Rapid Polymerase Chain Reaction Research Articles

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources.

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples

IntroductionSpillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.MethodsOur study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs.ResultsSequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods.DiscussionOur proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important.

Ultrafast Metaphotonic PCR Chip with Near-Perfect Absorber.

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification and quantification in diverse fields such as life sciences, global health, medicine, agricultural science, forensic science, and environmental science for global sustainability. However, implementing a cost-effective PCR remains challenging for rapid preventive medical action to the widespread pandemic diseases due to the absence of highly efficient and low-cost PCR chip-based POC molecular diagnostics. Here, this work reports an ultrafast metaphotonic PCR chip as a solution of a cost-effective and low-power-consumption POC device for the emerging global challenge of sustainable healthcare. This work designs a near-perfect photonic meta-absorber using ring-shaped titanium nitride to maximize the photothermal effect and realize rapid heating and cooling cycles during the PCR process. This work fabricates a large-area photonic meta-absorber on a 6-inch wafer cost-effectively using simple colloidal lithography. In addition, this work demonstrates 30 thermocycles from 65 (annealing temperature) to 95°C (denaturation temperature) within 3min 15s, achieving an average 16.66°Cs-1 heating rate and 7.77°Cs-1 cooling rate during thermocycling, succeeding rapid metaphotonic PCR. This work believes a metaphotonic PCR chip can be used to create a low-cost, ultrafast molecular diagnostic chip with a meta-absorber.

Asymptomatic malaria in pregnancy and associated risk factors in Majang Zone, Gambella Region, Southwest Ethiopia: a hard-to-reach malaria hotspot

BackgroundAsymptomatic malaria in pregnancy (AMiP) is a daunting public health problem with multifaceted adverse outcomes for mothers, fetuses, newborns and beyond. This study aimed to assess the prevalence and risk factors of AMiP and anaemia in Majang Zone, Gambella, Southwest Ethiopia.MethodsA facility-based cross-sectional study was conducted among 425 pregnant women attending the antenatal care (ANC) clinics of five health facilities in the Majang Zone from November 2022 to February 2023. Sociodemographic, obstetric, and anti-malarial intervention data were collected using an interviewer-administered questionnaire. A capillary blood specimen was collected to diagnose malaria and anaemia as well as determine the blood group. Malaria was diagnosed by rapid diagnostic test (RDT), microscopy, and quantitative polymerase chain reaction (qPCR). Statistical analyses were done by Statistical Package for Social Science (SPSS) version 26.0. The association between dependent and independent variables was assessed by multivariable binary logistic regression, considering P < 0.05 statistically significant. The magnitude of associations was quantified with the adjusted odds ratio (AOR) along with the corresponding 95% confidence interval (CI).ResultsThe overall prevalence of AMiP was 15.3% (95% CI 12.1, 18.9). It was 11.3% (95% CI 8.4, 14.7) by RDT, 11.8% (95% CI 8.9, 15.2) by microscopy and 17.6% (95% CI 11.7, 24.9) by qPCR. Plasmodium falciparum, moderate parasitaemia and submicroscopic infection accounted for 55.4% of the AMiP prevalence, 50.8% of the parasite density, and 41.6% of the qPCR-positive AMiP, respectively. Nearly 32.3% of pregnant women with AMiP carried gametocytes. Risk factors of AMiP were: not utilizing insecticide-treated net (ITN) within the previous week (AOR: 9.43 95% CI 1.57, 56.62), having a history of malaria within the previous year (AOR: 2.26 95% CI 1.16, 4.42), lack of indoor residual spraying (IRS) within the previous year (AOR: 3.00 95% CI 1.50, 6.00), and ANC contact below two rounds (AOR: 4.28 95% CI 2.06, 8.87). The prevalence of anaemia was 27.7% (95% CI 23.6, 32.1), and it was higher among AMiP-positives (56.9%) than the negatives (22.5%) (P: 000).ConclusionThe prevalence of AMiP and anaemia was high, and remained as a critical public health problem in the study area. Focus on the identified risk factors and introduction of more sensitive diagnostic tools should be considered to mitigate AMiP in the study area.

Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni.

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire's Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.

