Positive qPCR Research Articles

Negative serum (1,3) -β-D-glucan has a low power to exclude Pneumocystis jirovecii pneumonia (PJP) in HIV-uninfected patients with positive qPCR

ObjectiveThe current study evaluated the diagnostic performance of serum (1,3)-beta-D Glucan (BDG) in differentiating PJP from P. jirovecii-colonization in HIV-uninfected patients with P. jirovecii PCR-positive results.MethodsThis was a single-center retrospective study between 2019 and 2021. The diagnosis of PJP was based on the following criteria: detection of P. jirovecii in sputum or BAL specimen by qPCR or microscopy; Meet at least two of the three criteria: (1) have respiratory symptoms of cough and/or dyspnea, hypoxia; (2) typical radiological picture findings; (3) receiving a complete PJP treatment. After exclusion, the participants were divided into derivation and validation cohorts. The derivation cohort defined the cut-off value of serum BDG. Then, it was verified using the validation cohort.ResultsTwo hundred and thirteen HIV-uninfected patients were enrolled, with 159 PJP and 54 P. jirovecii-colonized patients. BDG had outstanding specificity, LR, and PPV for PJP in both the derivation (90.00%, 8.900, and 96.43%) and the validation (91.67%, 9.176, and 96.30%) cohorts at ≥ 117.7 pg/mL. However, it had lower sensitivity and NPV in the derivation cohort (89.01% and 72.97%), which was even lower in the validation cohort (76.47% and 57.89%). Of note, BDG ≥ 117.7 pg/mL has insufficient diagnostic efficacy for PJP in patients with lung cancer, interstitial lung disease (ILD) and nephrotic syndrome. And although lymphocytes, B cells, and CD4+ T cells in PJP patients were significantly lower than those in P. jirovecii-colonized patients, the number and proportion of peripheral blood lymphocytes did not affect the diagnostic efficacy of serum BDG.ConclusionsSerum BDG ≥ 117.7 pg/mL could effectively distinguish P. jirovecii-colonization from infection in qPCR-positive HIV-uninfected patients with infectious diseases, solid tumors (excluding lung cancer), autoimmune or inflammatory disorders, and hematological malignancies. Of note, for patients with lung cancer, ILD, and nephrotic diseases, PJP should be cautiously excluded at BDG < 117.7 pg/mL.

Study on genotyping and coinfection rate of human parvovirus 4 among the HTLV-I/II infected blood donors in Khorasan Razavi, Iran

BackgroundHuman Parvovirus 4 (PARV4) is an emerging virus infecting individuals with other blood-borne diseases. This study aimed to determine the prevalence of PARV4 in confirmed HTLVI/II positive samples from blood donors, assessing PARV4 viral load (DNA) and genotyping. Methods: A novel qReal-Time PCR, based on a plasmid construct, was developed to simultaneously detect all three PARV4 genotypes using in-house primers and probes. Positive qPCR samples were subjected to nested PCR amplification and subsequent sequencing. Phylogenetic trees were constructed using the Neighbor-joining (N.J.) method. Results: The coinfection rate of PARV4-DNA in HTLVI/II confirmed infected donors, who were previously deferred, was 14.4 % (13 out of 90), with no observed association with donation status (p = 1.0).Phylogenetic analysis indicated that PARV4-positive samples closely resembled genotype 2 in Iran.qPCR quantification demonstrated significant PARV4 viral loads in positive samples, ranging between 104 and 106 DNA copies/mL of serum. Conclusion: This study presents the first evaluation of HTLVI/II and PARV4coinfection rates among blood donors. Notably, elevated PARV4-DNA titers were detected in HTLVI/II-positive donors. Given PARV's resistance to standard plasma refinery inactivation methods and the absence of its targeted inactivation, its potential impact remains a concern.

Evaluation of the New Multi-HTLV Serological Assay: Improvement for HTLV-2 Detection.

