Chronic intervillositis of unknown etiology (CIUE) is a rare, poorly understood, histopathological diagnosis of the placenta that is frequently accompanied by adverse pregnancy outcomes including miscarriage, fetal growth restriction, and intrauterine fetal death. CIUE is thought to have an immunologically driven pathophysiology and may be related to human leukocyte antigen mismatches between the mother and the fetus. Dizygotic twins with one-sided CIUE provide an interesting context to study the influence of immunogenetic differences in such cases. The main immune-cell subsets were investigated using immunohistochemistry. We identified three dizygotic twin pregnancies in which CIUE was present in only one of the two placentas. Two of the pregnancies ended in term delivery and one ended in preterm delivery. Presence of CIUE was correlated with lower placental weight and lower birthweight. Relative number of CD68, CD56, CD20, and CD3 positive cells were comparable between co-twins. The presence of one-sided CIUE in dizygotic twin pregnancy was associated with selective growth restriction in the affected twin. This suggests a unique fetal immunogenetic contribution to the pathogenesis of CIUE. Further study of dizygotic and monozygotic placentas affected by CIUE could identify new insights into its pathophysiology and into the field of reproductive immunology. Chronic intervillositis of unknown etiology (CIUE) is an uncommon histopathological lesion first described by Labarrere and Mullen in 1987 as massive chronic intervillositis . It has The pathophysiology of CIUE is poorly understood, but it appears to be immunologically driven ,5. FactoThe phenomenon of dizygotic twins with one-sided CIUE, which occurs despite an identical intrauterine environment, suggests a significant contribution from fetal immunogenetics: the mother mounts an immune response against paternally inherited antigens present in only one of the two placentas. This series describes three cases of dizygotic twins with one-sided CIUE, reporting clinical outcomes, associated placental abnormalities and immunohistochemical findings.Clinical characteristics of the dizygotic twin pregnancies are described in Placental characteristics are described in The number of cells positive for CD3, CD20, CD56, and CD68 in the intervillous space was quantified A,B. MostThe fetuses with CIUE in their placenta had a lower birthweight compared to the fetuses without CIUE. In Case 1, the fetus with CIUE was admitted to NMCU; in Case 3, the fetus with CIUE in the placenta was admitted to NICU.This case series describes three dizygotic twin pregnancies in which the placentas were one-sidedly affected by CIUE, the first published report of this phenomenon. Overall, FGR is more common in twin pregnancies than in singleton pregnancies . DiscordOne-sided CIUE in the placenta of a dizygotic twin could suggest that CIUE is driven by maternal alloimmune sensitization against a particular fetoplacental histocompatibility antigen which differs between the twins. This is further supported by the presence of CIUE in cases of fetal and neonatal alloimmune thrombocytopenia, which is characterized by maternal allo-antibodies directed against paternally inherited antigens on the fetal platelets ,21. The One obvious limitation of this series is that it only describes three cases. However, given the rarity of CIUE, which affects approximately 1 in 10,000 pregnancies , and thePatient samples were selected from the pathology department of the University Medical Center Utrecht (UMCU) between 2000 and 2015 using the hospital’s pathology registry. During this period, approximately 13,300 placentas were studied, of which 1059 were dichorionic twin placentas. In total, 45 placentas with an intervillous infiltrate were identified, of which three were dizygotic dichorionic twin placentas (see: ). SlidesTerm delivery was defined as birth from 37 completed weeks of gestation onward, and preterm delivery was defined as birth between 24 and 37 completed weeks of gestation. Fetal growth restriction (FGR) was defined as a birthweight below the third percentile. Discordant growth was defined as a birthweight discrepancy of >25%.2O2 0.12%. Prior to incubation with the primary antibody, the slides were incubated for 1 h at room temperature with normal goat serum. The mouse monoclonal anti-CD20 (cy) antibody  was incubated for 1 h at room temperature. Binding of the primary antibody was visualized using a PO-labelled goat-anti-mouse polymer  and diaminobenzidine as a chromogen. Hematoxylin was used for counterstaining before slides were dehydrated and covered using mounting medium.Tissue samples were taken from the umbilical cord, fetal membranes, and placental parenchyma according to the Amsterdam Placental Consensus Statement . Formali2 were made per slide. Two annotations were placed subchorial, two were placed central, and two were placed basal (The number of cells positive for CD3 (T cells), CD20 (B cells), CD56 (NK cells), and CD68 (macrophages) was quantified using the Philips IMS viewer system. The stained slides were aligned and annotations of approximately 0.75 mmed basal . The numed basal . In a toThe presence of one-sided CIUE in dizygotic twin pregnancy suggests that CIUE is related to immunological incompatibility between the mother and the affected fetus. Studying one-sided histopathological changes in dizygotic twins and monozygotic twins could reveal novel insights into the pathophysiology of CIUE and other alloimmune gestational diseases.


The present study examined the etiology of childhood diarrhea and its comorbidity with malaria in a rural area of Burkina Faso.conventional culture techniques, direct stools examination, and viruses´ detection by rapid tests were performed on the fresh stools and microscopy was used to diagnose malaria. Some risk factors were also assessed.on a total of 191 samples collected, at least one pathogen was identified in 89 cases (46.6%). The proportions of pathogens found on the 89 positive stool samples were parasites 51.69% (46 cases), viruses 39.33% (35 cases), and bacteria 14.61% (13 cases), respectively. The relationship between malaria and infectious diarrhea was significant in viral and parasites causes (p=0.005 and 0.043 respectively). Fever, vomiting and abdominal pain were the major symptoms associated with diarrhea, with 71.51%, 31.72% and 23.66% respectively. The highest viral diarrhea prevalence was reported during the dry season  while parasite diarrhea was more encountered during the rainy season .Giardia spp and rotavirus were the leading cause of acute diarrhea in Nanoro, Burkina Faso with a predominance of rotavirus in children less than 2 years. Parasite and viral diarrhea were the most pathogens associated with malaria. However, the high rate of negative stool samples suggests the need to determine other enteric microorganisms. Diarrheal disease remains a worldwide public health problem and represents the second leading cause of death in children under five years old. Indeed, diarrhea kills around 760 000 children under five yearly worldwide, with the majority of cases in sub-Saharan Africa , 2. StudIn Burkina Faso, few studies have been conducted and consistent data are still scarce. In addition, most of the available data are from studies carried out in urban or semi-urban areas, therefore the results cannot be applied in rural areas , 11-13. Study setting, design and procedures: a cross-sectional descriptive study was conducted from March 2012 to March 2014 in the rural health district of Nanoro to investigate the etiologies of diarrhea in children aged below five years old, and their coinfection with malaria. The study was carried out in two health centers located in the Nanoro Health and Demographic Surveillance System (HDSS) catchment area. The Nanoro health district had around 150,000 inhabitants with 22.9% of children under five years living in an area of highly seasonal malaria transmission which overlaps with the rainy season [y season . All chiLaboratory procedures: all samples collected were proceeded within 1 hour. Thick and thin blood films were Giemsa stained and examined for detection of Plasmodium species according to standard procedures [Shigella spp. and Salmonella spp. isolates were serotyped using specific antisera from BioRad, France, following the Kauffmann-White scheme [ocedures . The resocedures . Shigelle scheme . All theStatistical methods and definitions: data were double entered, validated and cleaned using Epidata version 3.1. Statistical analysis was performed using STATA Version 10 . Diarrhea was categorized as follow: acute diarrhea (lasting less than 14 days), dysentery (presence of blood or mucus), and persistent diarrhea (more than 14 days) [14 days) . Fever wGiardia spwith 45.65% of cases in mono-infection, followed by Entamoeba coli and Hymenolepis nana. Among the viruses detected, rotavirus represented one-third of the infections while the proportion of adenovirus was less than 10%. The most frequent isolated bacteria were Shigella spp. and the enteropathogenic Escherichia coli (EPEC) in 8% and 6% respectively. Fever (71.51%) was the first reason of health facility attendance followed by vomiting (31.7%) and abdominal pain (23.7%)  .Plasmodium falciparum. The relationship between malaria and infectious diarrhea was statistically significant in viral and parasites causes (P = 0.005 and 0.043 respectively). A seasonal pattern of the distribution of associated pathogens was seen with parasite infection which was more associated with rainy season and the virus infection which was associated with the cold and dry season,  (The search of association between diarrhea and the described factors showed that very younger age (< 2 years) was strongly associated with viral diarrhea  while children above 2 years old were more infected with parasites than virus . Bacterial infection was not associated with patient age . Malariactively) . In oppoGiardia spamong our study samples supports the hypothesis of general poor hygienic conditions in rural areas [The objective of the present study was to determine the prevalence of infectious acute diarrhea and its associated pathogens among children under five years old in rural Burkina Faso. The results showed that parasitic infection was present in about half of the acute diarrhea cases. This finding contrasts with results from studies conducted in urban areas of the country, where viruses and bacteria were more prevalent , 8. If tal areas , 22, moral areas , 23, 24.al areas -8. This al areas , 13, 23.Salmonella spp, Shigella spp. and EPEC were investigated. Out of the thirteen cases of bacterial diarrhea reported in our study, Shigella spp. was predominant. The role of Shigella spp. in childhood diarrhea deserves further attention [Finally, very few bacteria species were isolated in our study regardless of the age of the patients. This finding was surprising because in previous studies of childhood diarrhea, bacterial causes were the most important after viral causes , 6, 23. ttention , 26. Furttention , 8.Fever along with abdominal pain and dehydration were the common symptoms reported by our study participants. Similar findings were already observed in a study conducted in Ouagadougou in 2010 and elsewhere . In otheIn our study, the occurrence of pathogens associated with diarrhea was significantly influenced by the season. Hence, viral diarrheas were more frequent in the dry and cold season, from November to February, while parasitic infections were more frequent during the rainy season, from June to October. Similar findings were highlighted by Bonkoungou et al for viral diarrhea, particularly with rotavirus in a study conducted in Ouagadougou . In TanzGiardia spp. respectively. These two pathogens are also the most associated with malaria. The bacterial isolates were rare. The main symptoms are also frequent with malaria infection making the biological diagnostic a crucial tool to be used, as well as a refining of well guideline for Integrated Management of Childhood Illness. Further investigations including the genotyping of virus strains encountered and the detection of a large spectrum of microorganisms are needed.Our study gives an overview of pathogens associated with childhood diarrhea in Nanoro, a rural setting of Burkina Faso. Thus, parasites and viruses were the more frequently incriminated pathogens with rotavirus and The prevalence of diarrhea in children mostly investigated in the urban areas in Burkina Faso;The common pathogens incriminated in diarrheal diseases.The infectious etiologies and their prevalence in childhood diarrhea in rural areas in Burkina Faso were different to the urban areas;The significance of the interaction between diarrheal pathogens and malaria in Burkina Faso, a malaria endemic area was seen in viral and parasite pathogens;The main etiologies were associated with season, mostly the parasites like Giardia spp in the rainy season.


