Blood Polymerase Chain Reaction Research Articles

Possible Concomitant Aggressive NK Cell Leukemia and EBV-positive T-cell lymphoma; Using the online beta version of WHO-HAEM5 and videoconferencing software to make diagnoses accessible in an emerging economy

BackgroundUsing the World Health Organization Classification 5th edition (beta version online; WHO-HAEM5bv) in emerging economies is key to global healthcare equity. Although there may be ongoing updates, hesitancy in accepting and reporting these diagnoses in publication conflicts with the WHO’s commitment to global accessibility. Aggressive NK cell leukemia (ANKL) and systemic EBV-positive T-cell lymphoma of childhood (SEBVTCL) with CD4-positive immunophenotype are both rare entities, are most described in Asians and East Asians, are associated with prior systemic chronic active EBV disease (CAEBV), and presentation with Hemophagocytic Lymphohistiocytosis (HLH). Recognizing and diagnosing any one of these entities requires not only training and experience in hematopathology, but good cooperation between clinical physicians and all areas of the laboratory. We describe a 30-year-old woman who presented to a Vietnam hospital and was rapidly diagnosed with ANKL, SEBVTCL, and HLH using WHO-HAEM5bv essential criteria, aided by expert consultation from a United States (US) board certified hematopathologist in real-time using video conferencing software.MethodsZoom™ videoconferencing software; Immunohistochemistry; flow cytometric immunophenotyping; polymerase chain reaction (PCR), Next Generation Sequencing (NGS).ResultsAt the time of hospital admission, automated complete blood count (CBC) with differential count showed slight anemia, slight lymphocytosis, and moderate thrombocytopenia. HIV serology was negative. Whole blood PCR for EBV was positive showing 98,000 copies/ml. A lymph node biopsy revealed histology and immunohistochemistry consistent with the online beta version WHO-HAEM5 classification of SEBVTCL arising in CAEBV. Blood and bone marrow studies performed for staging revealed no histologic or immunohistochemical evidence of T-cell lymphoma in the bone marrow core, however, atypical blood smear lymphocyte morphology and blood immunophenotyping by flow cytometry were consistent with WHO-HAEM5 classification of ANKL. NGS revealed no evidence of genetic variant(s) associated with HLH in Vietnam. All laboratory studies were performed at Blood Transfusion Hematology Hospital (BTHH) in Ho Chi Minh City Vietnam.ConclusionAlthough Vietnam, an emerging economy, currently lacks the laboratory infrastructure to more rigorously confirm a rare synchronous presentation of two distinct EBV-driven T/NK cell neoplasms, these two concomitant diagnoses were made using only laboratory techniques available in Vietnam with the help of WHO-HAEM5bv and real-time video consultation by a US hematopathologist.

Proteomics identification of overexpressed serum proteins in dogs with Babesiacanis infection.

Canine babesiosis, caused by the protozoan parasite Babesia canis, is characterized by clinical manifestations, including hemolytic anemia, thrombocytopenia, multiple organ failure, and may result in death. This disease is detected using conventional blood smears, which are time-consuming and have low sensitivity. This study aimed to investigate a more rapid and sensitive method for detecting B. canis infection in dogs by examining the expressed serum protein profiles using proteomics. We collected six sera samples from three healthy and three B. canis-infected dogs diagnosed using blood smear and polymerase chain reaction. We analyzed the proteins using two-dimensional gel electrophoresis. The candidate spots from the gel were subjected to protein identification using a nano-liquid chromatography system coupled to an ion-trap mass spectrometer equipped with an electrospray ionization nano-sprayer. We found that 10 protein spots were overexpressed in the serum samples from infected dogs compared with healthy dogs, which corresponded to three proteins: serotransferrin, serotransferrin isoforms X1, and hemopexin. Furthermore, analysis of the protein-protein interaction network confirmed that they strongly interacted with each other. This study suggests that high levels of serotransferrin and hemopexin are related to B. canis infection, making these proteins potential candidates for the development of diagnostic molecules or vaccines.

