Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naive hosts, P. falciparum parasites of the 3D7 strain produce 1.4 x 10(-)(1)(3) g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/muL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

Malaria rapid diagnostic test performance : results of WHO product testing of malaria RDTs : round 5 (2013)

WHO estimates that half the world’s population is at risk of malaria. In 2012, there were an estimated 207 million cases (with an uncertainty range of 135 million to 287 million) and an estimated 627 000 deaths (with an uncertainty range of 473 000 to 789 000). Approximately 90% of all malaria deaths occur in sub-Saharan Africa, and 77% occur in children under 5 years. Malaria remains endemic in 104 countries, and, while parasite-based diagnosis is increasing, most suspected cases of malaria are still not properly confirmed, resulting in over-use of antimalarial drugs and poor disease monitoring (1)...

Malaria rapid diagnostic test performance: Results of WHO product testing of malaria RDTs: Round 6 (2014-2015)

Overview This report, published in conjunction with a summary overview of results of rounds 1–6, is the sixth in a series of laboratory-based evaluations of rapid diagnostic tests (RDTs) for malaria. It provides a comparative measure of their performance in a standardized way to distinguish between well and poorly performing tests. It can be used by malaria control programmes and guide WHO procurement recommendations for these diagnostic tools. The evaluation reported here was a joint project of the WHO Global Malaria Programme, the Foundation for Innovative New Diagnostics (FIND) and the United States Centers for Disease Control and Prevention (CDC) within the WHO-FIND Malaria RDT Evaluation Programme. The project was financed by FIND through a grant from UNITAID.

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F-V/F-M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F-V/F-M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks

Abstract Background Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. Methods The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Results Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). Conclusions An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.

Cost effectiveness of OptiMal® rapid diagnostic test for malaria in remote areas of the Amazon Region, Brazil

Abstract Background In areas with limited structure in place for microscopy diagnosis, rapid diagnostic tests (RDT) have been demonstrated to be effective. Method The cost-effectiveness of the Optimal® and thick smear microscopy was estimated and compared. Data were collected on remote areas of 12 municipalities in the Brazilian Amazon. Data sources included the National Malaria Control Programme of the Ministry of Health, the National Healthcare System reimbursement table, hospitalization records, primary data collected from the municipalities, and scientific literature. The perspective was that of the Brazilian public health system, the analytical horizon was from the start of fever until the diagnostic results provided to patient and the temporal reference was that of year 2006. The results were expressed in costs per adequately diagnosed cases in 2006 U.S. dollars. Sensitivity analysis was performed considering key model parameters. Results In the case base scenario, considering 92% and 95% sensitivity for thick smear microscopy to Plasmodium falciparum and Plasmodium vivax, respectively, and 100% specificity for both species, thick smear microscopy is more costly and more effective, with an incremental cost estimated at US$549.9 per adequately diagnosed case. In sensitivity analysis, when sensitivity and specificity of microscopy for P. vivax were 0.90 and 0.98, respectively, and when its sensitivity for P. falciparum was 0.83, the RDT was more cost-effective than microscopy. Conclusion Microscopy is more cost-effective than OptiMal® in these remote areas if high accuracy of microscopy is maintained in the field. Decision regarding use of rapid tests for diagnosis of malaria in these areas depends on current microscopy accuracy in the field.

Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains : implications for rapid diagnostic tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

Blood transfer devices for malaria rapid diagnostic tests : evaluation of accuracy, safety and ease of use

Background: Malaria rapid diagnostic tests (RDTs) are increasingly used by remote health personnel with minimal training in laboratory techniques. RDTs must, therefore, be as simple, safe and reliable as possible. Transfer of blood from the patient to the RDT is critical to safety and accuracy, and poses a significant challenge to many users. Blood transfer devices were evaluated for accuracy and precision of volume transferred, safety and ease of use, to identify the most appropriate devices for use with RDTs in routine clinical care. Methods: Five devices, a loop, straw-pipette, calibrated pipette, glass capillary tube, and a new inverted cup device, were evaluated in Nigeria, the Philippines and Uganda. The 227 participating health workers used each device to transfer blood from a simulated finger-prick site to filter paper. For each transfer, the number of attempts required to collect and deposit blood and any spilling of blood during transfer were recorded. Perceptions of ease of use and safety of each device were recorded for each participant. Blood volume transferred was calculated from the area of blood spots deposited on filter paper. Results: The overall mean volumes transferred by devices differed significantly from the target volume of 5 microliters (p < 0.001). The inverted cup (4.6 microliters) most closely approximated the target volume. The glass capillary was excluded from volume analysis as the estimation method used is not compatible with this device. The calibrated pipette accounted for the largest proportion of blood exposures (23/225, 10%); exposures ranged from 2% to 6% for the other four devices. The inverted cup was considered easiest to use in blood collection (206/ 226, 91%); the straw-pipette and calibrated pipette were rated lowest (143/225 [64%] and 135/225 [60%] respectively). Overall, the inverted cup was the most preferred device (72%, 163/227), followed by the loop (61%, 138/227). Conclusions: The performance of blood transfer devices varied in this evaluation of accuracy, blood safety, ease of use, and user preference. The inverted cup design achieved the highest overall performance, while the loop also performed well. These findings have relevance for any point-of-care diagnostics that require blood sampling.

Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests

Background Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. Methods The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. Results Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. Conclusions These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Pan-Plasmodium band sensitivity for Plasmodium falciparum detection in combination malaria rapid diagnostic tests and implications for clinical management

Background: Malaria rapid diagnostic tests (RDTs) are appropriate for case management, but persistent antigenaemia is a concern for HRP2-detecting RDTs in endemic areas. It has been suggested that pan-pLDH test bands on combination RDTs could be used to distinguish persistent antigenaemia from active Plasmodium falciparum infection, however this assumes all active infections produce positive results on both bands of RDTs, an assertion that has not been demonstrated. Methods: In this study, data generated during the WHO-FIND product testing programme for malaria RDTs was reviewed to investigate the reactivity of individual test bands against P. falciparum in 18 combination RDTs. Each product was tested against multiple wild-type P. falciparum only samples. Antigen levels were measured by quantitative ELISA for HRP2, pLDH and aldolase. Results: When tested against P. falciparum samples at 200 parasites/μL, 92% of RDTs were positive; 57% of these on both the P. falciparum and pan bands, while 43% were positive on the P. falciparum band only. There was a relationship between antigen concentration and band positivity; ≥4 ng/mL of HRP2 produced positive results in more than 95% of P. falciparum bands, while ≥45 ng/mL of pLDH was required for at least 90% of pan bands to be positive. Conclusions: In active P. falciparum infections it is common for combination RDTs to return a positive HRP2 band combined with a negative pan-pLDH band, and when both bands are positive, often the pan band is faint. Thus active infections could be missed if the presence of a HRP2 band in the absence of a pan band is interpreted as being caused solely by persistent antigenaemia.

Impact of the availability of an influenza virus rapid antigen test on diagnostic decision making in a pediatric emergency department

OBJECTIVES: Fever is one of the most commonly seen symptoms in the pediatric emergency department. The objective of this study was to observe how the rapid testing for influenza virus impacts on the management of children with fever. METHODS: We performed a review of our pediatric emergency department records during the 2008/2009 annual influenza season. The BinaxNow Influenza A+B test was performed on patients with the following criteria: age 1.0 to 16.0 years, fever greater than 38.5 °C, fever of less than 96 hours' duration after the onset of clinical illness, clinical signs compatible with acute influenza, and nontoxic appearance. Additional laboratory tests were performed at the treating physician's discretion. RESULTS: The influenza rapid antigen test was performed in 192 children. One hundred nine (57%) were influenza positive, with the largest fraction (101 patients) positive for influenza A. The age distribution did not differ between children with negative and positive test results (mean, 5.3 vs. 5.1 years, not statistically significant). A larger number of diagnostic tests were performed in the group of influenza-negative patients. Twice as many complete blood counts, C-reactive protein determinations, lumbar punctures, and urinalyses were ordered in the latter group. CONCLUSIONS: Rapid diagnosis of influenza in the pediatric emergency department affects the management of febrile children as the confirmation of influenza virus infection decreases additional diagnostic tests ordered.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Impact of a Point-of-Care Rapid Influenza Test on Antibiotic Prescribing Patterns in Southern Sri Lanka

Background: Acute febrile respiratory illnesses, including influenza, account for a large proportion of ambulatory care visits worldwide. In the developed world, these encounters commonly result in unwarranted antibiotic prescriptions; data from more resource-limited settings are lacking. The purpose of this study was to describe the epidemiology of influenza among outpatients in southern Sri Lanka and to determine if access to rapid influenza test results was associated with decreased antibiotic prescriptions.

Methods: In this pretest- posttest study, consecutive patients presenting from March 2013- April 2014 to the Outpatient Department of the largest tertiary care hospital in southern Sri Lanka were surveyed for influenza-like illness (ILI). Patients meeting World Health Organization criteria for ILI-- acute onset of fever ≥38.0°C and cough in the prior 7 days--were enrolled. Consenting patients were administered a structured questionnaire, physical examination, and nasal/nasopharyngeal sampling. Rapid influenza A/B testing (Veritor System, Becton Dickinson) was performed on all patients, but test results were only released to patients and clinicians during the second phase of the study (December 2013- April 2014).

Results: We enrolled 397 patients with ILI, with 217 (54.7%) adults ≥12 years and 188 (47.4%) females. A total of 179 (45.8%) tested positive for influenza by rapid testing, with April- July 2013 and September- November 2013 being the periods with the highest proportion of ILI due to influenza. A total of 310 (78.1%) patients with ILI received a prescription for an antibiotic from their outpatient provider. The proportion of patients prescribed antibiotics decreased from 81.4% in the first phase to 66.3% in the second phase (p=.005); among rapid influenza-positive patients, antibiotic prescriptions decreased from 83.7% in the first phase to 56.3% in the second phase (p=.001). On multivariable analysis, having a positive rapid influenza test available to clinicians was associated with decreased antibiotic use (OR 0.20, 95% CI 0.05- 0.82).

Conclusions: Influenza virus accounted for almost 50% of acute febrile respiratory illness in this study, but most patients were prescribed antibiotics. Providing rapid influenza test results to clinicians was associated with fewer antibiotic prescriptions, but overall prescription of antibiotics remained high. In this developing country setting, a multi-faceted approach that includes improved access to rapid diagnostic tests may help decrease antibiotic use and combat antimicrobial resistance.

A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients.

Purpose: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. Methods: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. Results: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of the hybrid, resulting in a lower TM (TM=51.27 °C) than that observed for the perfectly matched mutant allele (TM=59.9 °C). The mutant allele was detected in only one of the 696 subjects, an affected AMD patient. Conclusions: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2%, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345Arg mutation and its association with AMD within other populations.