Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon.

Malaria elimination efforts in Peru have dramatically reduced the incidence of cases in the Amazon Basin. To achieve the elimination, the detection of asymptomatic and submicroscopic carriers becomes a priority. Therefore, efforts should focus on tests sensitive enough to detect low-density parasitemia, deployable to resource-limited areas and affordable for large screening purposes. In this study, we assessed the performance of the Malachite-Green LAMP (MG-LAMP) using heat-treated DNA extraction (Boil & Spin; B&S MG-LAMP) on 283 whole blood samples collected from 9 different sites in Loreto, Peru and compared its performance to expert and field microscopy. A real-time PCR assay was used to quantify the parasite density. In addition, we explored a modified version of the B&S MG-LAMP for detection of submicroscopic infection in 500 samples and compared the turnaround time and cost of the MG-LAMP with microscopy. Compared to expert microscopy, the genus B&S MG-LAMP had a sensitivity of 99.4% (95%CI: 96.9%- 100%) and specificity of 97.1% (95%CI: 91.9%- 99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%- 100%) and specificity of 99.2% (95.5%- 100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%- 100%) and specificity of 99.3% (95%CI: 97.3%- 99.8%). The modified genus B&S MG-LAMP assay detected eight submicroscopic malaria cases (1.6%) which the species-specific assays did not identify. The turnaround time of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. The modified B&S MG-LAMP offers a simple and sensitive molecular test of choice for the detection of submicroscopic infections that can be used for mass screening in resources limited facilities in endemic settings nearing elimination and where a deployable test is required.

Field validation of a magneto-optical detection device (Gazelle) for portable point-of-care Plasmodium vivax diagnosis.

A major challenge for malaria is the lack of tools for accurate and timely diagnosis in the field which are critical for case management and surveillance. Microscopy along with rapid diagnostic tests are the current mainstay for malaria diagnosis in most endemic regions. However, these methods present several limitations. This study assessed the accuracy of Gazelle, a novel rapid malaria diagnostic device, from samples collected from the Peruvian Amazon between 2019 and 2020. Diagnostic accuracy was compared against microscopy and two rapid diagnostic tests (SD Bioline and BinaxNOW) using 18ssr nested-PCR as reference test. In addition, a real-time PCR assay (PET-PCR) was used for parasite quantification. Out of 217 febrile patients enrolled and tested, 180 specimens (85 P. vivax and 95 negatives) were included in the final analysis. Using nested-PCR as the gold standard, the sensitivity and specificity of Gazelle was 88.2% and 97.9%, respectively. Using a cutoff of 200 parasites/µl, Gazelle's sensitivity for samples with more than 200 p/uL was 98.67% (95%CI: 92.79% to 99.97%) whereas the sensitivity for samples lower than 200 p/uL (n = 10) was 12.5% (95%CI: 0.32% to 52.65%). Gazelle's sensitivity and specificity were statistically similar to microscopy (sensitivity = 91.8, specificity = 100%, p = 0.983) and higher than both SD Bioline (sensitivity = 82.4, specificity = 100%, p = 0.016) and BinaxNOW (sensitivity = 71.8%, specificity = 97.9%, p = 0.002). The diagnostic accuracy of Gazelle for malaria detection in P. vivax infections was comparable to light microscopy and superior to both RDTs even in the presence of low parasitemia infections. The performance of Gazelle makes it a valuable tool for malaria diagnosis and active case detection that can be utilized in different malaria-endemic regions.

Molecular surveillance of the Plasmodium vivax multidrug resistance 1 gene in Peru between 2006 and 2015.

BACKGROUND: The high incidence of Plasmodium vivax infections associated with clinical severity and the emergence of chloroquine (CQ) resistance has posed a challenge to control efforts aimed at eliminating this disease. Despite conflicting evidence regarding the role of mutations of P. vivax multidrug resistance 1 gene (pvmdr1) in drug resistance, this gene can be a tool for molecular surveillance due to its variability and spatial patterns. METHODS: Blood samples were collected from studies conducted between 2006 and 2015 in the Northern and Southern Amazon Basin and the North Coast of Peru. Thick and thin blood smears were prepared for malaria diagnosis by microscopy and PCR was performed for detection of P. vivax monoinfections. The pvmdr1 gene was subsequently sequenced and the genetic data was used for haplotype and diversity analysis. RESULTS: A total of 550 positive P. vivax samples were sequenced; 445 from the Northern Amazon Basin, 48 from the Southern Amazon Basin and 57 from the North Coast. Eight non-synonymous mutations and three synonymous mutations were analysed in 4,395 bp of pvmdr1. Amino acid changes at positions 976F and 1076L were detected in the Northern Amazon Basin (12.8%) and the Southern Amazon Basin (4.2%) with fluctuations in the prevalence of both mutations in the Northern Amazon Basin during the course of the study that seemed to correspond with a malaria control programme implemented in the region. A total of 13 pvmdr1 haplotypes with non-synonymous mutations were estimated in Peru and an overall nucleotide diversity of π = 0.00054. The Northern Amazon Basin was the most diverse region (π = 0.00055) followed by the Southern Amazon and the North Coast (π = 0.00035 and π = 0.00014, respectively). CONCLUSION: This study showed a high variability in the frequencies of the 976F and 1076L polymorphisms in the Northern Amazon Basin between 2006 and 2015. The low and heterogeneous diversity of pvmdr1 found in this study underscores the need for additional research that can elucidate the role of this gene on P. vivax drug resistance as well as in vitro and clinical data that can clarify the extend of CQ resistance in Peru.