ObjectiveTo assess the frequency of severe acute malnutrition (SAM) and to determine the validity of mid-upper arm circumference (MUAC) as compared to weight for length z-score (WLZ-score) as an indicator of the nutritional status in this age group.MethodsA cross-sectional study, with a purposive sampling was conducted from March 2018 to November 2018 to enroll 540 infants ≤6 months of age from three different sites in Karachi, Pakistan. The anthropometric measurements  were taken by experienced community health workers. The data were analyzed using SPSS. MUAC was compared with WLZ-score for sensitivity and specificity to observe the concordant among the two diagnostic measures. The Youden Index was used to determine the ideal cut-off for infants less than 6 months of age in this population and the Kappa coefficient was also calculated to assess the agreement between MUAC and WLZ-score.ResultsThe study findings revealed that SAM was found in 13.6% (n=74) of the children. MUAC cut-off ≤11.5 cm yielded the Youden Index of 0.31 with 59.5% sensitivity and 71.4% specificity. The total area under receiver operating characteristic curve was 0.70 . The degree of agreement between mid-upper arm circumference and weight for length z-score to diagnose SAM ranged from 0.2 to 0.3.ConclusionThe Youden index implied that a MUAC cut-off of ≤11.5 cm can be used as an indicator with acceptable validity for diagnosing SAM in children ≤6 months of age in a low middle income developing country like Pakistan. There are approximately 3.8 million severely and moderately wasted children under the age of 6 months, mainly in low- and middle-income countries . MoreoveDespite this suggestion, the conclusions drawn from relevant surveys are not all in agreement. A survey from neighboring India showed that MUAC had low sensitivity 17.5%) and positive predictive value (30.4%) at the recommended cut-off of 11.5 cm for identifying severe acute malnutrition (SAM) in children aged 6-36 months old, arising question on its validity as a screening tool for identifying SAM at the community level .5% and p.On the other hand, MUAC has not been considered a reliable measure for diagnosing severe acute malnutrition in infants ≤6 months old because of a lack of data about its inter-observer reliability . MUAC haIn infants under 6 months, the World Health Organization defines SAM as those having a WLZ-score of <-3 standard deviations or the presence of bipedal edema . It has Availability of adequate data on measurement can make MUAC a simple, affordable and reliable screening tool for severe acute malnutrition in infants up to 6 months of age. This study therefore was conducted with the objective of assessing the frequency of severe acute malnutrition and to determine the validity of mid-upper arm circumference as compared to weight for length z-score as an indicator of the nutritional status in this age group. This study was rationalized to develop MUAC as a standard tool in diagnosing SAM in infants up to 6-month-old by comparing it with the current gold standard WLZ score.This was a cross-sectional observational study conducted in Karachi from March 2018 to November 2018 based on secondary data analysis of an earlier study titled “Building the evidence base for care of acutely ill, undernourished children in limited-resource setting-cohort study” with ethical approval number 4317-Ped-ERC-16. Karachi is located on the southern coastline of Pakistan and is the commercial hub of Pakistan. It is the most populous ethnically diverse city in the country representing communities from all over Pakistan. The study participants in the parent study were enrolled using purposive sampling because of age restriction enrolment in the study, from three sites in Karachi. The sites included, (i) Cattle Colony (Bhains colony), a peri-urban site catering to lower-income families; (ii) Pediatric ward, Civil Hospital Karachi, one of the main public sector hospitals serving a large population pool of lower-income families; (iii) the vaccination center at Aga Khan University, a private tertiary hospital largely population pool of middle to higher-income families. All infants who were under-6 months of age and visiting any of the three study sites for medical checkup with informed consent were included. Pre-term infants and those with severe congenital abnormalities, such as heart defects, neural tube defects and Down’s syndrome, that are associated with growth defaulting were excluded from the study. The rationale of having a hospital, a community and a vaccination center is obtaining the community representative sample.Under the supervision of a research assistant, all measurements were taken by experienced field workers who had been trained by the principal investigator in anthropometric assessment of weight, length and MUAC. Senior research staff monitored the whole data collection process to protect the integrity of the data collected.Weight was measured using a digital basin measuring scale (SECA 376). The length was measured by placing the child horizontal on a Shorr board and the MUAC was measured using standard UNICEF MUAC tape in the left arm at a flexed position. Weight was taken once with clothes removed, with two observers. Weight, length and MUAC were each taken twice by independent observers and an average of each value is taken as final measurement. If the inter-observer difference was more than 0.5 cm, measurements were repeated. These measurements were then used to calculate weight for length (WLZ) score, weight for age (WAZ) score and length for age (LAZ) scores using the WHO growth charts and the WHO anthropometry calculator .Sample size was calculated using 33% as the percentage frequency of severe acute malnutrition in Pakistan, with a confidence level of 95% and precision of 4%, to be 531 . AgainstStatistical Package for Social Sciences (SPSS) was used for data analysis.MUAC was compared with WHZ for sensitivity and specificity to diagnose severe acute malnutrition. The Youden Index was calculated to determine the ideal cut-off for infants less than 6 months of age. The Kappa coefficient was also calculated to calculate the agreement between MUAC and WLZ for diagnosing severe acute malnutrition.The study results showed that out of total of 540 children, aged 1-6 months, 57.2% (n=309) were male and 50.9% (n=275)were on exclusive breastfeeding (n=276). Severe acute malnutrition (WLZ Score <3 S.D.) were found in n=74 (13.7%) of the study population out of which n=52 (70.3%) were from a government hospital, n=12 (16.2%) were from tertiary care hospital while n=10 (13.5%) were from the peri-urban site. The mean weight, length and MUAC at 6 months were 5.35±1.49 grams; 59.51±5.76 cm; and 12.10±1.71 cm respectively   (data not shown).Among study participants with severe acute malnutrition, the mean MUAC was found to be lowest at the age of 2 months (9.60±1.24 cm) and highest at the age of 5 months (11.76±2.19 cm)  though still not good or excellent Table . HoweverThe study findings revealed that severe acute malnutrition (WHZ score <3 S.D.) was found in 13.6% of the study population. Moreover, the MUAC cut-off ≤11.5 cm yielded the Youden index of 0.31 with sensitivity and specificity of 59.5% and 71.4% respectively to diagnose severe acute malnutrition in infants up to 6 months of age. Furthermore, the degree of agreement between MUAC and WHZ as shown by kappa coefficient to diagnose severe acute malnutrition was slight and ranged from 0.2 to 0.3 in the same age group.The WHO defines severe acute malnutrition in infants who are 0-5 months of age as either weight-for-length less than -3 Z-score, or the presence of bilateral pitting edema . The useThe study results showed that MUAC cut off of ≤11.5 cm can be used with reasonable sensitivity and good specificity to diagnose severe acute malnutrition in infants up to 6 months of age. A study from South Africa in 2015 reported that weight for height measurements identified 7.7% of children as malnourished as compared to 6.6% by MUAC in 0 to 59 month age group, concluding weight for height measurements as more sensitive than MUAC for diagnosing acute malnutrition, but at the same time recommending further studies using both indicators at the community level for diagnosing moderate and severe malnutrition in infants aged 0 to 6 months .The study results further revealed a slight degree of agreement between MUAC and WHZ to diagnose severe acute malnutrition in infants up to 6 months of age as shown by a kappa coefficient that ranged from 0.2 to 0.3. The African study cited above also reported a kappa statistic value of 0.27 constituting a slight agreement between WHZ and MUAC in children aged 0 to 59 months . This siUnfortunately in our local and regional context, to the best of our knowledge, any relevant published data for a meaningful comparison with our study findings is almost non-existent. Unless the evidence base is broadened and MUAC as a screening and diagnosing tool for severe acute malnutrition in young infants is rigorously compared with current standards and its pros and cons are weighed carefully, questions on its potential usage as an indicator of severe acute malnutrition in young infants will remain unanswered.Developing a cut-off for MUAC for diagnosing SAM in infants 0-6 months may be beneficial as MUAC cut-offs of ≤11.5 cm and ≤11.0 cm have earlier been shown to effectively identify infants between the ages of 6-14 weeks with increased risk of death  .It is acknowledged that this study has certain limitations. The prime limitations of the study were non-exclusion of small for gestational age infants from the study along with the use of purposive sampling method. Moreover, the applicability of MUAC as an indicator of childhood malnutrition is limited as children grow rapidly in the first 6 months of life and more specific cut-offs may be needed for children under 3 months of age. Furthermore, evidence does not suggest occurrence of wasting below two weeks of age so the results cannot be generalized to that population as well.The MUAC cut-off of ≤11.5 cm yielded a Youden Index with reasonable sensitivity and good specificity implying that MUAC can be used as an indicator with acceptable validity for diagnosing childhood malnutrition children up to 6 months of age in low middle income developing country like Pakistan that has a moderate prevalence of stunting in this age group. It is recommended that the usefulness of MUAC as an indicator to predict malnutrition-associated outcomes other than SAM such as the risk of infections, morbidity and mortality in infants ˂ 6 months of age should be assessed in future studies.