Despite the accuracy of confirmatory tests for the diagnosis of human T cell lymphotropic virus (HTLV), inconclusive or false-negative results still occur when diagnosing human T cell lymphotropic virus type 2 (HTLV-2)-positive patients. The goal of this study was to evaluate the sensitivity and accuracy of a confirmatory immunoassay, the Multi-HTLV assay. A total of 246 plasma samples were tested by real-time polymerase chain reaction (qPCR) and used to calculate the sensitivity and typing accuracy of the Multi-HTLV assay. Of the 246 plasma samples, 127 were positive for human T cell lymphotropic virus type 1 (HTLV-1), 112 were positive for HTLV-2, and 7 were positive for both HTLV-1 and HTLV-2. Thereafter, the nonparametric Mann-Whitney U test was used to calculate the concordance between the qPCR test and Multi-HTLV assay in 12 samples with discrepant and inconclusive qPCR results. The Multi-HTLV assay showed high performance in identifying HTLV-1 and HTLV-2 with sensitivities of 97% [95% confidence interval (CI): 0.92-0.98] and 94% (0.87-0.96), respectively. However, due to typing performance (98% for HTLV-1 and 94% for HTLV-2), it had 95% agreement with positive HTLV-1 qPCR results (95% CI: 90.07-97.81) and 86% (78.04-91.01) of HTLV-2 qPCR results were positive. Moreover, this test was able to recognize 80% of indeterminate samples and all HTLV-2 positive samples that showed false-negative qPCR results. Our findings, derived from a substantial number of HTLV-positive samples, underscore the inherent reliability and feasibility of the Multi-HTLV assay, regardless of the molecular testing facilities. Furthermore, the distinctive multiparametric nature of this assay, combined with its straightforward procedural execution, introduces novel perspectives for analyzing specific serological profiles in each patient, as well as the potential for immunological monitoring of disease progression.

Molecular Surveillance of Yersinia pestis from Stray Dogs and Cats and their Fleas in Algiers.

In recent years, plague has re-emerged in several countries around the world and remains endemic in some regions. In a natural environment and in contact with rodents and their fleas, stray carnivores are most at risk of catching the disease and maintaining the spread of the bacillus. The present study aimed to demonstrate the presence or absence of Yersinia pestis in stray dogs and cats in the Algiers region by molecular methods and thus determine their role in epidemiology of this disease. Molecular research of Yersinia pestis has also been conducted on fleas from these carnivores. Preliminary identification of ectoparasites to genus and species level was performed. Real-time polymerase chain reaction targeting Yersinia pestis pla gene was used to survey the plague agent in fleas and carnivores captured as stray animals in Algiers (Algeria). Positive qPCR results were tested by PCR sequencing using glpD gene. Among 327 fleas captured from 107 dogs and 365 fleas from 140 cats, prevalence of Ctenocephalides felis was higher in cats (86,96%), whereas that of Ctenocephalides canis and Xenopsylla cheopis were higher in dogs (90,57% and 92,63%, respectively). While internal and external PCR positive controls were positive, none of the 107 dogs spleens and 140 cats spleens and none of the 256 analyzed fleas were positive for Y. pestis. These results suggested that stray cats and dogs are unlikely sources of plague in Algeria, contrary to what has been reported in other plague-endemic countries. This observation illustrates that the plague epidemiological chain varies from one region to another.

Alternative Tissue Sampling for Improved Detection of Candidatus Liberibacter asiaticus.

Early detection and prompt response are key factors in the eradication of 'huanglongbing' (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of 'Candidatus Liberibacter asiaticus' (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.

Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland

BackgroundTicks carry microbes, some of which are pathogenic for humans and animals. To assess this One Health challenge, 342 ticks were collected from pet dogs and cats at 10 veterinary clinics in Finland as part of the European project “Protect Our Future Too”.MethodsThe tick species were identified, and ticks were screened with quantitative PCR (qPCR) for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Ehrlichia canis, Anaplasma spp., Candidatus Neoehrlichia mikurensis, tick-borne encephalitis virus (TBEV), and Babesia spp. For comparison, a subset of tick DNA (20 qPCR-positive samples) was analysed with 16S next-generation sequencing (NGS).ResultsMost ticks were Ixodes ricinus (289, 84.5%), followed by Ixodes persulcatus (51, 14.9%). One hybrid tick (I. ricinus/I. persulcatus, 0.3%) and one Rhipicephalus sanguineus tick (0.3%) were identified. We found one or more of the analysed pathogens in 17% (59/342) of the ticks. The most prevalent pathogen was B. burgdorferi s.l. (36, 10.5%), followed by Anaplasma phagocytophilum (12, 3.5%), B. miyamotoi (5, 1.5%), Babesia venatorum (4, 1.2%), and TBEV (1, 0.3%). Candidatus Neoehrlichia mikurensis DNA was amplified from three (0.9%) ticks. Ehrlichia canis was not detected. In the 16S NGS, six samples produced enough reads for the analysis. In these six samples, we confirmed all the positive qPCR findings of Borrelia spp. and Ca. N. mikurensis.ConclusionsThe high prevalence of pathogenic microorganisms in the ticks of this study emphasizes the importance of awareness of ticks and tick-borne diseases and prevention. Furthermore, the results show that veterinary surveillance can facilitate early detection of tick-borne pathogens and new tick species and draw attention to possible co-infections that should be considered both in symptomatic humans and animals after tick bites.Graphical

