Baseline prevalence of molecular marker of sulfadoxine/pyrimethamine resistance in Ebonyi and Osun states, Nigeria: amplicon deep sequencing of dhps-540.

BACKGROUND: Chemoprevention plays an important role in malaria control strategy. Perennial malaria chemoprevention (PMC) using sulfadoxine/pyrimethamine (SP) is a WHO-approved strategy to combat malaria in young children and may lead to drug pressure. Introducing SP-PMC may therefore be compromised due to the emergence of Plasmodium falciparum resistant to SP, particularly mutation at K540E of the dihydropteroate synthase (dhps) gene. Molecular surveillance of resistance markers can support assessment of antimalarial efficacy and effectiveness. High prevalence of 540E is associated with reduced effectiveness of SP, and areas with more than 50% prevalence are considered unsuitable for intermittent preventative treatment in pregnancy (IPTp) implementation. Assessing 540E prevalence is an important undertaking before implementation of SP-PMC. METHODS: We conducted a rapid surveillance of dhps-540E to assess the suitability of SP as PMC in field studies from Ebonyi and Osun states in Nigeria. We used an in-house developed amplicon deep-sequencing method targeting part of the dhps gene. RESULTS: Our data reveal that 18.56% of individuals evaluated carried the 540E mutation mixed with the WT K540. Mutant variant 540E alone was not found, and 80% of isolates harboured only WT (K540). Clonal analysis of the sequencing data shows a very low proportion of 540E circulating in both states. CONCLUSIONS: Our data show that both states are suitable for SP-PMC implementation and, based on this finding, SP-PMC was implemented in Osun in 2022. Continuous monitoring of 540E will be required to ensure the chemoprevention effectiveness of SP in Nigeria.

Effect of sulfadoxine-pyrimethamine chemoprophylaxis in pregnant women on selection of the new P. falciparum dhps quintuple mutant carrying the I431V mutation.

BACKGROUND: A new mutation in the Plasmodium falciparum dihydropteroate synthetase gene (pfdhps), I431V, has been identified in several countries of Central and West Africa. This mutation is mostly found in association with four other SNPs on pfdhps (S436A, A437G, A581G and A613S), forming a quintuple mutant (vagKgs) and almost always associated with the Plasmodium falciparum dihydrofolate reductase gene (pfdhfr) CirnI (C50R, N51I, S108N) triple mutant. To date, nothing is known about the impact of this new pfdhps genotype on sulfadoxine-pyrimethamine (SP) resistance. OBJECTIVES: We sought to assess the prevalence of this pfdhps vagKgs quintuple mutant in two groups of pregnant women with malaria, one that took intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and one that did not. METHODS: The pfdhfr and pfdhps genes from Plasmodium falciparum isolates collected in Yaoundé (Cameroon) from pregnant women with symptomatic malaria under IPTp-SP or not, were sequenced. RESULTS: Of 159 patients evaluated, 70 had already taken SP during pregnancy and 89 had never taken SP. Only the vagKgs allele was significantly overrepresented in the SP+ group (21.4% versus 3.4%; P < 0.001), whereas the ISgKAA mutant, widely distributed in this area and known to be less susceptible to SP, tended to be less abundant in this group (48.6% versus 64.0%; P = 0.0503). CONCLUSIONS: We found a strong overrepresentation of the CirnI/vagKgs haplotype in the IPTp-SP pregnant group, suggesting a high level of resistance of this mutant to SP. This could compromise not only the effectiveness of IPTp-SP but also the seasonal malaria chemoprevention of young children, now widely implemented.

Analysis of random mutations in Salmonella Gallinarum dihydropteroate synthase conferring sulfonamide resistance.