Plasmodium. Cerebral malaria (CM) is a rapidly progressive and severe form of Plasmodium falciparum infection, characterized by a greater accumulation of red blood cells parasitized by Plasmodium falciparum in the brain. The diagnosis of malaria is usually made in living patients from a blood sample taken in the course of a fever on return from an endemic country, whereas CM, often associated with fatal outcomes even in treated subjects, is usually diagnosed at autopsy.Malaria is the first parasitic infection endemic in the world caused by parasites species of Plasmodium falciparum infection, with numerous parasitized erythrocytes containing dots of hemozoin pigment  in all examined brain sections and all other organs. Death was attributed to cerebral malaria with multiple organ failure.We present the case of a 36-year-old man who died a few days after returning from a business trip to the Ivory Coast. As a result of an unclear cause of death, a medicolegal autopsy was ordered. Autopsy findings revealed massive congestion and edema of the brain with no other macroscopic abnormalities at organ gross examination. Histology and laboratory tests were conducted revealing a This report summarizes several features for the diagnosis of malaria and how postmortem investigations, as well as histology and laboratory diagnosis, may lead to a retrospective diagnosis of a fatal complicated form with cerebral involvement. Plasmodium (single-celled parasites) that occurs in a person returning from a stay in a malaria-endemic area , due to its neurological damage, known as “cerebral malaria (CM)” or “neuromalaria”  test for Covid-19 had previously been performed (before traveling), which was negative. Mr. N. did not seek medical advice and resorted to self-medication including antipyretic and analgesics. On the 5th day, Mr. N.’s condition worsened as he presented a generalized tonic-clonic convulsive seizure, after which he did not regain consciousness. His family members called for medical assistance, but Mr. N. died before the arrival of the emergency units. As a result of an unclear cause of death, a medicolegal autopsy was ordered by the prosecutor, and the body was thus transferred to our department for autopsy. Malaria infection was already suspected at this point upon investigating the death circumstances with his relatives. A postmortem blood sample, on ethylenediamine tetraacetic acid (EDTA) preserved blood sample, was hence referred to the Parasitology Laboratory in order to confirm our assumptions before autopsy. Malaria rapid diagnostic test (RDT)  was positive. In both thick and thin Giemsa-stained blood smears  were found, showing high parasitemia. The percentage of parasitemia was though difficult to assess due to the hemolysis state of the postmortem samples. External examination revealed cyanosis with frank mucocutaneous icterus. Autopsy findings revealed massive congestion and edema of the brain, weighing 1550 g . Since his return to Tunisia, he presented a symptomatology associating fever, headache, asthenia, and diarrhea. A 0 g Fig. , lungs  after returning from a stay in a malaria-endemic area (Bruneel et al. In our case, the external examination found mucocutaneous jaundice as was the case in an autopsy series of 18 sudden deaths due to severe malaria-discovered postmortem (Djodjo et al. Plasmodium falciparum (Djodjo et al. Histology is the main means of diagnosing malaria postmortem (Burel-Vandenbos et al. Analysis of malaria case reports in the literature reveals the great scientific contribution of autopsies, as most of the data related to CM have been drawn from autopsy observations. Pathogenic mechanisms leading to cerebral malaria were at the beginning poorly defined as studies have been hampered by limited access to human tissues. This is also because limited studies can be performed in humans, and common models conducted on mouses do not reproduce all aspects of CM. The first attempts to discover the pathogenesis of this syndrome relied significantly on the histopathology of brain tissue from deceased CM patients (Rénia et al. This article reports a case of CM-discovered postmortem. This case report illustrates the fact that travelers, often through the lack of advice, fail to take preventive treatment when making frequent trips to endemic malaria areas. It also confirms the importance of informing these travelers of the rapid fatal outcome and hence of the need for early treatment and follow-up. This report summarizes several features for the diagnosis of malaria and how postmortem investigations may lead to a retrospective diagnosis of a fatal complicated form, with cerebral involvement. The proper diagnosis was made possible, thanks to close collaboration between the different pathologists.


Procalcitonin is an inflammatory marker strongly associated with the presence of bacterial infection. It has been considered raised in severe malaria infection as opposed to uncomplicated malaria. There are suggestions that it may be raised only when there is concomitant unnoticeable bacterial infection during a malaria crisis. We aimed to assess the difference in plasma procalcitonin levels between children affected by severe and uncomplicated malaria.p values < 0.05.We assessed plasma procalcitonin levels in 83 children diagnosed with malaria with no clinical and biological evidence of concomitant bacterial infection. Severity of malaria was established using WHO guidelines. Procalcitonin was determined using the ELISA method. Non-parametric Mann-Whitney U test was used to compare medians across the 2 groups. Statistical significance was set for all p = 0.916]. The parasite density was lower in the uncomplicated malaria group than in the severe malaria group, but not statistically significant; . There was no correlation between the parasite density in the general study population and PCT levels .Of the 83 participants, 28 had uncomplicated malaria, and 55 had severe malaria. PCT levels were obtained in 24 and 40 subjects of each group, respectively, and were similar in both groups; [2.76 (2.52–2.93) vs 2.74 (2.52–2.98) ng/ml, In the absence of overt bacterial infection, procalcitonin levels are not different between children affected with uncomplicated malaria and those with severe malaria. Therefore, bacterial infection should be thoroughly checked for in children with raised serum procalcitonin diagnosed with severe malaria. Procalcitonin (PCT) is currently known to be the standout marker for the diagnosis of bacterial infections as higher levels of serum PCT are found in patients with severe bacterial infections in comparison with patients with viral infections and other inflammatory conditions relative to viral infections and nonspecific inflammatory diseases . PCT is Serum PCT has previously been shown to be raised in malaria infections and serves as a good discriminatory tool between uncomplicated malaria and severe malaria in the adult population –10. WhetThe study was a cross-sectional, descriptive, and analytical study, carried out at the Mother and Child Centre (MCC), Chantal Biya Foundation, Yaoundé, Cameroon, over a period of 7 months (November 2018 to June 2019). A total of 126 children (aged 6 months to 15 years) were initially selected based on presenting complaints. The children to take part in the study were selected by a single paediatrician who thoroughly examined the children for a concomitant clinical focus of bacterial infection. Those with overt evidence of clinical bacterial infections were excluded. These included anomalies to various organ systems such as bulging tympanic membranes, hyperemic conjunctiva, erythematous oropharynx, lung crackles, lymphadenopathy and present costovertebral and supra-pubic tenderness. A complete blood count was also performed to exclude all cases with hyper leukocytosis. A total of 83 children were retained for the study. Figure Standard Diagnostics BIOLINE®, Malaria Ag. P.f/Pan) with peripheral venous blood which had been collected into a 3-5 ml EDTA tube. Whole blood (0.5 mL) was pipetted into the EDTA tube and dropped in the sample hole quickly enough to avoid blood clotting. Two drops of the buffer were put into the appropriate hole. After waiting for 15–20 minutes, the results were read. The RDT was considered valid with the presence of the control line.This was done using rapid diagnostic test (RDT) kits for malaria . Thin films were examined only for malaria parasite species. Severe malaria was defined using the WHO malaria severity criteria . All other full blood count parameters  showed no significant changes between groups.All cases enrolled into the study had a positive RDT result for malaria and showed a positive smear for p = 0.916) as shown in Table p = 0.57). In the uncomplicated malaria group, there was no meaningful correlation  between the parasite density and serum PCT values (data not shown).Serum PCT values were finally obtained in 64 patients; 24 and 40 cases with uncomplicated and severe malaria. Nineteen (19) cases had undetectable serum PCT levels (below the limit of detection of the kit used) Among those with undetectable serum PCT levels, 10 had severe malaria and 9 had uncomplicated malaria. The median (IQR) serum PCT level in the study was 2.75 (2.52–2.96) ng/ml. The median (IQR) serum PCT level in the cases with uncomplicated malaria was 2.76 (2.52–2.93) ng/ml while in those with severe malaria was 2.74 (2.52–2.98) ng/ml. There was no statistically significant difference in the serum PCT levels between both malaria groups  ng/mL vs 2.82 (2.55–2.97) ng/mL, p = 0.53. However, the parasite density was significantly lower among those with severe malaria who had received prior antimalarial treatment compared to those who had not; 18 869 (5025–32 737) parasite/μl vs 49 989 (22 641–108 475) parasite/μl, p = 0.005. The white blood cell count, plate count and haemoglobin level did not show any significant changes.Table We aimed to assess the difference in serum PCT levels in children aged between 6 months to 15 years diagnosed with uncomplicated and severe malaria in a clinical setting in sub-Saharan Africa. We found that 33.7 and 66.3% of our participants had uncomplicated and severe malaria, respectively, and that there was no significant difference in the serum PCT levels between these 2 groups. Serum PCT levels are usually very high in bacterial infections and therefore it can be used to confidently discriminate between bacterial infections and severe malaria given that high serum PCT in severe malaria cases is not supported by this finding, contrary to other studies.P. falciparum in all malaria cases recorded in Cameroon during the year 2017 [Most participants in our study presented with fever at consultation as was reported by Trampuz et al. to be the most common presenting symptom in about 92% of malaria cases . The majear 2017 . We obseear 2017 . The onsear 2017 , 17., Righi et al, Te Witt et al and Hesselink et al [The median serum PCT value in our study population was higher than the value in a normal subject (< 0.2 ng/ml) as seen in most studies done earlier , 17–19. nk et al , 21, 22.nk et al . This coOur study has some limitations. The small sample size of the study could have affected the ability to show significant differences between subjects with uncomplicated and severe malaria. In addition, PCT serum levels have been shown to possess rapid kinetics, with an early rise and fall in serum levels during a given infection , 24. ThiNevertheless, the findings presented in this study are of clinical interest to health practitioners in this region of the study; in the absence of overt bacterial infection, serum PCT levels are not different between patients diagnosed with uncomplicated malaria and those with severe malaria. This finding may therefore inform the practice of diligently seeking to prove an associated bacterial infection in patients diagnosed with uncomplicated/severe malaria who have high PCT levels as a high PCT level may not be as a result of the severity of the malaria infection. Further larger sample size studies including children with confirmed bacterial infections and those with confirmed malaria infections or both are needed to better inform on the association between procalcitonin and malaria infections.In the absence of overt bacterial infection, serum procalcitonin levels are not different between children affected with uncomplicated malaria and those with severe malaria. Therefore, bacterial infection should be thoroughly checked for in children with raised serum procalcitonin diagnosed with severe malaria.


In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1β (IL-1β) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1β release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1β release) in Δmag1 parasites completely inhibited all IL-1β release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection.Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δ Toxoplasma gondii is an obligately intracellular parasite that chronically infects up to a third of the human population -like pattern recognition receptor 3 (NLRP3) and induce interleukin 1β (IL-1β) secretion  that are either present in the interface between the parasitophorous vacuole (PV) and host cytosol or secreted into the host cell modulate innate immune responses in tachyzoites. Some GRAs have immunosuppressive roles; e.g., GRA7 complexes with pseudokinases and deactivates immunity-related GTPase a6 (IRGA6), GRA18 is secreted into host cytosol and induces anti-inflammatory cytokines (CCL17/22) , and botecretion –13.T. gondii infection , and caspase-1, resulting in proteolytic cleavage and secretion of proinflammatory cytokines IL-1β and IL-18 . The keynfection , and mic. gondii . Therefost stage . Howeverin vivo CRISPR knockout screen identified MAG1 as a key protein necessary for either dissemination to the brain or establishment of brain cysts , infection with Δmag1 parasites induced a significant increase in IL-1β secretion, which is a hallmark of inflammasome activation. Heterologous expression of MAG1 in BMDM prevented this Δmag1 parasite-induced IL-1β increase, indicating that secreted MAG1 dampens the inflammasome activation in the host cytosol. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, MAG1 would favor the survival of this parasite and the establishment of a latent infection.In this study, we demonstrate that MAG1 is secreted beyond the PV into the host cytosol in both tachyzoites and bradyzoites. In the murine infection model, ΔT. gondii. These antibodies were screened against in vitro cysts by an immunofluorescent assay (IFA) to evaluate the localization of their cognate antigens. Among the isolated monoclonal antibodies (MAbs), clone bB6 localized to the in vitro cyst wall of PruΔku80 parasites  was deleted from the genome of T. gondii in strain PruΔku80 . Cyst wall staining and the 65-kDa band are absent in the Δmag1 strain and restored in the complemented Δmag1::mycmag1 strain  Immunoblot of immunoprecipitated parasite lysate probed with bB6. (B) Immunoblot of parasite lysate probed with polyclonal anti-MAG1 antibody and bB6. Download Copyright © 2021 Tomita et al.2021Tomita et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro cysts were quantitatively assessed by IFA. In the tachyzoite stage, MAG1 is mostly present in the parasitophorous vacuolar matrix (LDH2 promoter), the majority of MAG1 migrates to the cyst wall (located at the PVM) and the matrix staining diminishes (in vitro cysts were probed with MAb bB6 (anti-MAG1) antibody and Dolichos biflorus lectin (DBA), which binds to the O-GalNAc glycans on the canonical cyst wall protein CST1 , and these infected mice were maintained until 35 dpi. While both the WT and Δmag1::mag1 parasites killed about half of the mice during this period of observation, there was no mortality in the Δmag1 parasite-infected mice  were injected with 2 × 10ted mice . This sumag1 parasites produced 98% fewer brain cysts than the WT parasites   were completely absent in Δmag1 strain-infected mice. Due to the paucity of Δmag1 brain cysts, we were not able to determine if the ultrastructure of the Δmag1 parasite cyst wall was altered, but cysts in tissue culture did not exhibit any obvious defects when examined under transmission electron microscopy  . While tin cysts , large bcroscopy . To rulets [HFF]  and mousts [HFF] ), indica10.1128/mBio.00603-21.2FIG S2in vitro cysts. Ultrastructure of in vitro cysts at day 3 postinfection. B, bradyzoites; W, cyst wall. Red arrow indicates the cyst wall membrane; green arrow indicates the cyst wall granular layer. Download FIG S2, TIF file, 1.9 MB.Transmission electron micrograph of Copyright © 2021 Tomita et al.2021Tomita et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00603-21.3FIG S3mag1 parasite-infected BMDM. BMDM culture supernatant infected with parasites were assessed for (A) IL-1β, (B) TNF-α, (C) IL-12p40, (D) nitrite, (E) LDH release, (F) NF-κB activity, and (G) nuclear intensity of p65. IFN-γ susceptibility was assessed on HFF in either 10% serum (H), 10% lipid-depleted medium (I), or 1% serum (J). Δmag1 parasites were much more susceptible to IFN-γ only in the 1% serum medium but not in the 10% medium. Blood-circulating IFN-γ (K), IL-12 (L), and TNF-α (M) at 3 dpi were measured with parasite-infected mice. In vivo cytokines were not different between WT and Δmag1 parasites. All multiple comparisons were performed with one-way ANOVA with Tukey’s HSD test, and paired comparison was performed with a t test. Download FIG S3, TIF file, 0.6 MB.Immunological screening identified IL-1β secretion was elevated in ΔCopyright © 2021 Tomita et al.2021Tomita et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mag1 parasites could be due to a defect of cyst generation and/or failure to disseminate into the brain. To distinguish these two possibilities, parasite expansion was monitored during the acute infection in mice using a novel red-shifted bioluminescence imaging system, AkaBLI , and parasite expansion was monitored at day 3 and day 7 using an in vivo imaging system (IVIS). At both 3 and 7 dpi, infected mice had parasite luminescence in their abdomens . Complementation of the MAG1 gene reduced the level of IL-1β back to the WT level. This demonstrates that the observed IL-1β increase is dependent on MAG1. To examine if regulation was present at the transcriptomic level, the mRNA levels of IL-1β were measured  MAG1 is a major vacuolar matrix protein in the tachyzoite stage, and disruption of the vacuolar matrix affects IL-1β secretion, or (ii) MAG1 in the host cell dampens the immune response, suppressing IL-1β release. To distinguish those two possibilities, we heterologously expressed MAG1 in BMDM and assessed if heterologous complementation of MAG1 in the host cell could inhibit IL-1β secretion. This allowed us to isolate the effect of MAG1 in the host cell on IL-1β secretion. MAG1 or mCherry (negative control) genes were transduced into BMDM using lentivirus. These cells were then infected with various mutant parasites to stimulate the inflammasome (P < 0.01), indicating that MAG1 in the host cytosol is involved directly in dampening the IL-1β secretion. This reduction of the IL-1β secretion in MAG1-expressing BMDM was also observed in WT and Δmag1::mag1 parasite-infected BMDM. This suggests an additive effect of heterologously expressed MAG1 and parasite-secreted MAG1 in BMDM in suppression of IL-1β release. To confirm that MAG1 is actually secreted from the parasitophorous vacuoles into BMDM cytosol, an immunofluorescent assay was performed on the transduced BMDM (Since lactate dehydrogenase (LDH) release was not different between WT- and Δammasome . Compareced BMDM . At 24 hgra15 parasites are more virulent    comparedmyr1 parasites still resulted in the secretion of MAG1 into the host cytosol  , 38. Howmag1 parasites was due to the reduction in their acute expansion based on the in vivo bioluminescent imaging of infected mice. We did, however, also observe a lack of large cysts in Δmag1 parasite-infected mice. While this was not statistically significant due to the scarcity of Δmag1 parasite brain cysts available for measurement, it suggests that MAG1 could also affect cyst formation. Ultrastructural studies of in vitro-derived Δmag1 parasites did not demonstrate any abnormalities in the cyst wall; unfortunately, due to the low numbers of cysts seen in vivo, we could not obtain any in vivo cysts for electron microscopy. Additional studies will be needed to discern whether MAG1 has any direct effect on cyst architecture.The effect of MAG1 on cyst number is striking. We