The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis.

Lack of available sensitive point-of-care testing is one of the primary obstacles to the rapid diagnosis of leptospirosis. The purpose of this study was to test the performance of two point-of-care tests, a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) fluorescence-based diagnostic assay (FBDA), a Leptospira immunoglobulin M (IgM) rapid diagnostic test (RDT), and the two tests combined. For the diagnosis of 171 clinical samples, a recombinase polymerase amplification (RPA)-CRISPR/Cas12a FBDA for whole blood and Leptospira IgM RDT (Medical Science Public Health, Thailand) for serum were used. The confirmed cases were determined by using any positive qPCR, microscopic agglutination test (MAT), and culture results. Diagnostic accuracy was assessed on the first day of enrollment and stratified by the day after symptom onset. The overall sensitivity of the Leptospira IgM RDT and RPA-CRISPR/Cas12a FBDA was 55.66% and 60.38%, respectively. When the two tests were combined, the sensitivity rose to 84.91%. The specificity of each test was 63.08% and 100%, respectively, and 63.08% when combined. The sensitivity of the Leptospira IgM RDT rose on days 4-6 after the onset of fever, while the RPA-CRISPR/Cas12a FBDA continued to decrease. When the two tests were combined, the sensitivity was over 80% at different days post-onset of fever. The combination of Leptospira IgM RDT and RPA-CRISPR/Cas12 FBDA exhibited significant sensitivity for the detection of leptospires at various days after the onset of fever, thereby reducing the likelihood of misdiagnosis. The combination of these assays may be suitable for early leptospirosis screening in situations with limited resources.

Antibody response following the fourth SARS-CoV-2 vaccine dose during the Omicron wave in Brazil.

We investigated the impact of the fourth dose with ChAdOx1 nCoV-19 (AstraZeneca) in the humoral immune response to SARS-CoV-2 during a 9-month follow-up period in which Omicron was the predominant variant in Brazil. IgG for the SARS-CoV-2 spike protein (S) and nucleocapsid (N) proteins were analyzed in samples collected before and after the fourth dose. All participants were tested monthly for SARS-CoV-2 infection by RT-qPCR. The antibody response induced by the fourth dose of the coronavirus disease 2019 vaccine was evaluated and compared with the response induced by the second and third doses. The additional antibody response to the viral S protein after the fourth dose was smaller than those after the third vaccine dose. In contrast, an increase in the N IgG levels could be observed after the fourth dose compared to other vaccine doses. In the comparison of the antibody response before and after the fourth dose, an increase in both S-and-N IgG was noted, mainly in the positive qPCR group. We did not observe a significant decline in IgG levels after the fourth dose, as observed after the second and third doses, therefore, a sustained humoral response to both S and N proteins seems to be achieved.

