Putative pathogen-selected polymorphisms in the PKLR gene are associated with mycobacterial susceptibility in Brazilian and African populations.

Pyruvate kinase (PK), encoded by the PKLR gene, is a key player in glycolysis controlling the integrity of erythrocytes. Due to Plasmodium selection, mutations for PK deficiency, which leads to hemolytic anemia, are associated with resistance to malaria in sub-Saharan Africa and with susceptibility to intracellular pathogens in experimental models. In this case-control study, we enrolled 4,555 individuals and investigated whether PKLR single nucleotide polymorphisms (SNPs) putatively selected for malaria resistance are associated with susceptibility to leprosy across Brazil (Manaus-North; Salvador-Northeast; Rondonópolis-Midwest and Rio de Janeiro-Southeast) and with tuberculosis in Mozambique. Haplotype T/G/G (rs1052176/rs4971072/rs11264359) was associated with leprosy susceptibility in Rio de Janeiro (OR = 2.46, p = 0.00001) and Salvador (OR = 1.57, p = 0.04), and with tuberculosis in Mozambique (OR = 1.52, p = 0.07). This haplotype downregulates PKLR expression in nerve and skin, accordingly to GTEx, and might subtly modulate ferritin and haptoglobin levels in serum. Furthermore, we observed genetic signatures of positive selection in the HCN3 gene (xpEHH>2 -recent selection) in Europe but not in Africa, involving 6 SNPs which are PKLR/HCN3 eQTLs. However, this evidence was not corroborated by the other tests (FST, Tajima's D and iHS). Altogether, we provide evidence that a common PKLR locus in Africans contribute to mycobacterial susceptibility in African descent populations and also highlight, for first, PKLR as a susceptibility gene for leprosy and TB.

Compromise of Second-Line Antiretroviral Therapy Due to High Rates of Human Immunodeficiency Virus Drug Resistance in Mozambican Treatment-Experienced Children With Virologic Failure.

BACKGROUND: Virologic failure (VF) is highly prevalent in sub-Saharan African children on antiretroviral therapy (ART) and is often associated with human immunodeficiency virus drug resistance (DR). Most children still lack access to routine viral load (VL) monitoring for early identification of treatment failure, with implications for the efficacy of second-line ART. METHODS: Children aged 1 to 14 years on ART for ≥12 months at 6 public facilities in Maputo, Mozambique were consecutively enrolled after informed consent. Chart review and caregiver interviews were conducted. VL testing was performed, and specimens with ≥1000 copies/mL were genotyped. RESULTS: Of the 715 children included, the mean age was 103 months, 85.8% had no immunosuppression, 73.1% were taking stavudine/lamivudine/nevirapine, and 20.1% had a history prevention of mother-to-child transmission exposure. The mean time on ART was 60.0 months. VF was present in 259 patients (36.3%); 248 (95.8%) specimens were genotyped, and DR mutations were found in 238 (96.0%). Severe immunosuppression and nutritional decline were associated with DR. M184V and Y181C were the most common mutations. In the 238 patients with DR, standard second-line ART would have 0, 1, 2, and 3 effective antiretrovirals in 1 (0.4%), 74 (31.1%), 150 (63.0%), and 13 (5.5%) patients, respectively. CONCLUSION: This cohort had high rates of VF and DR with frequent compromise of second-line ART. There is urgent need to scale-up VL monitoring and heat-stable protease inhibitor formulations or integrase inhibitorsfor a more a durable first-line regimen that can feasibly be implemented in developing settings.

Performance characteristics of finger-stick dried blood spots (DBS) on the determination of human immunodeficiency virus (HIV) treatment failure in a pediatric population in Mozambique.