reasoned that more than 98% of the reduction in the cyst burden in Δgra15 parasites demonstrated that a lack of GRA15 resulted in significantly higher acute parasite burden  pathway . We havein vivo, but more study is needed to define its role in cyst biology.In summary, we have demonstrated that MAG1 functions as a secreted effector that modulates inflammasome activity and acts as a counterbalance to GRA15-induced inflammasome activity. The effect of MAG1 on the inflammatory response supports the development of latency. The mechanism by which MAG1 is released from the cyst into its host cell is currently unknown. MAG1 may also have direct effects on cyst development ku80)  at 0.5% CO2.The type II strain Prugniaud with a deletion in the KU80 gene (PruΔku80)  was pass3 to 1 × 105 parasites per mouse. Viability of the parasite inoculant was verified by plaque assay. Mice were monitored daily and maintained until 35 dpi, at which point any surviving mice were euthanized. All procedures were done under an animal use protocol approved by the Albert Einstein College of Medicine IACUC.Parasites were lysed out of HFF by passage through a 27-gauge needle and filtered with a 5-μm filter. C57BL/6 mice were intraperitoneally (i.p.) injected with tachyzoites at 2 × 10dm1 mice  using standard techniques.BALB/cdm1 mice  were infechnique . The emuku80 parasites at a multiplicity of infection (MOI) of 1. Parasites were differentiated to bradyzoites and lysed in radioimmunoprecipitation assay (RIPA) buffer . Bradyzoite lysates were precleared by incubation with protein G agarose beads for 30 min at 4°C to remove nonspecifically bound proteins. Then, precleared samples were incubated with protein G agarose beads covalently cross-linked with MAb bB6 using bis(sulfosuccinimidyl)suberate (Thermo Scientific) according to the manufacturer’s instructions. Precleared samples were incubated with MAb bB6 beads overnight at 4°C while rotating. Beads were washed twice with RIPA buffer before elution in Laemmli sample buffer. MAb bB6, lysate input, bead flowthrough, RIPA washes, and bead eluates were resolved on a 4-to-15% polyacrylamide gel (Bio-Rad) and stained with Coomassie blue. Bands in the eluate lanes were excised and analyzed by mass spectrometry.HFF were infected with PruΔ18 reversed-phase (RP) chromatography using an Ultimate 3000 RSLCnano system  equipped with an Acclaim PepMap RSLC C18 column . The ultra-high-performance liquid chromatograph (UHPLC) was connected to a TriVersa NanoMate nano-electrospray source  and an LTQ-XL linear ion trap  mass spectrometer with the ESI source operated in the positive ionization mode. Automated protein identification was performed by Mascot search engine v. 2.5.1 (Matrix Science) against the ToxoV12_uniprot_20150225 database  with the following search parameters: trypsin, three missed cleavages, peptide charges of +2 and +3, peptide tolerance of 2.0 Da, MS/MS tolerance of 0.8 Da, carbamidomethylation (Cys) for fixed modification, and deamidation (Asn and Gln) and oxidation (Met) for variable modifications. A decoy database search was also performed to measure the false discovery rate. Mascot protein identification results were further analyzed by Scaffold software v. 4.4.5 based on 99% protein and 95% peptide probabilities.Excised gel bands were reduced, alkylated, and digested with trypsin. LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis of the peptide digests was done by Cmag1 line used in the immunological response screening were generated in the T. gondii strain Prugniaud (type II), which has a deletion of the ku80 gene (PruΔku80) for increased homologous recombination  immune response screening, and (C) transmission electron microscopy. Download Text S1, DOCX file, 0.01 MB.Detailed descriptions of (A) production of mutant strains of Copyright © 2021 Tomita et al.2021Tomita et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the T. gondii-infected HFF were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and then permeabilized with PBS containing 0.2% Triton X-100 for 20 min. The coverslips were then washed twice with 0.2% bovine serum albumin (BSA) in PBS and incubated with 1% BSA in PBS as a blocking solution at room temperature for at least 1 h. Primary antibodies were diluted in 1% BSA in PBS, and then slides were incubated with the antibody at 37°C for 90 min. bB6 antibody was used at 1:500, and mouse anti-Myc tag  was used at 1:200. The coverslips were then washed twice with 0.2% BSA in PBS and incubated in a secondary antibody solution in 1% BSA in PBS for 37°C for 90 min, after which the coverslips were washed twice with 0.2% BSA in PBS. Coverslips were then mounted onto slides using ProLong Gold with DAPI  and left to dry in the dark.Coverslips containing post hoc Tukey HSD test was used to determine the difference in the translocation of MAG1.For the quantification of MAG1 signal, images were taken with a Leica SP5 confocal microscope. Using ImageJ custom macros , the PVM10.1128/mBio.00603-21.5DATA SET S2in vitro cysts MAG1 images with annotated segments. Download Data Set S2, DOCX file, 0.5 MB.ImageJ macro and an example of image segmentation for MAG1 IFA. The ImageJ script was used to analyze the MAG1 intensity in the IFA images. The actual example of segmented Copyright © 2021 Tomita et al.2021Tomita et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the post hoc test of Tukey's HSD test.HFF monolayers were grown to confluence on 6-well plates and infected with 5-μm-filter-purified parasites. Parasites were allowed to grow without disturbance for 7 days. After incubation, cells were fixed, stained with a staining solution  for 30 min, and washed with water 10 times. Plaques were imaged and size was measured with ImageJ with the Trainable Weka Segmentation plugin . StatistT. gondii were grown in DMEM medium  at 5% CO2 for tachyzoites or in differentiation medium  at 0% CO2 for bradyzoites. Cells were harvested at 3 dpi lysed with RIPA buffer containing protease inhibitor cocktail. The samples were resolved by 10% SDS-PAGE and transferred using a semidry transfer system (Bio-Rad) onto Immobilon-FL (Millipore) polyvinylidene difluoride (PVDF) membranes. The membrane was blocked overnight with 5% nonfat dry milk (NFDM) in PBST and then probed with bB6 MAb at 1:500 in 1% BSA–PBS, polyclonal rabbit anti-MAG1 (gift from S. Parmley [HFF infected with  Parmley ) at 1: 5in vitro cysts is described in The method used for the ultrastructural assay of in vitro IFN-γ susceptibility measurement by plaque assay, measurement of NF-κB activation, LDH release assay, and in vivo infection for cytokine measurement, in vitro cytokine ELISA, and determination of nitrite, are described in Immune response screening methods, including Culturing of BMDM was performed according to a previous publication  with mod3 freshly liberated Akaluc-expressing parasites from HFF and filtered with 5-μm filters. At 3 and 7 days after infection, mice were injected with 1.5 μmol of Tokeoni/Akalumin-HCl (Sigma-Aldrich) in 100 μl PBS i.p. and imaged at 20 min after injection using IVIS Spectrum (PerkinElmer) under anesthesia.Six- to eight-week-old female albino C57BL/6  mice were injected i.p. with 103 freshly liberated Akaluc-expressing WT and Δmag1 parasites as described above. At 1 dpi, mice were injected i.p. with 10 μg of either neutralizing antibodies against IL-1β or normal goat IgG  as previously described (Albino C57BL/6 mice were injected i.p. with 10escribed .5/well in 96-well tissue culture plates overnight. At day 13 (10 days after differentiation), BMDM were infected with freshly lysed parasites filtered with 5-μm membrane at an MOI of 1 to 2 . After 24 h of infection, supernatants were removed and assayed for IL-1β secretion with a mouse IL-1β/IL-1F2 DuoSet ELISA  following the manufacturer's protocol.At 12 days postharvest, fully differentiated BMDM were seeded at 10Ex Taq II (TaKaRa) with iQ5 (Bio-Rad), following the manufacturer's protocols. Gene expression of IL-1 β was normalized against a housekeeping gene RPL27 .


Plasmodium species are common in many malaria endemic settings. Mean multiplicity of infection (MOI) and the proportion of polyclonal infections are often reported as surrogate marker of transmission intensity, yet the relationship with traditional measures such as parasite prevalence is not well understood. We have searched Pubmed for articles on P. falciparum and P. vivax multiplicity, and compared the proportion of polyclonal infections and mean MOI to population prevalence. The impact of the genotyping method, number of genotyping markers, method for diagnosis (microscopy/RDT vs. PCR), presence of clinical symptoms, age, geographic region, and year of sample collection on multiplicity indices were assessed. For P. falciparum, 153 studies met inclusion criteria, yielding 275 individual data points and 33,526 genotyped individuals. The proportion of polyclonal infections ranged from 0–96%, and mean MOI from 1–6.1. For P. vivax, 54 studies met inclusion criteria, yielding 115 data points and 13,325 genotyped individuals. The proportion of polyclonal infections ranged from 0–100%, and mean MOI from 1–3.8. For both species, the proportion of polyclonal infections ranged from very low to close to 100% at low prevalence, while at high prevalence it was always high. Each percentage point increase in prevalence resulted in a 0.34% increase in the proportion of polyclonal P. falciparum infections (P<0.001), and a 0.78% increase in the proportion of polyclonal P. vivax infections (P<0.001). In multivariable analysis, higher prevalence, typing multiple markers, diagnosis of infections by PCR, and sampling in Africa were found to result in a higher proportion of P. falciparum polyclonal infections. For P. vivax, prevalence, year of study, typing multiple markers, and geographic region were significant predictors. In conclusion, polyclonal infections are frequently present in all settings, but the association between multiplicity and prevalence is weak.Multiple infections of genetically distinct clones of the same  Malaria remains a global public health threat with over 200 million confirmed cases and over 400,000 deaths recorded in 2019 [P. vivax, relapses of heterologous hypnozoites during ongoing blood-stage infection can also result in polyclonal infections [Individuals in malaria endemic regions are often concurrently infected with several clones or strains of the same parasite species. Polyclonal infections can be the result of a single mosquito bite transmitting a genetically diverse inoculum (coinfection), or of repeated bites transmitting different clones (superinfection) . In P. vfections , 5.Plasmodium falciparum and Plasmodium vivax. A large number of genotyping protocols is used to determine multiplicity. The most common methods include typing of size-polymorphic markers (antigens or microsatellites) and sizing on agarose gel or by capillary electrophoresis (CE) [Multiplicity of infection , and the proportion of polyclonal infections  are among the most frequently reported measures of molecular malaria surveillance studies of sis (CE) , 7, SNP sis (CE) , and morsis (CE) , 10.P. vivax and P. falciparum approximately halved over a 14-year period. The proportion of polyclonal infections fell from 70% to 35% for P. vivax, and from 28 to 18% for P. falciparum [P. falciparum infections from 63% to 14% [MOI is generally assumed to be higher in high-transmission settings, while in low-transmission settings most individuals might carry single clone infections. Multiplicity has thus been proposed as surrogate marker to measure changes in transmission intensity in time and space. Numerous studies found a correlation between transmission intensity and MOI. For example, in West Papua, Indonesia, clinical incidence of lciparum . Along t% to 14% . In Sene% to 14% . In Vene% to 14% . In othe% to 14% , or abse% to 14% .P. falciparum diagnose the HRP2 protein. Increasing numbers of parasites are being reported carrying a deletion of the hrp2 gene, which results in false negative tests. Molecular surveillance of hrp2 deletion is a high priority [Understanding the extent of polyclonal infections is important for molecular malaria surveillance activities beyond measuring transmission intensity. Typing of molecular markers for drug resistance is an important aspect of malaria surveillance . Polyclopriority . In casepriority . Changespriority . As a fupriority –25.Despite the large number of studies reporting multiplicity data, the relationship between MOI or the proportion of polyclonal infections and transmission intensity across a wide range of studies remains poorly characterized. Likewise, little effort has been made to systematically assess the impact on multiplicity estimates of characteristics of the study population  or the method used for genotyping (e.g. gel electrophoresis vs. capillary electrophoresis for sizing of polymorphic markers). We have conducted a systematic literature search and compared the proportion of polyclonal infections and mean multiplicity to population prevalence of infection as common measure of transmission intensity. Furthermore, we studied the effect of laboratory procedures and characteristics of the study population on multiplicity indices.Plasmodium falciparum”, “Complexity of infection Plasmodium falciparum”, and “Multiple clone infection Plasmodium falciparum”. Articles were eligible if they reported data on mean MOI and/or the proportion of polyclonal infections. From clinical trials, data was included only when reported separately for the baseline. 265 studies were identified as eligible for assessment out of which 153 were included in the pooled analysis , 27P. vihttps://malariaatlas.org). MAP provides modeled prevalence of infection by microscopy in 1-10-year-old children at the country or sub country level. For the current study, data from the lowest administrative level available was recorded. With very few exceptions, data points for which prevalence data was taken from MAP had only genotyped children. Thus, the age range of the individuals genotyped matched the age range of the data in MAP. For studies conducted before the year 2000, data from 2000 was recorded (the earliest available from MAP).From each study, data on multiplicity of infection and/or the percentage of polyclonal infections were recorded. If available, population prevalence of infection was recorded from the article. If no prevalence data was reported, data was obtained from the Malaria Atlas Project (MAP) website  Geographic locations: Studies were grouped as conducted either in Africa, the Asia-Pacific, or the Americas. (vii) Year: Studies conducted from 1990 to 2019 were included in the analysis. Over this period, the number of P. falciparum and P. vivax cases globally has decreased [(i) Number of genotyping markers: A larger number of molecular markers increases the probability to detect additional clones. Studies were distinguished between using a single marker or multiple markers. SNP panels always include multiple markers, but only two alleles can be distinguished for each SNP. Whole genome sequencing (WGS) covers the entire genome, but read depth is generally lower than amplicon sequencing. Thus, SNP panels and WGS were excluded from this analysis. (ii) Diagnosis: In all transmission settings, a large proportion of infections remains below the limit of detection of microscopy or rapid diagnostic test (RDT) and can only be diagnosed by sensitive molecular methods (e.g. PCR) , 29. DetP-value >0.05, until the final model included only variables with P≤0.05.Linear regression models were used to analyze the impact of the above-described variables and the proportion of polyclonal infections or mean multiplicity. Multivariable regression was conducted, with step-wise removal of variables with a The literature search yielded 153 studies that met the inclusion criteria, resulting in 275 data points with a total of 33,526 genotyped infections. Summary statistics are presented in P<0.001). At low prevalence, the proportion of infections carrying multiple clones ranged from very low to almost 100% . In the Asia-Pacific region, the proportion of polyclonal infections was 21.9% lower than the proportion in Africa  . Typing multiple markers instead of only one marker resulted in detection of 14.1% more polyclonal infections (P<0.001). Diagnosis of infections by PCR instead of microscopy or RDT resulted in 9.4% fewer polyclonal infections detected (P = 0.013). Compared to Africa, the proportion of polyclonal infections in the Asia-Pacific region was 17.8% lower, and in Latin America, it was 28.0% lower (P<0.001).In multivariable analysis, the year the study was conducted, sizing by CE vs. gel, age group, and whether samples were from clinical or asymptomatic individuals did not reach significance and were excluded from the final model . In the P<0.001, P = 0.241, In univariate analysis, each percentage point increase in prevalence resulted in an increase in mean MOI of 0.010 . A trend towards lower mean MOI was observed in studies using PCR for diagnosis compared to microscopy or RDT . In studies including children <15 years (n = 90), mean MOI was 0.350 higher compared to studies including all ages . No difference was observed between studies including only adults >15 years (n = 14) and all ages (P = 0.988). Typing of size-polymorphic markers by CE instead of gel resulted in a trend toward lower mean MOI .Mean MOI did not differ significantly between studies on clinical and asymptomatic individuals . Over the time period analyzed (1996–2018), a trend toward a lower proportion of polyclonal infections was observed  and method of diagnosis  did not impact mean MOI. Mean MOI was higher in studies enrolling only children  compared to all ages . In multivariable analysis, the same predictors as for the proportion of polyclonal infections were significant, namely prevalence, year of sample collection, typing multiple markers, geographic region, and age group . For P. vivax, 76% of studies used CE for sizing. The choice of gel vs. CE can affect multiplicity estimates for two reasons. First, capillary electrophoresis increases detectability of minority clones. Second, it allows distinction of fragments of similar size compared to visualization on gel. By CE, fragments differing as little as 3 base pairs in size can be distinguished. In contrast, when binning using gels, resolution might be as low as 50 base pairs [P. falciparum polyclonal infections, and the method was not a significant predictor in multivariable analysis. The result from the univariate analysis could be explained by the more widespread use of CE in the Asia-Pacific compared to Africa, where transmission and multiplicity are generally lower than in Africa.Estimates of multiplicity are heavily impacted by the ability to detect minority clones and by the discrimination power of the genotyping method selected. Size polymorphic markers were the preferred method for both species, but pronounced differences in the methods used for se pairs . Previouse pairs , 32. In P. falciparum genotyping, 49% of studies typed a single marker. In multivariable analysis, typing multiple P. falciparum markers increased the proportion of polyclonal infections detected by 14%. An even stronger increase of 35% was observed for P. vivax, where only 9% of studies typed a single marker.For P. falciparum, the proportion of polyclonal infections was highest in Africa, and for P. vivax, in the Asia-Pacific. In both cases, this mirrors high transmission intensity.Pronounced differences were observed among geographic regions. For msp2 has been widely used for P. falciparum genotyping, allowing to repeat the analysis on this marker only. Results mirrored those of the entire dataset. The relationship between the proportion of polyclonal infections and prevalence remained poor. Despite its wide use, inclusion criteria for minority clones when typing msp2 differ among laboratories, and are often not reported. When using CE, the criteria applied include an arbitrary cut off of 33% of the height of the main peak [Marker ain peak , a cut oain peak , or inclain peak . This vaIn many studies, in particular those typing samples from clinical patients, no data on transmission intensity was given, such as population prevalence, clinical incidence, or test positive rate. In order to conduct more detailed analysis of the relationship between multiplicity and transmission intensity, it is recommended to report such measures along with genotyping data.hrp2 deletion. In polyclonal infections the presence of deletions can be masked by wild-type infections [In conclusion, the weak correlation between mean MOI or the proportion of polyclonal infections and prevalence indicates these measures do not accurately stratify transmission intensity when different studies are combined. Further studies will be required to understand the factors affecting the presence of polyclonal infections when prevalence is low. The possibility of a high proportion of polyclonal infections even in low transmission sites should be considered when typing markers of drug resistance or fections , and thefections .S1 File(XLSX)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.