One Health monitoring reveals invasive freshwater snail species, new records, and undescribed parasite diversity in Zimbabwe

BackgroundSnail-borne trematodes afflict humans, livestock, and wildlife. Recognizing their zoonotic potential and possible hybridization, a One Health approach is essential for effective control. Given the dearth of knowledge on African trematodes, this study aimed to map snail and trematode diversity, focusing on (i) characterizing gastropod snail species and their trematode parasites, (ii) determining infection rates of snail species as intermediate hosts for medically, veterinary, and ecologically significant trematodes, and (iii) comparing their diversity across endemic regions.MethodsA cross-sectional study conducted in 2021 in Chiredzi and Wedza districts in Zimbabwe, known for high human schistosomiasis prevalence, involved malacological surveys at 56 sites. Trematode infections in snails were detected through shedding experiments and multiplex rapid diagnostic polymerase chain reactions (RD-PCRs). Morphological and molecular analyses were employed to identify snail and trematode species.ResultsAmong 3209 collected snail specimens, 11 species were identified, including schistosome and fasciolid competent snail species. We report for the first time the invasive exotic snail Tarebia granifera in Zimbabwe, which was highly abundant, mainly in Chiredzi, occurring at 29 out of 35 sites. Shedding experiments on 1303 snails revealed a 2.24% infection rate, with 15 trematode species identified through molecular genotyping. Five species were exclusive to Chiredzi: Bolbophorus sp., Schistosoma mansoni, Schistosoma mattheei, Calicophoron sp., and Uvulifer sp. Eight were exclusive to Wedza, including Trichobilharzia sp., Stephanoprora amurensis, Spirorchid sp., and Echinostoma sp. as well as an unidentified species of the Plagiorchioidea superfamily. One species, Tylodelphys mashonensis, was common to both regions. The RD-PCR screening of 976 non-shedding snails indicated a 35.7% trematode infection rate, including the presence of schistosomes (1.1%) Fasciola nyanzae (0.6%). In Chiredzi, Radix natalensis had the highest trematode infection prevalence (33.3%), while in Wedza, R. natalensis (55.4%) and Bulinus tropicus (53.2%) had the highest infection prevalence.ConclusionsOur xenomonitoring approach unveiled 15 trematode species, including nine new records in Zimbabwe. Schistosoma mansoni persists in the study region despite six mass deworming rounds. The high snail and parasite diversity, including the presence of exotic snail species that can impact endemic species and biomedically important trematodes, underscores the need for increased monitoring.Graphical

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Comparison of malaria diagnostic methods for detection of asymptomatic Plasmodium infections among pregnant women in northwest Ethiopia

BackgroundMalaria in pregnancy remains a major public health problem in the globe, especially in sub-Saharan Africa. In malaria endemic areas, most pregnant women remain asymptomatic, but malaria could still cause complications on the mother and her offspring; as well as serve as reservoirs to transmit infection. Despite these effects, no attention is given to the diagnosis of asymptomatic Plasmodium infections (APIs) using highly sensitive and specific laboratory diagnostic tools in Ethiopia. Therefore, the goal of this study was to compare the performance of Rapid Diagnostic Test (RDT), microscopy and real-time polymerase chain reaction (RT-PCR) to detect APIs among pregnant women.MethodsA health facility based cross -sectional study was conducted among pregnant women attending antenatal care at Fendeka town health facilities Jawi district, northwest Ethiopia from February to March, 2019. A total of 166 participants were enrolled by using convenient sampling technique. Socio-demographic features were collected using a semi structured questionnaire. Dried blood spot (DBS) samples were collected for molecular analysis. Asymptomatic Plasmodium infection on pregnant women was diagnosed using RDT, microscopy and RT-PCR. Descriptive statistics were used to determine the prevalence of APIs. Method comparison was performed, and Cohen’s kappa coefficient (k) was used to determine the degree of agreement among the diagnostic methods. Parasite densities were also calculated.ResultsThe prevalence of API was 9.6%, 11.4% and 18.7% using RDT, microscopy and RT-PCR, respectively. The overall proportion of API was 19.3%. Sensitivity of the RDT was 83.3% as compared with microscopy. Rapid Diagnostic Test and microscopy also showed sensitivity of 50% and 60%, respectively, as compared with RT-PCR. The mean parasite density was 3213 parasites/µl for P falciparum and 1140 parasites/µl of blood for P. vivax.ConclusionPrevalence of API in the study area was high. Both RDT and microscopy had lower sensitivity when compared with RT-PCR. Therefore, routine laboratory diagnosis of API among pregnant women should be given attention and done with better sensitive and specific laboratory diagnostic tools.

Cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.).