Quantitative plasma viral load (VL) at 1000 copies /mL was recommended as the threshold to confirm antiretroviral therapy (ART) failure by the World Health Organization (WHO). Because of ongoing challenges of using plasma for VL testing in resource-limited settings (RLS), especially for children, this study collected 717 DBS and paired plasma samples from children receiving ART ≥1 year in Mozambique and compared the performance of DBS using Abbott's VL test with a paired plasma sample using Roche's VL test. At a cut-off of 1000 copies/mL, sensitivity of DBS using Abbott DBS VL test was 79.9%, better than 71.0% and 63.9% at 3000 and 5000 copies/mL, respectively. Specificities were 97.6%, 98.8%, 99.3% at 1000, 3000, and 5000 copies/mL, respectively. The Kappa value at 1000 copies/mL, 0.80 (95% CI: 0.73, 0.87), was higher than 0.73 (95% CI: 0.66, 0.80) and 0.66 (95% CI: 0.59, 0.73) at 3000, 5000 copies/mL, respectively, also indicating better agreement. The mean difference between the DBS and plasma VL tests with 95% limits of agreement by Bland-Altman was 0.311 (-0.908, 1.530). Among 73 children with plasma VL between 1000 to 5000 copies/mL, the DBS results were undetectable in 53 at the 1000 copies/mL threshold. While one DBS sample in the Abbott DBS VL test may be an alternative method to confirm ART failure at 1000 copies/mL threshold when a plasma sample is not an option for treatment monitoring, because of sensitivity concerns between 1,000 and 5,000 copies/ml, two DBS samples may be preferred accompanied by careful patient monitoring and repeat testing.

Evaluation of a high-throughput diagnostic system for detection of HIV-1 in dried blood spot samples from infants in Mozambique.

We performed a comparative analysis between Roche Amplicor HIV-1 DNA test and CAPTAQ assay for the detection of HIV in 830 dried blood spot (DBS) pediatric samples collected in Mozambique. Our results demonstrated no statistical difference between these assays. The CAPTAQ assay approached nearly 100% repeatability/accuracy. The increased throughput of testing with minimal operator interference in performing the CAPTAQ assay clearly demonstrated that this method is an improvement over the Roche Amplicor HIV-1 DNA test, version 1.5.

Human leukocyte antigen-A, -B, and -DRB1 allele and haplotype frequencies in the Mozambican population: a blood donor-based population study.

Human leukocyte antigen (HLA) has been used for several decades as genetic markers for analyzing diversity of gene pool origin, platelet transfusion, tissue transplantation, disease susceptibility or resistance, and forensic and anthropological studies. In the present study, the allele and haplotype frequencies of HLA-A, -B, and -DRB1 were studied in 250 unrelated Mozambican individuals (black African from south of Mozambique Basin) by using a low-medium resolution polymerase chain reaction-Luminex typing method. A total of 18 A, 25 B, and 13 DRB1 alleles were identified. The most frequent HLA-A, -B, and -DRB1 alleles were HLA-A*30 (23.9%), HLA-B*15 (15.6%), and HLA-DRB1*13 (19.8%), respectively. The most frequent two-locus haplotypes were HLA-A*30-B*42 (7.4%) and HLA-B*42-DRB1*03 (5.4%), and three-locus haplotypes were HLA-A*30-B*42-DRB1*03 (4.9%), and HLA-A*02-B*58-DRB1*11 (4.1%). Allele distribution and haplotype analysis demonstrated that Mozambican population shares HLA patterns with sub-Saharan populations.

Polimorfismos em genes de citocinas em dadores de sangue moçambicanos / Polymorphisms in cytokine genes in Mozambique blood donors