Plasmodium knowlesi, a simian malaria parasite responsible for all recent indigenous cases of malaria in Malaysia, infects humans throughout Southeast Asia. There are two genetically distinct subpopulations of Plasmodium knowlesi in Malaysian Borneo, one associated with long-tailed macaques (termed cluster 1) and the other with pig-tailed macaques (cluster 2). A prospective study was conducted to determine whether there were any between-subpopulation differences in clinical and laboratory features, as well as in epidemiological characteristics. Over 2 years, 420 adults admitted to Kapit Hospital, Malaysian Borneo with knowlesi malaria were studied. Infections with each subpopulation resulted in mostly uncomplicated malaria. Severe disease was observed in 35/298 (11.7%) of single cluster 1 and 8/115 (7.0%) of single cluster 2 infections (p = 0.208). There was no clinically significant difference in outcome between the two subpopulations. Cluster 1 infections were more likely to be associated with peri-domestic activities while cluster 2 were associated with interior forest activities consistent with the preferred habitats of the respective macaque hosts. Infections with both P. knowlesi subpopulations cause a wide spectrum of disease including potentially life-threatening complications, with no implications for differential patient management. Plasmodium knowlesi was found to be the commonest cause of human malaria infections in the Kapit Division of Sarawak, Malaysian Borneo in 20041. Subsequent studies have shown that zoonotic malaria cases occur throughout Southeast Asia and in the Andaman and Nicobar islands of India5. The highest incidence is in Malaysia, where 13,612 knowlesi malaria cases were reported between 2017 to 2020, with 87% from the Malaysian Borneo states of Sabah and Sarawak . All the indigenous malaria cases in Malaysia in 2018 and 2019 were due to P. knowlesi6. Human knowlesi malaria infections have been increasing in Malaysia since they were first reported in 20041. From the 120 cases detected in Sarawak over a 32-month period at that time1, the annual number of reported cases in Malaysia has increased to 912 in 2009, and to between 1600 and 4131 since 20127. This increase may reflect improved diagnostic capacity, decrease in cross-species immunity due to decrease in malaria caused by human malaria species, and increased interaction between humans, macaques and mosquito vectors. This latter explanation is based on land-use changes leading to alterations in mosquito abundance and composition, and to greater proximity of the movement of the reservoir macaque hosts to human habitation8. The increase in zoonotic malaria cases is of public health concern and a threat to the elimination of malaria.The simian malaria parasite, P. knowlesi isolates from wild macaques in Kapit, Malaysian Borneo and humans across Malaysia has identified two simian host-associated genetically distinct subpopulations9. Two-thirds of human infections were of one subpopulation (termed cluster 1) associated with long-tailed macaques (Macaca fascicularis), while the second subpopulation (cluster 2) was associated with pig-tailed macaques (Macaca nemestrina). Subsequent whole genome sequence analysis of P. knowlesi isolates confirmed these two subpopulations in Malaysian Borneo and further identified a third distinct subpopulation (cluster 3) occurring in Peninsular Malaysia11. Microsatellite genotyping of additional human and macaque samples from Sarawak, Sabah and Peninsular Malaysia confirmed these divergent P. knowlesi subpopulations across the three regions12. A recent study, using allele-specific PCR to define further the molecular epidemiology of subpopulations, identified cluster 1 infections to be the commonest in all locations across Malaysian Borneo13. Furthermore, it showed that, amongst 1204 knowlesi malaria patients admitted to Kapit Hospital between 2000 and 2018, cluster 1 accounted for two-thirds of cases with no evidence of seasonal or temporal influence on this proportion.Micro-satellite genotyping of 15. In camera trapping surveys conducted in 5 localities in Sarawak, Mohd-Azlan et al. noted that pig-tailed macaques had a higher occupancy in both primary and secondary forests compared to long-tailed macaques16. Furthermore, long-tailed macaques were more likely to be found in open habitats such as Beach and Kerangas forests than pig-tailed macaques. There are currently no data relating to the relative abundance of the long-tailed and pig-tailed macaques in Sarawak. The higher proportion of patients seen at Kapit Hospital with cluster 1 P. knowlesi infections may be due to differences in abundance of the reservoir macaque hosts or to rural communities predominantly conducting activities such as subsistence farming and fishing in close proximity to their longhouses, and overlapping with the preferred habitats of long-tailed macaques. P. knowlesi parasites appear in the blood between 7 and 9 days after a person has been bitten by an infected mosquito17, so a history of travel within 14 days of hospital admission would indicate the most likely place a patient acquired the infection.The Kapit Division of Sarawak is mainly composed of primary and secondary forests, with over 600 rural communities living in longhouses accommodating between 5 and over 50 families. These longhouses are situated on river banks in close proximity to forests which are the habitats of long-tailed and pig-tailed macaques. Long-tailed macaques are found in primary, secondary and riparian forests, while pig-tailed macaques mainly prefer to live and forage in primary forestsP. knowlesi infections covered a wide spectrum of disease. Although most cases presented with uncomplicated infections, 6.5% were severe and there was a case fatality rate of 1.8%18. Subsequent studies in Sarawak19 and Sabah21 have also found mainly uncomplicated disease, and have identified older age, higher parasitaemia and proportions of schizonts, thrombocytopenia, abdominal pain and breathlessness as features associated with severe infections. Not all P. knowlesi infections result in disease as shown by community-based studies which have identified asymptomatic individuals with P. knowlesi infections in Vietnam22, Malaysian Borneo24 and Peninsular Malaysia25.In our first prospective clinical study of 107 cases of knowlesi malaria presenting to Kapit Hospital from 2006 to 2008, we found that P. knowlesi from specific macaque hosts raises the possibility that they represent two independent zoonoses with distinct clinical and laboratory features26. Therefore, a prospective study was undertaken at Kapit Hospital to determine whether i) the baseline characteristics and clinical course of patients differ by P. knowlesi subpopulation, and ii) any between-subpopulation differences have implications for clinical management. In addition, demographic and behavioural data were obtained from each patient to determine whether there were any between-subpopulation epidemiological differences.The recognition of two divergent subpopulations of P. knowlesi monoinfection , of which 298/413 (72.2%) cases were cluster 1 and 115/413 (27.8%) cluster 2. While there was no significant age difference between subpopulations, patients with cluster 2 infections were more likely to be male (p = 0.043) and to have reported recent jungle activity (p = 0.023) than those with cluster 1. After adjusting for age, types of activities remained associated with subpopulations, with patients with cluster 1 infections 1.8-fold  more likely to report peri-domestic activity than those with cluster 2 (Table p < 0.001) more likely to report peri-domestic activity compared to males and each year increase in age increasing the odds of reported peri-domestic activity (OR 1.04 (95% CI 1.03–1.06), p < 0.01)  and severe anaemia.The median duration of illness before presentation and route of hospital referral in the two groups were similar Table . Of the p < 0.001) and serum sodium concentrations (p = 0.031), and had higher serum total bilirubin (p = 0.049) and blood urea concentrations (p = 0.002), than those with cluster 2 infections . In the seven patients infected with both cluster 1 and 2 subpopulations, the median parasite count was similar to those in single subpopulation infections, specifically 1488  parasites/µL with 2/7 (28.6%) > 20,000/µL and 1/7 (14.3%) > 100,000/µL.The median parasite count for all Optimal thresholds for platelet count, serum concentrations of sodium, total bilirubin and urea controlled for age, sex and ln (parasitaemia) in multivariable logistic regression remained independently significant for differences between subpopulations  met 2 or more severe criteria . A higher incidence of severe disease was present in patients with cluster 1 infections (11.7% [95% CI: 8.3–16%] versus 7.0% [95% CI 3.1–13.3%] in cluster 2) but this did not reach statistical significance , with 50% (4/8) of severe cluster 2 cases having hyperparasitaemia  compared to 34% (12/35) in the severe cluster 1 group (p = 0.443)  after adjustment for age, sex and ln (parasitaemia).Ordinal regression was used to assess associations between cluster types and markers of end organ disease, and identified a significant association between clusters for renal dysfunction Fig. . ClusterOverall 12 patients developed one or more severe criteria, with 4 developing anaemia (two from each cluster respectively with one cluster 1 uncomplicated upon presentation), 2 developed ARDS (cluster 1 and mixed cluster), 1 developed ARDS with anaemia (cluster 2), 1 developed anaemia and acidosis (cluster 1), 1 developed anaemia and abnormal bleeding , 1 developed AKI and anaemia (cluster 1), 1 developed AKI and 1 developed hypotension respectively   (95% CI 0.04–1.3%).There were no deaths but two patients who were not recruited to the present study died during the study period; one identified posthumously as P. knowlesi in Kapit arising from different zoonotic hosts, one associated with long-tailed macaques (cluster 1) and the other with pig-tailed macaques (cluster 2)9, were prospectively characterised. Infections with each subpopulation resulted in a wide spectrum of disease but the clinical presentation was predominantly with uncomplicated malaria. Furthermore, the incidence of severe disease, the proportions