Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

P. malariae is found worldwide and causes chronic parasitism in its human hosts. We developed a P. malariae (Pm) diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies /µL (~1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was 35 minutes. Combined with simplified DNA extraction methods, the assay has potential for future field-deployable point-of-care use to detect a parasite species that remains largely undiagnosed.

Micro-heterogeneity of transmission shapes the submicroscopic malaria reservoir in coastal Tanzania.

Background: Asymptomatic malaria may be patent (visible by microscopy) and detectable by rapid malaria diagnostic tests (RDTs), or it may be submicroscopic and only detectable by polymerase chain reaction (PCR). Methods: To characterize the submicroscopic reservoir in an area of declining malaria transmission, asymptomatic persons >5 years of age in Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir across villages, determined factors associated with submicroscopic parasitemia, and assessed the natural history of submicroscopic malaria over four weeks. Results: Among 6,076 participants, Plasmodium falciparum prevalence by RDT, microscopy, and PCR was 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female gender, dry season months, screened windows, and bednet use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over four weeks, 23% (61/266) of submicroscopic carriers became RDT-positive and were treated, with half exhibiting symptoms. This occurred more frequently in villages with higher malaria prevalence. Conclusions: Micro-heterogeneity in transmission impacts the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Similar Prevalence of Plasmodium falciparum and Non-P. falciparum Malaria Infections among Schoolchildren, Tanzania1.

Achieving malaria elimination requires considering both Plasmodium falciparum and non-P. falciparum infections. We determined prevalence and geographic distribution of 4 Plasmodium spp. by performing PCR on dried blood spots collected within 8 regions of Tanzania during 2017. Among 3,456 schoolchildren, 22% had P. falciparum, 24% had P. ovale spp., 4% had P. malariae, and 0.3% had P. vivax infections. Most (91%) schoolchildren with P. ovale infections had low parasite densities; 64% of P. ovale infections were single-species infections, and 35% of those were detected in low malaria endemic regions. P. malariae infections were predominantly (73%) co-infections with P. falciparum. P. vivax was detected mostly in northern and eastern regions. Co-infections with >1 non-P. falciparum species occurred in 43% of P. falciparum infections. A high prevalence of P. ovale infections exists among schoolchildren in Tanzania, underscoring the need for detection and treatment strategies that target non-P. falciparum species.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri species infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining malaria species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI( 2.7- 18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 10° copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to 14 oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovate-infected persons, mixed Poc/Pow infections were detected in 11 (79%). Based on these results, 8/9 P. ovate carriers transmitted both P. ovate species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Seasonality and transmissibility of Plasmodium ovale in Bagamoyo District, Tanzania.

BACKGROUND: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed. METHODS: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development. RESULTS: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae. CONCLUSIONS: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum.

Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in the United States: Living Density, Viral Load, and Disproportionate Impact on Communities of Color.

BACKGROUND: Households are hot spots for severe acute respiratory syndrome coronavirus 2 transmission. METHODS: This prospective study enrolled 100 coronavirus disease 2019 (COVID-19) cases and 208 of their household members in North Carolina though October 2020, including 44% who identified as Hispanic or non-White. Households were enrolled a median of 6 days from symptom onset in the index case. Incident secondary cases within the household were detected using quantitative polymerase chain reaction of weekly nasal swabs (days 7, 14, 21) or by seroconversion at day 28. RESULTS: Excluding 73 household contacts who were PCR-positive at baseline, the secondary attack rate (SAR) among household contacts was 32% (33 of 103; 95% confidence interval [CI], 22%-44%). The majority of cases occurred by day 7, with later cases confirmed as household-acquired by viral sequencing. Infected persons in the same household had similar nasopharyngeal viral loads (intraclass correlation coefficient = 0.45; 95% CI, .23-.62). Households with secondary transmission had index cases with a median viral load that was 1.4 log10 higher than those without transmission (P = .03), as well as higher living density (more than 3 persons occupying fewer than 6 rooms; odds ratio, 3.3; 95% CI, 1.02-10.9). Minority households were more likely to experience high living density and had a higher risk of incident infection than did White households (SAR, 51% vs 19%; P = .01). CONCLUSIONS: Household crowding in the context of high-inoculum infections may amplify the spread of COVID-19, potentially contributing to disproportionate impact on communities of color.

