Clinical significance of mutations in dihydropteroate synthase in Pneumocystis jirovecii pneumonia among non-HIV-infected patients.

BACKGROUND: Dihydropteroate synthase (DHPS) mutations may be associated with trimethoprim-sulfamethoxazole resistance in Pneumocystis jirovecii pneumonia (PCP) and worse clinical outcomes. However, the clinical significance of DHPS mutations in PCP among non-human immunodeficiency virus (HIV)-infected patients remains unclear. METHODS: Patients with PCP in three tertiary referral hospitals in Taiwan between 2016 and 2020 were retrospectively enrolled. Two point mutations, Thr55Ala and Pro57Ser, in the DHPS protein were analysed. Patients with invalid DHPS mutations in the respiratory specimen, chronic respiratory failure, receiving endotracheal intubation for surgical intervention, HIV infection, Pneumocystis jirovecii colonisation, and no lactate dehydrogenase (LDH) data were excluded. The primary outcome was 30-day survival. RESULTS: A total of 215 patients were analysed. Mutants inside DHPS were found in 78 patients (36.3%) and 68 patients (31.6%) died within 30 days. A total of 214 patients (99.5%) received trimethoprim-sulfamethoxazole as the first-line treatment. The rates of mechanical ventilation, 30-day, and in-hospital mortality were similar between wild-type and mutant DHPS PCP. After adjusting for important confounders, LDH > 500 µ/L (adjusted hazard ratio [aHR] = 2.448, P = 0.001), pneumonia severity index > 135 mg/dL (aHR = 1.689, P = 0.049), and having solid tumours (aHR = 1.832, P = 0.034) were independently associated with higher mortality. In subgroup analysis, mutant DHPS PCP patients had less 30-day mortality among patients aged > 65 years (P = 0.049), with lymphopenia (P = 0.040), and those without solid tumour (P = 0.045). CONCLUSIONS: In non-HIV-infected PCP, point mutants inside DHPS may not be associated with trimethoprim-sulfamethoxazole treatment outcomes. Further prospective large-scale studies are warranted.

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.

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.

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.

Distinct pattern and prevalence of Plasmodium falciparum dihydropteroate synthase gene mutations in children with sickle cell anaemia and haemoglobin AA in Benin City, Nigeria: the impact of HbAA.

Introduction: specific mutations on the Plasmodium falciparum dihydropteroate synthase (Pfdhps) gene mediate sulphadoxine/pyrimethamine (SP) resistance and thus, pose a threat to the efficacy of SP-Intermittent Preventive Therapy (SP-IPT) in malaria chemoprevention in children, including those with sickle cell anaemia (SCA). This study determined the distinct pattern and prevalence of Pfdhps mutations in children with SCA and in those with homozygous haemoglobin A (HbAA) in Benin City, Nigeria; showing the impact of haemoglobin phenotype. Methods: this was a cross-sectional study involving children with SCA and HbAA. Those with successfully amplified Pfdhps genes were included in the study. Point mutations and mutant haplotypes of the Pfdhps gene were identified. Parasite density (PD) was determined by estimating the parasite numbers/µl of blood from the thick film. Descriptive, univariable and multivariable analysis were used appropriately. Results: a total of 146 children: 71 with SCA and 75 with HbAA were recruited, with a mean age of 46.6 ± 13.0 and 36.4 ± 17.6 respectively; proportion of males were 45(63.4%) and 43(57.3%) respectively. I431V, S436A, A437G, A581G, and A613G mutations were present; but the K540E mutation was absent. ISGKAA 41(28.1%) and VAGKGS 61(41.8%) were the most prevalent mutant haplotypes in this study. The prevalence of VAGKGS haplotype 43(57.3%) was significantly higher in HbAA group compared to that 18(25.4%) in the SCA group (p < 0.001). The prevalence of ISGKAA in SCA group 25(35.2%) was significantly higher than that 16(21.3%) in the HbAA group (p=0.032). HbAA phenotype was the only significant predictor for the presence of the VAGKGS mutant haplotype (aOR: 3.0, 95%CI: 1.375 to 6.499; p=0.006). Conclusion: the HbAA phenotype was a significant predictor for the occurrence of the quintuple mutant haplotype (VAGKGS). The K540E mutation was absent; thus, SP-IPT can be explored in children younger than five years with SCA.