In bacteria and primitive eukaryotes, sulfonamide antibiotics block the folate pathway by inhibiting dihydropteroate synthase (FolP) that combines para-aminobenzoic acid (pABA) and dihydropterin pyrophosphate (DHPP) to form dihydropteroic acid (DHP), a precursor for tetrahydrofolate synthesis. However, the emergence of resistant strains has severely compromised the use of pABA mimetics as sulfonamide drugs. Salmonella enterica serovar Gallinarum (S. Gallinarum) is a significant source of antibiotic-resistant infections in poultry. Here, a sulfonamide-resistant FolP mutant library of S. Gallinarum was generated through random mutagenesis. Among resistant strains, substitution of amino acid Arginine 171 with Proline (R171P) in the FolP protein conferred the highest resistance against sulfonamide. Substitution of Phe28 with Leu or Ile (F28L/I) led to modest sulfonamide resistance. Structural modeling indicates that R171P and Phenylalanine 28 with leucine or isoleucine (F28L/I) substitution mutations are located far from the substrate-binding site and cause insignificant conformational changes in the FolP protein. Rather, in silico studies suggest that the mutations altered the stability of the protein, potentially resulting in sulfonamide resistance. Identification of specific mutations in FolP that confer resistance to sulfonamide would contribute to our understanding of the molecular mechanisms of antibiotic resistance.

Sub-microscopic Plasmodium falciparum parasitaemia, dihydropteroate synthase (dhps) resistance mutations to sulfadoxine-pyrimethamine, transmission intensity and risk of malaria infection in pregnancy in Mount Cameroon Region.

BACKGROUND: Plasmodium falciparum resistance to intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) continues to spread throughout sub-Saharan Africa. This study assessed the occurrence of microscopic and sub-microscopic P. falciparum parasitaemia, dihydropteroate synthase mutations associated with resistance to SP and maternal anaemia in the Mount Cameroon area. METHODS: Consenting pregnant women living in semi-rural and semi-urban/urbanized settings were enrolled in this cross-sectional study. Socio-demographic, antenatal and clinical data were documented. Microscopic and sub-microscopic parasitaemia were diagnosed using peripheral blood microscopy and nested polymerase chain reaction (PCR) respectively. The dhps mutations were genotyped by restriction fragment length polymorphism analysis. The presence of A437G, K540E, and A581G was considered a marker for high-level resistance. Haemoglobin levels and anaemia status were determined. RESULTS: Among the women, the prevalence of microscopic and sub-microscopic P. falciparum infection were 7.7% (67/874) and 18.6% (93/500) respectively. Predictors of microscopic infection were younger age (< 21 years) (AOR = 2.89; 95% CI 1.29-6.46) and semi-rural settings (AOR = 2.27; 95% CI 1.31-3.96). Determinants of sub-microscopic infection were the rainy season (AOR, 3.01; 95% CI 1.77-5.13), primigravidity (AOR = 0.45; 95% CI 0.21-0.94) and regular ITN usage (AOR = 0.49; 95% CI 0.27-0.90). Of the145 P. falciparum isolates genotyped, 66.9% (97) carried mutations associated with resistance to SP; 33.8% (49), 0%, 52.4% (76) and 19.3% (28) for A437G, K540E, A581G and A437G + A581G respectively. The A581G mutation was associated with ≥ 3 SP doses evident only among sub-microscopic parasitaemia (P = 0.027) and multigravidae (P = 0.009). Women with microscopic infection were more likely from semi-rural settings (AOR = 7.09; 95% CI 2.59-19.42), to report history of fever (AOR = 2.6; 95% CI 1.07-6.31), to harbour parasites with double resistant mutations (AOR = 6.65; 95% CI 1.85-23.96) and were less likely to have received 2 SP doses (AOR = 0.29; 95% CI 1.07-6.31). Microscopic infection decreased Hb levels more than sub-microscopic infection. CONCLUSION: The occurrence of sub-microscopic P. falciparum parasites resistant to SP and intense malaria transmission poses persistent risk of malaria infection during pregnancy in the area. ITN usage and monitoring spread of resistance are critical.

A snapshot of the prevalence of dihydropteroate synthase-431V mutation and other sulfadoxine-pyrimethamine resistance markers in Plasmodium falciparum isolates in Nigeria.