Genomic surveillance of Plasmodium falciparum and Plasmodium vivax cases at the University Hospital in Tegucigalpa, Honduras.

Malaria continues to be an important health problem in Honduras despite major progress achieved reducing its incidence in the last two decades. In a context of case reduction, continuing surveillance of parasite diversity and drug resistance is an important component to assist effective malaria control strategies and support risk assessments. In this study, we employed next generation sequencing on collected Plasmodium vivax and P. falciparum samples from the Hospital Escuela (University Hospital) in Honduras between 2005 and 2017. Hospital Escuela is the main public health hospital in Honduras and receives suspected malaria cases from endemic regions within the country. The resulting sequencing data was used to assess complexity of infections, parasite population structure, parasite diversity and drug resistance profiling. All P. vivax samples and all autochtonous P. falciparum samples were monoclonal and presented a low intra population diversity (π = 0.25 and 0.07, respectively). Genotyping of drug resistance markers showed that three P. falciparum samples presented the chloroquine resistant haplotype SVMNT on pfcrtr (positions 72-76). Epidemiological data suggested that two of these samples were imported cases from Africa whereas the third one was a local case. Three suspected imported cases (two of which were also pfcrt mutants) presented the pfmdr1 86Y mutation that further enhances the CQ resistant genotype. No evidence was found for kelch13 artemisinin resistance associated mutations nor parasite genetic background mutations. Discriminant analysis of principal components and phylogenetic analysis showed two P. vivax and two P. falciparum parasite sub-populations with limited recombination between them. It also confirmed the closer relationship of the three imported cases with African strains. Our findings showed that local Honduras P. falciparum strains do not hold CQ resistance polymorphisms which aligns with clinical data reported by the country and supports the continuity of CQ based treatment in Honduras. In addition, our findings highlight the need of using genomic approaches to provide key information about parasite biology including drug resistance, population structure and HRP2/HRP3 deletions which are becoming relevant as the country move towards elimination.

Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum.

The current context of malaria elimination requires urgent development and implementation of highly sensitive and specific methods for prompt detection and treatment of malaria parasites. Such methods should overcome current delays in diagnosis, allow the detection of low-density infections and address the difficulties in accessing remote endemic communities. In this study, we assessed the performance of the RealAmp and malachite-green loop mediated isothermal amplification (MG-LAMP) methodologies, using microscopy and conventional nested-PCR as reference techniques. Both LAMP techniques were performed for Plasmodium genus, P. falciparum, and P. vivax identification using 136 whole blood samples collected from three communities located in the Peruvian Amazon basin. Turnaround time and costs of performing the LAMP assays were estimated and compared to that of microscopy and nested-PCR. Using nested-PCR as reference standard, we calculated the sensitivity, specificity and 95% confidence interval (CI) for all methods. RealAmp had a sensitivity of 92% (95% CI: 85-96.5%) and specificity of 100% (95% CI: 89.1-100%) for species detection; sensitivity and specificity of MG-LAMP were 94% (95% CI: 87.5-97.8%) and 100% (89.1-100%), respectively. Whereas microscopy showed 88.1% sensitivity (95% CI: 80.2-93.7%) and 100% specificity (95%: 89.1-100%). The turnaround time and costs of performing the LAMP assays were lower compared to those associated with nested-PCR but higher than those associated with microscopy. The two LAMP assays were shown to be more sensitive and simple to implement than microscopy. Both LAMP methodologies could be used as large-scale screening tests, but the MG-LAMP assay uses a simple, portable heat-block while the RealAmp requires a RealAmp machine or a real-time PCR machine. This makes the MG-LAMP an appropriate choice for malaria surveillance studies in endemic sites. Use of LAMP tests in active case detection of Plasmodium parasites could help to detect positive malaria cases early.

A cluster of the first reported Plasmodium ovale spp. infections in Peru occuring among returning UN peace-keepers, a review of epidemiology, prevention and diagnostic challenges in nonendemic regions.

