Most Plasmodium falciparum infections that are detected in community surveys are characterized by low-density parasitaemia and the absence of clinical symptoms. Molecular diagnostics have shown that this asymptomatic parasitic reservoir is more widespread than previously thought, even in low-endemic areas. In this Opinion article, we describe the detectability of asymptomatic malaria infections and the relevance of submicroscopic infections for parasite transmission to mosquitoes and for community interventions that aim at reducing transmission. We argue that wider deployment of molecular diagnostic tools is needed to provide adequate insight into the epidemiology of malaria and infection dynamics to aid elimination efforts.

Asymptomatic malaria infections: detectability, transmissibility and public health relevance

Background
Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data.

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Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

In this review we examine the literature related to emerging technologies that will help to reshape the clinical microbiology laboratory. These topics include nucleic acid amplification tests such as isothermal and point-of-care molecular diagnostics, multiplexed panels for syndromic diagnosis, digital PCR, next-generation sequencing, and automation of molecular tests. We also review matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry methods and their role in identification of microorganisms. Lastly, we review the shift to liquid-based microbiology and the integration of partial and full laboratory automation that are beginning to impact the clinical microbiology laboratory.

Emerging Technologies for the Clinical Microbiology Laboratory

Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

https://www.pnas.org/content/pnas/116/11/4834.full.pdf

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

In the last decade, there has been an upsurge of interest in developing malaria rapid diagnostic test (RDT) kits for the detection of Plasmodium species. Three antigens – Plasmodium
falciparum histidine-rich protein 2 (PfHRP2), plasmodial aldolase and plasmodial lactate dehydrogenase (pLDH) – are currently used for RDTs. Tests targeting HRP2 contribute to more than 90 % of the malaria RDTs in current use. However, the specificities, sensitivities, numbers of false positives, numbers of false negatives and temperature tolerances of these tests vary considerably, illustrating the difficulties and challenges facing current RDTs. This paper describes recent developments in malaria RDTs, reviewing RDTs detecting PfHRP2, pLDH and plasmodial aldolase. The difficulties associated with RDTs, such as genetic variability in the Pfhrp2 gene and the persistence of antigens in the bloodstream following the elimination of parasites, are discussed. The prospect of overcoming the problems associated with current RDTs with a new generation of alternative malaria antigen targets is also described.

Malaria rapid diagnostic tests: challenges and prospects.

(See the major article by Polley et al on pages 637–44.)

Accurate detection of malaria is of increasing importance as the malaria prevalence declines across much of its range [1, 2], with surveillance and screening becoming increasingly important in program management [3–5]. Microscopy and antigen-detecting rapid diagnostic tests (RDTs), when performed correctly, provide accurate diagnosis for case management [6] but cannot reliably detect lower-density parasitemia that may occur in asymptomatic individuals, who then represent reservoirs of infection. Such low-density infections with Plasmodium species are widely documented [7–12] and may contribute to transmission [13–15]. To eliminate malaria and prevent reintroduction, sustaining the capacity to detect such infections will be critical. The development of field-ready assays that can detect foci of infection in a way timely enough to enable treatment is therefore recognized as a major priority for malaria elimination [3, 16, 17]. Such highly sensitive assays could also benefit antimalarial drug efficacy monitoring, vaccine studies, and screening of vulnerable populations, such as pregnant women, in which low-density infections may have significant clinical consequences [18–20].

Polymerase chain reaction (PCR) detects parasite DNA, can identify infections below the threshold of detection for microscopy and RDTs, and is commonly considered the gold standard to detect malaria infection. However, PCR requires sophisticated laboratory infrastructure and advanced training, making it challenging and costly to implement in most malaria-endemic areas. Although PCR is used in some cases for focal screening and treatment strategies [11, 21], its restriction to central reference laboratories, often far from the sampled population, greatly limits its usefulness.

Loop-mediated isothermal amplification (LAMP) may offer a practical alternative. Like PCR, LAMP is a molecular technique that amplifies nucleic acids but uses simpler equipment and is less time intensive. A prototype LAMP assay designed for use in resource-constrained settings was developed through a public/private collaboration between the Foundation for Innovative New Diagnostics, Switzerland, and Eiken Chemical Co., Ltd., Japan. The assay forms the basis for a CE-marked product commercially released in July 2012 as the Loopamp Malaria Pan/Pf Detection Kit, list number LMC562 (Eiken Chemical). It is consistent with a recent description of “an ideal LAMP detection format” [22], and the kit consists of vacuum-dried reagents stable at ambient temperature. The assay's primers target Plasmodium genus or P. falciparum–specific sequences. Assay performance includes either of 2 simple blood-processing methods, a 40-minute reaction time in a closed amplification unit, and a visual readout. The result is essentially qualitative. The relative simplicity and robustness of LAMP opens the potential for sustainable nucleic acid amplification in laboratories and near-patient locations in malaria-endemic countries. An evaluation of the LAMP test kit in a United Kingdom reference laboratory, reported in the article by Polley et al in this issue of the Journal, showed that LAMP sensitivity approximated that of nested PCR [23]. This article presents results of the same kit evaluated in a rural clinic in Uganda.

