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Abstract Background Insecticide-treated bed nets (ITNs) reduce malaria transmission and are an important prevention tool. However, there are still information gaps on how the reduction in malaria transmission by ITNs affects parasite genetics population structure. This study examined the relationship between transmission reduction from ITN use and the population genetic diversity of Plasmodium falciparum in an area of high ITN coverage in western Kenya. Methods Parasite genetic diversity was assessed by scoring eight single copy neutral multilocus microsatellite (MS) markers in samples collected from P. falciparum-infected children (< five years) before introduction of ITNs (1996, baseline, n = 69) and five years after intervention (2001, follow-up, n = 74). Results There were no significant changes in overall high mixed infections and unbiased expected heterozygosity between baseline (%MA = 94% and He = 0.75) and follow up (%MA = 95% and He = 0.79) years. However, locus specific analysis detected significant differences for some individual loci between the two time points. Pfg377 loci, a gametocyte-specific MS marker showed significant increase in mixed infections and He in the follow up survey (%MA = 53% and He = 0.57) compared to the baseline (%MA = 30% and He = 0.29). An opposite trend was observed in the erythrocyte binding protein (EBP) MS marker. There was moderate genetic differentiation at the Pfg377 and TAA60 loci (FST = 0.117 and 0.137 respectively) between the baseline and post-ITN parasite populations. Further analysis revealed linkage disequilibrium (LD) of the microsatellites in the baseline (14 significant pair-wise tests and ISA = 0.016) that was broken in the follow up parasite population (6 significant pairs and ISA = 0.0003). The locus specific change in He, the moderate population differentiation and break in LD between the baseline and follow up years suggest an underlying change in population sub-structure despite the stability in the overall genetic diversity and multiple infection levels. Conclusions The results from this study suggest that although P. falciparum population maintained an overall stability in genetic diversity after five years of high ITN coverage, there was significant locus specific change associated with gametocytes, marking these for further investigation.

Abstract Background An evaluation of the short-term impact on childhood malaria morbidity of mass distribution of free long-lasting insecticidal nets (LLINs) to households with children aged 9-59 months as part of the Togo National Integrated Child Health Campaign. Methods The prevalence of anaemia and malaria in children aged zero to 59 months was measured during two cross-sectional household cluster-sample surveys conducted during the peak malaria transmission, three months before (Sept 2004, n = 2521) and nine months after the campaign (Sept 2005, n = 2813) in three districts representative of Togo's three epidemiological malaria transmission regions: southern tropical coastal plains (Yoto), central fertile highlands (Ogou) and northern semi-arid savannah (Tone). Results In households with children 65% in all 3 districts. Reported ITN use by children during the previous night was 35.9%, 43.8% and 80.6% in Yoto, Ogou and Tone, respectively. Rainfall patterns were comparable in both years. The overall prevalence of moderate to severe anaemia (Hb < 8.0 g/dL) was reduced by 28% (prevalence ratio [PR] 0.72, 95% CI 0.62-0.84) and mean haemoglobin was increased by 0.35 g/dL (95% CI 0.25-0.45). The effect was predominantly seen in children aged 18-59 months and in the two southern districts: PR (95% CI) for moderate to severe anaemia and clinical malaria: Yoto 0.62 (0.44-0.88) and 0.49 (0.35-0.75); Ogou 0.54 (0.37-0.79) and 0.85 (0.57-1.27), respectively. Similar reductions occurred in children Conclusions A marked reduction in childhood malaria associated morbidity was observed in the year following mass distribution of free LLINs in two of the three districts in Togo. Sub-national level impact evaluations will contribute to a better understanding of the impact of expanding national malaria control efforts.

Abstract Background Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined. Methods Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya. Results An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21). Conclusions This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

Abstract Background Information on the burden of malaria in early infancy is scarce. Young infants are relatively protected against clinical malaria during the first six months of life due to the presence of maternal antibodies and foetal haemoglobin, and have received relatively little attention with respect to research and treatment guidelines. The World Health Organization provides treatment guidelines for children from six months onwards, without specific treatment guidelines for the younger infants. A number of recent reports however suggest that the burden in this young age group may be underestimated. Methods A retrospective review of paediatric hospital records at the Queen Elizabeth Central Hospital in Blantyre from 1998 to 2008 from three data sources was carried out. The number of admitted infants Results Retrospective analysis of hospital records showed that over the course of these years, the average annual proportion of paediatric admissions in children ≤ 15 years with confirmed malaria aged Conclusions These findings are consistent with recent reports suggesting that the burden of malaria during the six first months of life may be substantial, and highlight that more research is needed on dose-optimization, safety and efficacy of anti-malarials that are currently used off-label in this vulnerable patient group.

Functional analyses are the main method to classify mismatch repair (MMR) gene variants of uncertain significance (VUSs). However, the pathogenicity remains unclear for many variants because of conflicting results between clinical, molecular, and functional data. In this study, we evaluated whether whole exome sequencing (WES) could add another layer of evidence to elucidate the pathogenicity of MMR variants with conflicting interpretations. WES was performed on formalin-fixed paraffin-embedded tumor tissue of eight patients with a constitutional MMR VUS (seven families), including eight colorectal and two endometrial carcinomas and one ovarian carcinoma. Cell-free CIMRA assays were performed to assign Odds of Pathogenicity to these VUSs. In four families, seven tumors showed MMR deficiency-associated mutational signatures, supporting the pathogenicity of the VUS. Moreover, somatic (second) MMR hits identified in the WES data were found to explain MMR staining patterns when the MMR staining was discordant with the reported germline MMR gene variant. In conclusion, WES did not significantly reclassify VUS in these cases but clarified some phenotypic aspects such as age of onset and explanations in case of discordant MMR stainings., Competing Interests: Declaration of competing interest On behalf of myself and the other authors, I declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pathogenesis of duodenal tumors in the inherited tumor syndromes familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) is poorly understood. This study aimed to identify genes that are significantly mutated in these tumors and to explore the effects of these mutations. Whole exome and whole transcriptome sequencing identified recurrent somatic coding variants of phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) in 19/70 (27%) FAP and MAP duodenal adenomas, and further confirmed the established driver roles for APC and KRAS. PIGA catalyzes the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Flow cytometry of PIGA-mutant adenoma-derived and CRISPR-edited duodenal organoids confirmed loss of GPI anchors in duodenal epithelial cells and transcriptional profiling of duodenal adenomas revealed transcriptional signatures associated with loss of PIGA., Implications: PIGA somatic mutation in duodenal tumors from patients with FAP and MAP and loss of membrane GPI-anchors may present new opportunities for understanding and intervention in duodenal tumorigenesis., (©2024 The Authors; Published by the American Association for Cancer Research.)

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Colibactin, a genotoxin produced by polyketide synthase harboring (pks + ) bacteria, induces double-strand breaks and chromosome aberrations. Consequently, enrichment of pks + Escherichia coli in colorectal cancer and polyposis suggests a possible carcinogenic effect in the large intestine. Additionally, specific colibactin-associated mutational signatures; SBS88 and ID18 in the Catalogue of Somatic Mutations in Cancer database, are detected in colorectal carcinomas. Previous research showed that a recurrent APC splice variant perfectly fits SBS88., Methods: In this study, we explore the presence of colibactin-associated signatures and fecal pks in an unexplained polyposis cohort. Somatic targeted Next-Generation Sequencing (NGS) was performed for 379 patients. Additionally, for a subset of 29 patients, metagenomics was performed on feces and mutational signature analyses using Whole-Genome Sequencing (WGS) on Formalin-Fixed Paraffin Embedded (FFPE) colorectal tissue blocks., Results: NGS showed somatic APC variants fitting SBS88 or ID18 in at least one colorectal adenoma or carcinoma in 29% of patients. Fecal metagenomic analyses revealed enriched presence of pks genes in patients with somatic variants fitting colibactin-associated signatures compared to patients without variants fitting colibactin-associated signatures. Also, mutational signature analyses showed enrichment of SBS88 and ID18 in patients with variants fitting these signatures in NGS compared to patients without., Conclusions: These findings further support colibactins ability to mutagenize colorectal mucosa and contribute to the development of colorectal adenomas and carcinomas explaining a relevant part of patients with unexplained polyposis., (© 2024. The Author(s).)

Purpose: BRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC., Methods: The functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status., Results: The RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test., Conclusion: Given its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification., (© 2023. The Author(s).)

Diagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This study aimed to identify the various causes of the discordant phenotypes of colorectal cancer (CRC) and endometrial cancer (EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6 variant diagnosed with CRC or EC were categorized based on an microsatellite instability (MSI)/IHC test outcome that might fail to result in a diagnosis of LS (eg, retained staining of all 4 mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6 variant carriers with CRC or EC (n = 590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, 2 out of 3 MSI/IHC cases were found to be concordant with the MSH6 variant, and NGS showed that 4 discordant IHC results were sporadic rather than LS-associated tumors. In 1 case, somatic events explained the discordant phenotype. The use of reflex IHC mismatch repair testing, the current standard in most Western countries, may lead to the misdiagnosis of germline MSH6 variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should be considered in case of a strong positive family history. Germline DNA analysis of the mismatch repair genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Minichromosome Maintenance 8 Homologous Recombination Repair Factor (MCM8) and Minichromosome Maintenance 9 Homologous Recombination Repair Factor (MCM9) are recently discovered minichromosome maintenance proteins and are implicated in multiple DNA-related processes and pathologies, including DNA replication (initiation), meiosis, homologous recombination and mismatch repair. Consistent with these molecular functions, variants of MCM8/MCM9 may predispose carriers to disorders such as infertility and cancer and should therefore be included in relevant diagnostic testing. In this overview of the (patho)physiological functions of MCM8 and MCM9 and the phenotype of MCM8/MCM9 variant carriers, we explore the potential clinical implications of MCM8/MCM9 variant carriership and highlight important future directions of MCM8 and MCM9 research. With this review, we hope to contribute to better MCM8/MCM9 variant carrier management and the potential utilization of MCM8 and MCM9 in other facets of scientific research and medical care., Competing Interests: None., (© 2023 The Author(s).)

Small intestine-neuroendocrine tumors (SI-NETs) are one of the most common tumors of the small bowel. Despite an increasing incidence, the exact mechanisms driving underlying pathology remain to be determined. Interestingly, recent studies linked the development of (SI-)NETs to both Lynch syndrome (LS) and MUTYH variants. If confirmed, these associations would have important consequences for treatment. In this study we therefore investigated the prevalence of mismatch repair (MMR) deficiency and MUTYH variants in 64 primary resected SI-NETs. Immunohistochemistry was used to assess the expression of the MMR genes, and competitive allele-specific PCR (KASPar) targeting two hotspot MUTYH variants [p.(Tyr179Cys), p.(Gly396Asp)] was performed to determine their prevalence in SI-NETs. Strikingly, all 64 SI-NETs stained positive for MSH6 and PMS2, indicating MMR proficiency. In addition, no MUTYH hotspot variant was found in any of the 64 SI-NETs. As such, these results do not support an association between SI-NET development and LS or MUTYH variants. In order to gain insight into SI-NET pathogenesis and optimally manage patients, future research should therefore focus on other candidate genes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Previous estimates of the prevalence of mismatch repair (MMR) deficiency and Lynch syndrome in small bowel cancer have varied widely. The aim of this study was to establish the prevalence of MMR deficiency and Lynch syndrome in a large group of small bowel adenocarcinomas., Methods: To this end, a total of 400 small bowel adenocarcinomas (332 resections, 68 biopsies) were collected through the Dutch nationwide registry of histopathology and cytopathology (Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief (PALGA)). No preselection criteria, such as family history, were applied, thus avoiding (ascertainment) bias. MMR deficiency status was determined by immunohistochemical staining of MMR proteins, supplemented by MLH1 promoter hypermethylation analysis and next generation sequencing of the MMR genes., Results: MMR deficiency was observed in 22.3% of resected and 4.4% of biopsied small bowel carcinomas. Prevalence of Lynch syndrome was 6.2% in resections and 0.0% in biopsy samples. Patients with Lynch syndrome-associated small bowel cancer were significantly younger at the time of diagnosis than patients with MMR-proficient and sporadic MMR-deficient cancers (mean age of 54.6 years vs 66.6 years and 68.8 years, respectively, p<0.000)., Conclusions: The prevalence of MMR deficiency and Lynch syndrome in resected small bowel adenocarcinomas is at least comparable to prevalence in colorectal cancers, a finding relevant both for treatment (immunotherapy) and family management. We recommend that all small bowel adenocarcinomas should be screened for MMR deficiency., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Lynch syndrome (LS) is a hereditary cancer syndrome that accounts for 3% of all new colorectal cancer (CRC) cases. Patients carry a germline pathogenic variant in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2), which encode proteins involved in a post-replicative proofreading and editing mechanism. The clinical presentation of LS is highly heterogeneous, showing high variability in age at onset and penetrance of cancer, which may be partly attributable to the molecular profiles of carcinomas. This review discusses the frequency of alterations in the WNT/B-CATENIN, RAF/MEK/ERK and PI3K/PTEN/AKT pathways identified in all four LS subgroups and how these changes may relate to the 'three pathway model' of carcinogenesis, in which LS CRCs develop from MMR-proficient adenomas, MMR-deficient adenomas or directly from MMR-deficient crypts. Understanding the specific differences in carcinogenesis for each LS subgroup will aid in the further optimization of guidelines for diagnosis, surveillance and treatment., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APC and MUTYH variant analysis between 1992 and 2017 were collected (n = 2082). Using the data provided on the application form, the APC and biallelic MUTYH prevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APC gene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50-99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged <60 and >20 adenomas aged <70. Patients with variants outside these criteria had FAP-related extracolonic manifestations, colorectal cancer aged <40, somatic KRAS c.34G > T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APC and MUTYH testing in patients with >10 adenomas aged <60 and with >20 adenomas aged <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing.