BACKGROUND: Plasmodium ovale curtisi and Plasmodium ovale wallikeri are regarded as less virulent forms of malaria with a geographic distribution including Southeast Asia, Central and West Africa, and is increasingly reported as an infection in returning travellers. A species of malaria that may have delayed or relapsing presentations similar to Plasmodium vivax, the clinical presentation of P. ovale spp. has been described to have prepatent periods of 2 weeks or slightly longer with reports of relapse following primary infection out to 8-9 months. This presentation may be obscured further in the setting of anti-malarial exposure, with report of delayed primary infection out to 4 years. Presented is a cluster of 4 imported P. ovale spp. cases in returning Peruvian military personnel assigned to United Nations peace-keeping operations in the Central African Republic. CASE PRESENTATION: From January to December 2016, Peruvian peace-keepers were deployed in support of United Nations (UN) operations in the Central African Republic (CAR). While serving abroad, Navy, Army, and Air Force members experienced 223 episodes of Plasmodium falciparum malaria following interruption of prophylaxis with mefloquine. Diagnosis was made using rapid diagnostics tests (RDTs) and/or smear with no coinfections identified. Cases of malaria were treated with locally-procured artemether-lumefantrine. Returning to Peru in January 2017, 200 peace-keepers were screened via thick and thin smear while on weekly mefloquine prophylaxis with only 1 showing nucleic acid within red blood cells consistent with Plasmodium spp. and 11 reporting syndromes of ill-defined somatic complaints. Between a period of 5 days to 11 months post return, 4 cases of P. ovale spp. were diagnosed using smear and polymerase chain reaction (PCR) following febrile complaints. All cases were subsequently treated with chloroquine and primaquine, with cure of clinical disease and documented clearance of parasitaemia. CONCLUSION: These patients represent the first imported cases in Peru of this species of malaria as well as highlight the challenges in implementing population level prophylaxis in a deployed environment, and the steps for timely diagnosis and management in a non-endemic region where risk of introduction for local transmission exists.

Exploration of Plasmodium vivax transmission dynamics and recurrent infections in the Peruvian Amazon using whole genome sequencing.

BACKGROUND: Plasmodium vivax poses a significant challenge to malaria elimination due to its ability to cause relapsed infections from reactivation of dormant liver parasites called hypnozoites. We analyzed 69 P. vivax whole genome sequences obtained from subjects residing in three different villages along the Peruvian Amazon. This included 23 paired P. vivax samples from subjects who experienced recurrent P. vivax parasitemia following observed treatment with chloroquine and primaquine. METHODS: Genomic DNA was extracted from whole blood samples collected from subjects. P. vivax DNA was enriched using selective whole genome amplification and whole genome sequencing. We used single nucleotide polymorphisms (SNPs) from the core P. vivax genome to determine characteristics of the parasite population using discriminant analysis of principal components, maximum likelihood estimation of individual ancestries, and phylogenetic analysis. We estimated the relatedness of the paired samples by calculating the number of segregating sites and using a hidden Markov model approach to estimate identity by descent. RESULTS: We present a comprehensive dataset of population genetics of Plasmodium vivax in the Peruvian Amazonian. We define the parasite population structure in this region and demonstrate a novel method for distinguishing homologous relapses from reinfections or heterologous relapses with improved accuracy. The parasite population in this area was quite diverse with an estimated five subpopulations and evidence of a highly heterogeneous ancestry of some of the isolates, similar to previous analyses of P. vivax in this region. Pairwise comparison of recurrent infections determined that there were 12 homologous relapses and 3 likely heterologous relapses with highly related parasites. To the best of our knowledge, this is the first large-scale study to evaluate recurrent P. vivax infections using whole genome sequencing. CONCLUSIONS: Whole genome sequencing is a high-resolution tool that can identify P. vivax homologous relapses with increased sensitivity, while also providing data about drug resistance and parasite population genetics. This information is important for evaluating the efficacy of known and novel antirelapse medications in endemic areas and thus advancing the campaign to eliminate malaria.

Bacterial Etiology and Risk Factors Associated with Cellulitis and Purulent Skin Abscesses in Military Trainees.

Military trainees are at high risk for skin and soft-tissue infections (SSTIs). Although Staphylococcus aureus is associated with purulent SSTI, it is unclear to what degree this pathogen causes nonpurulent cellulitis. To inform effective prevention strategies and to provide novel insights into SSTI pathogenesis, we aimed to determine the etiology of SSTI in this population. We conducted a prospective observational study in US Army Infantry trainees with SSTI (cutaneous abscesses and cellulitis) from July 2012 through December 2014. We used standard microbiology, serology, and high-throughput sequencing to determine the etiology of SSTI. Furthermore, we compared purported risk factors as well as anatomic site colonization for S. aureus. Among 201 SSTI cases evaluated for SSTI risk factors, cellulitis was associated with lower extremity blisters (P = 0.01) and abscess was associated with methicillin-resistant S. aureus (MRSA) colonization (P<0.001). Among the 22 tested cellulitis cases that were part of the microbiome analysis, only 1 leading edge aspirate was culturable (Coagulase-negative Staphylococcus). Microbiome evaluation of aspirate specimens demonstrated that Rhodanobacter terrae was the most abundant species (66.8% average abundance), while abscesses were dominated by S. aureus (92.9% average abundance). Although abscesses and cellulitis share the spectrum of clinical SSTI, the bacterial etiologies as determined by current technology appear distinct. Furthermore, the presence of atypical bacteria within cellulitis aspirates may indicate novel mechanisms of cellulitis pathogenesis. CLINICAL TRIALS REGISTRATION: NCT01105767.

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model.

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.