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

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients.</p> <p>Methods</p> <p>475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.</p> <p>Results </p> <p>136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms.</p> <p>Conclusion</p> <p>Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.</p

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

Novel approaches to the diagnosis of Strongyloides stercoralis infection

AbstractStrongyloides stercoralis differs from the other soil-transmitted helminths because it puts infected subjects at risk of a fatal syndrome (in cases of immunosuppression for medical conditions, immunosuppressant therapies, or both). Chronic strongyloidiasis is often a non-severe condition, or is sometimes even asymptomatic, but diagnosis and effective therapy are essential in order to eradicate the infection and the life-long risk involved. Therefore, diagnostic methods need to be highly sensitive. Stool microscopy and the Kato–Katz technique are commonly used in prevalence studies, but they are inadequate for S. stercoralis detection. This is probably the main reason why the global prevalence has long been underestimated. Concentration methods, the Baermann technique and Koga agar plate culture have better, but still unsatisfactory, sensitivity. Serological tests have demonstrated higher sensitivity; although some authors have concerns about their specificity, it is possible to define cut-off values over which infection is almost certain. In particular, the luciferase immunoprecipitation system technique combined with a recombinant antigen (NIE) demonstrated a specificity of almost 100%. ELISA coproantigen detection has also shown promising results, but still needs full evaluation. Molecular diagnostic methods are currently available in a few referral centres as in-house techniques. In this review, on the basis of the performance of the different diagnostic methods, we outline diagnostic strategies that could be proposed for different purposes, such as: prevalence studies in endemic areas; individual diagnosis and screening; and monitoring of cure in clinical care and clinical trials

A retrospective study comparing agar plate culture, indirect immunofluorescence and real-time PCR for the diagnosis of Strongyloides stercoralis infection

Strongyloides stercoralis is a parasite that can cause death in immunocompromised people. A proper diagnosis is hence essential. The real-time polymerase-chain reaction (RT-PCR) is a novel, promising diagnostic method, that detects the DNA of the parasite in stool samples. In this retrospective study, we compared the sensitivity of agar plate coproculture (APC), an in-house immunofluorescence test (IFAT) and an in-house RT-PCR for the diagnosis of S. stercoralis infection. The study sample was composed by 223 samples. Samples resulting positive to APC, IFAT and RT-PCR were 20, 140 and 25, respectively. When sensitivity was calculated against a composite reference standard, serology confirmed the best performance (sensitivity 95%), followed by RT-PCR (57%) and APC (45%). In conclusion, in a non-endemic setting, serology is the best screening method, while the combination of APC and RT-PCR does not seem a reasonable approach to increase sensitivity. Both methods can have a role as confirmatory tests for selected cases

Proteção do dano oxidativo hepático induzido por ferro pelo extrato aquoso da planta Plectranthus barbatus

Plectranthus barbatus Andrews (Lamiaceae) &#233; uma planta muita utilizada na medicina popular para o tratamento de doen&#231;as gastrointestinais e hep&#225;ticas. O objetivo do presente trabalho foi estudar o efeito protetor do extrato aquoso de P. barbatus (EAPB) sobre os danos hep&#225;ticos causados pela sobrecarga de ferro provocada pelo ferro-dextran em ratos. O tratamento com ferro-dextran induziu uma redu&#231;&#227;o significativa na concentra&#231;&#227;o de glutationa reduzida nos animais tratados em rela&#231;&#227;o ao grupo controle e o tratamento pr&#233;vio dos animais com o EAPB protegeu o f&#237;gado do efeito provocado pelo ferro neste par&#226;metro. Com rela&#231;&#227;o &#224; lipoperoxida&#231;&#227;o, houve aumento significativo na concentra&#231;&#227;o de malondialde&#237;do (MDA) nos animais tratados em rela&#231;&#227;o ao controle, entretanto, quando os animais receberam o tratamento pr&#233;vio com o EAPB, houve redu&#231;&#227;o significativa na concentra&#231;&#227;o do MDA. A an&#225;lise histopatol&#243;gica mostrou que o grupo tratado com ferro-dextran apresentou gr&#226;nulos de ferro no citoplasma das c&#233;lulas de Kupffer com alargamento das mesmas e algumas com os n&#250;cleos hipertr&#243;ficos. O tratamento pr&#233;vio com EAPB resultou no desaparecimento dos sinais de danos &#224;s c&#233;lulas de Kupffer sem nenhum n&#250;cleo hipertr&#243;fico, mas com a presen&#231;a de gr&#226;nulos de ferro totalmente fagocitados, o que demonstra uma apar&#234;ncia morfol&#243;gica normal. Portanto, o EAPB pode ser &#250;til na preven&#231;&#227;o de danos hep&#225;ticos induzidos por sobrecarga de ferro

Screening for Neglected Tropical Diseases and other infections in African refugees and asylum seekers in Rome and Lazio region, Italy

Background: Few reliable data are available on Neglected Tropical Diseases (NTDs) and other infections among African refugees and asylum seekers in Italy.We aimed to estimate the prevalence of NTDs and other infections in a large cohort of African refugees and asylum seekers living in reception centers in Lazio, Italy. Material and methods: This is an observational, prospective prevalence study on infectious diseases in a large population of African refugees and asylum seekers (936 overall) consecutively enrolled for screening purpose at the Infectious and Tropical diseases outpatient clinic of the National Institute of Migrant and Poverty (INMP), Rome from August 2019 to December 2020. Results: We found a prevalence of 8.8 % and 31 % for Strongyloides and schistosoma infection, respectively, while the prevalence of human immunodeficiency virus (HIV) infection was 0.7 %, HCV antibodies2.5%, hepatitis B virus surface antigen 10.8 % and syphilis serological tests 2.9 %. Conclusion: Strongyloidiasis and schistosomiasis are highly prevalent among African refugees and asylum seekers in Italy, in contrast to communicable diseases (with the exception of hepatitis B).Raising awareness of NTDs among health professionals and implementing guidelines seems to be of paramount importance to prevent these diseases and their sufferers from becoming even more “neglected”