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.

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.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique.

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

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.

Molecular markers of resistance to amodiaquine plus sulfadoxine-pyrimethamine in an area with seasonal malaria chemoprevention in south central Niger.

BACKGROUND: In Niger, malaria transmission is markedly seasonal with most of the disease burden occurring in children during the rainy season. Seasonal malaria chemoprevention (SMC) with amodiaquine plus sulfadoxine-pyrimethamine (AQ + SP) is recommended in the country to be administered monthly just before and during the rainy season. Moreover, clinical decisions on use of SP for intermittent preventive treatment in pregnancy (IPTp) now depend upon the validated molecular markers for SP resistance in Plasmodium falciparum observed in the local parasite population. However, little is known about molecular markers of resistance for either SP or AQ in the south of Niger. To address this question, clinical samples which met clinical and biological criteria, were collected in Gabi, Madarounfa district, Maradi region, Niger in 2011-2012 (before SMC implementation). Molecular markers of resistance to pyrimethamine (pfdhfr), sulfadoxine (pfdhps) and amodiaquine (pfmdr1) were assessed by DNA sequencing. RESULTS: Prior to SMC implementation, the samples showed a high proportion of clinical samples that carried the pfdhfr 51I/59R/108N haplotype associated with resistance to pyrimethamine and pfdhps 436A/F/H and 437G mutations associated with reduced susceptibility to sulfadoxine. In contrast mutations in codons 581G, and 613S in the pfdhps gene, and in pfmdr1, 86Y, 184Y, 1042D and 1246Y associated with resistance to amodiaquine, were less frequently observed. Importantly, pfdhfr I164L and pfdhps K540E mutations shown to be the most clinically relevant markers for high level clinical resistance to SP were not detected in Gabi. CONCLUSIONS: Although parasites with genotypes associated with the highest levels of resistance to AQ + SP are not yet common in this setting, their importance for deployment of SMC and IPTp dictates that monitoring of these markers of resistance should accompany these interventions. This study also highlights the parasite heterogeneity within a small spatial area and the need to use caution when extrapolating results from surveys of molecular markers of resistance in a single site to inform regional policy decisions.

Different origin and dispersal of sulfadoxine-resistant Plasmodium falciparum haplotypes between Eastern Africa and Democratic Republic of Congo.

Sulfadoxine/pyrimethamine (SP) is still used for malaria control in sub-Saharan Africa; however, widespread resistance is a major concern. This study aimed to determine the dispersal and origin of sulfadoxine resistance lineages in the Democratic Republic of the Congo compared with East African Plasmodium falciparum dihydropteroate synthetase (Pfdhps) haplotypes. The analysis involved 264 isolates collected from patients with uncomplicated malaria from Tanzania, Uganda and DR Congo. Isolates were genotyped for Pfdhps mutations at codons 436, 437, 540, 581 and 613. Three microsatellite loci (0.8, 4.3 and 7.7 kb) flanking the Pfdhps gene were assayed. Evolutionary analysis revealed a shared origin of Pfdhps haplotypes in East Africa, with a distinct population clustering in DR Congo. Furthermore, in Tanzania there was an independent distinct origin of Pfdhps SGEGA resistant haplotype. In Uganda and Tanzania, gene flow patterns contribute to the dispersal and shared origin of parasites carrying double- and triple-mutant Pfdhps haplotypes associated with poor outcomes of intermittent preventive treatment during pregnancy using SP (IPTp-SP). However, the origins of the Pfdhps haplotypes in DR Congo and Eastern Africa sites are different. The genetic structure demonstrated a divergent and distinct population cluster predominated by single-mutant Pfdhps haplotypes at the DR Congo site. This reflects the limited dispersal of double- and triple-mutant Pfdhps haplotypes in DR Congo. This study highlights the current genetic structure and dispersal of high-grade Pfdhps resistant haplotypes, which is important to guide implementation of SP in malaria chemoprevention strategies in the region.

Molecular determinants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum in Nigeria and the regional emergence of dhps 431V.