BACKGROUND: Malaria is a major public health issue with substantial risks among vulnerable populations. Currently, the World Health Organization (WHO) recommends SP-IPTp in the second and third trimesters. However, the efficacy of SP-IPTp is threatened by the emergence of sulfadoxine-pyrimethamine resistant malaria parasites due to single nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. This study aimed to assess the current prevalence of Pfdhfr/Pfdhps mutations in P. falciparum isolates collected from individuals residing in Ile-Ife, Nigeria, and also present maps of the prevalence of Pfdhps 431V and 581G within Nigeria and surrounding countries. METHODS: Between October 2020 and April 2021, samples were collected as dried blood spots among 188 participants who showed malaria positivity with a histidine-rich-protein-based rapid diagnostic test (RDT). Nested PCR assays were used to confirm falciparum in the samples with RDT positivity, and to amplify fragments of the Pfdhfr/Pfdhps genes followed by targeted amplicon sequencing. Published data since 2007 on the prevalence of the Pfdhps genotypes in Nigeria and the neighbouring countries were used to produce maps to show the distribution of the mutant genotypes. RESULTS: Only 74 and 61 samples were successfully amplified for the Pfdhfr and Pfdhps genes, respectively. At codons resulting in N51I, C59R, and S108N, Pfdhfr carried mutant alleles of 97.3% (72/74), 97.3% (72/74) and 98.6% (73/74), respectively. The Pfdhps gene carried mutations at codons resulting in amino acid changes at 431-436-437-540-581-613; I431V [45.9%, (28/61)], A581G [31.1% (19/61)] and A613S [49.2% (30/61)]. Constructed haplotypes were mainly the triple Pfdhfr mutant 51I-59R-108N (95.9%), and the most common haplotypes observed for the Pfdhps gene were the ISGKAA (32.8%), ISGKGS (8.2%), VAGKAA (14.8%), VAGKAS (9.8%) and VAGKGS (14.8%). In the context of the previously published data, a high prevalence of 431V/581G mutations was found in the study population. It seems quite evident that the Pfdhps 431V, 581G and 613S often co-occur as Pfdhps-VAGKGS haplotype. CONCLUSION: This study showed that the prevalence of VAGKGS haplotype seems to be increasing in prevalence. If this is similar in effect to the emergence of 581G in East Africa, the efficacy of SP-IPTp in the presence of these novel Pfdhps mutants should be re-assessed.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance.

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.

Molecular Analysis of Dihydropteroate Synthase Gene Mutations in Pneumocystis jirovecii Isolates among Bulgarian Patients with Pneumocystis Pneumonia.

Pneumocystis jirovecii pneumonia (PCP) is a significant cause of morbidity and mortality in immunocompromised people. The widespread use of trimethoprim-sulfamethoxazole (TMP-SMZ) for the treatment and prophylaxis of opportunistic infections (including PCP) has led to an increased selection of TMP-SMZ-resistant microorganisms. Sulfa/sulfone resistance has been demonstrated to result from specific point mutations in the DHPS gene. This study aims to investigate the presence of DHPS gene mutations among P. jirovecii isolates from Bulgarian patients with PCP. A total of 326 patients were examined via real-time PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene and further at the DHPS locus. P. jirovecii DNA was detected in 50 (15.34%) specimens. A 370 bp DHPS locus fragment was successfully amplified in 21 samples from 19 PCP-positive patients, which was then purified, sequenced, and used for phylogenetic analysis. Based on the sequencing analysis, all (n = 21) P. jirovecii isolates showed DHPS genotype 1 (the wild type, with the nucleotide sequence ACA CGG CCT at codons 55, 56, and 57, respectively). In conclusion, infections caused by P. jirovecii mutants potentially resistant to sulfonamides are still rare events in Bulgaria. DHPS genotype 1 at codons 55 and 57 is the predominant P. jirovecii strain in the country.

Global Analysis of Plasmodium falciparum Dihydropteroate Synthase Variants Associated with Sulfadoxine Resistance Reveals Variant Distribution and Mechanisms of Resistance: A Computational-Based Study.

