treatment reduces the expression of pro-inflammatory cytokines and chemokines in the CNS as well as reduction the activation markers (Iba1 and GFAP) on CNS-resident cells7. However, the precise mechanism by which clozapine ameliorates EAE has not been identified, and because EAE involves not only myelin-specific CD4 + T cells but other peripheral immune and CNS-resident cells, there are many potential targets for clozapine’s protective effects.The ability of clozapine to regulate inflammation has been shown to reduce disease in experimental autoimmune encephalomyelitis (EAE), a mouse model useful for the study of neuroinflammatory aspects of multiple sclerosis (MS), a disease characterised by demyelination of the neurons in the CNS8. The CRISP trial results highlight that clozapine’s actions may be altered during MS such that people with progressive MS have an enhanced sensitivity to clozapine. Due to the early withdrawal of all participants in the clozapine group, this trial could not determine whether clozapine was able to impact MS disease as suggested by pre-clinical studies7. This surprising finding is at the heart of our rationale for this study, which is to understand why this sensitivity to clozapine occurred  so that it can be appropriately used in a therapeutic trial. While EAE does not recapitulate all aspects of MS, it does model CD4 + T cell-driven neuroinflammation9. As such, EAE is a useful model to dissect clozapine’s mechanism of action and to understand how neuroinflammation itself may alter clozapine’s effects. Thus, we investigated which tissues are targeted by clozapine by determining its tissue localisation and distribution as well as the expression of its target receptors.A recent phase 1b, randomized, controlled trial in progressive MS patients using low doses of clozapine  found that all participants treated with clozapine were withdrawn within 9 days of starting treatment due to adverse events10. All DRs are highly expressed in the brain10 and on immune cells11, and have shown to be involved in mental disorders and neuroinflammatory diseases like schizophrenia, depression, Parkinson's and Huntington’s disease13. Given clozapine’s affinity for DRs, it has been reported to displace dopamine from the receptor within less than one minute15, directing it towards other receptors and causing it to have unanticipated effects. Importantly, previous research has shown that selective DR1 antagonism reduces EAE16 while selective DR2 antagonism exacerbates EAE16 indicating that dopamine receptors are involved in EAE. Thus, we have focused our investigation on dopamine.Clozapine antagonises dopamine receptors (DR), which can be separated into two subtypes, D1 subtypes (DR1 and DR5) and D2 subtypes , based on modulation of cAMP production17 while depletion worsened disease18. This could be due to the immunomodulatory effects of dopamine on T cells leading to increased production of tumour necrosis factor-α and interleukin-1019. However, the context of the tissue and the inflammatory state seems to determine the effect dopamine on a given T cell subset and function20. The modulation of the function and phenotype of monocytes and macrophages is another way how dopamine might improve symptom of EAE21. Incubation of monocytes and macrophages in vitro with dopamine improved their phagocytic activity. Thus, these studies highlight an important role for dopamine as immunomodulator in EAE and suggest a change in dopamine levels by clozapine might be one means by which clozapine reduces EAE disease severity.Dopamine is a neurotransmitter with many distinct functions including in the reward pathway and motor control. Previous studies using the EAE model showed that increasing dopamine levels improved the symptoms of EAE22. However, LC–MS only measures absolute levels without any information about localisation and distribution. In contrast, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) analyses tissue sections for the distribution of a range of unlabelled targets23, and thus provides spatial information.Liquid chromatography-coupled mass spectrometry (LC–MS) is an important tool for drug metabolism and pharmacokinetic studies and is commonly used for metabolite identification, quantification, toxicology and pharmacokinetics due to its high sensitivity, rapid analysis and high specificityIn this study, we used MALDI IMS in combination with quantitative LC–MS time of flight analysis to investigate the localisation and distribution of clozapine and dopamine in brain regions and peripheral organs. In addition, we analysed dopamine and DRs in the CNS to better understand clozapine’s actions during EAE-associated neuroinflammation.24. Previous studies have shown that clozapine administered orally at a concentration of 60 mg/kg; reduced the onset and the severity of EAE in the mice25; therefore, we assessed the distribution and localisation of clozapine in the brains of healthy and EAE mice treated with vehicle or clozapine. As reported previously, clozapine treatment reduced the severity of disease as measured by the disease score and the area under curve of the disease course . No significant differences between groups was seen in the brain or the lung Fig. e,f, but 12.1142. 21, which could change in vivo dopamine levels. Therefore, we analysed the concentration of dopamine in the serum and brain of healthy and EAE mice with or without clozapine treatment in the drinking water, because dopamine exerts its main effect as a hormone and neurotransmitter in the brain. Interestingly, we found that serum levels were similar between all groups  by clozapineIn healthy mice, DR1 expression Fig. a was mos+T cells by EAE while the increase on macrophages did not reach significance  expression on brain-isolated immune cells was significantly and differentially altered by EAE and clozapine suggesting that clozapine may act differently during EAE due to an altered expression of its target receptor. This study is the first in-depth report assessing clozapine, dopamine and dopamine receptors in the brain during EAE and after chronic clozapine administration and highlights how EAE-associated neuroinflammation may alter clozapine’s target pathways.MS is characterised by demyelination of neuronal axons targeted by infiltrating autoreactive CD437, with the free fraction of clozapine being less than 10%38. Additionally, higher concentrations of α-1-acid glycoprotein have been reported in the serum and plasma of MS patients with different stages of disease40. Furthermore, a membrane bound α-1-acid glycoprotein can be synthesized by leucocytes and subsequently cleaved and released as the soluble serum form41. Because, leucocytes are activated in EAE, it is possible that immune cells are responsible for an increase in α-1-acid glycoprotein, which binds and retains clozapine in the blood. Peak serum levels of clozapine are reached 1–6 h after oral dosing and the half-life is about 4 to 16 h in healthy adults42, and our findings suggest this half-life may be extended during a neuroinflammatory response.The finding that clozapine levels were significantly higher in the serum of EAE compared to healthy mice is intriguing because there were no differences detected in any other tissue. The higher levels were not due to impaired metabolism as norclozapine concentrations were also significantly elevated during EAE. Instead, it is possible that clozapine and norclozapine are retained in the serum, and previous research has shown that clozapine and its metabolites are strongly bound to the serum protein, α-1-acid glycoprotein23. We also found DHB gave the best results compared to other matrices (data not shown); however, in our study, the oral-administered clozapine dose was too low to detect consistently in the brain. Therefore, we injected 10 mg/kg clozapine 30 min before euthanasia and were able to detect clozapine reproducibly. Similarly, Hsieh et al. showed by MALDI IMS that intravenous injections of clozapine into rats resulted in a high detection of clozapine in the brain after 45 min43 and Baldessarini et al. showed by reversed-phase liquid chromatographic separation, that detection levels peak at 30 min after 10 mg/kg intraperitoneal injection26.MALDI IMS was used for the first time to analyse the distribution of unlabelled clozapine within the brain tissue from healthy and EAE mice. Previously, Quiason and Shahidi-Latham used a DHB matrix on brain samples from rats treated orally with clozapine and reported a good detection of the drug in the brain tissue38. Brain PET scan imaging from healthy humans injected with11C-labelled clozapine showed that clozapine seems to localize to specific brain regions with the highest intensity in the insula and striatum and the lowest in the cerebellum27, and our results are consistent with these findings.With the segmentation of the brain into six different brain regions, we showed that clozapine localises to specific brain regions, with highest intensity in the cortex and thalamus and lowest in the cerebellum and medulla. Using desorption electrospray ionization IMS, clozapine was also detected in healthy rat brains after oral dose of 50 mg/kg clozapine, with the highest levels in the cortical regionsN-oxide44. Experiments in CYP1A2-null mice showed a significant longer half-life in the serum and a reduced time of clearance45. This finding likely explains the high clozapine concentrations in the liver after acute exposure. Over time, clozapine concentrations reduce due to its metabolism and only low levels can be detected in the continuously treated animals. While an ion suitable for clozapine-N-oxide was detected in some liver samples, it’s presence and concentration were inconsistent. Interestingly, we did see increased norclozapine intensity in liver samples from mice continuously treated with clozapine in the drinking water. This leads to the conclusion that clozapine metabolites accumulate in the liver over time and have a longer half-life.The high concentrations in the liver compared to other organs are most likely because the liver is the major site where clozapine is metabolized. The cytochrome P450 system, in particular CYP1A2, converts clozapine into the two main primary metabolites norclozapine and clozapine-46. At the time we began this study, previous research had shown that selective DR1 antagonism reduces EAE while selective DR2 antagonism exacerbates EAE indicating that regulation of the dopamine receptor pathways is important16. In contrast, serotonin reuptake inhibitors as well as a novel 5-HT1A/D2 ligand have been shown