There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd ß-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015.

Frequencies of dhfr/dhps multiple mutations and Plasmodium falciparum submicroscopic gametocyte carriage in Gabonese pregnant women following IPTp-SP implementation.

This study analyzed the relationship between intermittent preventive treatment with sulfadoxine-pyrimethamine (SP) (IPTp-SP), the rate of multiple resistant parasites and of submicroscopic gametocyte carriage among pregnant women at the beginning of IPTp implementation in Gabon (2005) and six years after (2011). The detection of pfdhfr and pfdhps gene mutations was performed by PCR-RFLP in Plasmodium (P.) falciparum positive samples collected from pregnant women in 2005 and 2011. Gametocytes carriage was detected by Pfs25mRNA amplification using QT-NASBA. Data were analyzed according to the time of collection (study period) and IPTp-SP doses. The proportion of isolates with at least a triple Pfdhfr mutation (n = 39/42, 92.9% versus 100%, n = 78/78)) and of those isolates with the S108N/C59R/N51I/S436A/A437G multiple mutation (17.9% versus 75.6%) significantly increased between 2005 and 2011 (p<0.01). Mutations I164L and A581G were not found, while higher proportions of 436 and 437 mutations were detected in 2011.A trend toward a higher frequency of isolates with five mutations was observed in women who received two SP doses (p<0.01). Pfs25mRNA was found in 6.8 % (n = 3/44) and 34.6% (n = 27/78) of the samples collected in 2005 and 2011 respectively (p<0.01). In 2011, 74.0% (n = 20/27) of women with detected submicroscopic gametocytes carried parasites with the S108N/C59R/N51/S436A/A437G multiple mutation. All the ten delivering women who received three IPTp-SP doses had a submicroscopic Plasmodium falciparum infection, but none had detected gametocytes. Following IPTp-SP implementation, an increase in the frequency of multiple mutant parasites and of submicroscopic gametocyte carriage was observed among pregnant women living in Gabon.

Clinical characteristics of hospital-onset Pneumocystis pneumonia and genotypes of Pneumocystis jirovecii in a single tertiary centre in Korea.

BACKGROUND: Pneumocystis pneumonia (PCP) may develop as a clinical manifestation of nosocomial pneumonia by means of either reactivation of resident P. jirovecii or de novo infection. However, there have been no studies describing the clinical characteristics of hospital-onset PCP. METHODS: A retrospective review of medical records was performed to identify episodes of hospital-onset PCP in a tertiary care centre in Korea between May 2007 and January 2013. We investigated whether human-to-human contact during hospitalisation contributed to PCP development by molecular analysis of the genes encoding mitochondrial large ribosomal subunit (mtLSU) rRNA and dihydropteroate synthase (DHPS) and a review of hospitalisation history. RESULTS: During the study period, 129 patients (130 episodes) were diagnosed with PCP. Of these, respiratory specimens from 94 patients during 95 PCP episodes were available for analysis. Sixteen episodes (16.8%) were categorised as hospital-onset PCP. There was a trend toward a higher proportion of haematological malignancy (43.8% [7/16] vs. 20.3% [16/79]; P = 0.058) in patients with hospital-onset PCP compared to patients with community-onset PCP. mtLSU genotype 1 was the most common, occurring in 41 (43.2%) patients. There were four possible cases of nosocomial transmission. Mutation in DHPS was not observed in any PCP episode. CONCLUSIONS: PCP can be one of the causes of nosocomial pneumonia, although the mode of acquisition and transmission of P. jirovecii remains uncertain. mtLSU genotype 1 is the predominant P. jirovecii strain in Korea.

Dihydropteroate synthase gene mutation rates in Pneumocystis jirovecii strains obtained from Iranian HIV-positive and non-HIV-positive patients.