The continual rise in sulfadoxine (SDX) resistance affects the therapeutic efficacy of sulfadoxine-pyrimethamine; therefore, careful monitoring will help guide its prolonged usage. Mutations in Plasmodium falciparum dihydropteroate synthase (Pfdhps) are being surveilled, based on their link with SDX resistance. However, there is a lack of continuous analyses and data on the potential effect of molecular markers on the Pfdhps structure and function. This study explored single-nucleotide polymorphisms (SNPs) in Pfdhps that were isolated in Africa and other countries, highlighting the regional distribution and its link with structure. In total, 6336 genomic sequences from 13 countries were subjected to SNPs, haplotypes, and structure-based analyses. The SNP analysis revealed that the key SDX resistance marker, A437G, was nearing fixation in all countries, peaking in Malawi. The mutation A613S was rare except in isolates from the Democratic Republic of Congo and Malawi. Molecular docking revealed a general loss of interactions when comparing mutant proteins to the wild-type protein. During MD simulations, SDX was released from the active site in mutants A581G and A613S before the end of run-time, whereas an unstable binding of SDX to mutant A613S and haplotype A437A/A581G/A613S was observed. Conformational changes in mutant A581G and the haplotypes A581G/A613S, A437G/A581G, and A437G/A581G/A613S were seen. The radius of gyration revealed an unfolding behavior for the A613S, K540E/A581G, and A437G/A581G systems. Overall, tracking such mutations by the continuous analysis of Pfdhps SNPs is encouraged. SNPs on the Pfdhps structure may cause protein-drug function loss, which could affect the applicability of SDX in preventing malaria in pregnant women and children.

Low prevalence of highly sulfadoxine-resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin.

BACKGROUND: In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated. METHODS: This study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6-59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance. RESULTS: The Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, Pfdhfr IRN/Pfdhps GE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites. CONCLUSIONS: This study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

Genetic diversity of Pneumocystis jirovecii isolates among Turkish population based on mitochondrial large subunit ribosomal RNA and dihydropteroate synthase gene typing.

Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause severe interstitial pneumonia in immunocompromised patients. In this study, mitochondrial large subunit ribosomal RNA (mtLSU-rRNA) and dihydropteroate synthase (DHPS) gene polymorphism in P. jirovecii isolates were investigated in Western Turkey's Izmir province and its surroundings. For this purpose, a total of 157 P. jirovecii isolates obtained from bronchoalveolar lavage samples of hospitalized cases and lung tissue samples of autopsy cases who died outside hospital were examined. Genotypes were identified by direct sequencing of mtLSU-rRNA restriction fragment length polymorphism analysis of the DHPS gene amplicons. The mtLSU-rRNA analysis revealed that genotype 2 was the most common genotype with 58%. The following genotypes were genotype 3 (13%), genotype 1 (11.6%) and genotype 4 (5.1%), while genotype 5 (0.7%) was detected in only one autopsy case. In addition, 16 (11.6%) cases had dual or triple different genotypes (mixed infection). It was observed that the genotype distribution was not affected by characteristics such as age, gender and immune status. However, the predominance of genotype 2 in solid organ tumors and the predominance of mixed infection in patients with chronic pulmonary disease were statistically significant. On the other hand, DHPS gene amplification was positive in 137 (87.3%) of 157 samples. While no mutation was observed in 135 samples, the association of wild-type and 57th codon mutation was detected in two hospitalized cases (1.5%). In this study, important epidemiological data on the distribution of mtLSU-rRNA genotypes were obtained. Also the existence of DHPS gene mutations associated with potential drug resistance in our community was shown for the first time. Further studies are needed to evaluate the possible effects of genotypes on the prognosis of the disease to help with the clinician's treatment decisions. LAY ABSTRACT: Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause life-threatening pneumonia in immunocompromised patients. In this study, we investigated the mtLSU-rRNA and DHPS gene polymorphisms in P. jirovecii isolates from both hospital and autopsy cases.

[Dihydropteroate Synthase (DHPS) Gene Mutations and Mitochondrial Large Subunit (mtLSU) rRNA Genotype Variations in Pneumocystis jirovecii Strains]. / Pneumocystis jirovecii Izolatlarinin Dihidropteroat Sentaz (DHPS) Gen Mutasyonlari ve Mitokondriyal Büyük Alt Birim (mtLSU) rRNA Genotip Varyasyonlarinin Belirlenmesi.