to ameliorate EAE suggesting a positive role for serotonin in MS49. These findings suggest that clozapine’s ability to reduce EAE is not principally through serotonin receptor antagonism although further research to assess the contribution of 5-HT2A and C is warranted, but that dopamine receptors look to be involved. Based upon these studies, we focused our investigation on dopamine and not serotonin.One mechanism of clozapine’s action is through antagonism of serotonin and dopamine receptors (DR). Our choice to investigate dopamine was based upon clozapine’s known targets and the involvement of those targets in EAE. Clozapine was developed to antagonize DR2 but has greater affinity for DR1, serotonin receptors (esp 5-HT2A and C subclasses), alpha adrenergic receptors, and histamine receptor 150. We have shown here that dopamine levels do not change in the serum of healthy or EAE mice despite continuous clozapine treatment in the drinking water. In addition, dopamine levels in the brain tissue measured by ELISA or by MALDI IMS were not different. However, we identified the striatum as the major site of dopamine localisation and it did not change in any condition tested. This finding agrees with several studies where high dopamine levels were detected in the striatum by HPLC53. Although Balkowiec-Iskra et al. reported higher concentrations than our study, these reports differ in tissue (isolated striatum vs whole brain) and method (HPLC vs MALDI IMS)44. Increased dopamine levels after clozapine injection were previously reported in the rat caudate, nucleus accumbens and the medial prefrontal cortex measured by HPLC48, which is a more sensitive method compared to MALDI IMS. Similarly, these studies assessed specific brain regions compared to our analysis in whole brains, which would dilute the dopamine levels. In addition, most of these studies injected clozapine i.p. and studied the dopamine concentration within one hour after injection while our treatment regime was in a continuous treatment of clozapine in the drinking water. Meltzer et al. have shown that dopamine levels peak 60 min after clozapine injection and then return to baseline levels46. This immediate and high response of dopamine to the clozapine injection may differ from the continuous lower levels of clozapine achieved by administration in the drinking water because it might depend on when the mice were last drinking. Future studies investigating the effect of chronic, low dose clozapine on dopamine, may need to assess not only dopamine but also its metabolites like DOPAC47.The specific role of dopamine and its receptors is still unclear but several previous studies have shown that the changes in dopamine might be related to inflammation in autoimmune diseases54. All DRs are reported to be widely expressed in the CNS and several studies have reported their expression on immune cells11, which play an important role in MS50. Moreover, DR expression on immune cells is also associated with some neurological and immune-related disorders56. But, to our knowledge, this study is the first to monitor DRs on brain-resident cells and infiltrating immune cells during EAE. Here, we show that all five DR are present on brain-resident microglia, although the levels were lower than on other myeloid cells . Furthermore, we found that EAE upregulated D2 subtypes (DR2 and DR3) and downregulated DR1. Interestingly, clozapine had an enhancing effect on DR1 and DR2 expression on cells from healthy and EAE animals. This study does not exclude any beneficial effects of clozapine on peripheral immune cells although our previous studies have shown that clozapine does not directly alter peripheral CD4+ T cells during EAE25. Instead, we have reported that clozapine modifies peripheral and CNS-resident innate immune cells with the greatest effects found in the CNS6. Therefore, this study has focused on DR expression on infiltrating and CNS-resident cells. Given the fascinating actions of dopamine outside of the CNS, further work needs to be performed to investigate DR expression on immune cells in the periphery and CNS at different stages of EAE, given that DR expression on immune cells is highly dynamic, context-sensitive and variable57.Interestingly, the regions with the highest clozapine levels are known to express a high density of DR1 and DR2, and the cerebellum, with low dopamine levels, has lower DR expression57, but the level of receptor expression differs between different T cell populations and activation states, and between resting and activated T cells. In healthy donors, resting T cells showed only a minimal expression of DR2-5 and no DR1 expression58. In line with our data, D1 subtypes are decreased on effector T cells from MS patients, and dopamine does not affect these cells57. Additionally, decreased DR5 expression could be detected, and DR5 performs an inhibitory role on T cells during MS56. Besides this evidence that DR are modulated during MS, dopamine showed only very limited therapeutic effects50.A solid body of evidence indicates that in MS, dopaminergic pathways are dysregulated in the peripheral immune system. In general, human T cells express all DRs on their surface8.Overall, this study is the first to show that clozapine localises to specific brain regions, which are known for high expression of the DR. While the localisation of clozapine was not altered by EAE, we found that clozapine levels were significantly elevated in the serum along with its metabolite norclozapine. Finally, immune cells in the CNS during EAE had decreased levels of DR1 but increased DR2, both of which were enhanced by clozapine administration. Overall, these findings indicate that clozapine may be retained for longer during neuroinflammation and mediate differential effects on immune cells through its target receptors, and together suggest that clozapine administration may have unexpected actions during MS due to modification of dopamine pathways, which is in line with a recent clinical trial assessing clozapine administration during MSAll methods were carried out in accordance with relevant guidelines and regulations.Eight-ten-week old female C57BL/6J mice were purchased from the Biomedical Research Unit of the Malaghan Institute of Medical Research  and housed in the Victoria University of Wellington PC2 animal facility at a 12 h dark–light cycle with food and water ad libitum.All animal experiments were carried out in the School of Biological Sciences Animal Facility at Victoria University of Wellington and were approved by the Victoria University of Wellington Animal Ethics Committee (2014-R23 and 25295). All methods were carried out in accordance with relevant guidelines and regulations and in compliance with the ARRIVE guidelines.35–55 peptide  in complete Freund’s adjuvant  containing 500 µg/mouse Mycobacterium tuberculosis . In addition, mice were injected i.p. with pertussis toxin  on days 0 and 2. Mice were weighed and scored daily as follows: 0, normal; 1, partial tail paralysis; 2, full tail paralysis; 3, paralysis in one hind limb; 4, paralysis in both hind limbs; and 5, moribund. One day before immunization drinking water was changed to 60 mg/kg/day clozapine  or vehicle (0.1 M acetic acid). Mice were injected i.p. with 10 mg/kg clozapine or vehicle 30 min prior to CO2 euthanasia. Spleen, liver, lung and brain were collected and immediately snap-frozen in liquid nitrogen. Serum was collected as described previously6. Organs and serum were stored at − 80 °C until analysis.Mice were immunized s.c. in the rear flanks with myelin oligodendrocyte glycoprotein (MOG)g). Brain sections 10 µm thick) were cut on a Leica CM3050 cryotome . Five sections/brain were homogenized in 100 µL 0.1 M acetic acid. Solutions were resuspended using a pipette, centrifuged for 5 min at 1000 g and supernatant was spin filtered through a 30 K Microcon Centrifugal Filter . Serum samples were mixed 1:1 with acetonitrile, vortexed 30 s and centrifuged 5 min at 14,000×g. All sample were transferred to vials containing a glass insert for small volumes . For the clozapine standard curves, clozapine was dissolved in 0.1 M acetic acid and standard curves were spiked in untreated healthy serum or tissue samples before sample preparation. All samples and standards were spiked with quetiapine  as an internal standard.Liver (70 mg) and lung tissue (20 mg) were homogenized in 1 mL 0.1 M acetic acid using a Polytron tissue homogenizer. Solutions were resuspended and centrifuged for 5 min at 1000 g, supernatant removed and centrifuged a second time  equipped with a Synergi Hydro-RP column  and 0.5 µl calibration mix was added outside of the tissue . A TM-sprayer  was used to spray-coat the sections with the DHB matrix as specified in Table 2 and 2.5 µg/mm2, respectively.TPP-TFB derivatisation matrix was sprayed on the tissue with the TM-sprayer as specified in Table The spray-coated sections were analysed with an AB Sciex TOF/TOF 5800 mass spectrometer  operated as specified in Table 59. Prior to loading the files into msIQuant, the data files were converted from the Analyze 7.5 format to the imzml format using the converter module from Spectviewer60, kindly provided by Jean-Pierre Both . Pre-processing of all images included background correction and spectral alignment. Image normalisation was based on the root mean square in msIQuant. Tissue sections used for clozapine detection were segmented into different brain regions based on three ion signals , while tissue sections used for dopamine detection were segmented based on the optical image.MALDI IMS data was processed with msIQuant softwareQTOF LC–MS data was analysed using the Qualitative Analysis B.06.00 from Agilent.g for 5 min at 4 °C and supernatants were assayed immediately. Dopamine was measured using mouse dopamine ELISA kit from CUSABIO  according to manufactures instructions61. Plates were read at 450 nm using a PerkinElmer  microplate reader.Serum was collected and used immediately. For the brain, one brain hemisphere was mashed in PBS (0.1 mL PBS per 100 mg tissue). Samples were stored at – 20 °C. Two freeze and thaw cycles were performed, lysates were centrifuged at 5000×6  or isotype control for 25 min. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analysed using FlowJo software version 10.1 .Each brain was processed into a single-cell suspension and stained as described previouslyAll graphs and statistical analyses were generated using GraphPad Prism 7 . For comparison of more than two groups one-way or two-way analysis of variance (ANOVA) was used with the recommended multiple comparison tests as indicated in the figure legend and as recommended by GraphPad Prism.Supplementary Information.


