Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

Background The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. Methods The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. Results Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. Conclusion The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Prevalence of SARS-Cov-2 antibodies and living conditions: the French national random population-based EPICOV cohort

Background We aimed to estimate the seroprevalence of SARS-CoV-2 infection in France and to identify the populations most exposed during the first epidemic wave. Methods Random selection of individuals aged 15 years or over, from the national tax register (96% coverage). Socio-economic data, migration history, and living conditions were collected via self-computer-assisted-web or computer-assisted-telephone interviews. Home self-sampling was performed for a random subsample, to detect IgG antibodies against spike protein (Euroimmun), and neutralizing antibodies with in-house assays, in dried blood spots (DBS). Results The questionnaire was completed by 134,391 participants from May 2nd to June 2st, 2020, including 17,441 eligible for DBS 12,114 of whom were tested. ELISA-S seroprevalence was 4.5% [95% CI 3.9–5.0] overall, reaching up to 10% in the two most affected areas. High-density residences, larger household size, having reported a suspected COVID-19 case in the household, working in healthcare, being of intermediate age and non-daily tobacco smoking were independently associated with seropositivity, whereas living with children or adolescents did not remain associated after adjustment for household size. Adjustment for both residential density and household size accounted for much of the higher seroprevalence in immigrants born outside Europe, twice that in French natives in univariate analysis. Conclusion The EPICOV cohort is one of the largest national representative population-based seroprevalence surveys for COVID-19. It shows the major role of contextual living conditions in the initial spread of COVID-19 in France, during which the availability of masks and virological tests was limited.

Poliovirus immunity among adults in the Democratic Republic of the Congo: a cross-sectional serosurvey

Background Vaccination efforts to eradicate polio currently focus on children under 5 years of age, among whom most cases of poliomyelitis still occur. However, in the Democratic Republic of the Congo (DRC), an outbreak of wild poliovirus type 1 occurred in 2010–2011 in which 16% of cases occurred among adults; in a related outbreak in the neighboring Republic of Congo, 75% of cases occurred among the same adult age-group. Given that infected adults may transmit poliovirus, this study was designed to assess adult immunity against polioviruses. Methods We assessed poliovirus seroprevalence using dried blood spots from 5,526 adults aged 15–59 years from the 2013–2014 Demographic and Health Survey in the DRC. Results Among adults in the DRC, 74%, 72%, and 57% were seropositive for neutralizing antibodies for poliovirus types 1, 2, and 3, respectively. For all three serotypes, seroprevalence tended to be higher among older age groups, those living in households with more children, and among women. Conclusions Protection against poliovirus is generally low among adults in the DRC, particularly for type 3 poliovirus. The lack of acquired immunity in adults suggests a potentially limited poliovirus circulation over the lifetime of those surveyed (spanning 1954 through 2014) and transmission of vaccine-derived poliovirus in this age group while underscoring the risk of these outbreaks among adults in the DRC.

Human Serum Albumin Cys34 Adducts in Newborn Dried Blood Spots: Associations With Air Pollution Exposure During Pregnancy

Background: Increasing evidence suggests that exposure to air pollution during pregnancy is associated with adverse pregnancy outcomes. However, biomarkers associated with air pollution exposure are widely lacking and often transient. In addition, ascertaining biospecimens during pregnacy to assess the prenatal environment remains largely infeasible.Objectives: To address these challenges, we investigated relationships between air pollution exposure during pregnancy and human serum albumin Cys34 (HSA-Cys34) adducts in newborn dried blood spots (DBS) samples, which captures an integration of perinatal exposures to small reactive molecules in circulating blood.Methods: Newborn DBS were obtained from a state archive for a cohort of 120 children born at one Kaiser Permanente Southern California (KPSC) hospitals in 2007. These children were selected to maximize the range of residential air pollution exposure during the entire pregnancy to PM2.5, PM10, NO2, O3, based on monthly estimates interpolated from regulatory monitoring sites. HSA-Cys34 adducts were selected based on previously reported relationships with air pollution exposure and oxidative stress.Results: Six adducts measured in newborn DBS samples were associated with air pollution exposures during pregnancy; these included direct oxidation products, adducts formed with small thiol compounds, and adducts formed with reactive aldehydes. Two general trends were identified: Exposure to air pollution late in pregnancy (i.e., in the last 30 days) was associated with increased oxidative stress, and exposure to air pollution earlier in pregnancy (i.e., not in the last 30 days) was associated with decreased oxidative stress around the time of birth.Discussion: Air pollution exposure occurring during pregnancy can alter biology and leave measurable impacts on the developing infant captured in the newborn DBS adductome, which represents a promising tool for investigating adverse birth outcomes in population-based studies.

