Polymerase Chain Reaction-based Diagnosis Research Articles

Role of Polymerase Chain Reaction-Based Diagnosis of Respiratory Viruses in Febrile Neutropenic Patients.

Background Neutropenic patients are commonly affected by respiratory infections, whereas respiratory viral infections causing high morbidity and mortality are routinely diagnosed in developing countries like India. Our study aimed to investigate the prevalence of respiratory viral infections in pediatric cancer patients with febrile neutropenia. Methods This prospective study was performed on 45 neutropenia patients with hematological malignancies. Nasal swabs were collected and analyzed by real-time multiplex polymerase chain reaction (PCR), covering the following viruses: influenza A virus, influenza B virus, human parainfluenza virus (subtypes 1-4), human respiratory syncytial virus A and B, enterovirus, human-coronavirus (HCoV: HKU1, NL63, 229E, and OC43), human bocavirus, adenovirus, human rhinovirus, human-metapneumovirus A and B, human paraechovirus, and a bacteriumMycoplasma pneumoniae. Patients enrolled in the study since the COVID-19 pandemic was also detected for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results Of the 45 cases included in our study, 26 cases showed the presence of at least one positivity by PCR (57.7%): 23 patients had monoinfection with only one virus, two patients were found positive for coinfection with two viruses, and one patient was found positive for three viruses. The most detected viruses were human rhinovirus (26.9%, n=7) and coronavirus 19 (19.2%, n=5). A total of 11.5% of the patients had multiple viral infections. About 19 (42.2%) of the patients enrolled in our study had no viral pathogen detected. Conclusion We found that respiratory viruses contribute significantly to the development of neutropenic fever, as evidenced by the results of our prospective study. Individualizing infection treatment can reduce antibiotic use in immunocompromised patients. Thus, routine screening for viremia may be warranted in this clinical setting.

Brain microstructural changes and fatigue after COVID-19.

Fatigue and cognitive complaints are the most frequent persistent symptoms in patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to assess fatigue and neuropsychological performance and investigate changes in the thickness and volume of gray matter (GM) and microstructural abnormalities in the white matter (WM) in a group of patients with mild-to-moderate coronavirus disease 2019 (COVID-19). We studied 56 COVID-19 patients and 37 matched controls using magnetic resonance imaging (MRI). Cognition was assessed using Montreal Cognitive Assessment and Cambridge Neuropsychological Test Automated Battery, and fatigue was assessed using Chalder Fatigue Scale (CFQ-11). T1-weighted MRI was used to assess GM thickness and volume. Fiber-specific apparent fiber density (FD), free water index, and diffusion tensor imaging data were extracted using diffusion-weighted MRI (d-MRI). d-MRI data were correlated with clinical and cognitive measures using partial correlations and general linear modeling. COVID-19 patients had mild-to-moderate acute illness (95% non-hospitalized). The average period between real-time quantitative reverse transcription polymerase chain reaction-based diagnosis and clinical/MRI assessments was 93.3 (±26.4) days. The COVID-19 group had higher total CFQ-11 scores than the control group (p < 0.001). There were no differences in neuropsychological performance between groups. The COVID-19 group had lower FD in the association, projection, and commissural tracts, but no change in GM. The corona radiata, corticospinal tract, corpus callosum, arcuate fasciculus, cingulate, fornix, inferior fronto-occipital fasciculus, inferior longitudinal fasciculus, superior longitudinal fasciculus, and uncinate fasciculus were involved. CFQ-11 scores, performance in reaction time, and visual memory tests correlated with microstructural changes in patients with COVID-19. Quantitative d-MRI detected changes in the WM microstructure of patients recovering from COVID-19. This study suggests a possible brain substrate underlying the symptoms caused by SARS-CoV-2 during medium- to long-term recovery.

Incidence of Pneumocystis jirovecii pneumonia utilizing a polymerase chain reaction-based diagnosis in patients receiving bendamustine.

Background Pneumocystis jirovecii pneumonia (PJP) is a life‐threatening infection occurring in patients receiving bendamustine. The poorly defined incidence, particularly when utilizing polymerase chain reaction (PCR)‐based diagnostic techniques, precipitates unclear prophylaxis recommendations. Our objective was to determine the cumulative incidence of PJP diagnosed by single copy target, non‐nested PCR in patients receiving bendamustine.MethodsPatients were evaluated for PJP from initiation of bendamustine through 9 months after the last administration. The cumulative incidence of PJP was estimated using the Aalen–Johansen method. Cox proportional hazard models were used to demonstrate the strength of association between the independent variables and PJP risk.ResultsThis single‐center, retrospective cohort included 486 adult patients receiving bendamustine from 1 January 2006 through 1 August 2019. Most patients received bendamustine‐based combination therapy (n = 461, 94.9%), and 225 (46.3%) patients completed six cycles. Rituximab was the most common concurrent agent (n = 431, 88.7%). The cumulative incidence of PJP was 1.7% (95% CI 0.8%–3.3%, at maximum follow‐up of 2.5 years), after the start of bendamustine (n = 8 PJP events overall). Prior stem cell transplant, prior chemotherapy within 1 year of bendamustine, and lack of concurrent chemotherapy were associated with the development of PJP in univariate analyses. Anti‐Pneumocystis prophylaxis was not significantly associated with a reduction in PJP compared to no prophylaxis (HR 0.37, 95% CI (0.05, 3.04), p = 0.36).ConclusionsOur incidence of PJP below 3.5%, the conventional threshold for prophylaxis implementation, indicates routine anti‐Pneumocystis prophylaxis may not be necessary in this population. Factors indicating a high‐risk population for targeted prophylaxis require further investigation.