This paper discusses the development of rapid, reliable, and accurate polymerase chain reaction (PCR) assays for detecting opium poppy (Papaver somniferum L.) in food. Endpoint, quantitative, and digital PCRs were compared based on the amplification of a newly developed DNA marker targeting the NADPH-dependent codeinone reductase (COR) gene. Designed assays were shown to be highly specific and sensitive in discriminating opium poppy from other plant species, even in heat-treated and food samples. Digital PCR was the most sensitive, with a detection limit of up to 5 copies, i.e., approximately 14 pg of target DNA per reaction. Quantitative and digital PCR further allowed the quantification of opium poppy in up to 1.5 ng and 42 pg (15 copies) of target DNA in a sample, respectively. In addition, two duplex PCRs have been developed for the simultaneous detection of opium poppy DNA and representatives of (i) the Papaveraceae family or (ii) the Plantae kingdom. Finally, all designed assays were successfully applied for analysis of 15 commercial foodstuffs; two were suspected of being adulterated. The study results have an important impact on addressing food fraud and ensuring the safety and authenticity of food products. Beyond food adulteration, the study may also have significant implications for forensics and law enforcement.

Rapid molecular identification of Rana dybowskii by species-specific primers

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific.

Dual testing for SARS Covid-19 in asymptomatic medico-legal cases: Experience and suggestions

ABSTRACT Context: A person caught by police known as a Medical Legal Case (MLC) should be negative for Covid-19 before they are presented in court. Police officers always insist on collecting a sample from MLC patients for both the rapid antigen test (RAT) and reverse transcriptase polymerase chain reaction (RTPCR). Using both tests for the same patient at the same time is a waste of resources. The aim of the study is to analyze and compare Covid-19 RAT and RTPCR testing results in MLC cases and to find out the need to prevent resource wasting due to dual tests. Methods: The study was conducted for 6 months on all the MLC patients who were brought by the police officer for Covid-19 testing at the center. Oropharyngeal or nasopharyngeal swabs were collected in viral transport media for Covid-19 RTPCR and RAT test. RAT is performed at the collection center, while viral transport media is transported to an authorized testing center for RTPCR. Result: All 156 patients’ RAT and RTPCR tests were performed as per requirements and rules. Only two specimens (1.28%) reported positive for Covid-19 in RTPCR and other samples (98.71%) reported negative for Covid-19 in RTPCR, while all 156 samples (100%) were reported negative for Covid-19 by RAT test. Conclusion: As per the current study, we can consider the RAT results for presenting the criminal person to court, but for sending them to prison, RTPCR is recommended to prevent the spread of disease.

Molecular Epidemiology of SARS-CoV-2 within Accra Metropolis Postlockdown.

Currently, sequencing has been the only tool for the identification of circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants. However, it is known to be an expensive and laborious approach involving high technical expertise. Considering the reduced adherence to preventive measures postlockdown in Accra, this study presents an alternative method that leverages polymerase chain reaction (PCR) to identify circulating SARS-CoV-2 variants in the Accra Metropolis postlockdown. This prospective cross-sectional study was conducted between July and December 2022. Nasopharyngeal samples were collected from 268 consenting participants. Samples were subjected to nucleic acid extraction and followed by real-time polymerase chain reaction for the detection and quantification of SARS-CoV-2 RNA. SARS-CoV-2 positive samples were subsequently subjected to variant identification using rapid PCR. Findings. The prevalence of SARS-CoV-2 within the Accra Metropolis was 30.2%. The majority of the SARS-CoV-2 infection was diagnosed in females, participants aged 41-50 years, and symptomatic participants. Participants aged ≤10 years and females recorded the highest viral load while participants aged 41-50 years recorded the highest number of infections. The SARS-CoV-2 variants detected were Alpha (64.2%), Delta (22.2%), and Omicron (13.6%). Predictors of SARS-CoV-2 infection identified were chills, cough, headache, body weakness, sore throat, and dyspnoea in order of decreasing association with SARS-CoV-2 infection. There was a strong association between symptom status, gender, age, and SARS-CoV-2 infection. There was a high prevalence of SARS-CoV-2 within the Accra Metropolis postlockdown within the sampling period. The Alpha variant of SARS-CoV-2 is the predominant circulating variant, and persons presenting with symptoms are most likely to be diagnosed with COVID-19. Children aged ≤10 years serve as a reservoir for infection transmission.

Diagnostic Approaches to Helicobacter pylori: A Comparative Study of Detection in Gastric Biopsy and Aspirates.

