Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays

BACKGROUND: In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations. METHODS: Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically. RESULTS: Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable. CONCLUSION: On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 10(9) International Units (IU) per ml. Each vial contains 5 x 10(8) IU, equivalent to 0.5 ml of material after reconstitution

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

BACKGROUND: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/μl) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/μl. METHODS: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. RESULTS: The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. CONCLUSION: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies

Imported Plasmodium falciparum malaria in HIV-infected patients: a report of two cases

As HIV becomes a chronic infection, an increasing number of HIV-infected patients are travelling to malaria-endemic areas. Association of malaria with HIV/AIDS can be clinically severe. Severe falciparum malaria is a medical emergency that is associated with a high mortality, even when treated in an Intensive Care Unit. This article describes two cases of HIV-positive patients, who returned from malaria-endemic areas and presented a parasitaemia > 5% of erythrocytes and clinical signs of severe falciparum malaria, both with > 350 CD4 cell count/μl, absence of chemoprophylaxis and successful response. Factors like drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections

Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea

Accurate diagnosis of Plasmodium infections is essential for malaria morbidity and mortality reduction in tropical areas. Despite great advantages of light microscopy (LM) for malaria diagnosis, its limited sensitivity is a critical shortfall for epidemiological studies. Robust molecular diagnostics tools are thus needed.; The present study describes the development of a duplex quantitative real time PCR (qPCR) assay, which specifically detects and quantifies the four human Plasmodium species. Performance of this method was compared to PCR-ligase detection reaction-fluorescent microsphere assay (PCR_LDR_FMA), nested PCR (nPCR) and LM, using field samples collected from 452 children one to five years of age from the Sepik area in Papua New Guinea. Agreement between diagnostic methods was calcualted using kappa statistics.; The agreement of qPCR with other molecular diagnostic methods was substantial for the detection of P. falciparum, but was moderate for the detection of P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax prevalence by qPCR was 40.9% and 65.7% respectively. This compares to 43.8% and 73.2% by nPCR and 47.1% and 67.5% by PCR_LDR_FMA. P. malariae and P. ovale prevalence was 4.7% and 7.3% by qPCR, 3.3% and 3.8% by nPCR, and 7.7% and 4.4% by PCR_LDR_FMA. Prevalence by LM was lower for all four species, being 25.4% for P. falciparum, 54.9% for P. vivax, 2.4% for P. malariae and 0.0% for P. ovale. The quantification by qPCR closely correlated with microscopic quantification for P. falciparum and P. vivax samples (R2 = 0.825 and R2 = 0.505, respectively). The low prevalence of P. malariae and P. ovale did not permit a solid comparative analysis of quantification for these species.; The qPCR assay developed proved optimal for detection of all four Plasmodium species. Densities by LM were well reflected in quantification results by qPCR, whereby congruence was better for P. falciparum than for P. vivax. This likely is a consequence of the generally lower P. vivax densities. Easy performance of the qPCR assay, a less laborious workflow and reduced risk of contamination, together with reduced costs per sample through reduced reaction volume, opens the possibility to implement qPCR in endemic settings as a suitable diagnostic tool for large epidemiological studies

Real-time PCR assay and rapid diagnostic tests for the diagnosis of clinically suspected malaria patients in Bangladesh

<p>Abstract</p> <p>Background</p> <p>More than 95% of total malaria cases in Bangladesh are reported from the 13 high endemic districts. <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>are the two most abundant malaria parasites in the country. To improve the detection and management of malaria patients, the National Malaria Control Programme (NMCP) has been using rapid diagnostic test (RDT) in the endemic areas. A study was conducted to establish a SYBR Green-based modified real-time PCR assay as a gold standard to evaluate the performance of four commercially-available malaria RDTs, along with the classical gold standard- microscopy.</p> <p>Methods</p> <p>Blood samples were collected from 338 febrile patients referred for the diagnosis of malaria by the attending physician at Matiranga</p> <p>Upazila Health Complex (UHC) from May 2009 to August 2010. Paracheck RDT and microscopy were performed at the UHC. The blood samples were preserved in EDTA tubes. A SYBR Green-based real-time PCR assay was performed and evaluated. The performances of the remaining three RDTs (Falcivax, Onsite Pf and Onsite Pf/Pv) were also evaluated against microscopy and real-time PCR using the stored blood samples.</p> <p>Result</p> <p>In total, 338 febrile patients were enrolled in the study. Malaria parasites were detected in 189 (55.9%) and 188 (55.6%) patients by microscopy and real-time PCR respectively. Among the RDTs, the highest sensitivity for the detection of <it>P. falciparum </it>(including mixed infection) was obtained by Paracheck [98.8%, 95% confidence interval (CI) 95.8-99.9] and Falcivax (97.6%, 95% CI 94.1-99.4) compared to microscopy and real-time PCR respectively. Paracheck and Onsite Pf/Pv gave the highest specificity (98.8%, 95% CI 95.7-99.9) compared to microscopy and Onsite Pf/Pv (98.8, 95% CI 95.8-99.9) compared to real-time PCR respectively for the detection of <it>P. falciparum</it>. On the other hand Falcivax and Onsite Pf/Pv had equal sensitivity (90.5%, 95% CI 69.6-98.8) and almost 100% specificity compared to microscopy for the detection of <it>P. vivax</it>. However, compared to real-time PCR assay RDTs and microscopy gave low sensitivity (76.9%, 95% CI 56.4-91) in detecting of <it>P. vivax </it>although a very high specificity was obtained (99- 100%).</p> <p>Conclusion</p> <p>The results of this study suggest that the SYBR Green-based real-time PCR assay could be used as an alternative gold standard method in a reference setting. Commercially-available RDTs used in the study are quite sensitive and specific in detecting <it>P. falciparum</it>, although their sensitivity in detecting <it>P. vivax </it>was not satisfactory compared to the real-time PCR assay.</p

Reduced Plasmodium vivax Erythrocyte Infection in PNG Duffy-Negative Heterozygotes

BACKGROUND: Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/−). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. METHODS AND FINDINGS: We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/−) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/− vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. CONCLUSIONS: Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p