Determinadas alteraçõesdos marcadores imunogenéticos são importantes para o controle de expressão gênica, ou aténa alteração da estrutura ou função de proteínas. Pequenas variações genéticas como os polimorfismos de nucleótidos únicos (SNPs, do inglês ­Single Nucleotide Polymorphism) estão associadas a susceptibilidade ou resistência do hospedeiro em doenças diferentes. Um mapeamento generalizado de SNPs em genes potencialmente envolvidos com a resposta imune pode ajudar no entendimento da fisiopatologia de doenças não apenas infecciosas, assim como de diversas etiologias em Moçambique. Neste contexto, um estudo foi desenvolvido no qual amostras de sangue foram colhidas em quinhentos dadores de sangue Moçambicanos. Este estudo teve como objectivos analisar à frequência demarcadores imunogenéticos com enfoque para os genes IL-10 (-819 C> T; -3575 A> T), TNF(-308G>A) eLTA (+252 A>G).O DNA genómico foi extraído de amostras de sangue total utilizando o QIAamp DNA mini kit (Qiagen, Germany);e a identificação das variantes polimórficas de todos os investigados foi realizada pelas técnicas de PCR em tempo real assim como PCR-RFLP. As frequências genotípicas das variantes alélicas de todos os genes analisados estavam em equilíbrio de Hardy-Weinberg.As frequências das variantes alélicas para o SNP LTA +252 (A>G)foram 0.47e 0,53 para os alelos G e A, enquanto que para o SNP TNF (-308G>A) foram 0,14 e 0,86 para os alelos A e G, respectivamente. Para o SNP -819 (T>C) as frequências foram: 0,59 para o alelo C e 0,41 para o alelo T, enquanto que para o SNP -3575 (A>T) foram 0,77 e 0,23 para os alelos T e A. As frequências genotípicas e alélicas dos quatro SNPs nos genes estudados não são diferentes entre os grupos de dadores quando estraficados quanto ao local de nascimento embora a frequência do alelo -308A seja maior no sul e diminui no centro e depois no norte do país. O mesmo acontece com a frequência do alelo -3575 A. A análise combinada dos SNPs LTA+252 A>G e TNF-308 G>A revelou um valor de D'= 0.691;r2= 0,078 para o desequilíbrio de ligação assim como valores de D'= 0,665,er2= 0,090para os SNPs IL 10-819 T>C e IL10-3575 A>T. Dos 4 haplótipos identificados, o haplótipo LTA+252/TNF-308 (A/G) foi o mais frequente com um percentual de 51% e para a combinação de SNPs IL10-3575/-819 o haplótipo AT foi o menos predominante com um percentual de 3.4%. As frequências dos SNPs estudados são muito parecidas com as frequências relatadas em populações Africanas e diferentes de populações Brasileiras, Europeias e Asiáticas. O presente estudo é o primeiro a descrever SNPs em genes de citocinas relevantes e possibilita que no futuro se efectuem estudos de associação a suscetibilidade a doenças infecciosas como a tuberculose, malária e HIV/SIDA em Moçambique.

Variations in the DNA sequence can influence gene expression and also change the protein structure or function. Small genetic variations such as single nucleotide polymorphisms (SNPs) are associated with susceptibility or host resistance in different diseases. Thus, a generalized mapping of SNPs in genes potentially involved in immune responses may help to understand the pathophysiology of infectious diseases and also disorders from other etiologies in Mozambique. In this context, the aim of this study was to analyze the frequency of immunogenetic markers, focusing on the genes IL10 (-819 C>T; -3575 A>T), TNF(-308 G>A) and LTA(+252 A>G).Genomic DNA was extracted from whole blood samples collected from five hundred Mozambican blood donors using the QIA amp DNA mini kit (Qiagen, Germany). The identification of the polymorphic variants was performed by PCR-RFLP and real-time PCR. The genotype frequencies of all SNPs analyzed were in Hardy-Weinberg equilibrium. The frequencies of allelic variants for the LTA +252 SNP (A>G) were 0.47 and 0.53for alleles Gand A, while for TNF SNPs (-308 G>A) were 0.14 and 0.86 for the alleles A and G, respectively. For SNP -819 (C>T) frequencies were0.59 for allele C and 0.41 for alleleT, while for the SNP -3575 (T>A) were 0.77 and 0.23 for the alleles T and A, respectively. The frequencies of the four SNP analyzed here were not statistically different when the volunteers were compared according to the birth place, although the frequency of -308A was higher in the south and decreased towards centre and then to north of the country. Combined analysis of SNPs LTA +252 A> G and TNF-308 G> A showed a value of D '= 0691, r2= 0.078 for linkage disequilibrium as well as values of D' = 0.665 and r2= 0.090 for SNPs IL 10-819 T> C and IL10-3575 A> T of the four haplotypes identified haplotype LTA +252 / TNF-308 (A/G) was the most frequent with an incidence of 51% and the combination of SNPs IL10-3575/-819 AT haplotype was less prevalent with a percentage of 3.4%.The SNP frequencies were also similar to the frequencies observed among African, but not to Brazilian, European and Asian populations. This study is the first to describe relevant SNPs in cytokine genes and enables association studies of susceptibility with infectious diseases as tuberculosis, malaria and HIV/AIDS in Mozambique.