Improving Diagnosis of Pulmonary Mucormycosis: Leads From a Contemporary National Study of 114 Cases

Pulmonary mucormycosis (PM) is a life-threatening invasive mold infection. Diagnosis of mucormycosis is challenging and often delayed, resulting in higher mortality. Are the disease presentation of PM and contribution of diagnosis tools influenced by the patient underlying condition? All PM cases from six French teaching hospitals between 2008 and 2019 were retrospectively reviewed. Cases were defined according to updated EORTC/MSG criteria with the addition of diabetes and trauma as host factors and positive serum or tissue PCR as mycological evidence. Thoracic CT scans were reviewed centrally. One hundred and fourteen cases of PM were recorded, including 40% disseminated forms. Main underlying conditions were hematological malignancy (49%), allogeneic hematopoietic stem-cell transplantation (21%), and solid organ transplantation (17%). When disseminated, main dissemination sites were the liver (48%), spleen (48%), brain (44%), and kidneys (37%). Radiological presentation included consolidation (58%), pleural effusion (52%), reversed halo sign (RHS, 26%), halo sign (24%), vascular abnormalities (26%), and cavity (23%). Serum qPCR was positive in 42/53 patients (79%) and bronchoalveolar lavage (BAL) in 46/96 patients (50%). Transthoracic lung biopsy was diagnostic in 8/11 (73%) patients with non-contributive BAL. Overall 90-day mortality was 59%. Neutropenic patients displayed more frequently an angioinvasive presentation including RHS and disseminated disease (p<0.05). Serum qPCR was more contributive in neutropenic patients (91% vs 62%, p=0.02) and BAL in non-neutropenic patients (69% vs 41%, p=0.02). Serum qPCR was more frequently positive in patients with a >3cm main lesion (91% vs 62%, p=0.02). Overall, positive qPCR was associated with an early diagnosis (p=0.03) and treatment onset (p=0.01). Neutropenia and radiological findings influence disease presentation and contribution of diagnostic tools during PM. Serum qPCR is more contributive in neutropenic patients and BAL examination in non-neutropenic patients. Lung biopsies are highly contributive in case of non-contributive BAL.

Validation of quantitative real-time polymerase chain reaction for detection of Legionella pneumophila in hospital water networks

Rapid monitoring of Legionella pneumophila (Lp) is essential to reduce the risk of Legionnaires' disease in healthcare facilities. However, culture results take at least eight days, delaying the implementation of corrective measures. Here, we assessed the performance of a qPCR method and determined qPCR action thresholds for the detection of Lp in hospital hot water networks (HWNs). Hot water samples (N= 459) were collected from a hospital HWN. Lp were quantified using iQ-Check® Quanti real-time PCR Quantification kits (Bio-Rad) and the results were compared with those of culture. qPCR thresholds corresponding to the culture action thresholds of 10 and 1000cfu/L were determined on a training dataset and validated on an independent dataset. Lp concentrations measured by culture and qPCR were correlated for both the training dataset (Spearman's correlation coefficient ρ= 0.687, P<0.0001) and the validation dataset (ρ= 0.661, P<0.0001). Lp qPCR positivity thresholds corresponding to culture action thresholds of 10cfu/L was 91 genome units (gu) per litre (sensitivity, 86.4%; negative predictive value - NPV, 93.3%) and that corresponding to culture action thresholds of 1000cfu/L was 1048 gu/L (sensitivity, 100%; NPV, 100%). Detection of Lp by qPCR could be implemented with confidence in hospitals as a complement to culture in the monitoring strategy to speed up the implementation of corrective measures.

Fatal intracranial haemorrhage in acute promyelocytic leukemia patients with short isoform of PML-RARα: Review of molecular and radiological data

Three major PML-RARα fusion gene transcripts (long [bcr1], variant [bcr2], and short [bcr3]) are currently used in clinical laboratories for the diagnosis and treatment monitoring of APL patients. Despite highly improved outcome, relapse and intracranial haemorrhage that may lead to early death is still an unsolved complication in APL. We reviewed APL patients confirmed by qPCR for the presence of PML-RARα transcripts (n = 27) and studied their outcome in relation to the isoform expression at diagnosis and follow-up in King Fahad Medical City. Eight in twenty-seven patients showed bcr3 and nineteen patients with bcr1 as major isoforms at diagnosis. Half of the bcr3 patients (n = 4/8) showed early mortality, prolonged qPCR positivity, 4-fold higher neutrophil/lymphocyte ratio, higher creatinine levels, and significantly reduced relapse free and overall survival time compared with bcr1 patients. Radiological findings in bcr3 patients revealed CNS involvement in the form of intracranial haemorrhage and periventricular microangiopathy and no CNS involvement in bcr1 patients. In conclusion, PML-RARα isoform expression at diagnosis in selective patients influences disease course over time and may even lead to early mortality due to haemorrhage. Thus, timely reporting of the specific PML-RARα isoform by clinical laboratories and CNS assessment by radiology can prevent complications leading to death in some APL patients.

