Effective high-throughput blood pooling strategy before DNA extraction for detection of malaria in low-transmission settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost-and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 μl in 1 sample, was optimal, and the parasite density as low as 2 p/μl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings.Publisher PDFPeer reviewe

Observational study of adult respiratory infections in primary care clinics in Myanmar: understanding the burden of melioidosis, tuberculosis and other infections not covered by empirical treatment regimes.

BACKGROUND: Lower respiratory infections constitute a major disease burden worldwide. Treatment is usually empiric and targeted towards typical bacterial pathogens. Understanding the prevalence of pathogens not covered by empirical treatment is important to improve diagnostic and treatment algorithms. METHODS: A prospective observational study in peri-urban communities of Yangon, Myanmar was conducted between July 2018 and April 2019. Sputum specimens of 299 adults presenting with fever and productive cough were tested for Mycobacterium tuberculosis (microscopy and GeneXpert MTB/RIF [Mycobacterium tuberculosis/resistance to rifampicin]) and Burkholderia pseudomallei (Active Melioidosis Detect Lateral Flow Assay and culture). Nasopharyngeal swabs underwent respiratory virus (influenza A, B, respiratory syncytial virus) polymerase chain reaction testing. RESULTS: Among 299 patients, 32% (95% confidence interval [CI] 26 to 37) were diagnosed with tuberculosis (TB), including 9 rifampicin-resistant cases. TB patients presented with a longer duration of fever (median 14 d) and productive cough (median 30 d) than non-TB patients (median fever duration 6 d, cough 7 d). One case of melioidosis pneumonia was detected by rapid test and confirmed by culture. Respiratory viruses were detected in 16% (95% CI 12 to 21) of patients. CONCLUSIONS: TB was very common in this population, suggesting that microscopy and GeneXpert MTB/RIF on all sputum samples should be routinely included in diagnostic algorithms for fever and cough. Melioidosis was uncommon in this population

Complexes Formed in Solution Between Vanadium(IV)/(V) and the Cyclic Dihydroxamic Acid Putrebactin or Linear Suberodihydroxamic Acid

The ability of microbial siderophores to coordinate metal ions, particularly Fe(III), continues to generate interest in the poten-tial applications of these bioligands in the environment and medicine.13 Siderophores produced by terrestrial and marin

Dinuclear [(V^VO(putrebactin))₂(μ-OCH₃)₂] Formed in Solution as Established from LC-MS Measurements Using ⁵⁰V‑Enriched V₂O₅

Analysis of 1:1 solutions of V­(V) and the macrocyclic dihydroxamic acid siderophore putrebactin (pbH₂) in 1:1 H₂O/CH₃OH using triple quadrupole liquid chromatography–mass spectrometry (LC-MS-QQQ) (pH ≈ 4) showed two well-resolved peaks (<i>t</i>_R(1) 10.85 min; <i>t</i>_R(2) 14.27 min) using simultaneous detection modes (absorbance, 450 nm; selective ion monitoring, <i>m</i>/<i>z</i> 437) characteristic of the previously identified oxidoV­(V) complex [V^VO­(pb)]⁺ ([M]⁺, <i>m</i>/<i>z</i>_calc 437.1). Peak 1 gave mass spectrometry (MS) signals consistent with [V^VO­(pb)]⁺, together with [V^VO­(pb)­(OH)] and the dinuclear complexes [(V^VO­(pb))₂(μ-OH)]⁺ and [(V^VO­(pb))₂(μ-OH)₂]. Peak 2 gave MS signals consistent with [V^VO­(pb)]⁺, together with [V^VO­(pb)­(OCH₃)] and the dinuclear complexes [(V^VO­(pb))₂(μ-OCH₃)]⁺ and [(V^VO­(pb))₂(μ-OCH₃)₂]. This analysis showed that two groups of V­(V)/pbH₂ complexes with water- or methanol-derived ancillary ligands were resolved by liquid chromatography (LC). The detection of [V^VO­(pb)]⁺ in both peaks could be accounted for by its production from dissociation (peak 1: [(V^VO­(pb))₂(μ-OH)]⁺ → [V^VO­(pb)]⁺ + [V^VO­(pb)­(OH)]; peak 2: [(V^VO­(pb))₂(μ-OCH₃)]⁺ → [V^VO­(pb)]⁺ + [V^VO­(pb)­(OCH₃)]). The assignment of the signal at <i>m</i>/<i>z</i>_obs 959.2 (100%) as the dinuclear complex [(V^VO­(pb))₂(μ-OCH₃)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 959.3) and not an ion cluster of mononuclear [V^VO­(pb)­(OCH₃)] ({2­[M] + Na⁺}⁺, <i>m</i>/<i>z</i>_calc 959.3) was made unequivocal by the use of ⁵⁰V-enriched V₂O₅, which gave a signal with an isotope pattern comprising the sum of the patterns of the three constituent ⁵¹V–⁵¹V, ⁵¹V–⁵⁰V, and ⁵⁰V–⁵⁰V species. Coordination of methoxide was confirmed upon the replacement of CH₃OH with CD₃OD, which generated [(V^VO­(pb))₂(μ-OCD₃)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 965.3, <i>m</i>/<i>z</i>_obs 965.3). Analysis of 1:1 solutions of Mo­(VI) and pbH₂ showed a single peak in the LC (<i>t</i>_R 16.04 min), which gave MS signals that were characterized as mononuclear [Mo^VI(O)₂(pb)] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 523.1, <i>m</i>/<i>z</i>_obs 523.1) and dinuclear [(Mo^VIO­(pb))₂(μ-O)₂] ([M + Na⁺]⁺, <i>m</i>/<i>z</i>_calc 1019.1, <i>m</i>/<i>z</i>_obs 1019.2). The steric and electronic effects of the <i>cis</i>-dioxido group(s) in [Mo^VI(O)₂(pb)] mitigated coordination of solvent-derived ancillary ligands. The work highlights the value of using isotopically enriched metal ion sources and deuterated solvents to deconvolute metal/siderophore solution speciation. The results have relevance for an improved understanding of the coordination chemistry of pbH₂ and other marine siderophores in V­(V)- and Mo­(VI)-rich surface ocean waters

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R

Proteomics of <i>Pseudomonas aeruginosa</i> Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire <i>P. aeruginosa</i> from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and <i>S</i>-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (<i>pchDFG</i> and <i>fptA</i>) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R

Effective high-throughput blood pooling strategy before DNA extraction for detection of malaria in low-transmission settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings