Multicopy targets for Plasmodium vivax and Plasmodium falciparum detection by colorimetric LAMP.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection. METHODS: The cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR. RESULTS: The LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method. CONCLUSION: The colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.

Assessment of an automated capillary system for Plasmodium vivax microsatellite genotyping.

BACKGROUND: Several platforms have been used to generate the primary data for microsatellite analysis of malaria parasite genotypes. Each has relative advantages but share a limitation of being time- and cost-intensive. A commercially available automated capillary gel cartridge system was assessed in the microsatellite analysis of Plasmodium vivax diversity in the Peruvian Amazon. METHODS: The reproducibility and accuracy of a commercially-available automated capillary system, QIAxcel, was assessed using a sequenced PCR product of 227 base pairs. This product was measured 42 times, then 27 P. vivax samples from Peruvian Amazon subjects were analyzed with this instrument using five informative microsatellites. Results from the QIAxcel system were compared with a Sanger-type sequencing machine, the ABI PRISM(®) 3100 Genetic Analyzer. RESULTS: Significant differences were seen between the sequenced amplicons and the results from the QIAxcel instrument. Different runs, plates and cartridges yielded significantly different results. Additionally, allele size decreased with each run by 0.045, or 1 bp, every three plates. QIAxcel and ABI PRISM systems differed in giving different values than those obtained by ABI PRISM, and too many (i.e. inaccurate) alleles per locus were also seen with the automated instrument. CONCLUSIONS: While P. vivax diversity could generally be estimated using an automated capillary gel cartridge system, the data demonstrate that this system is not sufficiently precise for reliably identifying parasite strains via microsatellite analysis. This conclusion reached after systematic analysis was due both to inadequate precision and poor reproducibility in measuring PCR product size.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = -0.52 & Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Malaria transmission structure in the Peruvian Amazon through antibody signatures to Plasmodium vivax.

BACKGROUND: The landscape of malaria transmission in the Peruvian Amazon is temporally and spatially heterogeneous, presenting different micro-geographies with particular epidemiologies. Most cases are asymptomatic and escape routine malaria surveillance based on light microscopy (LM). Following the implementation of control programs in this region, new approaches to stratify transmission and direct efforts at an individual and community level are needed. Antibody responses to serological exposure markers (SEM) to Plasmodium vivax have proven diagnostic performance to identify people exposed in the previous 9 months. METHODOLOGY: We measured antibody responses against 8 SEM to identify recently exposed people and determine the transmission dynamics of P. vivax in peri-urban (Iquitos) and riverine (Mazán) communities of Loreto, communities that have seen significant recent reductions in malaria transmission. Socio-demographic, geo-reference, LM and qPCR diagnosis data were collected from two cross-sectional surveys. Spatial and multilevel analyses were implemented to describe the distribution of seropositive cases and the risk factors associated with exposure to P. vivax. PRINCIPAL FINDINGS: Low local transmission was detected by qPCR in both Iquitos (5.3%) and Mazán (2.7%); however, seroprevalence indicated a higher level of (past) exposure to P. vivax in Mazán (56.5%) than Iquitos (38.2%). Age and being male were factors associated with high odds of being seropositive in both sites. Higher antibody levels were found in individuals >15 years old. The persistence of long-lived antibodies in these individuals could overestimate the detection of recent exposure. Antibody levels in younger populations (<15 years old) could be a better indicator of recent exposure to P. vivax. CONCLUSIONS: The large number of current and past infections detected by SEMs allows for detailed local epidemiological analyses, in contrast to data from qPCR prevalence surveys which did not produce statistically significant associations. Serological surveillance will be increasingly important in the Peruvian Amazon as malaria transmission is reduced by continued control and elimination efforts.

Effect of out-of-village working activities on recent malaria exposure in the Peruvian Amazon using parametric g-formula.

In the Amazon Region of Peru, occupational activities are important drivers of human mobility and may increase the individual risk of being infected while contributing to increasing malaria community-level transmission. Even though out-of-village working activities and other mobility patterns have been identified as determinants of malaria transmission, no studies have quantified the effect of out-of-village working activities on recent malaria exposure and proposed plausible intervention scenarios. Using two population-based cross-sectional studies in the Loreto Department in Peru, and the parametric g-formula method, we simulated various hypothetical scenarios intervening in out-of-village working activities to reflect their potential health benefits. This study estimated that the standardized mean outcome (malaria seroprevalence) in the unexposed population (no out-of-village workers) was 44.6% (95% CI: 41.7%-47.5%) and 66.7% (95% CI: 61.6%-71.8%) in the exposed population resulting in a risk difference of 22.1% (95% CI: 16.3%-27.9%). However, heterogeneous patterns in the effects of interest were observed between peri-urban and rural areas (Cochran's Q test = 15.5, p < 0.001). Heterogeneous patterns were also observed in scenarios of increased prevalence of out-of-village working activities and restriction scenarios by gender (male vs. female) and age (18 and under vs. 19 and older) that inform possible occupational interventions targetting population subgroups. The findings of this study support the hypothesis that targeting out-of-village workers will considerably benefit current malaria elimination strategies in the Amazon Region. Particularly, males and adult populations that carried out out-of-village working activities in rural areas contribute the most to the malaria seropositivity (recent exposure to the parasite) in the Peruvian Amazon.

Drug resistance and population structure of Plasmodium falciparum and Plasmodium vivax in the Peruvian Amazon.

Malaria is a major health problem in Peru despite substantial progress achieved by the ongoing malaria elimination program. This study explored the population genetics of 63 Plasmodium falciparum and 170 P. vivax cases collected in the Peruvian Amazon Basin between 2015 and 2019. Microscopy and PCR were used for malaria detection and positive samples were genotyped at neutral and drug resistance-associated regions. The P. falciparum population exhibited a low nucleotide diversity (π = 0.02) whereas the P. vivax population presented a higher genetic diversity (π = 0.34). All P. falciparum samples (n = 63) carried chloroquine (CQ) resistant mutations on Pfcrt. Most P. falciparum samples (53 out of 54) carried sulfadoxine (SD) resistant mutations on Pfdhfr and Pfdhps. No evidence was found of artemisinin resistance mutations on kelch13. Population structure showed that a single cluster accounted for 93.4% of the P. falciparum samples whereas three clusters were found for P. vivax. Our study shows a low genetic diversity for both species with significant differences in genetic sub-structuring. The high prevalence of CQ-resistance mutations could be a result of indirect selection pressures driven by the P. vivax treatment scheme. These results could be useful for public health authorities to safeguard the progress that Peru has achieved towards malaria elimination.

Diagnosis of Plasmodium vivax by Loop-Mediated Isothermal Amplification in Febrile Patient Samples from Loreto, Perú.

Plasmodium vivax is co-endemic with Plasmodium falciparum in Peru, and optimum management requires distinguishing these two species in the blood of patients. For the differential identification of P. vivax and other Plasmodium spp., the LoopampTM Malaria Pan Detection Kit in combination with the Loopamp Malaria Pv Detection Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) was used to evaluate 559 whole blood samples collected in 2017 from febrile patients with suspected malaria attending different health facilities in the Loreto region. The Loopamp Malaria Pan Detection Kit showed a sensitivity of 87.7% (95% CI: 83.5-91.9) and a specificity of 94.4% (95% CI: 91.9-96.9) and good agreement with PCR (Cohen's kappa 0.8266, 95% CI: 0.7792-0.874). By comparison, the Loopamp Malaria Pv Detection Kit showed a similar sensitivity (84.4%, 95% CI: 79.0-89.7) and specificity (92.4%, 95% CI: 89.7-95.0) and substantial agreement with PCR (Cohen's kappa: 0.7661, 95% CI: 0.7088-0.8234).