Clinical, Haemato-biochemical Evaluation and Comparison of Polymerase chain reaction with Conventional Blood Smear Examination for the Detection of Babesia gibsoni Infection in Dogs

The objective of the present study was to detect Babesia gibsoni infection in dogs which are presented to Veterinary Clinical Complex, Rajiv Gandhi Institute of Veterinary Education and Research, Puducherry. A total of 85 dogs with clinical signs such as inappetence, fever, enlarged lymph nodes and tick infestation were screened. The diagnostic efficacy of polymerase chain reaction (PCR) technique has been compared with blood smear examination to detect B. gibsoni infection in dogs. By blood smear and PCR examination, six (7.1%) dogs and 28 (42.4%) dogs were positive for B. gibsoni infection respectively. Clinical examination of dogs with B. gibsoni showed tick infestation, anorexia, dullness, pyrexia, pale/congested conjunctival mucus membrane, tachypnoea and swollen lymph nodes. In addition, few dogs had icterus and haemoglobinuria. Laboratory examination of blood samples of B. gibsoni dogs revealed significant reduction in haemoglobin, PCV, erythrocyte count, platelet count and serum albumin levels. Significant increase in levels of ALP, globulin, bilirubin, BUN and creatinine were recorded.

Development of a multiplex reverse transcription qPCR assay for identification of saliva, blood, and semen

ABSTRACT The identification of body fluids can provide important information for case processing in forensic investigations. Accurate identification through messenger RNA (mRNA) is effective, especially in cases of sexual assault, where body fluids from one or more contributors are mixed. In this study, a saliva, blood, and semen simultaneous identification reverse transcription-quantitative polymerase chain reaction (SBS RT-qPCR) assay using mRNA from a DNA/RNA co-extraction method was developed. The body fluid-specific mRNA markers histatin 3 (HTN3), haemoglobin subunit beta (HBB), and protamine 1 (PRM1) were selected for identification of saliva, blood, and sperm cells, respectively. The assay accurately identified saliva, blood, and semen without cross-reaction with 10 body fluids. The limits of detection (LoD) were 1/102-, 1/105-, and 1/103-fold dilutions of total RNA in saliva, blood, and semen, respectively. Furthermore, the assay accurately identified trace amounts of body fluids in mixed samples at a 58:1:1 ratio. Finally, it was possible to analyse the origin of body fluids by obtaining an STR profile using the DNA/RNA co-extraction method. The SBS RT-qPCR assay was able to simultaneously identify each body fluid with efficiency in sample consumption. Therefore, it is advisable to use the DNA/RNA co-extraction method to identify mixed body fluids in sexual assault cases.

Prevalence of Different Courses of Feline Leukaemia Virus Infection in Four European Countries.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

Effect of cytomegalovirus infection on post-transplant hospitalization days among children undergoing allogeneic hematopoietic cell transplantation: A marginal structural model approach.

Cytomegalovirus (CMV) commonly reactivates after allogeneic hematopoietic cell transplant (HCT), potentially leading to CMV disease and significant morbidity and mortality. To reduce morbidity and mortality, many centers conduct weekly CMV blood polymerase chain reaction (PCR) surveillance testing with subsequent initiation of antiviral therapy upon CMV DNAemia detection. However, the impact of CMV DNAemia on subsequent hospitalization risk has not been assessed using models accounting for the time-varying nature of the exposure, outcome, and confounders. All allogeneic HCTs at the Children's Hospital of Philadelphia from January 2004-April 2017 were considered for inclusion. Patients were monitored with CMV surveillance via PCR testing for up to 105 days after HCT receipt. We estimated the association between CMV DNAemia and rate of hospitalization using marginal structural models (MSM). There were 343 allogeneic HCT episodes in 330 with CMV surveillance; median age was 9.0 (range: 0.1-26.2) and 46.5% were female. And 24.1% of HCT patients had at least one positive CMV blood PCR during the follow-up period. Median time to CMV DNAemia detection was 19 days (range: 4-97). The MSM estimated the incidence rate ratios for an association of CMV DNAemia with hospitalization to be 1.24, (95% confidence interval: 1.04-1.47). CMV DNAemia was associated with an increased hospitalization in the post-HCT period. The MSM accounted for time-varying nature of the outcome, exposure and confounders. The findings support prevention of CMV DNAemia in this population. We recommend further investigation into the effectiveness and safety of prophylaxis versus pre-emptive CMV prevention approaches.