Background Helicobacter pyloriis one of the most common bacterial pathogens in humans. It is a microaerophilic bacteria with multiple unipolar flagella. It is associated with the development of various lesions like chronic gastritis, gastric ulcers, adenocarcinoma, and mucosa-associated lymphomas. The aim of this study was a comparative evaluation of the rapid urease test (RUT) and polymerase chain reaction (PCR) in gastric biopsy and aspirates for the detection ofH. pyloriinfection and to further determine the sensitivity and specificity of RUT and PCR. Method Endoscopic guided biopsy tissue and gastric aspirate specimens were collected from 110 patients with symptoms like gastritis, dyspepsia, etc., and subjected to RUT and PCR for detection ofH. pyloriinfection. Results A total of 110 samples, including both biopsy tissue (77) and gastric aspirate (33) were subjected to RUT and PCR. RUT for biopsy tissue showed the highest sensitivity (97.18%), compared to gastric aspirate (78.94%). Comparing RUT with PCR, the sensitivity and specificity of PCR were 93.33% and 90.0%, respectively. The positive predictive value (PPV) of PCR was 97.67%, the negative predictive value (NPV) was 75.0%, and the accuracy was 92.73%. Conclusion The present study showed that RUT is a rapid and accurate invasive test for the detection ofHelicobacter pyloriinfection in biopsy tissue as compared to gastric aspirate specimens, which are more sensitive to PCR. The study also showed that biopsy tissue was found to be a superior specimen for the detection ofHelicobacter pylorias compared to gastric aspirate.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Clinical Presentation, Risk Factors, and Comparison of Laboratory Diagnostics for Seasonal Influenza Virus Among Cambodians From 2007 to 2020.

Despite its global significance, challenges associated with understanding the epidemiology and accurately detecting, measuring, and characterizing the true burden of seasonal influenza remain in many resource-poor settings. A prospective observational study was conducted in Cambodia at 28 health facilities between 2007 and 2020 utilizing passive surveillance data of patients presenting with acute undifferentiated febrile illness (AUFI) to describe the prevalence of influenza A and B and characterize associated risk factors and symptoms using a questionnaire. A comparison of rapid influenza diagnostic tests (RIDTs) and real-time reverse transcription polymerase chain reaction (rRT-PCR) results was also conducted. Of 30 586 total participants, 5634 (18.4%) tested positive for either influenza A or B, with 3557 (11.6%) positive for influenza A and 2288 (7.5%) positive for influenza B during the study. Influenza A and B were strongly associated with the rainy season (odds ratio [OR], 2.30; P < .001) and being from an urban area (OR, 1.45; P < .001). Analysis of individual symptoms identified cough (OR, 2.8; P < .001), chills (OR, 1.4; P < .001), and sore throat (OR, 1.4; P < .001) as having the strongest positive associations with influenza among patients with AUFI. Analysis comparing RIDTs and rRT-PCR calculated the overall sensitivity of rapid tests to be 0.492 (95% CI, 0.479-0.505) and specificity to be 0.993 (95% CI, 0.992-0.994) for both influenza type A and B. Findings from this 14-year study include describing the epidemiology of seasonal influenza over a prolonged time period and identifying key risk factors and clinical symptoms associated with infection; we also demonstrate the poor sensitivity of RIDTs in Cambodia.

Prevalence of Malaria Infection in Pregnant Women Attending Antenatal Clinics in Southern Senegal.

Despite marked progress in Senegal, three regions in the southeast part continue to have a high burden of malaria, but there have been no recent studies assessing the prevalence of malaria associated with pregnancy. This study aimed to determine the prevalence of malaria infection in pregnant women attending antenatal clinics in Senegal. During the malaria transmission season of 2019, pregnant women attending 11 health care facilities for a scheduled visit and those presenting unwell with signs of malaria were invited to participate in a malaria screening study. A finger prick blood sample was taken for malaria diagnosis by rapid diagnosis test (RDT) and polymerase chain reaction (PCR). A total of 877 pregnant women were enrolled, 787 for a scheduled antenatal consultation and 90 for an unscheduled consultation with signs of malaria. The prevalence of Plasmodium falciparum among the first group was 48% by PCR and 20% by RDT, and that among the second group was 86% by PCR and 83% by RDT. RDT sensitivity in capturing asymptomatic, PCR-positive infections was 9.2% but ranged from 83% to 94% among febrile women. The prevalence of infection by PCR in women who reported having received at least three doses of sulfadoxine pyrimethamine (SP) was 41.9% compared with 58.9% in women who reported they had not received any SP doses (prevalence ratio adjusted for gravidity and gestational age, 0.54; 95% CI, 0.41-0.73). The burden of P. falciparum infections remains high among pregnant women, the majority of which are not captured by RDT. More effective measures to prevent malaria infection in pregnancy are needed.