Mycobacterium avium subsp. Paratuberculosis in Different Environmental Samples from a Dairy Goat Barn-Implications for Sampling Strategies for Paratuberculosis Diagnostic and Prevention.

Environmental samples are often used to classify the paratuberculosis status of cattle herds. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), predominantly through oral ingestion during infancy. In this explorative study, the presence of MAP was determined in the barn environment of a paratuberculosis-infected vaccinated dairy goat herd. A total of 256 bedding, dust, feed, and water samples were collected at eight time points and examined using culture and qPCR. Detection rates of both methods were compared, and factors determining MAP confirmation were identified. MAP was cultured from 28 bedding and one dust sample, while MAP DNA was detected in all materials (117/256). Samples from high animal traffic areas and those collected during the indoor season were more likely to yield positive culture and qPCR results. Cultivation of MAP from kidding pens indicated this area as a possible infection site. Dust proved to be the most suitable material for detecting MAP DNA, as bedding was for MAP culture. Environmental sampling was demonstrated to be an effective way to detect MAP in a dairy goat herd. qPCR results could confirm herd infection, while culture results provided insight into crucial areas for MAP transmission. These findings should be considered when designing farm-specific paratuberculosis control plans.

Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs.

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

Feline leishmaniosis with focus on ocular manifestation: a case report

BackgroundIn Europe, feline leishmaniosis is commonly caused by Leishmaniainfantum. There is little knowledge regarding pathogenesis, ocular manifestations and long-term follow-ups in cats with leishmaniosis.FindingsA 6-year-old female, spayed European Shorthair cat was imported from Spain to Germany 2 years prior to its first clinical presentation. The cat showed lethargy, weight loss, ulcerative lesions on the front limbs and high-grade chronic uveitis. The diagnosis of L. infantum infection was based on the cytological finding of amastigotes in skin lesions, positive qPCR of EDTA-blood and positive PCR of a cyto-brush sample from the conjunctiva. Supportive findings included positive serology by IFAT, serum protein capillary electrophoresis with peaks in alpha2- and gamma-globulin sections and marked elevation of SAA. Enucleation had to be performed on day 288 on both eyes because of blindness, glaucoma and high-grade uveitis. Histologically, high numbers of Leishmania spp. amastigotes were found in histiocytes. IFAT and PCR were positive in the aqueous humor in both eyes, respectively. Feline leukemia virus antigen and feline immunodeficiency virus antibody testings were positive. Hematological and biochemical results revealed mild leukocytosis with lymphocytosis, monocytosis and eosinopenia as well as marked elevation of SAA and hyperglobulinemia. The cat was treated with allopurinol, responded well and was still alive at follow-up on day 288 after first presentation. However, enucleation was necessary because of refractory glaucoma and uveitis. ConclusionFor the first time, ocular evidence of Leishmania IgG antibodies was demonstrated in the aqueous humor of both eyes in cats. There is limited knowledge about the pathogenesis, treatment options and outcomes in cats infected with L. infantum. This case report supports the hypothesis that immunosuppression increases the risk of clinical signs of leishmaniasis in cats. Alpha2- and gamma-globulin peaks in serum protein capillary electrophoresis are supportive criteria for the diagnosis of L. infantum infection. SAA is valuable for monitoring. Regarding ophthalmology, uveitis and glaucoma may have a poor prognosis. Graphical abstract

Monitoring SARS-CoV-2 incidence and seroconversion among university students and employees: a longitudinal cohort study in California, June–August 2020

ObjectivesTo identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students...

