Evaluation of Sulfa Drugs against Recombinant Pneumocystis carinii Dihydropteroate Synthetase and In vivo

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74851/1/j.1550-7408.1996.tb04976.x.pd

Sulfa Resistance in Mouse-Derived Pneumocystis carinii

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74674/1/j.1550-7408.1996.tb04975.x.pd

Rapid Diagnostic Test Performance Assessed Using Latent Class Analysis for the Diagnosis of Plasmodium falciparum Placental Malaria.

Placental malaria causes low birth weight and neonatal mortality in malaria-endemic areas. The diagnosis of placental malaria is important for program evaluation and clinical care, but is compromised by the suboptimal performance of current diagnostics. Using placental and peripheral blood specimens collected from delivering women in Malawi, we compared estimation of the operating characteristics of microscopy, rapid diagnostic test (RDT), polymerase chain reaction, and histopathology using both a traditional contingency table and a latent class analysis (LCA) approach. The prevalence of placental malaria by histopathology was 13.8%; concordance between tests was generally poor. Relative to histopathology, RDT sensitivity was 79.5% in peripheral and 66.2% in placental blood; using LCA, RDT sensitivities increased to 93.7% and 80.2%, respectively. Our results, if replicated in other cohorts, indicate that RDT testing of peripheral or placental blood may be suitable approaches to detect placental malaria for surveillance programs, including areas where intermittent preventive therapy in pregnancy is not used

Spatial and social factors drive anemia in Congolese women

Anemia is common in women of child-bearing age in the Democratic Republic of the Congo (DRC). As part of the 2007 DRC Demographic and Health Survey (DHS), 4,638 women of childbearing age (including 526 pregnant women) were tested for HIV and had the hemoglobin content of their blood recorded. We assessed malaria prevalence using laboratory methods. The DHS provided extensive information for individuals, as well as household cluster coordinates which enabled us to derive several spatial variables. Multilevel analyses were conducted to determine individual and contextual risk factors for anemia. Prevalence varied geographically and was associated with both one's ethnic group and the amount and type of nearby agriculture. In contrast, prevalence was not affected by HIV or malaria status

Comparison of real-time PCR and microscopy for malaria parasite detection in Malawian pregnant women

Abstract Background New diagnostic tools for malaria are required owing to the changing epidemiology of malaria, particularly among pregnant women in sub-Saharan Africa. Real-time PCR assays targeting Plasmodium falciparum lactate dehydrogenase (pfldh) gene may facilitate the identification of a high proportion of pregnant women with a P. falciparum parasitaemia below the threshold of microscopy. These molecular methods will enable further studies on the effects of these submicroscopic infections on maternal health and birth outcomes. Methods The pfldh real-time PCR assay and conventional microscopy were compared for the detection of P. falciparum from dried blood spots and blood smears collected from the peripheral blood of 475 Malawian women at delivery. A cycle threshold (Ct) of the real-time PCR was determined optimizing the sensitivity and specificity of the pfldh PCR assay compared to microscopy. A real-time PCR species-specific assay was applied to identify the contribution to malaria infections of three Plasmodium species (P. falciparum P. ovale and P. malariae) in 44 discordant smear and pfldh PCR assay results. Results Of the 475 women, P. falciparum was detected in 11 (2.3%) by microscopy and in 51 (10.7%) by real-time PCR; compared to microscopy, the sensitivity of real-time PCR was 90.9% and the specificity 91.2%. If a Ct value of 38 was used as a cut-off, specificity improved to 94.6% with no change in sensitivity. The real-time PCR species-specific assay detected P. falciparum alone in all but four samples: two samples were mixed infections with P. falciparum and P. malariae, one was a pure P. malariae infection and one was a pfldh PCR assay-positive/species-specific assay-negative sample. Of three P. malariae infections detected by microscopy, only one was confirmed by the species-specific assay. Conclusions Although microscopy remains the most appropriate method for clinical malaria diagnosis in field settings, molecular diagnostics such as real-time PCR offer a more reliable means to detect malaria parasites, particularly at low levels. Determination of the possible contribution of these submicroscopic infections to poor birth outcomes and maternal health is critical. For future studies to investigate these effects, this pfldh real-time PCR assay offers a reliable detection method

Increased prevalence of dhfr and dhps mutants at delivery in Malawian pregnant women receiving intermittent preventive treatment for malaria