Comparison of C-Reactive Protein in Dried Blood Spots and Saliva of Healthy Adolescents

Background/AimDetermining C-reactive protein (CRP) by non-invasive methods is of great interest for research addressing inflammation in young people. However, direct comparisons of such methods applied in children and adolescents are lacking so far. This study aimed to evaluate the association between CRP measured in dried blood spots (DBS CRP) and in saliva (sCRP), two less invasive alternatives to venipuncture, in 12- to 14-year-old adolescents. To evaluate the validity of both measurements in the context of biobehavioral studies, the potential of DBS CRP and sCRP to discriminate between defined BMI subgroups was assessed.Materials and MethodsCRP levels in DBS and saliva collected from 87 healthy adolescents (M = 13.25 years, SD = 0.30, 51.7% females) were determined using high sensitive CRP ELISA for serum and salivary CRP ELISA, respectively. Characteristics and correlation of both measurements were assessed for the total sample and for three subgroups classified by BMI percentile ranges (A: ≤ 25; B: 26–74; C: ≥ 75).ResultsIn the total sample, DBS CRP and sCRP were significantly associated (r = 0.59, p &lt; 0.001). Splitting the sample into BMI-dependent subgroups revealed similarly strong associations of DBS CRP with sCRP for all three groups (A: r = 0.51; B: r = 0.61; C: r = 0.53). However, comparing the mean CRP values per BMI subgroup, one-way ANOVA reported significant differences for DBS CRP, but not for sCRP mean values.ConclusionsThe significant correlation of DBS CRP with sCRP was independent of the investigated BMI range groups, yet BMI-dependent distinction was only provided by DBS CRP mean values. Overall, our results suggest that DBS CRP is likely to reflect systemic inflammation more precisely. Salivary CRP can be alternatively determined in studies with adolescents when conditions require it, given the oral health status is assessed. Considering that DBS CRP and sCRP share only 35% of common variance, further studies should examine their specific validity.

HIV Phylogenetics Reveals Overlapping Transmission Networks among Cities and Key Populations in Pakistan

The first case of HIV in Pakistan was documented in 1987, with multiple subtypes and circulating recombinant forms being introduced and currently circulating in the country. Since then, there has been a shift in the country from a low prevalence/high-risk to a high-risk concentrated epidemic. Pakistan's epidemic is concentrated among key populations at greater risk of HIV infection including people who inject drugs (PWID), Hijra sex workers (HSW), female sex workers (FSW), male sex workers (MSW), and men who have sex with men (MSM). This study focused on the geographical aspect as well as on the interactions between key populations at higher risk of contracting HIV. We aimed at understanding the behavior of these key populations at a molecular level with high granularity as well as investigating the possibility of multiple HIV-1 introductions in Pakistan. In this cross-sectional biological and behavioral survey, we collected dried blood spots (DBS) for the purposes of seroprevalence estimates and molecular epidemiology from individuals in 17 cities in Pakistan representing four key populations: PWID, HSW, MSW, and FSW. A total of 1153 envelope sequences (reference positions in HXB2: 7860-8274) of HIV were sequenced using a Sanger-based sequencing approach. To identify clusters based on the introduction of the virus in Pakistan from foreign countries we added 3623 publicly available HIV envelope sequences to our dataset. Phylogeographic inference suggests at least 15 independent introductions of the virus into Pakistan, with a total of 12 clusters ranging from 3 to 675 sequences in size containing sequences from Pakistan and neighboring countries exclusively. Our phylogenetic analysis shows a significant degree of connectivity and directionality suggesting broad and overlapping networks of HIV-1 transmission among cities and key populations in Pakistan.