Clinical evaluation of a 16S ribosomal RNA polymerase chain reaction test for the diagnosis of lymph node tuberculosis.

UNLABELLED: Reports on the sensitivity of polymerase chain reaction (PCR) for the diagnosis of lymph node tuberculosis (TB) show divergent results. We evaluated the accuracy of the Roche Amplicor Mycobacterium tuberculosis PCR test with lymph node aspirate and biopsy samples. METHODS: The study was conducted at a public reference hospital in Lima, Peru. From the period of January 2003 to January 2004, we included patients who had lymphadenopathy and in whom the attending physician suspected TB. Aspirate and biopsy samples were submitted for culturing in Lowenstein-Jensen medium, for histopathologic testing, and for PCR. The sensitivity and specificity of PCR were calculated against a reference standard based on histopathologic findings and culture. RESULTS: Our study included 154 patients. Median age was 29 years (interquartile range, 21-40 years); 97 patients (62.9%) were men. Twenty-nine patients (18.8%) had acid fast bacilli-positive histopathologic findings, and 44 (28.6%) had a positive culture result. Using the combination of histopathologic findings and culture as reference standard, 55 patients (35.7%) had a diagnosis of tuberculous lymphadenitis. The sensitivity of the PCR test was 58.2%, and the specificity was 93.9%. For biopsy tissue only, the sensitivity of PCR was 52.7%, and the specificity was 97.0%. For aspirate samples only, the sensitivity of PCR was 47.3%, and the specificity was 96.0%. CONCLUSION: The Amplicor PCR test revealed low sensitivity and high specificity for the diagnosis of lymph node TB. The sensitivity was higher in cases in which the bacillary load was high--in acid fast bacilli-positive samples and among HIV-infected patients. Considering the results of microbiological and PCR tests together, there was still a patient group in whom no final diagnosis could be established.

Newly developed primers for comprehensive amplification of the rpoB gene and detection of rifampin resistance in Mycobacterium tuberculosis.

New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif(r) isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.

Diagnóstico temprano en un brote epidémico del virus dengue en Piura usando RT-PCR y NESTED-PCR / Early diagnosis test in a epidemic outbreak of dengue virus in Piura using RT and NESTED-PCR

Un test de diagnóstico temprano (RT-PCR y Nested-PCR) fue evaluado y comparado con métodos convencionales (Cultivo in vitro, IFI y MAC-ELISA). Treinta y cuatro sueros de pacientes correspondientes de un brote epidémico de la costa norte peruana (Mancora, Piura) en mayo de 1997 fueron incluídos en este estudio. Todos los sueros obtenidos de pacientes que presentaron en los primeros cinco días manifestaciones clínicas siendo diagnosticados luego como dengue serotipo 1. Asimismo, RT-PCR permitió diagnosticar 82 de los sueros (28/34), sin embargo Mac-ELISA y cultivo in vitro reconocieron unicamente 41 de los sueros (14/34) y 38 de los sueros (13/34) respectivamente. Por lo tanto, el uso de esta herramienta molecular (RT-PCR y Nested-PCR) permitirá dar un diagnóstico temprano a estos pacientes y actuar inmediatamente ante la presencia de un brote epidémico

Diagnóstico molecular para leishmaniasis / Molecular diagnosis for leishmaniasis

El potencial diagnóstico de epitopes inmunodominantes seleccionados fue ensayado satisfactoriamente a fin de obtener una prueba serodiagnóstica alternativa para la Leishmaniasis Tegumentaria Americana. Dos proteínas recombinantes prometedoras de L. (v.) peruviana referidas como T-26-U2/T26-U4 fueron reconocidas por sueros individuales de pacientes con Leishmaniasis Tegumentaria Americana usuando Western Blot. La sensibilidad de la prueba fue de 86 con sueros permanetes con Leishmaniasis peruana