Human Plasmodium infection produces a robust adaptive immune response. Time courses for 104 children followed for 42 days after initiation of Plasmodium falciparum chemotherapy were assayed for antibody levels to the five isotypes of human immunoglobulins (Ig) and 4 subclasses of IgG for 32 P. falciparum antigens encompassing all 4 parasite stages of human infection. IgD and IgE against these antigens were undetectable at 1:100 serum concentration, but other Ig isotypes and IgG subclasses were consistently observed against all antigens. Five quantitative parameters were developed to directly compare Ig response among isotypes and antigens: Cmax, maximum antibody level; ΔC, difference between Cmax and the antibody level at Day 0; tmax, time in days to reach Cmax; t1/2, Ig signal half-life in days; tneg, estimated number of days until complete loss of Ig signal. Classical Ig patterns for a bloodborne pathogen were seen with IgM showing early tmax and IgG production highest among Ig isotypes. However, some unexpected trends were observed such as IgA showing a biphasic pattern for many antigens. Variability among these dynamics of Ig acquisition and loss was noted for different P. falciparum antigens and able to be compared both quantitatively and statistically. This parametrization methodology allows direct comparison of Ig isotypes produced against various Plasmodium antigens following malaria infection, and the same methodology could be applied to other longitudinal serologic studies from P. falciparum or different pathogens. Specifically for P. falciparum seroepidemiological studies, reliable and quantitative estimates regarding the IgG dynamics in human populations can better optimize modeling efforts for serological outputs.
Rapid diagnostic tests (RDTs) that detect the Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) antigen are the primary methods for malaria diagnosis in Mozambique. However, these tests do not detect infections with non-falciparum malaria or Pfhrp2- and Pfhrp3-deleted P. falciparum parasites. To assess the appropriateness of conventional PfHRP2-only RDTs for malaria diagnosis in Mozambique, samples collected during a health facility survey conducted in three provinces of Mozambique were screened using antigen detection methods and further characterized by molecular techniques. Samples from 1,861 outpatients of all ages and symptoms attending 117 randomly selected public health facilities in 2018 were analyzed with an ultrasensitive bead-based immunoassay for the presence of PfHRP2, pan-Plasmodium aldolase (pAldo), and pan-Plasmodium lactate dehydrogenase (pLDH). The presence of PfHRP2 in patient blood detected using the bead-based assay was compared to the results of PfHRP2-based RDTs performed during the routine health facility consult and during the survey reexamination at the exit interview. Samples with discordant antigen profiles (negative for PfHRP2 but positive for pAldo and/or pLDH) were further characterized by photoinduced electron transfer PCR (PET-PCR). Using the bead-based laboratory assay as the gold standard, the sensitivities of the conventional RDTs administered during the routine health facility consult and the exit interview were 90% and 83%, respectively, and the specificities were 91% and 97%, respectively. Of 710 samples positive for at least one antigen, 704 (99.2%) were positive for PfHRP2. Six (0.8% of total) discordant samples lacked PfHRP2 but were positive for pAldo and/or pLDH; 3 of these (0.4% of total) were Plasmodium ovale monoinfections or coinfections where P. ovale was the dominant species. The remaining 3 discordant samples were negative by PET-PCR. The sensitivity and specificity of the conventional RDTs performed in the routine health facility consults and survey exit interviews were acceptable, and there was no evidence of Pfhrp2- and Pfhrp3-deleted parasites. Monoinfections with non-falciparum malaria species comprised <1% of the total malaria infections. Nearly all malaria antigen-positive patients had detectable PfHRP2, confirming that this antigen remains an appropriate malaria diagnostic target in the surveyed provinces.
Background Lingering post-treatment parasite antigen in blood complicates malaria diagnosis through antigen detection. Characterization of antigen clearance dynamics is important for interpretation of positive antigen detection tests. Results We used a bead-based serological assay to measure lactate dehydrogenase (LDH), aldolase (Aldo), and histidine-rich protein 2 (HRP2) levels in 196 children with Plasmodium falciparum malaria treated with effective antimalarials and followed for 28 to 42 days as part of therapeutic efficacy studies in Angola. Compared to pre-treatment levels, antigen concentrations two days after treatment declined by 99.7% for LDH, 96.3% for Aldo, and 54.6% for HRP2. After Day 2, assuming a first-order kinetics clearance model, half-lives of the antigens were 1.8 days (95% CI: 1.5–2.3) for LDH, 3.2 days (95% CI: 3.0–3.4) for Aldo, and 4.8 days (95% CI: 4.7–4.9) for HRP2. Conclusions LDH and Aldo show substantially different clearance rates than HRP2, and their presence is largely indicative of active infection. Electronic supplementary material The online version of this article (10.1186/s13071-019-3549-x) contains supplementary material, which is available to authorized users.