In the context of an Intermittent preventive treatment (IPTp) trial for pregnant women in Malawi, P. falci-parum samples from 85 women at enrollment and 35 women at delivery were genotyped for mutations associated with sulfadoxine-pyrimethamine resistance. The prevalence of the highly resistant haplotype with mutations at codons 51 and 108 of dihydrofolate reductase (dhfr) and codons 437 and 540 of dihydropteroate synthase (dhps) increased from 81% at enrollment to 100% at delivery (p=0.01). Pregnant women who were smear-positive at enrollment were more likely to have P. falciparum parasitemia at delivery. These results lend support to concerns that IPTp use may lead to increased drug resistance in pregnant women during pregnancy and emphasize the importance of screening pregnant women for malaria parasites in areas with prevalent SP resistance even when they are already on IPTp

A Cross-Sectional Survey of Plasmodium falciparum pfcrt Mutant Haplotypes in the Democratic Republic of Congo

In the Democratic Republic of the Congo (DRC), artesunate-amodiaquine is first-line therapy for falciparum malaria; little is known about the prevalence of molecular markers of parasite drug resistance. Across the DRC, we genotyped 166 parasites in Plasmodium falciparum chloroquine resistance transporter (pfcrt) using polymerase chain reaction (PCR) and sequencing. Of these parasites, 73 (44%) parasites were pure wild-type CVMNK, 55 (31%) parasites were chloroquine-resistant CVIET, 35 (21.1%) parasites were mixed CVMNK and CVIET, and 3 parasites were other genotypes. Ninety-two infections (55.4%) harbored the pfcrt K76T substitution that is highly correlated with chloroquine failure. The amodiaquine-resistant SVMNT haplotype was absent. Geographically, pfcrt haplotypes were not clearly clustered. Chloroquine accounted for 19.4% of antimalarial use, and amodiaquine accounted for 15.3% of antimalarial use; there were no associations between drug use and mutant haplotype prevalence. In the DRC, our molecular survey indicates that resistance to chloroquine is substantial but that resistance to amodiaquine is absent. These contrasting findings highlight the need for molecular surveillance of drug resistance to inform malaria control policies

Malaria surveillance in the Democratic Republic of the Congo: comparison of microscopy, PCR, and rapid diagnostic test

Malaria surveillance is critical for control efforts, but diagnostic methods frequently disagree. Here we compare microscopy, PCR, and a Rapid Diagnostic Test in 7,137 samples from children in the Democratic Republic of the Congo using Latent Class Analysis. PCR had the highest sensitivity (94.6%) and microscopy had the lowest (76.7%)

Pooled Deep Sequencing of Plasmodium falciparum Isolates: An Efficient and Scalable Tool to Quantify Prevailing Malaria Drug-Resistance Genotypes

Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies

Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis jirovecii

Pneumocystis is an important opportunistic pathogen in immunocompromised patients. Molecular epidemiology studies are needed to understand transmission. This article evaluates non-invasive sampling and a new strain typing tool, both of which could be used for this purpose.Pneumocystis jirovecii is a symbiotic respiratory fungus that presents in 2 clinical forms: pneumonia in immunocompromised patients or colonization, defined by the presence of the organism without associated clinical symptoms. Currently, diagnosis requires invasive bronchoscopy, which may not be available in some settings and is inappropriate for detecting colonization in healthy individuals. Noninvasive diagnostic techniques and molecular strain typing tools that can be used on these samples are critical for conducting studies to better understand transmission. We evaluated 2 real-time polymerase chain reaction (PCR) assays targeting dihydropteroate synthase and the major surface glycoprotein for detection in 77 oropharyngeal washes (OPWs) from 43 symptomatic human immunodeficiency virus-infected patients who underwent bronchoscopy. We also evaluated the ability of a new microsatellite (MS) genotyping panel to strain type infections from these samples. Each PCR used individually provided a high sensitivity (>80%) for detection of pneumonia but a modest specificity (<70%). When used in combination, specificity was increased to 100% with a drop in sensitivity (74%). Concentration of organisms by PCR in the OPW tended to be lower in colonized individuals compared with those with pneumonia, but differences in concentration could not clearly define colonization in symptomatic individuals. Oropharyngeal wash samples were genotyped using 6 MSs with ≥4 alleles successfully genotyped in the majority of colonized patients and ≥5 alleles in patients with pneumonia. The MS profile was consistent over time within patients with serial OPWs analyzed. Microsatellite genotyping on noninvasive samples may aid in studying the molecular epidemiology of this pathogen without requiring invasive diagnostic techniques