Mycobacterium bovis isolated in central Ethiopia. A total of 65 dairy farms and 654 cattle were tested for bTB using a single intradermal comparative cervical tuberculin (SICCT) test. Data on farm management, animal-related characteristics, and the owner's knowledge of the zoonotic importance of bTB were collected using a structured questionnaire. In addition, a total of 16 animals from different farms were identified for postmortem examination. Lowenstein Jensen (LJ) culture was also conducted, and spoligotyping was used to type the M. bovis strains isolated. Chi-square test and logistic regression models were used to analyze the herd- and animal-level risk factors. Herd- and animal-level prevalence rates of bTB were 58.5% (95% CI: 46.2%−69.2%) and 39.3% (95% CI: 35.5%−43.5%), respectively. At the herd level, poor farm management was the predictor for bTB positivity (p < 0.05). Animal breed, poor BCS, farm type, and poor farm management conditions were significant predictors of bTB positivity (p < 0.05) at an individual animal level. All animals identified for postmortem examination were found to have gross TB-like lesions. A total of 14 M. bovis strains were identified from 12 animals that were positive for LJ culture. The strain with the largest number of clusters (five isolates) was SB1176, followed by SB0134 (three isolates), SB0192 (two isolates), and SB2233 (two isolates), and two new strains, each consisting of only one isolate. The majority (58.5%) of the respondents did not know the zoonotic importance of bTB. The result of this study showed a high prevalence of bTB in the Addis Ababa milkshed and a low level of consciousness of the owners on its transmission to humans. Therefore, the launching of acceptable control measures of bTB and the creation of public awareness about its zoonotic transmission and prevention measures are required.Bovine tuberculosis (bTB) continues to be one of the most widely distributed chronic infectious diseases of zoonotic importance, which causes a significant economic loss in animal production. A cross-sectional study was conducted to estimate the prevalence of bTB and its associated risk factors and type the  Mycobacterium bovis, a member of Mycobacterium tuberculosis complex (MTBc). It is a chronic infectious disease of animals characterized by the formation of granulomas primarily in the lungs, lymph nodes, intestine, and kidney. M. bovis has the widest host ranges of all MTBc organisms and can readily be transmitted to humans or a variety of domestic and wild animals  is caused by  animals . The mos animals . In 2019Many developed nations have reduced or eliminated bTB from their cattle population by implementing effective control strategies that include testing and culling of infected animals, active surveillance, and restrictions of movement in affected areas –7. HowevEthiopia has the largest cattle population in Africa  and is aM. bovis strains isolated, and assess the knowledge of farm owners or workers about bTB and its zoonotic importance in Addis Ababa milkshed, the capital of Ethiopia.Therefore, understanding the magnitude of bTB infection and the molecular epidemiology in animal and human populations in the peri-urban area that supplies the major city of Addis Ababa is a key priority. Additionally, information about the knowledge of dairy farm owners or farm workers about the bTB and its zoonotic importance is essential in designing the control strategy and for policy recommendation. To this effect, the objectives of this study were to determine the prevalence of bTB both at the herd and animal level, type the The study was conducted between December 2017 and March 2019 in the milkshed of the capital Addis Ababa, central Ethiopia, situated in a range of 80 km toward the North West and North East, namely, Chanco Woreda, Laga-Tafo Laga-Dadi Town, Muka Turi Town, Sandafa-Bake Woreda, and Suluta Town . The areThe study subjects were dairy cattle managed in the selected dairy farms in the study area. The farms were characterized by a mix of small holders at household level and intensive and semi-intensive farms owned by members of private investors . Dairy cFarms were included if they had been established for over a year, owned at least five cattle, gave written informed consent for cattle to have SICCT test, and agreed that at least one strong reactor could be slaughtered in return for financial compensation. Individual animals were excluded if they were calves younger than 4 weeks, clinically sick cattle with diseases not suggestive of bTB, or cows in the last 2 months of pregnancy.A cross-sectional study was conducted in the farms located in the milkshed of Addis Ababa City, central Ethiopia. Lists of intensive dairy farms with more than five cattle with HF and/or crossbreed were obtained from the local Livestock and Fishery Department offices in the study area. The farms were grouped into three categories; small , medium , and large . From each study area, the farms and the animals were randomly selected. The farms were approached for their willingness to participate in bTB testing and those farms that agreed were tested. New farms with less than 1 year since establishment were excluded.M. bovis and Mycobacterium avium tuberculin to cattle on the day of consent and returned 72 h later to read the tests. A total of 16 highly reactive cows using SICCT test were purchased , and further laboratory examinations were performed.A total of 65 farms and 654 individual animals were screened for bTB infection using a single intradermal comparative cervical tuberculin (SICCT) test. After the farms were identified and written informed consent was obtained from the owners for their cattle to undergo SICCT test, study staff administered intradermal injections of purified protein derivatives (PPDs) from Risk factors associated with bTB positivity both at animal and herd levels were recorded before PPD injection. Body condition score (BCS) of the animals was determined as good, medium, or poor according to Nicholson and Butterworth . Good BCThe management condition of the farms was categorized based on Ameni et al.  as poor,Data were collected by study staff using a paper Case Report Format (CRF). Each page of the CRF bears a study ID number unique for each participant farm. Either the owner or the farm manager was interviewed using a predesigned questionnaire about the awareness of bTB transmission, habits of raw milk and meat consumption, and recent TB cases identified from family or workers.M. bovis (PPDb) and M. avium (PPDa) according to a published protocol  for further processing. At ALIPB laboratory, samples were stored at −22°C, and all samples were processed and cultured for mycobacteria as previously described by the World Organization for Animal Health protocols . The tisM. tuberculosis complex by polymerase chain reaction (PCR) using oligonucleotide biotin-labeled primers derived from the DR sequence, RDa (5′GGTTTTGGGTTTGAACGAC3′) and RDb (5′CCGAGAGGGGACG GAAAC3′) . Spoligotyping was performed following the standard operating procedure that was used by Berg et al.  and primGAAAC3′) .2, 200 μM of each deoxynucleotide triphosphate, 2 μl of each primer (20 pmol each), 5 μl suspension of heat-killed cells (approximately 10–50 ng), and 3.5 μl distilled water were used. The mixture was heated for 15 min at 96°C and then subjected to 30 cycles of 1 min denaturation at 96°C, annealing at 55°C for 1 min and extension at 72°C for 30 s. And the final stabilization stage at 72°C for 10 min. Immediately before running spoligotyping, the PCR product was denatured using thermocyler at 96°C for 10 min and then removed from the thermocycler and kept on ice so as to prevent renaturing of the PCR products. Thereafter, the denatured PCR product was loaded onto a membrane covalently bonded with a set of 43 oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus of M. tuberculosis complex and then hybridized at 60°C for 1 h. After hybridization, the membrane was washed twice for 10 min in 2× SSPE -0.5% sodium dodecyl sulfate (SDS) at 60°C and then incubated in 1:4,000 diluted streptavidin peroxidase (Boehringer) for 1 h at 42°C. The membrane was washed twice for 10 min in 2× SSPE-0.5% SDS at 42°C and rinsed with 2× SSPE for 5 min at room temperature. Hybridizing DNA was detected by the enhanced chemiluminescence (ECL) method  and by exposure to X-ray film . A mixture of 10 ml of ECL reagent 1 and 10 ml of ECL reagent 2 was prepared and then added onto the membrane, and the membrane was rinsed in the solution for 5 min at room temperature. Then, the membrane was attached onto a film in the dark room and placed in the cassette and incubated for 15 min at room temperature. The film was removed and placed in a developer solution for 2 min, removed from the developer, and rinsed with tap water for 15 s and then placed in a fixer solution for 1 min. Finally, the film was dried and used for interpretation of the result. The presence of the spacer was identified as a black square, while absence of the spacer was identified as a white square on the film. The black squares were converted to 1 while the white squares were converted to 0 and then transferred to the spoligotype international type (SIT)-VNTR international type (VIT) database for the identification of the SITs and the lineages of the isolates.A total volume of 25 μl and reaction mixtures of 12.5 μl of HotStarTaq Master Mix (Qiagen), a final concentration of 1.5 mM MgClp-values <0.05.SICCT test and other questionnaire data were entered and analyzed by SPSS version 21.0. Descriptive statistics like mean, median, and standard deviation were used to summarize data. For both herd and animal levels, prevalence was calculated by dividing the number of reactors to the total number tested. One-sample nonparametric test was used to compute the 95% confidence interval of the prevalence. A chi-square test was used to compare the proportions. Univariable and multivariable logistic regressions were used to rule out the risk factors associated with bTB at the herd and animal levels. Statistical significance was indicated using 95% confidence intervals and The study obtained ethical approval from the Armauer Hansen Research Institute (AHRI) Ethics Review Committee (Ref P018/17); the Ethiopian National Research Ethics Review Committee (Ref 310/253/2017); the Queen Mary University of London Research Ethics Committee, London UK (Ref 16/YH/0410); and the ALIPB, Addis Ababa