Histidine-rich protein 2 (HRP2)–based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 ( pfhrp2/3 ) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016–2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan- Plasmodium lactate dehydrogenase, and pan- Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%–4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%–1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%–5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.
D iagnosis and appropriate case management ofPlasmodium falciparum infection has greatly improved in many malaria-endemic settings through the use of rapid diagnostic tests (RDTs) that detect the histidine-rich protein 2 (HRP2) antigen (1). As the only Plasmodium species infecting humans to produce this antigen, the P. falciparum parasite expresses HRP2 in abundance and releases it into the bloodstream during blood-stage infection, making this marker a very sensitive and specific target for falciparum malaria (1,2). The pfhrp2 gene is located on chromosome 8 of the parasite genome, and a paralogous gene (pfhrp3) is located on chromosome 13. The 2 protein products share common epitopes for diagnostic antibodies, enabling the HRP3 antigen to also be detected to some extent by HRP2based RDTs (3-6).P. falciparum produces large quantities of these antigens during human blood-stage infection, but their biologic functions are not well elucidated, and pfhrp2-deleted and pfhrp3-deleted parasites still complete the human-mosquito lifecycle successfully (7). Reports of these gene deletions have increased over the past decade from multiple countries in Africa, South America, and Asia (https://apps.who.int/ malaria/maps/threats) (8). For countries that rely on HRP2-based RDTs for diagnosis of P. falciparum infection, those reports affirm the need to monitor the performance of this tool because deleted parasites could emerge and elicit false-negative results.P. falciparum infection represents ≈99.7% of all malaria cases in sub-Saharan Africa, and ≈300 million HRP2-based RDTs are used in this region annually (9). Studies in the east Africa countries of Eritrea (10) and Ethiopia (11,12) have found high prevalence of pfhrp2/pfhrp3 deletions, forcing changes away from HRP2-based RDTs to accurately diagnose P. falci-
Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia. This study investigated whether mixed infections were missed by microscopy from a 2017 therapeutic efficacy study at two health facilities in Ethiopia. All patients (N = 304) were initially classified as having single-species P. falciparum (n = 148 samples) or P. vivax infections (n = 156). Dried blood spots were tested for Plasmodium antigens by bead-based multiplex assay for pan-Plasmodium aldolase, pan-Plasmodium lactate dehydrogenase, P. vivax lactate dehydrogenase, and histidine-rich protein 2. Of 304 blood samples, 13 (4.3%) contained both P. falciparum and P. vivax antigens and were analyzed by polymerase chain reaction for species-specific DNA. Of these 13 samples, five were confirmed by polymerase chain reaction for P. falciparum/P. vivax co-infection. One sample, initially classified as P. vivax by microscopy, was found to only have Plasmodium ovale DNA. Plasmodium falciparum/P. vivax mixed infections can be missed by microscopy even in the context of a therapeutic efficacy study with multiple trained readers.
Laboratory detection of malaria antigens has proved valuable for research and epidemiological purposes. We recently developed a bead-based multiplex antigen assay for pan-Plasmodium and Plasmodium falciparum targets. Here, we report integration of a Plasmodium vivax-specific target to this multiplex panel: P. vivax lactate dehydrogenase (PvLDH). Within the multiplex panel, assay signal for purified PvLDH antigen titrated into the single-digit picogram range. Against a panel of polymerase chain reaction (PCR)-confirmed samples from acute P. vivax infections (n = 36), sensitivity was 91.7% in using PvLDH detection for identifying the presence of parasites. Specificity against a panel of persons with no Plasmodium infection (n = 44) was 100%, and specificity against a panel of PCR-confirmed P. falciparum, Plasmodium malariae, or Plasmodium ovale infections (n = 164) was 90.2%. Addition of this PvLDH capture and detection system into the multiplex antigen panel will now allow for sensitive screening for species identification of both P. falciparum and P. vivax in the laboratory.
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