Assessment of the Field Utility of a Rapid Point-of-Care Test for SARS-CoV-2 Antibodies in a Household Cohort.

Point-of-care (POC) tests to detect SARS-CoV-2 antibodies offer quick assessment of serostatus after natural infection or vaccination. We compared the field performance of the BioMedomics COVID-19 IgM/IgG Rapid Antibody Test against an ELISA in 303 participants enrolled in a SARS-CoV-2 household cohort study. The rapid antibody test was easily implemented with consistent interpretation across 14 users in a variety of field settings. Compared with ELISA, detection of seroconversion lagged by 5 to 10 days. However, it retained a sensitivity of 90% (160/177, 95% confidence interval [CI] 85-94%) and specificity of 100% (43/43, 95% CI 92-100%) for those tested 3 to 5 weeks after symptom onset. Sensitivity was diminished among those with asymptomatic infection (74% [14/19], 95% CI 49-91%) and early in infection (45% [29/64], 95% CI 33-58%). When used appropriately, rapid antibody tests offer a convenient way to detect symptomatic infections during convalescence.

Practical strategies for SARS-CoV-2 RT-PCR testing in resource-constrained settings.

Alternatives to nasopharyngeal sampling are needed to increase capacity for SARS-CoV-2 testing. Among 275 participants, we piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including polyester flocked swabs as well as 3D-printed plastic lattice swabs, placed into viral transport media or an RNA stabilization agent. Flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), missing those with low viral load (<106 viral copies/mL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into RNA preservative, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. We also evaluated pooling strategies that involved pooling specimens in the lab versus pooling swabs at the point of collection, finding both successfully detected samples with >105 viral copies/mL.

Practical strategies for SARS-CoV-2 RT-PCR testing in resource-constrained settings.

BACKGROUND: Standard nasopharyngeal swab testing for SARS-CoV-2 detection by PCR is not always feasible due to limitations in trained personnel, personal protective equipment, swabs, PCR reagents, and access to cold chain and biosafety hoods. METHODS: We piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including both standard polyester flocked swabs as well as 3D printed plastic lattice swabs, placed into either viral transport media or an RNA stabilization agent. Quantitative SARS-CoV-2 viral detection by RT-qPCR was compared to that obtained by nasopharyngeal sampling as the reference standard. Pooling specimens in the lab versus pooling swabs at the point of collection was also evaluated. RESULTS: Among 275 participants, flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), mostly missing those with low viral load (<10^3 viral copies/uL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into an RNA stabilizer, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. Pooling sample specimens or swabs both successfully detected samples >102 viral copies/uL. CONCLUSIONS: Nasal swabs are likely adequate for clinical diagnosis of acute infections to help expand testing capacity in resource-constrained settings. When collected into an RNA preservative that also inactivates infectious virus, nasal swabs yielded quantitative viral loads approximating those obtained by nasopharyngeal sampling.

High household transmission of SARS-CoV-2 in the United States: living density, viral load, and disproportionate impact on communities of color.