University (Ref ALIPB/IRB/011/2017/18). Written informed consent was obtained from all the owners of the farms.A total of 65 dairy farms in the milkshed of Addis Ababa City, central Ethiopia, were screened for bTB using SICCT test. The overall prevalence of bTB at herd level was 58.5% (95% CI: 45.6–70.6) at a cutoff value >4.0 mm. When the study sites were considered, 50.0% (19/38) of the positive herds were recorded at Sandafa followed by Sululta (23.7%), Chancho (13.2%), Laga-Tafo (10.5%), and Muka-Turi (2.6%). The overall animal-level prevalence was 39.3% (95% CI: 35.5–43.2) at a cutoff value >4.0 mm. The highest proportion (63.8%) of bTB-positive animals was reported from Sandafa area, followed by Sululta area 19.8% (51/257), Laga-Tafo area 10.5% (27/257), Chancho area 3.1% (8/257), and Muka-Turi area 2.7% (7/257) .Results on the farms' characteristics including the type of farms whether they are traditional or commercial, herd size of the farms, and the farms' management conditions are summarized in p < 0.05) . The multivariable logistic regression analysis showed that farm management conditions remain the predictors for bTB positivity at the herd level ( < 0.05) .The mean age of the animals included in this study was 5.1 years (SD = 2.21), the majority of them were in the age group 4.0–9.0 years of age. Eighty-eight percent of the animals were cows, and 93.0% of the animals belong to crossbreed between either HF or Jersey and the local Zebu .p < 0.05). According to the multivariable logistic regression location of the animals, breed, poor body condition, animals in a commercial farm, and farm management conditions were significant predictors of bTB positivity (p < 0.05)  .M. bovis culture on LJ medium with a culture positivity rate of 75.0%. All the samples have shown growth on the LJ media supplemented with sodium pyruvate, and only three samples have shown growth on the LJ media supplemented with glycerol. The spoligotype pattern of 14 isolates was shown in M. bovis strains of which two were new strains. The genotype with the largest of isolates (five isolates) was SB1176, followed by SB0134 (three isolates), SB0192 (two isolates), and SB2233 (two isolates) and two new strains each with one isolate.A total of 16 cows, one cow from each farm were purchased based on their higher SICCT test result. The mean SICCT score was 22.5 mm (SD = 15.59), 8.7 and 71.0 mm being the lowest and the highest score, respectively. Out of the 16 animals that showed gross TB lesions, 12 of them were positive for Farm owners or managers were interviewed to assess their awareness about bTB and its zoonotic transmission. Out of 65 respondents, 31.0% did not know what bTB was, and 58.5% did not know the zoonotic importance of bTB. The participants responded that they had a habit of eating raw meat 83.0% and drinking raw milk 86.2%. Similarly, 87.7% of owners' families or their workers share the same room with their cattle. Finally, 13.8% reported a history of TB in either the owners' family or their workers .This study reports the epidemiology of bTB and its zoonotic implication in Addis Abba milkshed, central Ethiopia. The country is known to have a high burden of TB in its human  and cattIn the present study, the herd and animal prevalence rates of bTB using SICCT test were 58.5 and 39.3%, respectively. This is one of the highest reports regarding bTB in the country. Previous studies reported lower results compared to the present study , 19–23. M. bovis infection, creating a conducive environment for easy proliferation and transmission.Based on multivariable logistic regression analysis of the risk factors in the present study, poor farm management condition was significantly associated with bTB positivity at the herd level. This observation was consistent with the results by Kemal et al.  from EasIn this study, breed of cattle was one of the predictors of bTB positivity. Previous studies reported by Ameni et al.  and VordIn the present study, animals from large commercial farms were more likely to be positive for bTB diseases when compared to traditional farms with smaller herds. This is consistent with the type of breeds that are largely found in such setup. In the study area, commercial farms are largely populated by European breeds and crossbreeds. Studies in Eastern Ethiopia also demonstrated that commercial farms are more likely to be positive for bTB . In the M. bovis, or a combination of both. Clinically, poor body condition is a typical sign that follows M. bovis infection in cattle.Similar to a previous study by Dejene et al. , animalsM. bovis and gross pathology  belong to the African 2 (AF2) clonal complex. The AF2 complex is known by the deletion of spacers from 3 to 7 in the spoligotype signatures. These strains of M. bovis are known by localized distribution in Eastern African countries including Ethiopia, Uganda, Burundi, and Tanzania  Ethics Review Committee (Ref P018/17), from the Ethiopian National Research Ethics Review Committee (Ref 310/253/2017), the Queen Mary University of London Research Ethics Committee, London UK (Ref 16/YH/0410); and by the Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University (Ref ALIPB/IRB/011/2017/18). Written informed consent was obtained from all the owners of the farms.BT and GA conceived the study. BT, AZ, MB, AM, and GA contributed to the study design and development of laboratory assays. BT, AZ, MZ, MG, MT, FI, MB, DJ, HM, MA, TB, BG, AM, and GA contributed to the implementation of the study and data acquisition. BT did statistical analyses, wrote the first draft of the manuscript, and had final responsibility for the decision to submit for publication. All authors reviewed the final draft and agreed with its content and conclusions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.The disease-causing blood-stage of the  Plasmodium. Here, the authors characterize the rhoptry-associated protein CERLI1 using quantitative super-resolution microscopy, showing that it is important for parasite invasion and secretion of rhoptry proteins including vaccine antigens.Rhoptries are essential organelles for invasion of erythrocytes by  Plasmodium spp. parasites, results in >400,000 deaths annually with Plasmodium falciparum being responsible for the majority of malaria mortality1. Plasmodium parasites have a complex lifecycle with human infection beginning with transmission of the liver-cell invading sporozoite from a female Anopheline mosquito to the human host. Thousands of daughter merozoites develop in the liver-stages and are released into the blood stream where they invade erythrocytes. In the case of P. falciparum, the blood-stage parasite multiplies over the next 48 h, forming 16–32 merozoites that rupture out of the schizont stage parasite and infect new erythrocytes2. This asexual blood-stage of the parasite lifecycle causes all disease symptoms.Malaria, caused by infection with 3. The merozoite then reorientates such that the apical tip, containing specialised invasion organelles known as the rhoptries and micronemes, contacts the erythrocyte membrane. Invasion ligands are released from the micronemes and rhoptries prior to formation of an irreversible tight-junction that binds the merozoite to the erythrocyte surface. The merozoite then engages an acto-myosin motor complex, anchored in an inner membrane complex (IMC) that lies under the merozoite plasma membrane, to pull the erythrocyte membrane around itself; thus completing invasion4.Erythrocyte invasion is an essential and highly co-ordinated process that begins with reversible attachment of the merozoite to the erythrocyte surface6. The contents of micronemes are released first and are responsible for coordinating initial attachment events7. The rhoptries, which are the largest of the invasion organelles, adopt a dual club-shape with bulbs further towards the basal side of the merozoite and necks at the apical tip8. During invasion, rhoptry neck contents are released first and initiate early interactions with erythrocyte receptors9. Rhoptry bulb contents are secreted later in the invasion process and are typically involved in parasitophorous vacuole establishment10.The merozoite invasion organelles harbour essential antigens that are the targets of therapeutic interventions aimed at preventing blood-stage parasite replication11. Secretion of rhoptry contents may be partially coordinated through changes in the organelle’s structure during invasion. The membranes at the tip of the rhoptry neck fuse with the parasite membrane early in invasion and the two rhoptries themselves also fuse beginning at the neck11. At the point of erythrocyte entry, the rhoptries fully fuse into a single rhoptry (neck and bulb). The mechanisms leading to rhoptry fusion and secretion are not understood.It is unclear what differentiates the rhoptry bulb from the neck, as no membrane separates them. Because the neck and bulb remain differentiated after plasma membrane fusion, it has been hypothesised that the structure of the rhoptry may be supported by a scaffold, but the components of such a scaffold are yet to be elucidated8. Despite the rhoptries being membrane-bound organelles with dynamic functions during secretion of invasion ligands, there remains only a single protein reported to localise to the luminal face (Rhoptry associated membrane antigen12) and two to the cytosolic face  of the P. falciparum rhoptry membrane. Therefore, the drivers of rhoptry secretion and compartmentalisation are unknown. Here we describe a highly conserved P. falciparum protein, Pf3D7_0210600 ), which lies on the cytosolic face of the rhoptry bulb and is essential for rhoptry secretion and merozoite invasion.Approximately 30 proteins have been linked to the rhoptry18. PfCERLI1 is highly conserved among Plasmodium spp., with >90% identity between P. falciparum, P. reichenowi and P. gaboni orthologues, >75% amino acid identity across human infecting species (including the zoonotic P. knowlesi) and >70% when compared to distantly related species   but no transmembrane domains or glycophosphatidylinositol (GPI) anchor Fig. . The RNAies Fig. . To deteP. falciparum parasites with a C-terminal haemagglutinin tagged (HA) PfCERLI1 with control of protein expression achieved through a glucosamine (GLCN) inducible GlmS ribozyme knockdown system (PfCERLI1HAGlmS)20 20 Fig. . The resion Fig. ; cloned ion Fig. , concordGlmS ribozyme could control PfCERLI1 protein expression, we treated PfCERLI1HAGlmS parasites with 2.5 mM GLCN and quantified changes in HA-tagged protein levels using Western blot. Treatment