Venovenous extracorporeal membrane oxygenation after cardiac arrest for acute respiratory distress syndrome caused by Legionella: a case report

BackgroundLegionella remains underdiagnosed in the intensive care unit and can progress to acute respiratory distress syndrome (ARDS), multiorgan failure and death. In severe cases, venovenous extracorporeal membrane oxygenation (VV-ECMO) allows time for resolution of disease with Legionella-targeted therapy. VV-ECMO outcomes for Legionella are favorable with reported survival greater than 70%. Rapid molecular polymerase chain reaction (PCR) testing of the lower respiratory tract aids in diagnosing Legionella with high sensitivity and specificity. We present a unique case of a patient with a positive COVID-19 test and ARDS who suffered a cardiac arrest. The patient was subsequently cannulated for VV-ECMO, and after lower respiratory tract PCR testing, Legionella was determined to be the cause. She was successfully treated and decannulated from VV-ECMO after eight days.Case presentationA 53-year-old female presented with one week of dyspnea and a positive COVID-19 test. She was hypoxemic, hypotensive and had bilateral infiltrates on imaging. She received supplemental oxygen, intravenous fluids, vasopressors, broad spectrum antibiotics, and was transferred to a tertiary care center. She developed progressive hypoxemia and suffered a cardiac arrest, requiring ten minutes of CPR and endotracheal intubation to achieve return of spontaneous circulation. Despite mechanical ventilation and paralysis, she developed refractory hypoxemia and was cannulated for VV-ECMO. Dexamethasone and remdesivir were given for presumed COVID-19. Bronchoscopy with bronchoalveolar lavage (BAL) performed with PCR testing was positive for Legionella pneumophila and negative for COVID-19. Steroids and remdesivir were discontinued and she was treated with azithromycin. Her lung compliance improved, and she was decannulated after eight days on VV-ECMO. She was discharged home on hospital day 16 breathing room air and neurologically intact.ConclusionsThis case illustrates the utility of rapid PCR testing to diagnose Legionella in patients with respiratory failure and the early use of VV-ECMO in patients with refractory hypoxemia secondary to Legionella infection. Moreover, many patients encountered in the ICU may have prior COVID-19 immunity, and though a positive COVID-19 test may be present, further investigation with lower respiratory tract PCR testing may provide alternative diagnoses. Patients with ARDS should undergo Legionella-specific testing, and if Legionella is determined to be the causative organism, early VV-ECMO should be considered in patients with refractory hypoxemia given reported high survival rates.

Prevalence of Asymptomatic Falciparum Malaria, Risk Factors, and Absence of PfHRP2 Gene Deletion in Makurdi, Nigeria

Background: Malaria remains a persistent public health challenge, particularly in sub-Saharan Africa, where asymptomatic falciparum malaria poses a significant threat. Asymptomatic cases serve as a crucial parasite reservoir, contributing to ongoing transmission.&#x0D; Aim: The study investigates the prevalence of asymptomatic falciparum malaria and assesses the occurrence of gene deletion in the Plasmodium falciparum Histidine Rich Protein 2 (Pf HRP2) gene in Makurdi, Benue State, Nigeria.&#x0D; Methodology: A cross-sectional study was conducted between September and October 2019, which involved 374 apparently healthy individuals from five communities. Malaria diagnosis utilized Rapid Diagnostic Test (RDT) kits, microscopy, and polymerase chain reaction (PCR) for Pf HRP2 gene assessment.&#x0D; Results: The study found a prevalence of 25.4% by RDT and 28.1% by microscopy. Asymptomatic falciparum malaria was significantly influenced by location, proximity to water bodies, bed net usage, and history of malaria treatment, while, age, and insecticide usage showed no significant impact. PCR results revealed amplified fragments with band sizes ranging from 600 to 900 base pairs in 40 positive isolates, eliminating Pf HRP2 gene deletion as a cause for false negatives observed between RDT and microscopy results.&#x0D; Discussion/Conclusion: The study highlights a high malaria transmission rate in Nigeria, emphasizing the role of location-specific factors and bed net usage in the proliferation of asymptomatic falciparum malaria. Importantly, no gene deletion was identified in the Pf HRP2 gene among the studied Plasmodium falciparum isolates.

