Investigation of the sequence profile of the Plasmodium falciparum 18SrRNA diagnostic target in isolates from naturally infected children with uncomplicated malaria

The gold standard for malaria diagnosis is evidence of parasitological confirmation but the traditional method by light microscopy and the routinely used rapid diagnostic tests (RDTs) have limitations. Molecular assays are known to have higher sensitivity and specificity but there are indications that they may also be compromised by genetic variability of the target sequence. The aim of this study therefore, was to evaluate the DNA sequence profile of the diagnostic target of the P. falciparum 18S rRNA PCR assay in field isolates from North-Central Nigeria. Blood samples were collected from 324 children presenting with acute febrile illness suspected to be uncomplicated malaria. Light microscopy and 18S rRNA PCR assay were employed to determine the presence of P. falciparum parasites. Sequence profile of the diagnostic target was evaluated by Sanger sequencing of the PCR products on ABI PRISM® 3100 DNA sequencer (PE Applied Biosystems). Of the 324 children enrolled into this study, 134 (41.4%) were positive for P. falciparum by microscopy while 218 (67.3%) were positive by PCR. The sensitivity of microscopy was 61.47%(95% CI= 57.88% - 69.64%) using the PCR assay as reference standard. The degree of agreement between microscopy and PCR as measured by Cohen's kappa was moderate (κ = 0.502, 95% CI = 0.463 - 0.715).Sequence analysis showed that the DNA target of the P. falciparum 18S rRNA PCR from the field isolates were highly conserved. Only one A>T single nucleotide polymorphism was found within the target sequence among the isolates in this study. This study showed that the DNA target sequence of the18S rRNA PCR assay is highly conserved in field isolates in the study region suggesting little or no impact of selective pressure acting on the locus and has implications for the enhanced sensitivity of the molecular assay.

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Molecular Factors and Biochemical Pathways Induced by Febrile Temperature in Intraerythrocytic Plasmodium falciparum Parasites

Infection and Immunity ◽

10.1128/iai.01236-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 2012-2025 ◽

Cited By ~ 83

Author(s):

Miranda S. M. Oakley ◽

Sanjai Kumar ◽

Vivek Anantharaman ◽

Hong Zheng ◽

Babita Mahajan ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Elevated Temperature ◽

Profile Analysis ◽

Expression Profiles ◽

Febrile Illness ◽

Dependent Manner ◽

Sequence Profile ◽

Host Cell Membrane ◽

Altered Expression ◽

Parasite Survival

ABSTRACT Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as “hypothetical.” We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the “crisis forms” and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

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Prone ventilation in a pregnant patient with scrub typhus-induced acute respiratory distress syndrome

BMJ Case Reports ◽

10.1136/bcr-2021-242870 ◽

2021 ◽

Vol 14 (4) ◽

pp. e242870

Author(s):

Meenupriya Arasu ◽

Nagalakshmi Swaminathan ◽

Anusha Cherian ◽

Magesh Parthiban

Keyword(s):

Acute Respiratory Distress Syndrome ◽

Respiratory Distress Syndrome ◽

Respiratory Distress ◽

Distress Syndrome ◽

Scrub Typhus ◽

Febrile Illness ◽

Pregnant Patient ◽

Lung Compliance ◽

Pcr Assay ◽

Oxygenation Indices

A 23-year-old primigravida at 20 weeks of gestation presented to our hospital with undifferentiated febrile illness and severe acute respiratory distress syndrome. She was intubated in the emergency department and transferred to the intensive care unit. Initial treatment included ventilatory care, vasopressor support and broad-spectrum antibiotics. Based on a positive PCR assay for scrub typhus, she was treated with intravenous doxycycline and azithromycin. Despite reduction in fever, her oxygenation further declined. Following a risk–benefits assessment, we decided to ventilate her in prone position for 8 hours a day for three consecutive days using a checklist-based protocol. Her oxygenation indices and lung compliance markedly improved over this period, and she was extubated a day later. She was eventually discharged home after 1 week.

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A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids

Journal of Medical Microbiology ◽

10.1099/jmm.0.47485-0 ◽

2008 ◽

Vol 57 (3) ◽

pp. 324-331 ◽

Cited By ~ 33

Author(s):

Alvin Stewart ◽

Benjamin Satterfield ◽

Marissa Cohen ◽

Kim O'Neill ◽

Richard Robison

Keyword(s):

Yersinia Pestis ◽

Real Time ◽

Real Time Pcr ◽

Yersinia Pseudotuberculosis ◽

Target Sequence ◽

Health Departments ◽

Pcr Assay ◽

Virulence Plasmids ◽

Taqman Probes ◽

Sporadic Disease

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

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Dihydroartemisinin-piperaquine for the routine treatment of uncomplicated malaria in Northern Ghana

International Journal of Clinical Trials ◽

10.18203/2349-3259.ijct20200200 ◽

2020 ◽

Vol 7 (1) ◽

pp. 1

Author(s):

Abraham R. Oduro ◽

Samuel Chatio ◽

Emmanuel Ayamba ◽

Thomas Anyorigiya ◽

Fred Binka ◽

...

Keyword(s):

Uncomplicated Malaria ◽

Haemoglobin Concentration ◽

Febrile Illness ◽

Northern Ghana ◽

First Line ◽

Intention To Treat ◽

Health Delivery ◽

Baseline Temperature ◽

Concentration Changes

Background: Dihydroartemisinin-piperaquine is a first line treatment for uncomplicated malaria in Ghana. A facility-based study was undertaken to examine the effectiveness of the treatment in the routine health care system.Methods: The study was undertaken at the Navrongo demographic surveillance area. Patients presenting with acute febrile illness were enrolled after informed consented and confirmation by microscopy. Patients were randomized into supervised group who received treatment under direct observation and unsupervised group which had only the first treatment given under supervision. Treatment was according to bodyweight and 42 days follow-up was undertaken.Results: A total of 194 patients were enrolled; 54.1% were females and 51% had supervised treatment. The median age and weight were 6.7 years and 20.0 kg respectively. Mean baseline temperature, haemoglobin concentration and parasite density were, 37.6 oC, 11.1 g/dl and 11,098 parasites per microliter of blood respectively. Study completion rate was 93.3%, day 42 polymerase chain reaction-unadjusted adequate clinical and parasitological responses rate (ACPR) was 93.4% by evaluable and 87.1 % by intention-to-treat (ITT). The day 42 ACPR by evaluable was 92.3% in the supervised arm compared to 94.4% in the unsupervised arm. The day 42 ACPR by ITT was 85.7% in the supervised and 88.5% in the unsupervised arms. The fever resolution and haemoglobin concentration changes for the two arms were similar.Conclusions: The results show that dihydroartemisinin-piperaquine is effective and good first-line antimalarial in the routine health delivery system.

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