of synchronous parasites with 2.5 mM GLCN for ~44 h from early ring stage led to a >80% reduction in PfCERLI1 expression, whereas expression of the loading control EXP2 was unaffected  of GLCN for 48 h and parasite growth assessed the following cycle. GLCN treatment led to dose-dependent growth inhibition, with 5 mM GLCN leading to an approximately 55% reduction in parasite growth  reporter plasmid (PfCERLI1HAGlmS/GFP) that allowed accurate quantitation of new merozoite invasion events by flow cytometry23. Since PfCERLI1 was expressed only at schizont stages, we limited the amount of time PfCERLI1HAGlms/GFP parasites were exposed to increasing concentrations of GLCN (0.125 to 5 mM) by only treating from early trophozoite stages (24 hpi) for 24 h before assessing parasite rupture and invasion. There was a dose-dependent inhibition of merozoite invasion   Fig. .p < 0.05)  Fig. . When thWe also assessed whether knockdown of PfCERLI1 protein expression could lead to reduced or inhibited growth in merozoites that managed to invade a new erythrocyte. We found that there was no difference between the number of early ring stages progressing through to late trophozoite stages (36 h post-invasion) between GLCN treated and untreated cultures, indicating that PfCERLI1 knockdown merozoites that do invade do not have reduced viability , inner membrane complex marker glideosome-associated protein 45 (GAP45), rhoptry neck marker rhoptry neck protein 4 (RON4) and rhoptry bulb marker rhoptry-associated protein 1 (RAP1) Fig. . Using ture Fig. . This flHA as detected by Western blot , treated with sodium carbonate  and Triton X-100 . The supernatants containing solubilised proteins with the different treatments, as well as the final Tx100 insoluble fraction  were used in Western blots , anti-RAP1 (rhoptry bulb), and anti-RON4 (rhoptry neck) antibodies prior to super-resolution microscopy. Enhanced spatial resolution of fully-formed merozoites revealed that RON4 is located closest to the apical tip of the merozoite , a Protein kinase G inhibitor that prevents parasitophorous vacuole membrane ruptureite Fig. , as expeulb Fig. 29, I-TASSER32, and COACH34. These analyses suggested that PfCERLI1 contains a C2 domain, which are known lipid binding regions (amino acids ∼50-170), a pleckstrin homology (PH) domain that typically binds calcium  from Arabidopsis thaliana, which has been shown to bind phospholipids in a calcium ion dependent manner38. The PH domain of PfCERLI1 was most structurally similar to the PH domain of Protein kinase B/Akt, and like Akt, was predicted to bind to inositol 1,3,4,5-tetrakisphosphate (IP4) , which commonly coordinates protein membrane localisation40. In addition, PfCERLI1 has previously been predicted to be palmitoylated41. Collectively, this suggests that PfCERLI1 contains potentially three different lipid-interacting moieties that may work cooperatively or independently to coordinate the binding of PfCERLI1 to the rhoptry bulb membrane.To investigate how PfCERLI1 may be binding to the surface of the rhoptry bulb and provide insights on protein function, we used protein structural prediction software Phyre242. The putative SP displayed on PlasmoDB43 is predicted using SignalP-3.044, however, the latest version of this program  no longer predicts that PfCERLI1 contains an SP  that lacked the N-terminal 26 amino acids. Pfcerli1NoSP was then cloned into the pArl1a vector, conjugating PfCERLI1NoSP to GFP and placing Pfcerli1NoSP under the merozoite-specific ama1 promoter  that all undergo proteolytic cleavage inside the parasite prior to invasion commencing , beginning as an 84 kDa precursor and being processed to a 67 kDa product that is contained in free merozoitesays Fig. , we notiays Fig. . No chanays Fig. . In addi18. In addition, PfCERLI1 was identified as a member of the ‘invadome’, an interaction network of putative invasion-associated proteins, and was localised to the merozoite apical tip in this study16. Here we show that PfCERLI1 localises to the rhoptries and is confined to the cytosolic face on the rhoptry bulb membrane, a localisation that would prevent exposure to antibodies during the natural course of invasion. Semi-quantitative super-resolution fluorescence microscopy showed a difference between the mean diameter of PfCERLI1 and the rhoptry bulb marker RAP1 around the bulb structure, indicating they occupy distinct compartments on/within the rhoptry. PfCERLI1 was sensitive to proteinase K after selective membrane solubilisation and RAP1, which appears to lie along the luminal face of the rhoptry bulb and is protected from cleavage, was insensitive, confirming that PfCERLI1 compartmentalises to the cytosolic face of the rhoptry bulb membrane. PfCERLI1 has been shown to be palmitoylated41, but was predominantly present in the carbonate fraction of the membrane solubility assay  members of the IMC53. C2 domains, as present in PfCERLI1, are predicted to bind calcium and known to form ionic bridges with lipid phosphates in membrane bilayers that are not easily disruptable54, providing a second potential membrane binding site for PfCERLI1. The presence of two distinct moieties that are predicted to interact with membranes may explain why PfCERLI1 partially solubilises in carbonate buffer while the remainder stays in the Tx100 insoluble fraction.Previous studies have identified PfCERLI1 (Pf3D7_0210600) as being a potential vaccine target on the basis of its predicted SP and late schizont stage of expression, a transcription profile typical of proteins involved in merozoite invasionsay Fig. , suggestPfcerli1 proved unsuccessful, confirming a previous random mutagenesis study that identified Pfcerli1 as essential55. However, we successfully integrated a GlmS-ribozyme sequence at the 3’ end of Pfcerli1 and were able to knockdown protein expression by >80% leading to >45% inhibition of parasite growth. While we were unable to achieve complete growth inhibition, it is possible that loss of PfCERLI1 function is compensated for by other proteins with similar function or that enough PfCERLI1 remains to enable invasion of some parasites. Indeed, bioinformatic searches identified a second protein, Pf3D7_0405200 , with similar structure and shared predicted epitopes that has previously been implicated in merozoite invasion56, providing a possible functional homologue that could compensate for loss of PfCERLI1.Our attempts to knock-out Knockout and knockdown studies implicate PfCERLI1 as having an important role in asexual stage parasite growth and we further confirmed that loss of this protein led to a defect in invasion. Initial investigation of the PfCERLI1 knockdown parasites showed that the number and morphology of merozoites was unaffected by loss of PfCERLI1 and parasites that did invade with PfCERLI1 knockdown developed normally. We determined that PfCERLI1 knockdown led directly to a significant reduction in newly invaded ring-stage parasites and a proportional build up in free merozoites in the culture, possibly through stopping invasion at a point prior to the formation of the tight junction and engagement of the acto-myosin motor. Quantitative assessment of immunofluorescence signal revealed a significant decrease in RAP1 staining intensity with PfCERLI1 knockdown, corresponding to increases in RAP1 volume and area, indicating knockdown of PfCERLI1 changes RAP1 distribution. While changes in area and volume for RAP1 were observed, the diameter of RAP1 rhoptry bulb staining and TEM analysis indicated that there were no large morphological changes in the rhoptry bulb itself. Thus, changes in the ultrastructure of the rhoptry with PfCERLI1 knockdown appear to be minimal and the strongest indication of a modified rhoptry shape achieved to date is increase prolate staining for RAP1, which may indicate a lengthening of the rhoptries that was not noticeable in the single sections of TEM images.57, disruption of rhoptry neck antigen secretion with PfCERLI1 knockdown points to a role for this protein in successful rhoptry antigen secretion during invasion. Furthermore, when PfCERLI1 was knocked down in free merozoites we saw a clear defect in the processing of the rhoptry bulb antigen RAP1. Given RAP1 processing occurs inside the rhoptry lumen, most likely after merozoite egress, and PfCERLI1 lies on the cytosolic face of the rhoptry bulb, we speculate that aberrant RAP1 processing is likely to be a consequence of general rhoptry dysfunction with PfCERLI1 knockdown.To assess whether the segregation of rhoptry antigens was affected by loss of PfCERLI1, we compared the colocalisation of RAP1 (bulb) and RON4 (neck). Knockdown of PfCERLI1 led to a significant decrease in the proportion of RAP1 overlapping with RON4. Using apical organelle secretion assays, we identified that knockdown of PfCERLI1 resulted in a decrease in secretion of the rhoptry antigens RH4 and RON4 but no change in secretion of the micronemal antigen EBA175. As antigens secreted from the rhoptry neck are essential for merozoite invasion18 reported functional analysis of a homologue of PfCERLI1, which they termed Rhoptry Apical Surface Protein 2 (RASP2), in the related Apicomplexan parasite Toxoplasma gondii. They identified that TgRASP2 localises to the cytosolic face of the rhoptry, is essential for tachyzoite invasion of the host cell and confirmed that the C2 domain of this protein binds to the rhoptry membrane through interactions with phospholipids. In the studies of T. gondii, TgRASP2 localised along the length of the tachyzoite rhoptries with foci at the rhoptry tip. Parallel studies in P. falciparum also identified an essential function for PfCERLI1 (PfRASP2) in invasion using a rapamycin inducible DiCre knockdown system. Although these two studies are highly complementary in the functional analysis of this protein, our quantitative analysis of the localisation of PfCERLI1 differs to that described by Suarez et al., with PfCERLI1 maintaining a clear rhoptry bulb localisation using both fluorescence and immuno-EM localisation in our study and a rhoptry tip localisation reported by Suarez et al. It is possible that PfCERLI1 and TgRASP2 have different localisations/functions, as while these organisms are related, they display markedly different host and cellular tropisms. However, further investigation will be required to tease out the specifics of these differences.A recent study by Suarez et al.,Taking all the data obtained together, we propose that PfCERLI1 has an important role in the function of the rhoptries during invasion, with PfCERLI1 knockdown leading to abnormal