The dihydropteroate sulfate (DHPS) gene is associated with resistance to sulfa/sulfone drugs in Pneumocystis jirovecii. We investigated the DHPS mutation rate in three groups of Iranian HIV-positive and HIV-negative patients by polymerase chain reaction-restricted fragment length polymorphism analysis. Furthermore, an association between P. jirovecii DHPS mutations and strain typing was investigated based on direct sequencing of internal transcribed spacer region 1 (ITS1) and ITS2. The overall P. jirovecii DHPS mutation rate was (5/34; 14.7%), the lowest rate identified was in HIV-positive patients (1/16; 6.25%) and the highest rate was in malignancies patients (3/11; 27.3%). A moderate rate of mutation was detected in chronic obstructive pulmonary disease (COPD) patients (1/7; 14.3%). Most of the isolates were wild type (29/34; 85.3%). Double mutations in DHPS were detected in patients with malignancies, whereas single mutations at codons 55 and 57 were identified in the HIV-positive and COPD patients, respectively. In this study, two new and rare haplotypes were identified with DHPS mutations. Additionally, a positive relationship between P. jirovecii strain genotypes and DHPS mutations was identified. In contrast, no DHPS mutations were detected in the predominant (Eg) haplotype. This should be regarded as a warning of an increasing incidence of drug-resistant P. jirovecii strains.

Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii / Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii

Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.

Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Dihydropteroate synthase mutations in Pneumocystis pneumonia: impact of applying different definitions of prophylaxis, mortality endpoints and mutant in a single cohort.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are well-reported. Although sulfa prophylaxis generally is associated with DHPS mutant infection, whether mutant infection is associated with poorer clinical outcomes is less clear. The differing definitions of sulfa prophylaxis and the different mortality endpoints used in these studies may be one explanation for the conflicting study results. Applying different definitions of prophylaxis, mortality endpoints and DHPS mutant to 301 HIV-infected patients with Pneumocystis pneumonia, we demonstrate that prophylaxis, irrespective of definition, increased the risk of infection with pure mutant (any prophylaxis: AOR 4.00, 95% CI: 1.83-8.76, P < 0.001) but not mixed genotypes (any prophylaxis: AOR 0.78, 95% CI: 0.26-2.36, P = 0.65). However, infection with mutant DHPS, irrespective of definition, was not associated with increased mortality (all-cause or PCP death) at the three time-intervals examined (all P > 0.05). Future studies should standardize key variables associated with DHPS mutant infection as well as examine DHPS mutant subtypes (pure mutant vs. mixed infections) - perhaps even individual DHPS mutant genotypes - so that data can be pooled to better address this issue.

Comparative efficacies of artemisinin combination therapies in Plasmodium falciparum malaria and polymorphism of pfATPase6, pfcrt, pfdhfr, and pfdhps genes in tea gardens of Jalpaiguri District, India.

In India, chloroquine has been replaced by a combination of artesunate and sulfadoxine-pyrimethamine (AS-SP) for uncomplicated P. falciparum malaria. Other available combinations, artemether-lumefantrine (AM-LF) and artesunate-mefloquine (AS-MQ), not included in the national program, are widely used by private practitioners. Little is known about the therapeutic efficacy of these artemisinin combinations and the prevalence of molecular markers associated with antimalarial drug resistance. A total of 157 patients with P. falciparum monoinfection were recruited and randomized into three study groups (AS-SP, AM-LF, and AS-MQ). All patients were followed up for 42 days to study the clinical and parasitological responses according to the WHO protocol (2009). We assessed the polymorphism of the pfATPase6, pfcrt, pfdhfr, and pfdhps genes by the DNA-sequencing method. The PCR-corrected therapeutic efficacies of AS-SP, AM-LF, and AS-MQ were 90.6% (95% confidence interval [CI], 0.793 to 0.969), 95.9% (95% CI, 0.860 to 0.995), and 100% (95% CI, 0.927 to 1.00), respectively. No specific mutational pattern was observed in the pfATPase6 gene. All isolates had a K76T mutation in the pfcrt gene. In the pfdhfr-pfdhps genotype, quadruple mutation was frequent, and quintuple mutation was documented in 6.3% of P. falciparum isolates. The significant failure rate of AS-SP (9.5%), although within the limit (10%) for drug policy change, was due to SP failure because of prevailing mutations in pfdhfr, I(51)R(59)N(108), with pfdhps, G(437) and/or E(540). The efficacy of this ACT needs periodic monitoring. Artemether-lumefantrine and artesunate-mefloquine are effective alternatives to the artesunate-sulfadoxine-pyrimethamine combination.

Molecular markers of resistance to sulphadoxine-pyrimethamine during intermittent preventive treatment of pregnant women in Benin.

BACKGROUND: The prevention of malaria faces with the repeated emergence of Plasmodium falciparum resistance to drugs, often involving point mutations of the target gene. In the pregnant woman, currently the WHO recommendation is the administration of an intermittent preventive treatment (IPTp) with sulphadoxine-pyrimethamine. Sulphadoxine-pyrimethamine (SP) resistance has increased for several years in Africa, stressing the need for alternative molecules. In this context, the first randomized clinical trial comparing the efficacy of SP and mefloquine for IPTp has been conducted recently in Benin. Using samples from this trial, the current study evaluated and quantified the prevalence of mutations on the pfdhfr and pfdhps genes as well as the copy number of the pfmdr1 gene in parasites from P. falciparum-infected pregnant women before first and second IPTp administration, and at delivery. METHODS: PCR-restriction fragment length polymorphism of polymorphic codons of the pfdhfr gene (51, 59, 108, and 164) was performed. The identification of mutations in three codons of the pfdhps gene (436, 437 and 540) was achieved by PCR and sequencing. Copy number quantification for pfmdr1 gene was performed using real-time PCR. RESULTS: Results show a high prevalence rate of mutant parasites in women taking IPTp with sulphadoxine-pyrimethamine or mefloquine. The prevalence of triple and quadruple mutants was high before first drug regimen administration (79/93, 85%), and remained similar until delivery. Infection with mutant parasites was not correlated with low birth weight nor placental infection. In all samples, the copy number of pfmdr1 gene was equal to one. CONCLUSIONS: The clinical trial comparing SP and mefloquine efficacy during IPTp showed SP remained efficacious in preventing low birth weight. The present study shows a high prevalence of triple and quadruple mutations implicated in SP resistance. Although the pfdhfr/pfdhps triple and quadruple mutations were frequent, there was no evidence of correlation between these genotypes and the lack of efficacy of SP in the context of IPTp. Nevertheless, it is now obvious that SP will soon be compromised in whole Africa. Molecular markers have been recommended to monitor SP efficacy for IPTp, but given the current prevalence of mutant parasites their usefulness is questionable.

Detection of mutations in folp1, rpoB and gyrA genes of M. leprae by PCR- direct sequencing--a rapid tool for screening drug resistance in leprosy.

UNLABELLED: Conventional Mouse foot-pad (MFP) assay for screening drug resistance in M. leprae is cumbersome and time-consuming (approximately 6 to 12 months). Molecular targets for different anti-leprosy drugs have been well defined. Molecular tools for rapid detection of drug resistance in M. leprae have been standardised. A study to compare molecular methods with MFP assay in determining the drug susceptibility of M. leprae was carried out. METHODS: Forty Bacteriological Index (BI) positive patients of leprosy with clinical features of relapse (25), new cases (11) and defaulters (4) were included in the study. A skin biopsy was done and the samples were processed using both MFP assay and Molecular method. PCR assays were carried out to amplify, 388 bp of folP1 gene for dapsone resistance, 305 bp of rpoB gene for rifampicin resistance and 342 bp of gyrA gene for ofloxacin resistance, followed by direct DNA sequencing. RESULTS: Significant growth in the MFP test was obtained in only 28 out of 40 biopsies processed (70%). Ten of these isolates were dapsone resistant; one isolate showed combined resistance against dapsone, rifampicin and clofazimine. Amplification for all three genes was successful in all the 40 (100%) samples. Among folP1 products sequenced, six isolates showed mutations at 53 (or) 55 amino acid positions. Those strains which showed high-level resistance with two log growth in MFP test, and/or showed growth in passage had mutations in folp1 gene. No mutation was detected in rpoB and gyrA products. Thus no molecular evidence of Rifampicin resistance was found in the DNA isolated from biopsies. CONCLUSION: Thus PCR-direct sequencing--the rapid and high sensitive molecular technique can be applied for detection of resistance against dapsone, rifampicin and ofloxacin in M. leprae, to over come the limitations of the conventional MFP assay.