The Mortality of Congenital Syphilis

To document the case-fatality rate (CFR) of congenital syphilis diagnosed by molecular tools and rabbit infectivity testing (RIT) of clinical specimens in addition to standard evaluation and to compare that with the CFR using the Centers for Disease Control and Prevention (CDC) surveillance case definition. Prospective, single site, cohort study of all cases of syphilis among mothers and their infants from 1984 to 2002. The diagnosis of congenital syphilis was determined using IgM immunoblotting, polymerase chain reaction, and RIT of fetal or infant specimens in addition to clinical, laboratory, and radiographic criteria. Data were retrospectively reviewed to ascertain fetal and neonatal mortality. During the 18-year study, there were 191 cases of congenital syphilis confirmed by abnormalities on clinical, laboratory, or radiographic evaluation and/or positive serum IgM immunoblot, blood polymerase chain reaction, or blood/cerebrospinal fluid RIT. Of the 191 cases, 59 died for a CFR of 31%. Of the 59 deaths, 53 (90%) were stillborn and 6 (10%) died in the neonatal period. The majority (74%, 39/53) of stillbirths occurred in the third trimester. The CDC surveillance case definition correctly identified all infants with congenital syphilis, but the CDC CFR was 10% which underestimated the CFR by more than 300%. Our findings corroborate the high sensitivity of the CDC surveillance definition for congenital syphilis but highlight its poor estimation of its associated mortality. The CFR among infected progeny of pregnant women with syphilis was 31%, due mostly to demise in the third trimester and as such highlights the need for detection and appropriate treatment of syphilis during pregnancy.

Data usefulness in determining cause of stillbirth in South Asia.

To evaluate the usefulness of data to determine cause of stillbirth in India and Pakistan. Prospective, observational study. Study hospitals in India and Pakistan. 200 fetal deaths with placental evaluation and minimally invasive tissue sampling (MITS) of internal organs and polymerase chain reaction (PCR) test for 75 pathogens. Data defined as useful to determine stillbirth causes. Placental pathology was the most useful to determine cause of stillbirth. Comparing placental and fetal weight with standard weights was useful in 44.5% and 48.5%, respectively. Lung histology was useful in 42.5%. Most of the other findings of internal organ histology were only occasionally useful. Signs of abruption, by maternal history or placental evaluation, were always deemed useful. Placenta, brain and cord blood PCR were also useful, but less often than histology. Based on this analysis, maternal clinical history, placental histology and fetal examination were most informative. Comparing the placental and fetal weights with recognised standards was useful in nearly half the cases. Fetal tissue histology and PCR were also informative. Of all the potential tests of MITS-obtained specimens, we would first recommend histological evaluation of the lungs, and using a multiplex PCR platform would determine pathogens in blood and brain/CSF. We recognise that this approach will not identify some causes, including some genetic and internal organ anomalies, but will confirm most common causes of stillbirth and most of the preventable causes of stillbirth in low- and middle-income countries.

Cytomegalovirus esophagitis associated with gastroesophageal reflux disease in an immunocompetent patient: case report

Cytomegalovirus infection has a wide variability of clinical presentations depending on the patient's immunity, ranging from asymptomatic infections in immunocompetent individuals to infections with significant morbidity and mortality in immunocompromised patients. We originally described a clinical case of severe cytomegalovirus esophagitis, confirmed by blood polymerase chain reaction (PCR) test and esophageal biopsy, in a young immunocompetent patient with a long-standing diagnosis of gastroesophageal reflux disease. Clinical improvement occurred only after the initiation of specific treatment with ganciclovir, providing a negative viral load. This report reinforces the importance of ruling out the etiology of cytomegalovirus infection in severe ulcerative esophagitis, even in immunocompetent individuals.

Molecular investigation of bovine tropical theileriosis outbreak in an organized dairy cattle farm in Madhya Pradesh, India.