distribution, secretion and processing of rhoptry antigens that are likely to be essential for merozoite invasion. While further studies need to be undertaken to determine the fine detail of how PfCERLI1 knockdown causes these changes in rhoptry function, identification of PfCERLI1’s direct association with release of rhoptry antigens is a key step in understanding the complex molecular events that control rhoptry secretion during invasion. By understanding how rhoptry secretion is controlled and the key proteins involved, we can identify targets for drug development that will stop merozoites invading and prevent replication of disease-causing parasites. In addition, this study makes extensive use of semi-automated quantitative immunofluorescence microscopy and highlights how this powerful tool can be used to study the process of invasion.P. reichenowi (PRCDC_0209500), P. gaboni (PGAB01_0208200), P. malariae (PmUG01_04021700), P. ovale (PocGH01_04019500), P. knowlesi (PKNH_0410600), P. vivax (PVX_003980), P. berghei (PBANKA_0307500), P. yoelii (PY17X_0308100) and P. chabaudi (PCHAS_0309700) were obtained by searching within the PlasmoDB.org database43. Sequence similarities were determined using Geneious 9.1.3 (Biomatters) and performing multiple pairwise alignments using the global alignment with free end gaps alignment algorithm with the Blosum62 cost matrix.PfCERLI1 (Pf3D7_0210600) and orthologous sequences in P. falciparum  parasites were cultured in human O+ erythrocytes 58. Parasites were grown in RPMI-HEPES culture medium at pH 7.4 (Gibco) supplemented with 50 μM hypoxanthine, 25 mM NaHCO3, 20 μM gentamicin and either 0.5% Albumax II (Gibco) or 0.25% w/v Albumax II, 5% v/v human serum . Parasite cultures were maintained in an atmosphere of 1% O2, 4% CO2 and 95% N2 at 37 °C.HAGlmS riboswitch transfection vector was prepared from the PTEX150HAglmS vector59. The final 767 base pairs of the 3’ end of the Pfcerli1 genomic sequence (excluding the stop codon) was PCR amplified using the primers Pfcerli1 5’ F RBW (GGTAGATCTCATATCAAATTTGGTTCTTGAAG) and Pfcerli1 3’ R RBW (GGTCTGCAGCATCACTATAGTTGTACATATTTTTGC). The resulting PCR fragment was cloned into the PTEX150-HAglmS vector using the restriction enzyme cloning sites Bgl II and Pst I (restriction enzyme sites in bold). To generate the cytosolic GFP expressing 3D7 ΔPfcerli1HAGlmS/GFP line, the pHGBrHrBl-1/2 GFP plasmid22 was used, without modification. For PfCERLI1 disruption, a source vector (Pf3D7_1463000 SLI-TGD) was digested with Not I and Mlu I with a 5’ Pfcerli1 flank being amplified using the primers (restriction enzyme sites in bold) Pfcerli1 SLI-TGD F (GGTGCGGCCGCGATACTCACAACATATTATATCTTGG) and Pfcerli1 SLI-TGD R (GGTACGCGTCATACCTCTATGTGTACTTTGTTCTG)19. To construct the PfCERLI1 signal peptide deletion plasmid, Pfcerli1 lacking the signal peptide was amplified using the forward primer Pfcerli1 No SP F (GGTGGTACCATGAGAAACCGTGAGTTATTTC) and the reverse primer Pfcerli1 R (GGTCCTAGGATCACTATAGTTGTACATATTTTTGC). The resulting PCR product was restriction digested using KpnI and AvrII and ligated into the pArl1a plasmid, containing the ama1 promoter60, to generate the Pfcerli1NoSP pArl1a plasmid.The PfCERLI1P. falciparum 3D7 parasites were transfected using erythrocyte loading61. Briefly, uninfected erythrocytes were centrifuged at 1500 rcf for 1 min, before removal of the supernatant and washing in cytomix . Erythrocytes were then resuspended in cytomix containing 200 μg of ethanol precipitated plasmid DNA, incubated in a 0.2 cm cuvette (Bio-Rad) on ice for 30 min and then electroporated (Bio-Rad) at 0.31 kV with a capacitance of 960 μF. Cells were washed 2× with culture media and transferred to a 10 mL dish containing magnet purified schizont stage parasites (3–5% parasitaemia). Integration of the HA-GlmS plasmid, or presence of the Pfcerli1NoSP pArl1a plasmid, was selected for using 3 cycles of 5 nM WR99210  drug treatment. For generation of the ΔPfcerli1HAGlmS/GFP line, PfCERLI1HAGlmS parasites were transfected with the an episomal cytosolic GFP expressing plasmid pHGBrHrBl-1/2 and maintenance of this plasmid was selected for using 5 μg/mL blasticidin-S-deaminase HCl (Merck Millipore). Maintenance of the SLI-TGD plasmid was selected for using 5 nM WR99210, with successful integrants then selected for using 400 μg/mL G418 sulphate .Pfcerli1HAGlmS into the parasite genome, the Pfcerli1 locus was amplified with the forward primer Pfcerli1 5’ F RBW (GGTAGATCTCATATCAAATTTGGTTCTTGAAG) and either the Pfcerli1 3’ R RBW (GGTCTGCAGCATCACTATAGTTGTACATATTTTTGC) reverse primer (amplifies wildtype gDNA sequence) or the Glms R (GAAATCCTTACGGCTGTGATCTG) reverse primer (amplifies DNA only upon Pfcerli1HAGlmS integration).To extract parasite gDNA and confirm whether transfected constructs were stably integrated, schizont stage parasite cultures were saponin lysed. gDNA was extracted from saponin lysed parasites using the Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer’s protocol. To confirm integration of To determine the number of merozoites produced by each fully formed schizont after knockdown of PfCERLI1 protein expression, thin blood smears were made of D-(+)-Glucosamine hydrochloride (Sigma-Aldrich) (GLCN) treated and untreated schizont stage cultures matured in the presence of E64 (Sigma-Aldrich) to prevent schizont rupture. Smears were methanol fixed and stained with Giemsa (Merck Millipore) before blinded assessment of the number of merozoites by light microscopy . All parasites assessed were fully matured, with individual segmented merozoites visible.Pfcerli1HAGlmS (growth) and 3D7 ΔPfcerli1HAGlmS/GFP (invasion) parasites were synchronised to ring stages using sorbitol lysis and assays were set up in 96-well U-bottom plates at 1% parasitaemia and 1% haematocrit in a volume of 45 μL62. Five microlitre of 10× concentration GLCN or complete media was added to make a final volume of 50 μL. Assays were stained with 10 μg/mL ethidium bromide (Bio-Rad) in PBS before assessment of parasitaemia using flow-cytometry . To assess merozoite development and invasion of 3D7 ΔPfcerli1HAGlmS/GFP parasites, GLCN treated and untreated cultures were grown for 36 h, until newly invaded rings were present (0–6 h post-invasion). Flow cytometry data was analysed using FlowJo (Tree Star). The gating strategy used to identify free merozoites and ring-stage parasites is detailed in Supplementary Fig. To assess the impact of PfCERLI1 knockdown, 3D7 ΔHAGlmS parasites were synchronised to ring-stages and set up into duplicate 96-well U-bottom plates, either in the presence or absence of GLCN, as described for growth and invasion assays. Using flow cytometry, the schizont-stage parasitaemia of both treatments was determined at an estimated pre-rupture timepoint for the first plates. After a further 6 h of incubation, the schizont-stage parasitaemia of the second (post-rupture) of the duplicate plates was determined. To determine the percentage of schizont rupture that occurred over the 6-h of incubation the following equation was used:To determine whether knockdown of PfCERLI1 altered the ability of schizonts to rupture, PfCERLI163), rabbit anti-GAP45 , mouse anti-RH5 (1:5000), mouse anti-RAP1 7H8 , rabbit anti-RON4 (1:5000), rabbit anti-EBA175 (1:500065), mouse anti-RH4 2E8 , rabbit anti-ERC (1:10000 67)) and secondary , IRDye ® 680RD goat anti-rabbit ) antibodies were incubated with membranes for 1 h at room temperature (prepared in Odyssey Blocking Buffer (TBS)), before washing in 0.05% v/v PBS Tween followed by a PBS wash after the secondary antibody incubation. Western blots were visualised using an Odyssey Infrared imaging system (LI-COR Biosciences). Western blot quantification was performed using Image Studio Lite 5.2.5 (LI-COR Biosciences). Uncropped images of all western blots can be found in Supplementary Figs. For protein samples, ~10 mL of high parasitaemia culture was lysed with 0.15% w/v saponin for 10 min on ice, parasite material was pelleted by centrifugation before washing once in 0.075% w/v saponin and three times in PBS. In the presence of protease inhibitors . Parasite lysates were resuspended in reducing sample buffer , 0.002% w/v bromophenol blue (Sigma-Aldrich)) and separated by size using SDS-PAGE 4-12% Bis-Tris Gels  at 130 V for 60 min. Proteins were then transferred to a nitrocellulose membrane  at 20 V for 7 min, before blocking the membrane for 1 h at room temperature in Odyssey Blocking Buffer (TBS) (LI-COR Biosciences). Primary , rabbit anti-EXP2  alone. A second tube with SOTE plus 0.02% w/v digitonin (Sigma-Aldrich) incubated for 10 min at 4 °C prior to washing in SOTE buffer. A third tube with digitonin treatment followed by digestion with 0.1 μg/μL Proteinase K (Sigma-Aldrich) in SOTE for 30 min at 4 °C. Proteinase K was inactivated by adding 50 μL of 100% v/v trichloroacetic acid followed by a PBS wash. All tubes were then resuspended in 500 μL acetone before cells were pelleted, the supernatant removed, and the pellet used for Western blot analysis of proteinase K sensitivity.For proteinase protection assays13, Saponin-lysed pellets from 10 mL of high-parasitaemia E64 treated schizonts were resuspended in 100 μL dH2O, snap-frozen at −80 °C four times, passed through a 26-gauge needle 5 times to disrupt the parasite membrane and then centrifuged at 15,000 rcf for 10 min with the water-soluble supernatant reserved. The pellet was washed twice in dH2O and once in 1× PBS before resuspension in 0.1 M sodium carbonate (Na2CO3) for 30 min at 4 °C with the supernatant reserved. Following carbonate treatment, samples were resuspended in 0.1% v/v Triton-X-100 for 30 min at 4 °C with the supernatant reserved and the resulting pellet washed and resuspended in 1× PBS. Samples were analysed by Western blotting.For protein solubility assaysHAGlmS late-stage schizonts were concentrated by centrifugation at 1700 rpm for 3 min, washed in PBS and fixed with 4% v/v paraformaledehyde , 0.0075% v/v glutaraldehyde  solution for 30 min at room temperature with gentle shaking. Fixed parasite suspensions were adjusted to 1% haematocrit prior to adhering onto 0.01% poly-L-lysine (Sigma-Aldrich) coated #1.5 H high-precision coverslips  for 1 h at room temperature before permeabilistion with 0.1% v/v Triton-X-100 for 10 min. Coverslips were incubated with fresh blocking solution  for at least 1 h. Primary antibodies (Chicken anti-HA (Abcam), mouse anti-RAP149, mouse anti-CyRPA 8A768, rabbit anti-RON469, rabbit anti-GAP4564) were diluted 1:500 in antibody diluent  and added to coverslips for 1 h at room temperature or overnight