Pneumocystis jirovecii is an atypical fungus that causes Pneumocystis pneumonia (PCP) in HIV/AIDS and immunocompromised patients. Antibiotics containing sulfa and sulfone groups are widely used in PCP prophylaxis and treatment. Especially, long-term use of trimethoprim sulfamethoxazole (TMP-SMX) is known to cause certain point mutations associated with drug resistance in the P.jirovecii dihydropteroate synthase (DHPS) gene. In addition, DHPS and mitochondrial large subunit (mtLSU) rRNA genotype characterization provides important data on the epidemiology of P.jirovecii. In this study, it was aimed to investigate the DHPS and mtLSU rRNA gene polymorphisms of P.jirovecii strains isolated from immunocompromised patients in Mersin University Hospital. In this study, 16 P.jirovecii positive samples, which isolated from 96 patients samples, between August 2016 and February 2018, were included. P.jirovecii mtLSU rRNA genotypes were determined by sequence analysis according to polymorphisms at the 85th and 248th nucleotide positions. Nested PCR and RFLP method was applied for mutation analysis of DHPS locus, 165th and 171st nucleotide positions. In the DHPS mutation analysis, 12/16 (75%) wild type (W165/W171) and 4/16 (25%) mutant type (M165/W171) were detected. Two mutant types belonged to HIV/AIDS positive patients with PCP and had a history of prophylaxis; the other 2 mutant types belonged to patients with colonization. In the study, a history of prophylaxis in 3 (19%) of the 16 patients were recorded, and mutant type was detected in these 2 of 3 patients. According to mtLSU-rRNA analysis, 3 different genotypes were obtained from 16 P.jirovecii isolates. In our region, genotype 2 (43.75%; n= 7) was the most common genotype, genotype 1 (37.5%; n= 6) was the second common and genotype 3 (18.75%; n= 3) was the least one. Genotype 4 was not detected in our region. When DHPS and mtLSU-rRNA were evaluated as multilocus, five different genotypes were observed. As a result, these findings provided important data on P.jirovecii epidemiology in our region and potential drug-resistant strains showed a risk of transmission in immunosuppressive patients. Multicenter studies involving more P.jirovecii isolates are needed to better define the epidemiology of P.jirovecii in our region and in our country.

Increased prevalence of pfdhfr and pfdhps mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates from Jazan Region, Southwestern Saudi Arabia: important implications for malaria treatment policy.

BACKGROUND: Despite significant progress in eliminating malaria from the Kingdom of Saudi Arabia, the disease is still endemic in the southwestern region of the country. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been used in Saudi Arabia since 2007 as a first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to artemisinin and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum parasites circulating in Jazan region, southwestern Saudi Arabia. METHODS: A total of 151 P. falciparum isolates were collected between April 2018 and March 2019 from 12 of the governorates in Jazan region. Genomic DNA was extracted from dried blood spots and amplified using nested PCR. Polymorphisms in the propeller domain of the P. falciparum k13 (pfkelch13) gene and point mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by sequencing. RESULTS: No mutations in the pfkelch13 propeller domain were found in any of the 151 isolates. However, point mutations in the pfdhfr and pfdhps genes were detected in 90.7% (137/151) of the isolates. The pfdhfr double mutations N51I + S108N (i.e. ACICNI haplotype) and triple mutations N51I + C59R + S108N (i.e. ACIRNI haplotype) were detected in 47% and 37.8% of the isolates, respectively. Moreover, the pfdhps single mutation at codon A437G and double mutations A437G + K540E (i.e. SGEAAI haplotype) were observed in 4.6% and 51.7% of the isolates, respectively. Interestingly, 23.8%, 25.1 and 12.6% of the isolates had quintuple, quadruple and triple mutated combined pfdhfr-pfdhps genotypes, respectively. Furthermore, significant associations were found between the prevalence of mutant haplotypes and the age, gender and nationality of the patients (P < 0.05). CONCLUSION: This study revealed a high prevalence of point mutations in the pfdhfr and pfdhps genes of P. falciparum isolates from Jazan region, with quintuple and quadruple mutant pfdhfr-pfdhps genotypes reported for the first time in Saudi Arabia and the Arabian Peninsula. Despite the absence of the pfkelch13 mutation in the isolates examined, the pfdhfr and pfdhps mutations undermine the efficacy of SP partner drug, thereby threatening the main falciparum malaria treatment policy in Saudi Arabia, i.e. the use of AS + SP. Therefore, the continuous molecular and in-vivo monitoring of ACT efficacy in Jazan region is highly recommended.