The present study aimed to investigate an outbreak of Theileria annulata (T. annulata) infection in an organized dairy cattle farm in Madhya Pradesh, India, using clinical and molecular techniques. Following the deaths of two crossbred cattle in March 2021, 43 blood samples were collected from infected and apparently healthy animals and examined by blood smear and polymerase chain reaction (PCR) techniques. The blood smear examination showed that 23.25% of samples were positive for Theileria organisms, while conventional PCR targeting the 18S ribosomal RNA (18S rRNA) and T. annulata merozoite surface antigen-1 (TAMS-1) genes revealed that 32.55% of samples were positive for T. annulata. PCR targeting cytochrome b (Cytb) gene showed 46.51% of samples were positive for T. annulata. Haematological analysis confirmed clinical signs of infection in affected animals, which were treated with buparvaquone @ 2.5mg/kg body weight intramuscularly along with supportive medicine. Two 18S rRNA gene amplicons were sequenced and analysed in a phylogenetic tree and haplotype network with 54 Indian and 38 foreign sequences. The phylogenetic tree revealed two groups with a high posterior probability and bootstrap value, while the haplotype network revealed 35 haplotypes, with haplotype 1 (H1) being the most abundant and several single haplotypes clustering around it, indicating fast and widespread expansion. Genetic diversity indices and neutrality tests confirmed that the population was expanding. These studies highlight the significance of prompt and precise diagnosis and management of T. annulata outbreaks and provide insights into its evolutionary history and population dynamics of T. annulata in India, which could aid improving disease preventive and control strategies.

Efficacy of buparvaquone in pcr confirmed concurrent infection of Trypansoma evansi and Theileria annulata in buffaloes

Five buffaloes, 5 to 7 years old in the month of July 2015 with history of high rise of temperature, nasal discharge, exophthalmia, excitement, lacrimation, salivation, dyspnea, generalised lymphadenopathy, especially in the prescapular lymph nodes were attended at a farm in Mahakaushal region of Madhypradesh. By blood smear and polymerase chain reaction analysis, it was determined that all the buffaloes had mixed infections of T. evansi and T. annulata. The treatment of all five buffaloes was initiated with single deep intramuscular injection of diminazine acceturate 3.5 mg/kg body weight along with three dosages of long acting oxytetracycline 20 mg/kg b.wt. on alternate day. A single injection of buparvaquone on the fourth day (in 3 cases) and two doses of buparvaquone (2.5 mg kg-1) at 48 h intervals (in 2 cases) were administered. Only 3 buffaloes showed uneventful recovery after a single injection with buparvaquone. When the blood samples of all the five buffaloes were again analyzed by PCR and found all the samples were negative for T. evansi and T. annulata except two buffaloes (Two cases hading two doses of buparvaquone) which were positive for T. annulata in spite of giving the second dose of buparvaquone. The study indicated that there might be reduced efficacy or resistance of T. annulata against buparvaquone.

Biochemical and genotyping analyses of camels (Camelus dromedaries) trypanosomiasis in North Africa

Camels are considered an important food source in North Africa. Trypanosomiasis in camels is a life-threatening disease that causes severe economic losses in milk and meat production. Therefore, the objective of this study was to determine the trypanosome genotypes in the North African region. Trypanosome infection rates were determined by microscopic examination of blood smears and polymerase chain reaction (PCR). In addition, total antioxidant capacity (TAC), lipid peroxides (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were determined in erythrocyte lysate. Furthermore, 18S amplicon sequencing was used to barcode and characterizes the genetic diversity of trypanosome genotypes in camel blood. In addition to Trypanosoma, Babesia and Thelieria were also detected in the blood samples. PCR showed that the trypanosome infection rate was higher in Algerian samples (25.7%) than in Egyptian samples (7.2%). Parameters such as MDA, GSH, SOD and CAT had significantly increased in camels infected with trypanosomes compared to uninfected control animals, while TAC level was not significantly changed. The results of relative amplicon abundance showed that the range of trypanosome infection was higher in Egypt than in Algeria. Moreover, phylogenetic analysis showed that the Trypanosoma sequences of Egyptian and Algerian camels are related to Trypanosoma evansi. Unexpectedly, diversity within T. evansi was higher in Egyptian camels than in Algerian camels. We present here the first molecular report providing a picture of trypanosomiasis in camels, covering wide geographical areas in Egypt and Algeria.

Bacterial coinfections in hospitalized children with COVID-19 during the SARS-CoV-2 Omicron BA.2 variant pandemic in Taiwan.