at 4 °C. Cells were washed three times with PBS-Tween-20 (0.1% v/v) and incubated with goat anti-chicken/mouse/rabbit Alexa Fluor coupled secondary antibodies  (Life Technologies) diluted 1:500 for 1 h at room temperature. After secondary antibody incubations, coverslips were washed three times with PBS-Tween-20 (0.1% v/v) then post-fixed with 4% w/v PFA for 5 min. PFA was washed off and coverslips dehydrated in ethanol at 70% v/v (3 min), 95% v/v (2 min) and 100% v/v (2 min) before being allowed to air dry and mounted on slides with ProLong® Gold antifade solution (refractive index 1.4) containing 4’, 6-diamidino-2phenylindole, dihydrochloride (DAPI) (ThermoFisher Scientific). Once the mountant had cured for 24 h, cells were visualised by either conventional confocal (Olympus FV3000) or Zeiss LSM 800 Airyscan super-resolution microscopy .Untreated and GLCN treated cultures (approximately 10 mL of 3% parasiteamia) of Compound 1 arrested PfCERLI1Conventional confocal microscopy was performed on Olympus FV3000 fluorescent microscope (Olympus) equipped with a ×100 MPLAPON oil objective (NA 1.4) using the 405 nm, 488 nm, 561 nm, and 633 nm lasers. Z-stacks were acquired with a step size of 0.43 μm using a sequential scan .TM Fluorescent Microspheres (Life Technologies) were mounted on #1.5H coverslips  with a density of ~2.3 × 1010 particles/ml and used as the 200 nm bead calibration sample to correct for chromatic and spherical aberrations. The super-resolution reconstructions of multi-labelled PfCERLI1GlmSHA schizonts were acquired, sequentially in four channels, as follows: channel 1 = 633 nm laser, channel 2 = 561 nm laser, channel 3 = 488 nm laser, channel 4 = 405 nm laser, with exposure times optimised from positive and negative control samples  to avoid saturation. Three-dimensional (3D) Z-stacks were acquired at a pixel resolution of 0.04 μm in XY and 0.16 μm intervals in Z using piezo drive prior to being Airyscan processed in 3D using batch mode in ZEN Black (Zeiss).Sub-diffraction microscopy was performed on a Zeiss LSM800 AxioObserver Z1 microscopy  fitted with an Airyscan detector and a Plan-Apochromat ×63 (NA 1.4) M27 oil objective. TetraSpeckHAGlmS labelled structures. Fluorescence curves between the two antipodal points on the surface of the ring-like organelle were exported as.csv Excel files and plotted against the distance in nanometres. Only rhoptries with homogeneously stained ring-like structures containing no gaps in RAP1 or PfCERLI1HAGlmS labelling and >100 nm in size were used for diameter measurements. Schizonts densely packed with merozoites with overlapping rhoptries were excluded from scoring. More than 800 individual organelles were scored and data from biological replicates plotted in Prism for comparison of diameters between untreated and glucosamine treated parasites.For further spatial exploration of the colocalised signals, 3D volumes captured with super-resolution microscopy after Airyscan processing were imported into ZEN Blue (Zeiss) for object-based colocalisation analysis. In this approach, volumetric colocalisation relies on manual identification of structures of interest and a subsequent measurement of their fluorescence intensity curves. Each rhoptry bulb immunolabelled with anti-RAP1 and anti-HA antibodies was manually quantified by a researcher blinded to experimental conditions, and the organelle diameter measured by drawing a vector through the centre of these structures and plotting the fluorescence intensities for the green and red channel against the length of the vector. Fluorescence intensity profiles of overlapping subcellular structures were then analysed in successive single sections from an image stack representing the two RAP1 and PfCERLI1Schizont stage parasites were Percoll purified and fixed in 1% glutaraldehyde in RPMI-HEPES buffer. Samples were dehydrated in increasing concentrations of ethanol and embedded in LRGold resin. Ultrathin sections were cut and then labelled with anti-HA antibody  and then with 18 nm Colloidal Gold . Sections were then post-stained with uranyl acetate and lead citrate and viewed using a Talos L120C Transmission Electron Microscope.70. Briefly, synchronous ring-stage cultures were obtained using sorbitol and either treated with 2.5 mM GLCN or left untreated and incubated in standard culture conditions for 24 h until early trophozoite stage. Cultures were then enzyme treated with neuraminidase (0.067 U/mL), chymotrypsin (1 mg/mL) and trypsin (1 mg/mL). Parasites were then allowed to rupture over the following 24 h. Following rupture, cultures were centrifuged at 13,000 rcf for 10 min in a benchtop centrifuge with some culture supernatant reserved, stored on ice, and the rest removed. The remaining pellet was then also placed on ice and saponin lysed to form a lysate containing the parasites that did not rupture and free merozoites, with this pellet and the culture supernatant analysed by Western blot.Apical organelle secretion assays were modified from a previous protocolGraphs and statistical analyses were performed using GraphPad PRISM 7 (GraphPad Software Inc.). In all figures where p-values were calculated, the corresponding statistical test is listed in the figure legend along with the number of experiments results are pooled from.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Hypothyroxinemia of prematurity increases the rate of false-positive results in total thyroxine (tT4)-based screening programs for congenital hypothyroidism. The use of specific cutoff values for preterm infants has been proposed, but data on tT4 reference ranges in this population are limited. The primary aim was to establish reference percentiles for tT4 in dried blood spots among Mexican preterm infants. Secondary aims included a comparison of the change of tT4 concentrations over time according to gestational age and to discuss its impact on tT4-based screening programs. This was a retrospective cohort study; 1561 preterm infants were included. Percentile 10th for tT4 concentration at 24–27, 28–30, 31–34, and 35–36 weeks of gestational age, measured in the first week of life was: 47.6, 56.6, 82.3, and 117.1 nmol/L, respectively. tT4 concentrations were compared in three different time points: first week of life, 2–3 weeks of life, and term-corrected gestational age (38 weeks of gestation), progressively increased in infants below 30 weeks, remained stable in infants from 31 to 34 weeks, and decreased in late preterm newborns (35–36 weeks). This study suggests that preterm infants may require the use of lower tT4 cutoff values in newborn screening. Newborn screening (NS) for congenital hypothyroidism (CH) is one of the most useful tools for preventing severe mental retardation . Most NSCentral congenital hypothyroidism (CCH) is less common than PCH, with a reported incidence of 1:16,000 to 1:21,600 ,5. This One of the principal disadvantages of tT4-based programs is the high false-positive rate, caused mainly by hypothyroxinemia of prematurity and thyroxine-binding globulin (TBG) deficiency . It is wThere is controversy around the world over which is the best newborn screening strategy for CH in preterm infants. Several studies have shown that serial TSH testing improves detection of CH ,9, but tUsing the same tT4 cutoffs for preterm and term neonates increases the recall rate in NS in preterm infants and could lead to misdiagnosis or overdiagnosis of CH . Data onThis is a retrospective cohort study performed in a tertiary care hospital in Mexico City. All preterm infants born before 37 weeks of gestation from January 2018 to December 2019 were included. The following infants were excluded: infants whose first NS was done before 24 h, infants diagnosed with primary or central CH, neonatal hyperthyroidism or persistent hyperthyrotropinemia, and infants who did not complete the institutional screening strategy or in whom confirmatory tests to rule out CH could not be performed for any reason. The study was performed according to the principles of the Declaration of Helsinki and was approved by the Ethic and Research Committees of the Instituto Nacional de Perinatología Isidro Espinoza de los Reyes; register number 2020-1-12, 7 May 2020.All newborn infants were screened for CH according to the institutional NS program, serial testing of simultaneous tT4 and TSH were conducted. The first specimen was collected between two and five days of life, the second specimen was collected in all preterm infants between the 2nd and 3rd weeks of life (2 weeks after the first screening) and the third and last specimen was collected at term-corrected gestational age (38 weeks of gestation). This last specimen was obtained only in preterm infants born before 34 weeks of gestation. The institutional screening program cutoff value for TSH is 5 mIU/L, lower than the national cutoff of 10 mIU/L , and thetT4 concentration was measured by solid-phase, time-resolved fluoroimmunoassay from dried blood spots (DBS) by using Perkin Elmer’s AutoDELFIA platform . Intra and interassay coefficients of variation (CV) were 5.9 and 7.9%, respectively, for a tT4 of 13.5 μg/dL, with a functional sensitivity of 1.5 μg/dL. The TSH and tT4 measurements were then converted to estimated serum concentration with the following conversion factor: a T4 blood level of 0.5 μg/dL = a serum level of 1 μg/dL, a TSH blood concentration of 1 mIU/mL = a serum concentration of 2 mIU/L according to the manufacturer’s recommendations (assuming 50–55% hematocrit).The samples were taken in the following settings: Neonatal Intensive Care Unit , Neonatal Intermediate Care Unit , Neonatal Minimal Care Unit , and obstetrics wards or ambulatory .Infant’s demographics, NS results, and confirmatory test results were obtained from the electronic medical record of each infant. All serial tT4 values of the study cohort were included in the analysis. tT4 values obtained between days 2 and 7 of life were recorded as tT4 values at first screening (week 1), tT4 values obtained between days 7 and 21 as tT4 values at second screening (week 2–3), and tT4 values obtained after these time periods as tT4 values at third screening . Collected specimens from other time points were excluded. Reference percentiles for preterm infants were presented in 4 groups according to differences in their thyroid hormone physiology ; Group 1Results were expressed either as means and standard deviations (SD) for continual variables or median and interquartile ranges when the distribution was not normal; the numerical count and the percentage for categorical variables were used. We studied the normal distribution of tT4 concentrations in the four study groups and found non-normal distribution in the following gestational age groups . tT4 percentiles were reported for the 4 gestational age groups in three-time points . The following percentiles were calculated: 5th, 10th, 25th, 50th, 75th, 90th, and 95th.