Characterization of putative drug resistant biomarkers in Plasmodium falciparum isolated from Ghanaian blood donors.

BACKGROUND: Plasmodium falciparum parasites, which could harbour anti-malaria drug resistance genes, are commonly detected in blood donors in malaria-endemic areas. Notwithstanding, anti-malaria drug resistant biomarkers have not been characterized in blood donors with asymptomatic P. falciparum infection. METHODS: A total of 771 blood donors were selected from five districts in the Greater Accra Region, Ghana. Each donor sample was screened with malaria rapid diagnostic test (RDT) kit and parasitaemia quantified microscopically. Dried blood spots from malaria positive samples were genotyped for P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum multi-drug resistance (Pfmdr1), P. falciparum dihydropteroate-synthetase (Pfdhps), P. falciparum dihydrofolate-reductase (Pfdhfr) and Kelch 13 propeller domain on chromosome 13 (Kelch 13) genes. RESULTS: Of the 771 blood donors, 91 (11.8%) were positive by RDT. Analysis of sequence reads indicated successful genotyping of Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes in 84.6, 81.3, 86.8, 86.9 and 92.3% of the isolates respectively. Overall, 21 different mutant haplotypes were identified in 69 isolates (75.8%). In Pfcrt, CVIET haplotype was observed in 11.6% samples while in Pfmdr1, triple mutation (resulting in YFN haplotype) was detected in 8.1% of isolates. In Pfdhfr gene, triple mutation resulting in IRNI haplotype and in Pfdhps gene, quintuple mutation resulting in AGESS haplotype was identified in 17.7% parasite isolates. Finally, five non-synonymous Kelch 13 alleles were detected; C580Y (3.6%), P615L (4.8%), A578S (4.8%), I543V (2.4%) and A676S (1.2%) were detected. CONCLUSION: Results obtained in this study indicated various frequencies of mutant alleles in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes from P. falciparum infected blood donors. These alleles could reduce the efficacy of standard malaria treatment in transfusion-transmitted malaria cases. Incorporating malaria screening into donor screening protocol to defer infected donors is therefore recommended.

Occurrence of septuple and elevated Pfdhfr-Pfdhps quintuple mutations in a general population threatens the use of sulfadoxine-pyrimethamine for malaria prevention during pregnancy in eastern-coast of Tanzania.

BACKGROUND: Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations compromise the effectiveness of sulfadoxine-pyrimethamine (SP) for treatment of uncomplicated malaria, and are likely to impair the efficiency of intermittent preventive treatment during pregnancy (IPTp). This study was conducted to determine the level of Pfdhfr-Pfdhps mutations, a decade since SP was limited for IPTp use in pregnant women in Tanzania. METHODS: P. falciparum genomic DNA was extracted from dried blood spots prepared from a finger prick. Extracted DNA were sequenced using a single MiSeq lane by combining all PCR products. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools whereas custom scripts were used to filter and translate genotypes into SP resistance haplotypes. RESULTS: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in 6 (1.3%) samples. Triple PfdhfrIRNI (mutations are bolded and underlined) haplotype was dominant, contributing to 84% (number [n] = 374) of haplotypes while 446 samples were studied for Pfdhps, WT for Pfdhps (SAKAA) was found in 6.7% (n = 30) in samples. Double Pfdhps haplotype (SGEAA) accounted for 83% of all mutations at Pfdhps gene. Of 447 Pfdhfr-Pfdhps combined genotypes, only 0.9% (n = 4) samples contained WT gene (SAKAA-NCSI). Quintuple (five) mutations, SGEAA-IRNI accounted for 71.4% (n = 319) whereas 0.2% (n = 1) had septuple (seven) mutations (AGKGS-IRNI). The overall prevalence of Pfdhfr K540E was 90.4% (n = 396) while Pfdhps A581G was 1.1% (n = 5). CONCLUSIONS: This study found high prevalence of Pfdhfr-Pfdhps quintuple and presence of septuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be not effective. Nonetheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp in the study area is recommended.