Bacterial coinfections have been widely recognized in adults with coronavirus disease 2019 (COVID-19). However, bacterial coinfections in hospitalized children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been sufficiently researched. This study aimed to determine the clinical presentations and risk factors for bacterial coinfections of pediatric inpatients during the SARS-CoV-2 Omicron BA.2 variant pandemic. This retrospective, observational study included patients younger than 18 years of age who were hospitalized for COVID-19 confirmed by polymerase chain reaction (PCR) or antigen rapid tests during the SARS-CoV-2 Omicron BA.2 variant pandemic. Data and outcomes of these patients with or without bacterial coinfections were compared. During this study period, 161 children with confirmed COVID-19 were hospitalized. Twenty-four had bacterial coinfections. The most frequently reported concurrent diagnosis was bacterial enteritis, followed by lower respiratory tract infections. Children with bacterial coinfections had higher white blood cell (WBC) counts and PCR cycle threshold values. The bacterial coinfection group comprised a relatively greater proportion of patients who required high-flow nasal cannula oxygen and remdesivir. The length of stay in the hospital and that in the intensive care unit were longer for children with COVID-19 with bacterial coinfections. Mortality was not observed in either group. Abdominal pain, diarrhea, and comorbidity with neurologic illnesses were risk factors for bacterial coinfections with COVID-19. This study provides clinicians with reference points for the detection of COVID-19 in children and its possible association with bacterial infections. Children with COVID-19 and neurologic diseases who present with abdominal pain or diarrhea are at risk of bacterial coinfections. Prolonged fever duration and higher PCR test cycle threshold values, WBC levels, and high-sensitivity C-reactive protein (hsCRP) levels may indicate bacterial coinfections in children with COVID-19.

Clinical usefulness of metagenomic next-generation sequencing for Rickettsia and Coxiella burnetii diagnosis

Rickettsia and Coxiella burnetii are zoonotic tick-borne pathogens that cause febrile illnesses in humans. Metagenomic next-generation sequencing (mNGS) is a new technology used to diagnose infectious diseases. However, clinical experience with applying the test to rickettsioses and Q fever is relatively limited. Therefore, this study aimed to explore the diagnostic performance of mNGS in detecting Rickettsia and C. burnetii. We retrospectively studied patients with rickettsioses or Q fever between August 2021 and July 2022. Peripheral blood mNGS and polymerase chain reaction (PCR) were performed for all patients. Clinical data were retrieved for analysis. Thirteen patients were included in this study (eleven confirmed cases and two suspected cases). Signs and symptoms included fever (13, 100%), rash (7, 53.8%), muscle soreness (5, 38.5%), headache (4, 30.8%), skin eschar (3, 23.1%), and disturbance of consciousness (2, 15.4%). In addition, eight patients (61.6%) had thrombocytopenia, ten (76.9%) had liver function impairment, and two (15.4%) had renal function impairment. The results of mNGS revealed seven patients with R. japonica (53.8%), five with C. burneti (38.5%), two with R. heilongjiangensis (15.4%), and one with R. honei (7.7%). PCR results were positive in 11 patients (84.6%). After receiving doxycycline-based treatment, 12 (92.3%) patients returned to a normal temperature within 72 h. All patients were discharged in better health. Therefore, mNGS can help diagnose Rickettsia and C. burnetii and shorten the diagnosis time, especially for patients with atypical clinical manifestations and unclear epidemiologic evidence of a tick bite or exposure.

Utility of Polymerase Chain Reaction Versus Serology-Based Testing in Immunocompromised Patients With Bartonella henselae Infection.

Bartonella henselae serologies can be nondiagnostic in immunocompromised individuals due to impaired humoral immunity. Blood polymerase chain reaction (PCR) is more useful diagnostically in immunosuppressed persons. We discuss 3 cases: 2 solid organ transplant (SOT) recipients and 1 person with human immunodeficiency virus (HIV) with positive blood PCR despite negative serology.

Prolonged Fever in a Child With Congenital Heart Disease and a Prosthetic Valve.

Prolonged Fever in a Child With Congenital Heart Disease and a Prosthetic Valve.

Late-Onset Sepsis Mortality among Preterm Infants: Beyond Time to First Antibiotics.