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Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda

BACKGROUND: Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative to intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. However, the antenatal parasitaemias are frequently very low, and the most appropriate screening test for IST has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a multi-center prospective study of 990 HIV-uninfected women attending ANC in two different malaria transmission settings at Tororo District Hospital, eastern Uganda and Colsama Health Center in western Burkina Faso. Women were enrolled in the study in the second or third trimester of pregnancy and followed to delivery, generating 2,597 blood samples for analysis. Screening tests included rapid diagnostic tests (RDTs) targeting histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) and microscopy, compared to nPCR as a reference standard. At enrolment, the proportion of pregnant women who were positive for P. falciparum by HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT, microscopy and PCR was 38%, 29%, 36% and 44% in Uganda and 21%, 16%, 15% and 35% in Burkina Faso, respectively. All test positivity rates declined during follow-up. In comparison to PCR, the sensitivity of the HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT and microscopy was 75.7%, 60.1% and 69.7% in Uganda, 55.8%, 42.6% and 55.8% in Burkina Faso respectively for all antenatal visits. Specificity was greater than 96% for all three tests. Comparison of accuracy using generalized estimating equation revealed that the HRP2- detecting RDT was the most accurate test in both settings. CONCLUSIONS/SIGNIFICANCE: The study suggests that HRP2-based RDTs are the most appropriate point-of-care test currently available for use during pregnancy especially for symptomatic women, but will still miss some PCR-positive women. The clinical significance of these very low density infections needs to be better defined.

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HRP2 and pLDH-Based Rapid Diagnostic Tests, Expert Microscopy, and PCR for Detection of Malaria Infection during Pregnancy and at Delivery in Areas of Varied Transmission: A Prospective Cohort Study in Burkina Faso and Uganda

Background

Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative to intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. However, the antenatal parasitaemias are frequently very low, and the most appropriate screening test for IST has not been defined. 

Methodology/Principal Findings

We conducted a multi-center prospective study of 990 HIV-uninfected women attending ANC in two different malaria transmission settings at Tororo District Hospital, eastern Uganda and Colsama Health Center in western Burkina Faso. Women were enrolled in the study in the second or third trimester of pregnancy and followed to delivery, generating 2,597 blood samples for analysis. Screening tests included rapid diagnostic tests (RDTs) targeting histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) and microscopy, compared to nPCR as a reference standard. At enrolment , the proportion of pregnant women who were positive for P. falciparum by HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT, microscopy and PCR was 38%, 29%, 36% and 44% in Uganda and 21%, 16%, 15% and 35% in Bukina Faso, respectively. All test positivity ratesdeclined during follow-up. In comparison to PCR, the sensitivity of the HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT and microscopy was 75.7 %, 60.1% and 69.7% in Uganda, 55.8%, 42.6% and 55.8% in Burkina Faso respectively for all antenatal visits. Specificity was greater than 96% for all three tests. Comparison of accuracy using generalized estimating equation revealed that the HRP2- detecting RDT was the most accurate test in both settings. 

Conclusions/Significance

The study suggests that HRP2-based RDTs are the most appropriate point-of-care test currently available for use during pregnancy especially for symptomatic women, but will still miss some PCR-positive women. The clinical significance of these very low density infections needs to be better defined.

HRP2 and pLDH-based rapid diagnostic tests, expert microscopy, and PCR for detection of malaria infection during pregnancy and at delivery in areas of varied transmission: A prospective cohort study in Burkina Faso and Uganda

Patrick Angutoko

Papers

Highly Sensitive Detection of Malaria Parasitemia in a Malaria-Endemic Setting: Performance of a New Loop-Mediated Isothermal Amplification Kit in a Remote Clinic in Uganda

HRP2 and pLDH-Based Rapid Diagnostic Tests, Expert Microscopy, and PCR for Detection of Malaria Infection during Pregnancy and at Delivery in Areas of Varied Transmission: A Prospective Cohort Study in Burkina Faso and Uganda

HRP2 and pLDH-based rapid diagnostic tests, expert microscopy, and PCR for detection of malaria infection during pregnancy and at delivery in areas of varied transmission: A prospective cohort study in Burkina Faso and Uganda