Substantial progress has been made in the control of malaria in Africa but much remains to be done before malaria elimination on the continent can be achieved. Further progress can be made by enhancing uptake of existing control tools but, in high transmission areas, additional tools will be needed. Development and evaluation of these new tools will require a substantial cadre of African scientists well trained in many different disciplines. This paper describes the activities undertaken by the Malaria Capacity Development Consortium (MCDC) to support the careers of PhD students and postdoctoral fellows undertaking research on malaria at five African universities. A systematic assessment of constraints on PhD training and research support systems was undertaken at each partner African university at the beginning of the programme and many of these constraints were remedied. The success of the programme is shown by the fact that 18 of the 21 PhD students recruited to the programme completed their theses successfully within a 4-year period and that all 27 scientists recruited to the postdoctoral programme were still working in Africa on its completion. The work of the consortium will be continued through Career Development Groups established at each partner university and at an affiliated institution at the University of Nairobi and through the Developing Excellence in Leadership, Training and Science award from the Wellcome Trust made to one of the African partners. Lessons learnt during the MCDC programme may help the planning and execution of other research capacity development programmes in Africa., Competing Interests: Competing interests: None declared.

Purpose: In preparation for a tight glycaemic control (TGC) clinical trial we assessed the agreement between methods used to measure blood glucose in critically ill children., Methods: Service evaluation comparing blood gas and main laboratory analysers with point-of-care (POC) devices PCX, ACCU-Chek and Hemocue., Results: Two hundred forty-five samples from 157 children measured on 2-4 devices provided 790 values. Marked variation was evident in glucose values between devices, time between tests, sample (whole blood/plasma) and source; 39% of paired values had >20% difference. The decision to start insulin at 7 mmol/L differed depending on the device used for 33% of samples. At low glucose values (<4 mmol/L), differences up to 1.8 mmol/L were evident. The blood gas analyser read lower than all POC models and the laboratory analyser (less risk of undetected hypoglycaemia). An inverse relationship was evident between haematocrit (Hct) and glucose error using POC devices. PCX values for samples with Hct <30% were higher (85%), whereas those for Hct values >38% were lower (66%). Glycolysis occurred during transfer of samples to the laboratory. Using the PCX at the bedside resulted in 0.5 mmol/L mean difference higher than laboratory values; locating the PCX in the laboratory reduced this to 0.2 mmol/L., Conclusions: Discrepancies between measurements may mask hypoglycaemia, and the potential benefits of controlling hyperglycaemia may not be achieved. Variation introduced by different devices, sample or source may have led to misclassification of treatment decisions contributing to the conflicting results of TGC studies.

Despite the clear public health benefit of insecticide-treated bednets (ITNs), the impact of malaria transmission-reduction by vector control on the spread of drug resistance is not well understood. In the present study, the effect of sustained transmission reduction by ITNs on the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) in children under the age of five years was investigated during an ITN trial in Asembo area, western Kenya. During the ITN trial, the national first line antimalarial treatment changed from CQ to SP. Smear-positive samples collected from cross sectional surveys prior to ITN introduction (baseline, n = 250) and five years post-ITN intervention (year 5 survey, n = 242) were genotyped for single nucleotide polymorphisms (SNPs) at dhfr-51, 59, 108, 164 and dhps-437, 540 (SP resistance), and pfcrt-76 and pfmdr1-86 (CQ resistance). The association between the drug resistance mutations and epidemiological variables was evaluated. There were significant increases in the prevalence of SP dhps mutations and the dhfr/dhps quintuple mutant, and a significant reduction in the proportion of mixed infections detected at dhfr-51, 59 and dhps-437, 540 SNPs from baseline to the year 5 survey. There was no change in the high prevalence of pfcrt-76 and pfmdr1-86 mutations. Multivariable regression analysis further showed that current antifolate use and year of survey were significantly associated with more SP drug resistance mutations. These results suggest that increased antifolate drug use due to drug policy change likely led to the high prevalence of SP mutations 5 years post-ITN intervention and reduced transmission had no apparent effect on the existing high prevalence of CQ mutations. There is no evidence from the current study that sustained transmission reduction by ITNs reduces the prevalence of genes associated with malaria drug resistance.