Objective: To investigate the impact of timing, in vitro activity and appropriateness of empirical antimicrobials on the outcome of late-onset sepsis among preterm very low birth weight infants that are at high risk of developing meningitis. Study design: This retrospective study included 83 LOS episodes in 73 very low birth weight infants born at ≤32 weeks' gestation with positive blood and/or cerebrospinal fluid culture or polymerase chain reaction at >72 h of age. To define the appropriateness of empirical antimicrobials we considered both their in vitro activity and their ideal delivery through the blood-brain barrier when meningitis was confirmed or not ruled out through a lumbar puncture. The primary outcome was sepsis-related mortality. The secondary outcome was the development of brain lesions. Timing, in vitro activity and appropriateness of empirical antimicrobials, were compared between fatal and non-fatal episodes. Uni- and multi-variable analyses were carried out for the primary outcome. Results: Time to antibiotics and in vitro activity of empirical antimicrobials were similar between fatal and non-fatal cases. By contrast, empirical antimicrobials were appropriate in a lower proportion of fatal episodes of late-onset sepsis (4/17, 24%) compared to non-fatal episodes (39/66, 59%). After adjusting for Gram-negative vs. Gram-positive pathogen and for other supportive measures (time to volume administration), inappropriate empirical antimicrobials remained associated with mortality (aOR, 10.3; 95% CI, 1.4-76.8, p = 0.023), while timing to first antibiotics was not (aOR 0.9; 95% CI, 0.7-1.2, p = 0.408; AUC = 0.88). The association between appropriate antimicrobials and brain sequelae was also significant (p = 0.024). Conclusions: The risk of sepsis-related mortality and brain sequelae in preterm very low birth weight infants is significantly associated with the appropriateness (rather than the timing and the in vitro activity) of empirical antimicrobials. Until meningitis is ruled out through lumbar puncture, septic very low birth weight infants at high risk of mortality should receive empiric antimicrobials with high delivery through the blood-brain barrier.

The application of nanopore targeted sequencing in the diagnosis and antimicrobial treatment guidance of bloodstream infection of febrile neutropenia patients with hematologic disease.

Traditional microbiological methodology has limited sensitivity, detection range, and turnaround times in diagnosis of bloodstream infection in Febrile Neutropenia (FN) patients. A more rapid and sensitive detection technology is urgently needed. Here we used the newly developed Nanapore targeted sequencing (NTS) to diagnose the pathogens in blood samples. The diagnostic performance (sensitivity, specificity and turnaround time) of NTS detection of 202 blood samples from FN patients with hematologic disease was evaluated in comparison to blood culture and nested Polymerase Chain Reaction (PCR) followed by sanger sequence. The impact of NTS results on antibiotic treatment modification, the effectivity and mortality of the patients under the guidance of NTS results were assessed. The data showed that NTS had clinical sensitivity of 92.11%, clinical specificity of 78.41% compared with the blood culture and PCR combination. Importantly, the turnaround time for NTS was <24 h for all specimens, and the pre-report time within 6 h in emergency cases was possible in clinical practice. Among 118 NTS positive patients, 98.3% patients' antibiotic regimens were guided according to NTS results. There was no significant difference in effectivity and mortality rate between Antibiotic regimen switched according to NTS group and Antibiotic regimen covering pathogens detected by NTS group. Therefore, NTS could yield a higher sensitivity, specificity and shorter turnaround time for broad-spectrum pathogens identification in blood samples detection compared with traditional tests. It's also a good guidance in clinical targeted antibiotic treatment for FN patients with hematologic disease, thereby emerging as a promising technology for detecting infectious disease.

Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

Infecção experimental por Trypanosoma vivax em caprinos no semiárido brasileiro: detecção de DNA de Trypanosoma vivax no colostro e avaliação da transmissão lactogênica

ABSTRACT: This study aimed to identify the presence of Trypanosoma vivax DNA in the colostrum of infected goats and to explore the possibility of transmission for neonates fed using colostrum collected from infected goats. We used twelve goats in the final third of gestation with an age of approximately 24 months. Six goats were inoculated intravenously with 0.5mL of blood containing approximately 1.25x105 trypomastigotes of T. vivax, and six remained uninfected. The presence of T. vivax in colostrum was evaluated by Polymerase Chain Reaction (PCR). The possibility of T. vivax transmission by colostrum was assessed by feeding six neonates born of serologically negative goats using colostrum from infected goats. Peripheral blood from neonates was collected daily for thirty days to assess the T. vivax presence through the examination of Giemsa-stained smears of leukocyte layers with the buffy coat technique (BCT) and by PCR. The results of a direct examination of colostrum were negative, but PCR confirmed the presence of T. vivax DNA in all infected goats. Additionally, lactogenic transmission by colostrum was not demonstrated once both BCT and PCR of neonate peripheral blood were negative.