Is There an Association between Concurrent Epstein–Barr Virus Infection and Sudden Hearing Loss?—A Case—Control Study in an East Asian Population

Viral infection serves as the crucial etiology for the development of sudden sensorineural hearing loss (SSNHL). We aimed to investigate whether there is an association between concurrent Epstein-Barr virus (EBV) infection and SSNHL in an East Asian population. Patients who were older than 18 years of age and met the criteria of sudden hearing loss without an identifiable etiology were enrolled from July 2021 until June 2022, followed by the serological testing of IgA antibody responses against EBV-specific early antigen (EA) and viral capsid antigen (VCA) with an indirect hemagglutination assay (IHA) and real-time quantitative polymerase chain reaction (qPCR) of EBV DNA in serum before the treatment was initiated. After the treatment for SSNHL, post-treatment audiometry was performed to record the treatment response and degree of recovery. Among the 29 patients included during enrollment, 3 (10.3%) had a positive qPCR result for EBV. In addition, a trend of poor recovery of hearing thresholds was noted for those patients with a higher viral PCR titer. This is the first study to use real-time PCR to detect possible concurrent EBV infection in SSNHL. Our study demonstrated that approximately one-tenth of the enrolled SSNHL patients had evidence of concurrent EBV infection, as reflected by the positive qPCR test results, and a negative trend between hearing gain and the viral DNA PCR level was found within the affected cohort after steroid therapy. These findings indicate a possible role for EBV infection in East Asian patients with SSNHL. Further larger-scale research is needed to better understand the potential role and underlying mechanism of viral infection in the etiology of SSNHL.

Impact of population based indoor residual spraying with and without mass drug administration with dihydroartemisinin-piperaquine on malaria prevalence in a high transmission setting: a quasi-experimental controlled before-and-after trial in northeastern Uganda

BackgroundDeclines in malaria burden in Uganda have slowed. Modelling predicts that indoor residual spraying (IRS) and mass drug administration (MDA), when co-timed, have synergistic impact. This study investigated additional protective impact of population-based MDA on malaria prevalence, if any, when added to IRS, as compared with IRS alone and with standard of care (SOC).MethodsThe 32-month quasi-experimental controlled before-and-after trial enrolled an open cohort of residents (46,765 individuals, 1st enumeration and 52,133, 4th enumeration) of Katakwi District in northeastern Uganda. Consented participants were assigned to three arms based on residential subcounty at study start: MDA+IRS, IRS, SOC. IRS with pirimiphos methyl and MDA with dihydroartemisinin- piperaquine were delivered in 4 co-timed campaign-style rounds 8 months apart. The primary endpoint was population prevalence of malaria, estimated by 6 cross-sectional surveys, starting at baseline and preceding each subsequent round.ResultsComparing malaria prevalence in MDA+IRS and IRS only arms over all 6 surveys (intention-to-treat analysis), roughly every 6 months post-interventions, a geostatistical model found a significant additional 15.5% (95% confidence interval (CI): [13.7%, 17.5%], Z = 9.6, p = 5e−20) decrease in the adjusted odds ratio (aOR) due to MDA for all ages, a 13.3% reduction in under 5’s (95% CI: [10.5%, 16.8%], Z = 4.02, p = 5e−5), and a 10.1% reduction in children 5–15 (95% CI: [8.5%, 11.8%], Z = 4.7, p = 2e−5). All ages residents of the MDA + IRS arm enjoyed an overall 80.1% reduction (95% CI: [80.0%, 83.0%], p = 0.0001) in odds of qPCR confirmed malaria compared with SOC residents. Secondary difference-in-difference analyses comparing surveys at different timepoints to baseline showed aOR (MDA + IRS vs IRS) of qPCR positivity between 0.28 and 0.66 (p < 0.001). Of three serious adverse events, one (nonfatal) was considered related to study medications. Limitations include the initial non-random assignment of study arms, the single large cluster per arm, and the lack of an MDA-only arm, considered to violate equipoise.ConclusionsDespite being assessed at long time points 5–7 months post-round, MDA plus IRS provided significant additional protection from malaria infection over IRS alone. Randomized trials of MDA in large areas undergoing IRS recommended as well as cohort studies of impact on incidence.Trial registration: This trial was retrospectively registered 11/07/2018 with the Pan African Clinical Trials Registry (PACTR201807166695568).

Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children.

To develop a rapid and low-cost method for 16S rDNA nanopore sequencing. This was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR. The turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively. The nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.

Prevalence and Impact of BK Polyomavirus in the Ureters of Kidney Donors: Research Letter.