Background: Insecticide-treated bed nets (ITNs) reduce malaria transmission and are an important prevention tool. However, there are still information gaps on how the reduction in malaria transmission by ITNs affects parasite genetics population structure. This study examined the relationship between transmission reduction from ITN use and the population genetic diversity of Plasmodium falciparum in an area of high ITN coverage in western Kenya., Methods: Parasite genetic diversity was assessed by scoring eight single copy neutral multilocus microsatellite (MS) markers in samples collected from P. falciparum-infected children (< five years) before introduction of ITNs (1996, baseline, n = 69) and five years after intervention (2001, follow-up, n = 74)., Results: There were no significant changes in overall high mixed infections and unbiased expected heterozygosity between baseline (%MA = 94% and He = 0.75) and follow up (%MA = 95% and He = 0.79) years. However, locus specific analysis detected significant differences for some individual loci between the two time points. Pfg377 loci, a gametocyte-specific MS marker showed significant increase in mixed infections and He in the follow up survey (%MA = 53% and He = 0.57) compared to the baseline (%MA = 30% and He = 0.29). An opposite trend was observed in the erythrocyte binding protein (EBP) MS marker. There was moderate genetic differentiation at the Pfg377 and TAA60 loci (FST = 0.117 and 0.137 respectively) between the baseline and post-ITN parasite populations. Further analysis revealed linkage disequilibrium (LD) of the microsatellites in the baseline (14 significant pair-wise tests and ISA = 0.016) that was broken in the follow up parasite population (6 significant pairs and ISA = 0.0003). The locus specific change in He, the moderate population differentiation and break in LD between the baseline and follow up years suggest an underlying change in population sub-structure despite the stability in the overall genetic diversity and multiple infection levels., Conclusions: The results from this study suggest that although P. falciparum population maintained an overall stability in genetic diversity after five years of high ITN coverage, there was significant locus specific change associated with gametocytes, marking these for further investigation.

Background: Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined., Methods: Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya., Results: An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21)., Conclusions: This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

A polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene, with a guanine to adenine nucleotide change at position -308, TNF2 is associated with increased TNF-alpha production. TNF2 homozygotes have a higher risk of severe disease and/or death due to cerebral malaria and other infectious diseases. We investigated the impact of this allele on malaria morbidity and mortality in young children who participated in an immuno-epidemiologic cohort study of malaria in an area of intense perennial Plasmodium falciparum transmission in western Kenya. A total of 1,048 children were genotyped. Poisson regression and Cox proportional hazards models were used to determine the relationship between TNF-308 variants and morbidity and mortality. The gene frequencies of the TNF1 and TNF2 alleles were 0.90 and 0.10, respectively. TNF2 homozygosity was associated with pre-term birth when compared with TNF1 homozygotes [relative risk (RR) 7.3, 95% CI, 2.85-18.9, P = 0.002) and heterozygotes (RR 6.7, 95% CI 2.0-23.0, P = 0.008). Among children born prematurely, the TNF2 allele was significantly associated with a higher risk of death in infancy compared with TNF1 (RR 7.47, 95% CI 2.36-23.6). The risk of death was higher among TNF2 homozygotes than among heterozygotes. The TNF2 allele was significantly associated with high density P. falciparum parasitemia (RR 1.11, 95% CI 1.0-1.24). Among low birth weight children, the TNF2 allele was associated with severe anemia (RR 2.16, 95% CI 1.17-4.01) and showed a trend toward a risk for severe malaria anemia (RR 1.99, 95% CI 0.89-4.46). These data suggest that TNF2 is a risk factor for pre-term birth and early childhood mortality and malaria morbidity in children in this region. Further understanding of the pathogenic mechanisms underlying this association is required., (Copyright 2001 Wiley-Liss, Inc.)