of patients with individual criteria for severity, and the distribution of parasitaemia did not differ significantly between subpopulations. Haematological and biochemical findings revealed minor, clinically insignificant differences between subpopulations with cluster 1 infections having lower platelet counts and sodium serum concentrations, together with higher serum total bilirubin and urea concentrations, compared with cluster 2.In the present study, the clinical and laboratory features of patients infected with one of two divergent subpopulations of P. knowlesi subpopulations, including renal dysfunction26. In that study, the driver of a severe phenotype was an association with the P. knowlesi normocyte binding protein (PkNBP) xa gene, which along with the Duffy binding protein (DBP) gene, has been shown to be highly differentiated between the subpopulations30. PkNBP belongs to a family of invasion ligands proteins known as reticulocyte binding-like proteins that are associated with recognition of erythrocyte receptors upon which DBP proteins are released to begin invasion32. The dimorphism observed in these two genes do suggest plausible differences in efficiency of host cell selection and invasion between the two subpopulations. Disease severity in malaria is a complex interplay between the host’s immune system, parasite virulence and also entomological inoculation rate which contribute to the wide clinical spectrum commonly seen in malaria34. Therefore, clinical disease severity and virulence extend beyond heterogeneity in these genes which may explain the lack of distinct clinical phenotypes observed.An ordinal logistic regression model was used to explore markers of end organ disease and found that, after adjusting for age, sex and ln (parasitaemia), cluster 1 had a greater association with renal dysfunction. A previous study in patients at Sibu and Sarikei in Sarawak has shown increased end organ disease with selected alleles in 20, severe disease was present in 10.5% of all knowlesi malaria cases regardless of subpopulation. Also consistent with previous reports35, AKI was the commonest severity criteria observed, while two or more severity criteria were present in half of our patients with severe disease. Anaemia following treatment occurred in eight of the patients. Post-treatment anaemia in uncomplicated malaria is well recognised and multifactorial, but has been attributed mainly to haemolysis of both parasitized and non-parasitized erythrocytes36. Delayed haemolysis has also been reported with artemisinin derivatives which could be a possible contributing factor in the present series38. This is thought to result from shorter lifespan in pitted erythrocytes following treatment and this has been found to correlate with hyperparasitaemia in falciparum malaria37.Consistent with previous prospective studies in Sarawak and Sabah20. This may reflect between-study differences in diagnostic criteria. Overall there were no differences in severe phenotype between subpopulations but, since there were only eight severely ill patients with cluster 2 infection, more cases will need to be studied before definitive conclusions can be reached. The clinical characteristics of the seven patients infected with both subpopulations were not compared to the single cluster infections due to their small number. However, it is of interest that all were males with one severe case having 4 severe criteria including hyperparasitaemia .The incidence of patients meeting severe respiratory criteria in the current study  was higher than seen in a recent study from Sabah p = 0.13)18. Despite an increased number of health staff, improved awareness of knowlesi malaria as a potential diagnosis, and prompt use of artemisinin-based combination therapies at Kapit Hospital compared with a decade ago, the two deaths that occurred were associated with delays in diagnosis29. Although late presentation associated with limited local transportation and health infrastructure may have been unavoidable, it was also frequently observed in the early descriptions of fatal cases39. As thrombocytopenia is almost universally detected at presentation in knowlesi malaria cases, it remains one of the most useful prompts for including P. knowlesi infection in the differential diagnosis of illness in endemic areas and in travellers returning from malaria-endemic countries18.None of the patients recruited into the study died but, if two fatal knowlesi malaria cases that were known to have occurred during the study period were included, this would represent a mortality rate of 0.35%. This is lower, but not statistically different to, the rate of 1.8% observed during our first prospective study conducted 10 years ago at Kapit Hospital 40. All individuals who are blood slide microscopy-positive for malaria are required to be hospitalised in Malaysia and discharged when clinically well and have had two malaria-negative blood films on two consecutive days.A prospective observational study was conducted at Kapit Hospital, in the Kapit Division of Sarawak, Malaysian Borneo. Kapit Division covers an area of 38,934 kmP. knowlesi or P. malariae and no travel history outside of Malaysia within the previous 28 days were eligible for enrolment. Patients with significant co-morbidities (established end organ disease or co-infection) or who had received antimalarial treatment within the previous 14 days were excluded. Patients diagnosed at a peripheral health care facility were included if they arrived at Kapit Hospital within one hour of starting antimalarial treatment. Only patients with a PCR confirmed P. knowlesi monoinfection were subsequently included in the study. All patients provided informed written consent to the study procedures which were approved by the Medical Research Ethics Committee of Universiti Malaysia Sarawak and the Medical Research and Ethics Committee of the Ministry of Health Malaysia (NMRR-16-943-31224 [IIR]). All procedures on patients were performed in accordance with relevant guidelines and regulations.All non-pregnant patients aged ≥ 15 years admitted to Kapit Hospital between September 2016 and October 2018 with a positive blood film for either 27 at the time of, or during course of admission. The criteria were: hyperparasitaemia ; severe anaemia (haemoglobin concentration < 7 g/dL); hypotension (systolic blood pressure < 80 mmHg); AKI (serum creatinine > 265 μmol/L or blood urea > 20 mmol/L); jaundice (serum bilirubin > 50 μmol/L) with parasite count > 20,000/μL; hypoglycaemia (serum glucose < 2.2 mmol/L); acidosis (base deficit > 8 mEq/L or plasma bicarbonate < 15 mmol/L or venous plasma lactate ≥ 5 mmol/L); acute pulmonary oedema/acute respiratory distress syndrome (ARDS) (respiratory rate > 30 breaths/minute plus oxygen saturation < 92% on air and/or pulmonary infiltrates on chest radiograph); cerebral malaria (unarousable coma). In assessing the presence of acidosis, serum bicarbonate or base excess was used when plasma lactate was unavailable, or vice versa, as per WHO 2014 research criteria.Patient demographics, recent travel history including activities within 14 days prior to hospital admission, clinical details and baseline laboratory data were recorded on a standardised case report form by study clinicians. Recent peri-domestic activities were defined as any outdoor activities that were conducted around the perimeter of residential households and towards the town centre, while recent jungle activities refer to any outdoor activities inside deep jungle including logging, activities around the logging camps, jungle trekking and hunting. Ten mL of venous blood was collected from each patient for biochemical and haematologic tests. Severe knowlesi malaria disease was defined according to the WHO research criteria 201441. All patients received artemether–lumefantrine and/or intravenous artesunate depending on disease severity. Clinical management, including the investigation and treatment of bacterial infection, was otherwise at the discretion of the attending clinician. Similarly, each patient was assessed at least daily by the treating team and baseline investigations only repeated if clinically indicated.Treatment followed the Malaysian Ministry of Health (MMoH) guidelines, including the diagnosis of severe malaria in patients with > 20,000 parasites/µL42 and nested PCR assays for identification of malaria species with primers for P. falciparum, P. malariae, P. vivax, P. ovale, P. knowlesi, P. cynomolgi and P. inui were utilised as described previously44. Each P. knowlesi subpopulation was identified using cluster-specific PCR assays13. Parasite counts were determined using the average readings of two independent experienced laboratory technicians derived from the number of parasites per 500 white cells, adjusted by the total white cell count for each patient.Blood spots were prepared on filter paper from the blood sample taken from each patient and subsequently processed at the Malaria Research Centre, Universiti Malaysia Sarawak. DNA was extracted from the blood spots using InstaGene™Haematological profiles on-site at the hospital laboratory were determined using semi-automated methods . Citrated blood samples were analysed for coagulation studies with Sysmex® Ca 500v . Biochemistry tests were conducted using Beckman-Coulter™ AU480 and a point-of-care meter was used for determination of lactate levels .45 using arsenal, finalfit, pROC, cutpointr, ggplot2 and ggupset packages. Pairwise deletion was used in analysing the data with number of observations for each variable listed in Supplementary Table t-test and Mann–Whitney test respectively. Parasite densities were natural logarithm (ln) transformed before analysis. Proportions were compared using Fisher’s exact test with post hoc pairwise comparison using Bonferroni correction. Ordinal logistic regression was used to detect differences between subpopulations with normal, abnormal and defining criteria for severe disease severity, and adjustment for confounders. The categorisation of variables used in ordinal logistic regression was normal (1), abnormal but not severe (2) and severe (3) as summarised in Supplementary Table p value of < 0.05 (two-tailed) as significant.All analyses were carried out in R Studio version 1.2.1335Supplementary Tables.