Background: Few prospective studies of SARS-CoV-2 transmission within households have been reported from the United States, where COVID-19 cases are the highest in the world and the pandemic has had disproportionate impact on communities of color. Methods and Findings: This is a prospective observational study. Between April-October 2020, the UNC CO-HOST study enrolled 102 COVID-positive persons and 213 of their household members across the Piedmont region of North Carolina, including 45% who identified as Hispanic/Latinx or non-white. Households were enrolled a median of 6 days from onset of symptoms in the index case. Secondary cases within the household were detected either by PCR of a nasopharyngeal (NP) swab on study day 1 and weekly nasal swabs (days 7, 14, 21) thereafter, or based on seroconversion by day 28. After excluding household contacts exposed at the same time as the index case, the secondary attack rate (SAR) among susceptible household contacts was 60% (106/176, 95% CI 53%-67%). The majority of secondary cases were already infected at study enrollment (73/106), while 33 were observed during study follow-up. Despite the potential for continuous exposure and sequential transmission over time, 93% (84/90, 95% CI 86%-97%) of PCR-positive secondary cases were detected within 14 days of symptom onset in the index case, while 83% were detected within 10 days. Index cases with high NP viral load (>10^6 viral copies/ul) at enrollment were more likely to transmit virus to household contacts during the study (OR 4.9, 95% CI 1.3-18 p=0.02). Furthermore, NP viral load was correlated within families (ICC=0.44, 95% CI 0.26-0.60), meaning persons in the same household were more likely to have similar viral loads, suggesting an inoculum effect. High household living density was associated with a higher risk of secondary household transmission (OR 5.8, 95% CI 1.3-55) for households with >3 persons occupying <6 rooms (SAR=91%, 95% CI 71-98%). Index cases who self-identified as Hispanic/Latinx or non-white were more likely to experience a high living density and transmit virus to a household member, translating into an SAR in minority households of 70%, versus 52% in white households (p=0.05). Conclusions: SARS-CoV-2 transmits early and often among household members. Risk for spread and subsequent disease is elevated in high-inoculum households with limited living space. Very high infection rates due to household crowding likely contribute to the increased incidence of SARS-CoV-2 infection and morbidity observed among racial and ethnic minorities in the US. Quarantine for 14 days from symptom onset of the first case in the household is appropriate to prevent onward transmission from the household. Ultimately, primary prevention through equitable distribution of effective vaccines is of paramount importance.

Direct Comparison of Standard and Ultrasensitive PCR for the Detection of Plasmodium falciparum from Dried Blood Spots in Bagamoyo, Tanzania.

Ultrasensitive PCR used in low-transmission malaria-endemic settings has revealed a much higher burden of asymptomatic infections than that detected by rapid diagnostic tests (RDTs) or standard PCR, but there is limited evidence as to whether this is the case in higher transmission settings. Using dried blood spots (DBS) collected among 319 schoolchildren in Bagamoyo, Tanzania, we found good correlation (Pearson's R = 0.995) between Plasmodium falciparum parasite densities detected by a DNA-based 18s rRNA real-time PCR (qPCR) and an RNA-based ultrasensitive reverse transcriptase (RT)-PCR (usPCR) for the same target. Whereas prevalence by usPCR was higher than that found by qPCR (37% versus 32%), the proportion of additionally detected low-density infections (median parasite density < 0.050 parasites/µL) represented an incremental increase. It remains unclear to what extent these low-density infections may contribute to the infectious reservoir in different malaria transmission settings.

Selection of Cytochrome b Mutants Is Rare among Plasmodium falciparum Patients Failing Treatment with Atovaquone-Proguanil in Cambodia.

Atovaquone-proguanil remains effective against multidrug-resistant Plasmodium falciparum in Southeast Asia, but resistance is mediated by a single point mutation in cytochrome b (cytb) that can arise during treatment. Among 14 atovaquone-proguanil treatment failures in a clinical trial in Cambodia, only one recrudescence harbored the cytb mutation Y268C. Deep sequencing did not detect the mutation at baseline or in the first 3 days of treatment, suggesting that it arose de novo Further sequencing across cytb similarly found no low-frequency cytb mutations that were up-selected from baseline to recrudescence. Copy number amplification in dihydroorotate dehydrogenase (DHODH) and cytb as markers of atovaquone tolerance was also absent. Cytb mutation played a minor role in atovaquone-proguanil treatment failures in an active comparator clinical trial.