® SPSS® Statistics software .We compared tT4 and TSH concentration between gestational age groups in the three different screening time points with the Kruskal–Wallis test. To search for a linear trend during the 3 screening times for each gestational age group we performed a Friedman test as the non-parametric equivalent of the repeated measures ANOVA test; for Group 4 (35–36 weeks) we performed a Wilcoxon signed-rank test, as this group only had 2 screening times. All tests were considered significant at an alpha level of 0.05. All statistical analysis was performed using the IBM5969 infants were screened at the institute between January 2018 and December 2019, of which 1614 (27%) were born before 37 weeks. Screening tests were positive in 81 preterm infants , 40 had TSH above the institutional cutoff in any of the three serial samples, 39 had tT4 values in their first and second screening below the 5th percentile, and two infants screened positive for both parameters, tT4 and TSH. The recall rate for a positive tT4 screening  was inversely proportional to the gestational age, being 14% in Group 1 (24–27 weeks), 11.6% in Group 2 (28–30 weeks), 3.3% in Group 3 (31–34 weeks), and only 0.2% in Group 4 (35–36 weeks).Eight cases of congenital hypothyroidism were confirmed during the study period, four PCH, two cases of CCH  and two with transient CCH associated with maternal Graves’ disease. The false-positive rate was 4.5%, the positive predictive value was 9.8% and the total incidence of CH in our population was 1 in 269 preterm births .The final cohort included 1561 preterm infants after excluding 53 infants on the basis of exclusion criteria: 2 infants whose first NBS was done before 24 h, 11 infants diagnosed with primary or central CH, neonatal hyperthyroidism or persistent hyperthyrotropinemia, and 40 infants without complete screening strategies or without confirmatory tests to rule out CH . 1549 (99.2%) of infants completed the first screening, 1430 (91.6%) completed the second screening, and from those who were born before 34 weeks of gestation, only 571 (81.3%) completed the third screening due to being lost to follow-up. Samples were obtained in the following settings: First screening, 15.5% in NICU, 32.2% in ICU, 23.6% in MCU, and 28.7% in obstetrics wards or ambulatory; Second screening, 11.7% in NICU, 31.6% in ICU, 2.2% in MCU, and 54.5% in obstetrics wards or ambulatory; Third screening, 5.4% in NICU, 40.5% in ICU, 1.7% in MCU, and 52.4% in obstetrics wards or ambulatory.p < 0.001. At second screening, Group 1 = 104.2 (90.1–124.8) nmol/L, Group 2 = 114.5 (95.2–139) nmol/L, Group 3 = 126.1 (108.1–150.6) nmol/L, and Group 4 = 130 (110.6–154.4) nmol/L; p < 0.001. In the third screening , tT4 concentration was similar in all gestational age groups: Group 1 = 127.4 (108.1–162.1) nmol/L, Group 2 = 133.8 (110.6–158.3) nmol/L and Group 3, = 130 (105.5–160.9) μg/dL; p = 0.76; tT4 concentration was higher at higher gestational age in first and second screening, tT4 medians (IQR) in first screening were as follows: Group 1 (24–27 weeks) = 75.9 (61.7–90.1) nmol/L, Group 2 (28–30 weeks) = 87.5 (73.3–113.2) nmol/L; Group 3 (31–34 weeks) = 132.5 (106.8–160.9) nmol/L, and Group 4 (35–36 weeks) = 164.7 (137.1–194.3) nmol/L; p < 0.001, and Group 2, infants born at 28 to 30 weeks, p < 0.001. However, in Group 3, infants born at 31 to 34 weeks, tT4 concentration was similar in the three screening times, p = 0.861. In Group 4, 35 to 36 weeks, tT4 concentration decreased in the second screening compared to the first screening, p < 0.001; tT4 concentrations progressively increased in the three screening time points in Group 1, infants born at 24 to 27 weeks, p < 0.001; Group 2 (28–30 weeks), first screening = 1.8 mIU/L, second screening = 2.8 mIU/L, third screening = 3.2 mIU/L, p < 0.001; Group 3 (31–34 weeks), first screening = 2 mIU/mL, second screening = 2.2 mIU/mL, third screening = 2.4 mIU/mL, p < 0.001; Group 4 (35–36 weeks), first screening = 2 mUI/L, second screening = 2.2 mIU/L, p = 0.086. TSH concentration progressively increased in the three screening time points in Group 1 to 3, in Group 4, TSH concentration was similar in the two screening time points. The medians of TSH were as follows: Group 1 (24–27 weeks), first screening = 1.2 mIU/L, second screening = 2.6 mIU/L, third screening = 3.2 mIU/L, This study allowed us to obtain tT4 reference ranges in DBS for Mexican preterm infants in three different time points ; to our knowledge, this is the first study in which serial tT4 values in DBS are described in preterm newborns.Few studies ,16,17,18In agreement with previous studies, we found that tT4 concentration increases at higher gestational ages ,14,19, hRecently, Hijman et al.  determinThis study showed that in preterm infants below 30 weeks, tT4 concentration increased progressively; in preterm infants from 31 to 34 weeks, tT4 concentrations were stable at three screening timepoints; and finally, in late preterm newborns (35–36 weeks) tT4 concentration decreased at the second screening (2 to 3 weeks of age) timepoint compared to the first screening (first week of life) timepoint. These results differ from those reported by Williams et al. , who fouWhen we analyzed the change over time in TSH concentrations in each gestational age group, we found that T4 medians increased progressively in premature infants born before 34 weeks and remained stable in infants born at 35 to 36 weeks. Although it is well known that term infants had a postnatal TSH surge in the first 24 h of life and that TSH concentrations then gradually decrease over time, it has been reported that this postnatal TSH surge may be abolished or attenuated in premature infants , which mPreterm infants represent a challenge for NS programs as they present higher false-positive and false-negative test results compared with term newborns, this has been attributed to factors such as the immaturity of the hypothalamic-pituitary-thyroid axis, reduced capacity for synthesis of thyroid hormones, presence of concomitant critical diseases, and the use of certain drugs  ,21. HypoThere is worldwide controversy about which is the best screening strategy for CH, especially in preterm infants. Serial TSH testing has shown to improve detection of CH, however, this strategy is not capable of detecting CCH ,21. KorzFew studies have evaluated the effectiveness of a primary tT4-based strategy specifically in preterm infants, a recent study conducted in the USA showed that a tT4 screening strategy consisting of two serial tT4 samples, first at 24 h of life and second at 10–14 days (cutoff of tT4 <10th percentile in the first sample a and tT4 <0.5th percentile and/or TSH >30 mIU/mL in the second sample), showed poor efficacy in preterm infants with a birth weight lower than 1500 g hospitalized in an NICU unit. In this cohort, almost 40% of infants <1500 g had a positive screening test with a calculated sensitivity of 62.5%, a specificity of 79.1%, and a positive predictive value of only 8.1% [Most NS programs with a simultaneous tT4 and TSH or tT4-based strategy uses the 5th or 10th percentile as the tT4 cutoff point ,25,26. SIn our cohort study, if we use tT4 cutoff from our total population of term and preterm infants in their first week of life, more than 70% of preterm infants of 24 to 27 weeks had a tT4 concentration below the 5th percentile (90 nmol/L in serum) and 85% below the 10th percentile (108 nmol/L in serum); among infants between 28 and 30 weeks, more than 50% had a tT4 concentration below the 5th percentile and more than 70% below the 10th percentile. To minimize recall and false-positive rates, our program has decided to request confirmatory thyroid profiles only in infants with two serial tT4 concentrations below the 5th percentile, even so, our recall rate in preterm infants continues to be high. It is also important to highlight that this strategy has allowed us to detect some cases of CCH in a timely manner (2 cases in the study period).Including tT4 in NS programs increases the operating costs of them, however, the benefit of CCH detection outweighs these costs, as countries with a screening strategy capable of detecting CCH have shown a higher prevalence of this condition than previously reported ,5,6, conn = 36), as the frequency of births at this gestational age is not so high. Furthermore, in almost 20% of infants below 34 weeks, the third screening sample was not available due to loss to follow-up, many of these lost infants were discharged before reaching this gestational age and they did not come back for the last screening. It is possible that infants who were in a more critical situation and remained hospitalized were more likely to be included at this time point and this could decrease the tT4 concentrations reported. We decided to include sick neonates (as those requiring ventilatory support or pressor agents) because we wanted the reference ranges to reflect the real situation of these preterm infants.Limitations of this study include its retrospective design, although the data were collected prospectively by the institutional NS program. Another limitation is that we include a few tT4 measurements of infants of 24–27 gestational weeks , and tT4 values were obtained directly in DBS, not in stored DBS cards or serum samples, as one of our objectives was to obtain reference ranges for tT4 in preterm infants that could be useful to define screening cutoffs in the future.To establish recommendations on which are the best cutoff points, multi-center studies with a higher number of preterm infants are required, as well as studies that directly compare the sensitivity and specificity of different cutoff points.The results of this study suggest that preterm infants may require the use of lower DBS tT4 cutoff values according to gestational age at birth. tT4 concentrations increase progressively in infants below 30 weeks, remained stable in infants from 31 to 34 weeks, and decrease in late preterm newborns (35–36 weeks). To improve the effectiveness of screening strategies in preterm infants, each country should design their own protocols to define which tT4 cutoff values best optimize sensibility and specificity considering their health care system resources.