More than 75% of the population is seropositive for BK polyomavirus (BKV), which remains quiescent in the urothelium in immunocompetent hosts. However, it can reactivate in kidney transplant recipients (KTRs), and up to 30% of them will develop BKV viremia in the 2 years following transplant, with a risk of developing BKV-associated nephropathy (BKVAN). Viral reactivation is associated with the level of immunosuppression, but there is currently no way to predict which patients are at high risk for reactivation. As BKV originates from kidney donors, our primary objective was to determine the prevalence of detectable BKV in donor ureters. Our secondary objective was to see if there is a correlation between the presence of BKV in donor urothelium and the development of BKV viremia and BKVAN in KTR. Prospective cohort study. Single-center academic kidney transplant program. Prospective sequential KTRs that received a kidney transplant between March 2016 and March 2017. The presence of BKV in donor ureters was determined by TaqMan-based quantitative polymerase chain reaction (PCR; qPCR). We performed a prospective study which was done on 35 out of the 100 donors initially foreseen to take part in the study. During surgery, the distal part of donor ureter was kept and analyzed by qPCR (to establish the presence of BKV in the urothelium). The primary outcome was the development of BKV viremia in KTR over a period of 2 years after transplant. Secondary outcome was the development of BKVAN. Out of 35 ureters analyzed, only one had a positive qPCR for BKV (2.86%, 95% confidence interval [CI]: [0.07-14.92]). Considering the primary objective would not be met, the study was interrupted after 35 specimens. After surgery, 9 recipients had a slow graft function and 4 had delayed graft function, one of which never recovered graft function. Over the 2-year follow-up, 13 patients developed BKV viremia, while 5 patients developed BKVAN. The patient who received a graft from a positive qPCR donor eventually developed BKV viremia and nephropathy. The specimen analyzed was a distal rather than a proximal portion of the ureter. However, BKV replication is known to concentrate in the corticomedullary junction. BK polyomavirus prevalence in the distal part of donor ureters is lower than previously reported. It cannot be used as a predictor for the development of BKV reactivation and/or nephropathy.

Quantificação por PCR em tempo real e achados histopatológicos da infecção por Mycoplasma hyopneumoniae em pulmão de suínos abatidos na cidade de São Luís, Maranhão, Brasil

ABSTRACT: Porcine enzootic pneumonia (PES), mainly caused by the bacteria Mycoplasma hyopneumoniae, is the main cause of respiratory problems in pigs. Infection by M. hyopneumoniae leads to production losses and the predisposition of affected animals to secondary infections, which may result in the condemnation of carcasses and organs due to lung lesions at the time of slaughter. The objective of the research was to evaluate the infection by M. hyopneumoniae in pigs submitted to slaughter in São Luís Island/MA, using molecular and histopathological diagnostic methods. One hundred fifty lung samples were collected from inspected (n=65) and non-inspected (n=85) slaughter pigs on São Luís Island, Maranhão, from July 2019 to August 2021. Of the 150 DNA samples collected, 121 showed an amplified product for Cyt B in the PCR assay. Thus, 121 samples were submitted to qPCR of M. hyopneumoniae, of which 44 (36.36%) showed positive results. The mean amount of bacterial load ranged from 1.20 × 101 to 7.20 × 104, with a mean of 1.73 × 104 copies. Of the reagent samples, 81.81% (36 samples) were obtained from non-inspected slaughter, while 18.18% (8 samples) were obtained from slaughterhouses. In the histopathological analysis, 44 positive qPCR samples were evaluated, of which 28 (63.63%) presented results compatible with the main inflammatory process associated with the presence of M. hyopneumoniae, that is, bronchial-associated lymphoid tissue hyperplasia (BALT). Three samples that showed the highest bacterial load (qPCR: 5.63 × 10³, 2.19 × 104 and 7.23 × 104) showed more evident lesions in this study. The microscopic findings associated with the quantifications indicated a relationship between the amount of bacterial load and the presence of microscopic lesions; higher bacterial load in lung tissue is associated with increased histopathologic staining for BALT hyperplasia. In conclusion, the results point to the circulation of the etiological agent in the sampled animals and the need for preventive measures on pig farms in Maranhão with the involvement of producers, sanitary defense and inspection agencies.