Immunochromatographic Detection of SARS CoV-2 Antigen In Comparison With the Polymerase Chain Reaction for Laboratory Diagnosis of Covid-19 in Karachi, Pakistan

Introduction: The Covid-19 pandemic raised the question of which laboratory test was relevant for a rapid and accurate diagnosis of the disease. The current two methods for the detection of SARS CoV-2 are the ICT (Immunochromatographic Technique) and the PCR (Polymerase Chain Reaction) tests; therefore comparative analysis of test performance has to be done. Objective: To perform a comparative analysis of two different techniques (Immunochromatographic technique for Antigen and Polymerase Chain Reaction for RNA) which are implicated for the detection of SARS CoV-2. Materials &amp; Methods: The Rapid Antigen Test (RAT) and Polymerase Chain Reaction (PCR) were performed for the detection of Covid-19 on individuals having the signs and symptoms of SARS Covid-19. Immunochromatographic technique (ICT) was performed by SD Biosensor kits, while for Polymerase Chain Reaction RNA extraction was performed by Liferiver® auto extraction analyzer, and amplification was performed by SLAN®-48P Real-Time PCR System. Results: Among of 100 samples tested, 62 (23.56%) were positive on ICT while PCR had 66 (22.44%) and 38 were negative on ICT while PCR had 34. Thus the sensitivity of ICT was 90.77% (95% CI, 80.98% - 96.54%) and specificity was 91.43% (95% CI 76.94%-98.20%). The Positive Predictive Value was 95.16% while the Negative Predictive Value was 84.21%. However the Cohen’s Kappa Index Value was recorded as 0.806. Conclusion: The clinical performance of SD Biosensor Roche kits was excellent. On the basis of sensitivity and specificity it was concluded that RAT have comparable results with RT PCR. Keywords: Antigen, COVID-19, diagnosis, immunoassay, PCR, SARS-CoV-2.

Malaria elimination in Ghana: recommendations for reactive case detection strategy implementation in a low endemic area of Asutsuare, Ghana.

Progress toward malaria elimination is increasing as many countries near zero indigenous malaria cases. In settings nearing elimination, interventions will be most effective at interrupting transmission when targeted at the residual foci of transmission. These foci may be missed due to asymptomatic infections. To solve this problem, the World Health Organization recommends reactive case detection (RACD). This case study was conducted to identify individuals with asymptomatic malaria, their predisposing risk factors and recommend RACD in Asutsuare, Ghana based on literature review and a cross sectional study. The study involved a search on PubMed and Google Scholar of literature published between 1st January, 2009-14th August, 2023 using the search terms "malaria" in "Asutsuare". Furthermore, structured questionnaires were administered to one hundred individuals without symptoms of malaria and screened using rapid diagnostic test (RDT) kits, microscopy and real-time polymerase chain reaction (rt-PCR). Malaria prevalence based on the three diagnostic techniques as well as potential malaria risk factors were assessed through questionnaires in a cross-sectional study. Cumulatively, sixty-four (64) studies (Google Scholar, 57 and PubMed, 7) were reviewed and 22 studies included in the literature on malaria in Asutsuare, Ghana. Significant risk factors were occupation, distance from a house to a waterbody, age group and educational level. Out of the 100 samples, 3 (3%) were positive by RDT, 6 (6%) by microscopy and 9 (9%) by rt-PCR. Ages 5-14.9 years had the highest mean malaria parasite densities of 560 parasites/µl with Plasmodium falciparum as the dominant species in 4 participants. Moreover, in the age group ≥ 15, 2 participants (1 each) harboured P. falciparum and Plasmodium malariae parasites. RDT had a higher sensitivity (76.54%; CI95 66.82-85.54) than rt-PCR (33.33%; CI95 4.33-77.72), while both rt-PCR and RDT were observed to have a higher specificity (92.55; CI95 85.26-96.95) and (97.30; CI95 93.87-99.13), respectively in the diagnosis of malaria. In Asutsuare, Ghana, a low endemic area, the elimination of malaria may require finding individuals with asymptomatic infections. Given the low prevalence of asymptomatic individuals identified in this study and as repleted in the literature review, which favours RACD, Asutsuare is a possible setting receptive for RACD implementation.