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance.

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China.

Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

Molecular Diagnosis of Pneumocystis jirovecii Pneumonia by Use of Oral Wash Samples in Immunocompromised Patients: Usefulness and Importance of the DNA Target.

Pneumocystis jirovecii pneumonia (PJP) is an important cause of pneumonia in the HIV-negative immunocompromised population, for whom the fungal load is low, the differential diagnosis is difficult, and a bronchoalveolar lavage (BAL) sample is often not readily available. Molecular techniques have improved the microbiological diagnosis in this scenario. The usefulness of two real-time PCR techniques targeting nuclear single-copy and mitochondrial multicopy genes, respectively, applied to oral wash specimens (OW) for PJP diagnosis was assessed, and its accuracy was compared to a BAL fluid-based diagnosis. Immunocompromised patients having PJP in the differential diagnosis of an acute respiratory episode, and from whom OW and BAL or lung biopsy specimens were obtained ≤48 h apart, were retrospectively included. PCRs targeting the dihydropteroate synthase gene (DHPS) and the mitochondrial small-subunit (mtSSU) rRNA gene were performed in paired OW-BAL specimens. Thirty-six patients were included (88.6% HIV negative). Fifteen patients (41.7%) were classified as PJP, and a further 8 were considered P. jirovecii colonized. Quantification of DHPS and mtSSU in BAL fluid showed an accuracy of 96.9% and 93.0%, respectively, for PJP diagnosis, whereas a qualitative approach performed better when applied to OW (accuracy, 91.7%) irrespective of the PCR target studied (kappa = 1). Qualitative molecular diagnosis applied to OW showed an excellent performance for PJP diagnosis regardless of the target studied, being easier to interpret than the quantitative approach needed for BAL fluid.

High prevalence of Pfdhfr-Pfdhps quadruple mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment of malaria in Africa. However, increasing SP resistance (SPR) affects the therapeutic efficacy of the SP. As molecular markers, Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthase) genes are widely used for SPR surveillance. This study aimed to assess the prevalence of Pfdhfr and Pfdhps genes mutations and haplotypes in Plasmodium falciparum isolates collected from Bioko Island, Equatorial Guinea (EG). METHODS: In total, 180 samples were collected in 2013-2014. The single nucleotide polymorphisms (SNPs) of the Pfdhfr and Pfdhps genes were identified with nested PCR and Sanger sequencing. The genotypes and linkage disequilibrium (LD) tests were also analysed. RESULTS: Sequences of Pfdhfr and Pfdhps genes were obtained from 92.78% (167/180) and 87.78% (158/180) of the samples, respectively. For Pfdhfr, 97.60% (163/167), 87.43% (146/167) and 97.01% (162/167) of the samples carried N51I, C59R and S108N mutant alleles, respectively. The prevalence of the Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 20.25% (32/158), 90.51% (143/158), 5.06% (8/158), 0.63% (1/158), and 3.16% (5/158) of the samples, respectively. In total, 3 unique haplotypes at the Pfdhfr locus and 8 haplotypes at the Pfdhps locus were identified. A triple mutation (CIRNI) in Pfdhfr was the most prevalent haplotype (86.83%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in Pfdhps. A total of 130 isolates with 12 unique haplotypes were found in the Pfdhfr and Pfdhps combined haplotypes, 65.38% (85/130) of them carried quadruple allele combinations (CIRNI-SGKAA), whereas only one isolate (0.77%, 1/130) was found to carry the wild-type (CNCSI-SAKAA). For LD analysis, the Pfdhfr N51I was significantly associated with the Pfdhps A437G (P < 0.05). CONCLUSION: Bioko Island possesses a high prevalence of the Pfdhfr triple mutation (CIRNI) and Pfdhps single mutation (SGKAA), which will undermine the pharmaceutical effect of SP for malaria treatment strategies. To avoid an increase in SPR, continuous molecular monitoring and additional control efforts are urgently needed in Bioko Island, Equatorial Guinea.