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As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination.

Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan- Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform’s performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.

Background Rapid diagnostic tests (RDTs) that rely on the detection of Plasmodium falciparum histidine-rich protein 2 ( Pf HRP2) have become key tools for diagnosing P. falciparum infection. The utility of RDTs can be limited by Pf HRP2 persistence, however it can be a potential benefit in low transmission settings where detection of persistent Pf HRP2 using newer ultra-sensitive Pf HRP2 based RDTs can serve as a surveillance tool to identify recent exposure. Better understanding of the dynamics of Pf HRP2 over the course of a malaria infection can inform optimal use of RDTs. Methods A previously published mathematical model was refined to mimic the production and decay of Pf HRP2 during a malaria infection. Data from 15 individuals from volunteer infection studies were used to update the original model and estimate key model parameters. The refined model was applied to a cohort of patients from Namibia who received treatment for clinical malaria infection for whom longitudinal Pf HRP2 concentrations were measured. Results The refinement of the Pf HRP2 dynamic model indicated that in malaria naïve hosts, P. falciparum parasites of the 3D7 strain produce 33.6 × 10 −15  g (95% CI 25.0–42.1 × 10 −15  g) of Pf HRP2 in vivo per parasite replication cycle, with an elimination half-life of 1.67 days (95% CI 1.11–3.40 days). The refined model included these updated parameters and incorporated individualized body fluid volume calculations, which improved predictive accuracy when compared to the original model. The performance of the model in predicting clearance of Pf HRP2 post treatment in clinical samples from six adults with P. falciparum infection in Namibia improved when using a longer elimination half-life of 4.5 days, with 14% to 67% of observations for each individual within the predicted range. Conclusions The updated mathematical model can predict the growth and clearance of Pf HRP2 during the production and decay of a mono-infection with P. falciparum , increasing the understanding of Pf HRP2 antigen dynamics. This model can guide the optimal use of Pf HRP2-based RDTs for reliable diagnosis of P. falciparum infection and re-infection in endemic settings, but also for malaria surveillance and elimination programmes in low transmission areas.

Background To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. Methods A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. Results The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95–99) and specificity 83 % (n = 15/18, 95 % CI 59–96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92–99) and specificity of 96 % (n = 151/157, 95 % CI 92–99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80–100) and specificity of 95 % (n = 123/128, 95 % CI 90–98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69–97). Conclusion This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination.

Background: Pneumocystis jirovecii pneumonia is increasingly diagnosed with highly sensitive PCR diagnostics in immunocompromised, HIV-negative individuals. We assessed the performance of our in-house quantitative PCR with the aim to optimise interpretation. Methods: Retrospective audit of all positive P. jirovecii qPCRs on induced sputum or BAL fluid at a single centre from 2012 to 2023. Medical and laboratory records were analysed and people with HIV were excluded. Cases were categorised as colonisation, high-probability PCP or uncertain PCP infection against a clinical gold standard incorporating clinico-radiological data. Quantitative PCR assay targeting the 5s gene was utilised throughout the time period. Results: Of the 82 positive qPCRs, 28 were categorised as high-probability PCP infection, 30 as uncertain PCP and 24 as colonisation. There was a significant difference in qPCR values stratified by clinical category but not respiratory sample type. Current assay performance with a cutoff of 2.5 × 105 copies/mL had a sensitivity of 50% (95% CI, 30.65–69.35%) and specificity of 83.33% (95% CI, 62.62–95.26%). Youden Index calculated at 6.5 × 104 copies/mL had a sensitivity of 75% (56.64–87.32%, 95% CI) and specificity of 66.67% (46.71–82.03%, 95% CI). High and low cutoffs were explored. Significant variables associated with infection were age > 70 years old, the presence of fever, hypoxia or ground glass changes. Conclusions: A single qPCR cutoff cannot reliably determine P. jirovecii infection from colonisation. Low and high cutoffs are useful, however, a large “possible infection” cohort will remain where interpretation of clinic-radiological factors remains essential. Standardisation of assays with prospective validation in specific immunocompromised groups will allow greater generalisability and allow large-scale prospective assay validation to be performed.

Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority. A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia. The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105). This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of P. vivax . It is widely used as a diagnostic biomarker and a measure of total-body parasite biomass in vivax malaria. However, the dynamics of PvLDH remains poorly understood. Here, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated key biological parameters characterising in vivo PvLDH dynamics based on longitudinal data of parasitemia and PvLDH concentration collected from P. vivax -infected humans, with the estimates informed by the ex vivo data as prior knowledge in a Bayesian hierarchical framework. We found that the in vivo accumulation rate of intraerythrocytic PvLDH peaks at 10–20 h post-invasion (late ring stage) with a median estimate of intraerythrocytic PvLDH mass at the end of the life cycle to be 9.4 × 10 −3 ng. We also found that the median estimate of in vivo PvLDH half-life was approximately 21.9 h. Our findings provide a foundation with which to advance our quantitative understanding of P. vivax biology and will facilitate the improvement of PvLDH-based diagnostic tools. A modelling framework characterising the dynamics of Plasmodium vivax lactate dehydrogenase provides estimates of key biological parameters to facilitate the development of improved diagnostic tools for the detection of P. vivax .

  There have been case reports of non-vector, healthcare-associated transmission of DENVs--four cases of percutaneous transmission via needlestick injuries, mucocutaneous transmission through a blood splash to the face, vertical transmission, and transmission via bone marrow transplant (summarised in [5]). [...]although north Queensland was experiencing concurrent outbreaks involving all four DENV serotypes at the time [11], the patient had not travelled to this region prior to developing dengue.

Increasing rates of syphilis in pregnancy (SiP) in Australia and other high-income countries, has led to the resurgence of congenital syphilis. Suboptimal syphilis screening during pregnancy has been identified as a key contributing factor. This study aimed to explore, from the perspective of multidisciplinary healthcare providers (HCPs), the barriers to optimal screening during the antenatal care (ANC) pathway. Semi-structured interviews conducted with 34 HCPs across multiple disciplines practising in south-east Queensland (SEQ) were analysed through a process of reflexive thematic analysis. Barriers were found to occur at the system level of ANC, through difficulties in patient engagement in care, limitations in the current model of health care delivery and limitations in the communication pathways across health care disciplines; and at the individual HCP level, through HCP knowledge and awareness of epidemiological changes in syphilis in SEQ, and adequately assessing patient risk. It is imperative that the healthcare systems and HCPs involved in ANC address these barriers to improve screening in order to optimise management of women and prevent congenital syphilis cases in SEQ.