An evaluation of patients with a previous endemic coronavirus infection during the COVID-19 pandemic.

Few studies exist on the clinical manifestation of coronavirus disease 2019 (COVID‐19) in patients who previously had a common cold due to an endemic coronavirus (eCoV). In a retrospective scan of the data obtained in our microbiology laboratory, 64 patients who were diagnosed with an eCoV infection between 2016 and 2020 were identified. National COVID‐19 surveillance data showed that four (6.2%) of 64 patients were infected with severe acute respiratory syndrome coronavirus 2 by the end of 2020, while, simultaneously, the COVID‐19 prevalence in the city of Malatya ranged from 7.8% (polymerase chain reaction‐based diagnosis) to 9.2% (total diagnosis). The differences were found statistically significant (6.2% vs. 7.8%, p < .01; 6.2% vs. 9.2%, p < .001). Patient interviews and evaluation of medical records revealed that these four patients did not manifest any severe COVID‐19 symptoms despite their substantial comorbidities, and they did not require hospitalization. Consequently, despite a low number of samples, we determined a lower frequency of COVID‐19 among the patients who had a prior eCoV infection, and the results of this study support the previous findings that people with a prior eCoV infection develop a milder case of COVID‐19. Our results may provide some insights for future studies aiming at vaccine development, but detailed investigations are still required.

Clinical Recommendations of the Russian Gastroenterological Association on Diagnosis and Treatment of Infectious Esophagitis

Aim. Current clinical recommendations aim to provide gastroenterologists, general practitioners (family doctors), endoscopists and infectionists with modern methods for diagnosis and treatment of infectious esophagitis.Key points. Infectious esophagitis is an esophageal disease of fungal, viral, bacterial or parasitic origin. Esophagus may be affected per se or conjointly in a common gastrointestinal infection. All patients suspected for infectious esophagitis, unless contraindicated, are advised an esophagogastroduodenoscopy with biopsy to confirm diagnosis. Selected incidents of infectious esophagitis require a histological, immunohistochemical examination or polymerase chain reaction-based diagnosis to verify the infectious agent. Uncomplicated infectious esophagitis requires a conservative therapy, mostly on an outpatient basis. However, cases of severe odynophagia, dysphagia, severe pain syndrome, high complication risks (e.g., esophageal ulcer bleeding in thrombocytopenia), severe immunodeficiency, generalised forms of disease and severe concomitant disorders are considered for hospital care. The clinical recommendations outline criteria for the medical care quality assessment and provide relevant information to the patient.Conclusion. Diagnosis of infectious esophagitis capitalises on the clinical picture (odynophagia, dysphagia), presence of immunosuppression, endoscopic and histological evidence. All patients with infectious esophagitis of verified origin are recommended a suitable etiotropic therapy.

Incidence, clinical presentation, and outcomes of Pneumocystis pneumonia when utilizing Polymerase Chain Reaction-based diagnosis in patients with Hodgkin lymphoma

A Polymerase Chain Reaction-based diagnosis of Pneumocystis Pneumonia (PCP) and the need for anti-Pneumocystis prophylaxis in Hodgkin lymphoma patients receiving chemotherapy requires further investigation. This retrospective, single-center, study evaluated 506 consecutive adult patients diagnosed with Hodgkin lymphoma receiving chemotherapy between January 2006 and August 2018. The cumulative incidence of PCP 1 year after start of chemotherapy was 6.2% (95% CI 3.8–8.5%). Mortality 30 days from PCP diagnosis was 8% (n = 2) with one death attributable to PCP. Bleomycin-containing combination chemotherapy regimen was not significantly associated with a higher risk for PCP when compared to other regimens (HR = 1.59, 95% CI 0.55–4.62 p = 0.40). Anti-Pneumocystis prophylaxis was not significantly associated with a decreased incidence of PCP (HR = 0.51, 95% CI 0.15–1.71, p = 0.28). As the overall incidence is above the commonly accepted 3.5% threshold, clinicians should consider the potential value of prophylaxis. The utility of universal vs. targeted anti-Pneumocystis prophylaxis requires prospective, randomized investigation.

"Proposals for Amendments in the Diagnosis and Treatment of Encephalitis caused by Free-living Amoebae".

Encephalitis caused by Free-living amoebae (FLA) has a mortality rate of around 95- 98%, a fraction that has not changed in the past decades. Pathogenic FLA include Acanthamoeba, Balamuthia mandrillaris, and Naegleria fowleri that are known to target the brain after an extra cerebral infection in the case of Acanthamoeba and Balamuthia mandrillaris, or directly the brain, as in the case of the Naegleria fowleri. The Acanthamoeba spp. and Balamuthia mandrillaris cause granulomatous amoebic encephalitis (GAE) while Naegleria fowleri, the so termed "brain eating amoeba" causes primary amoebic meningoencephalitis (PAM). The attempts to obtain a speedy diagnosis and an aggressive treatment protocol are the areas where advances can make a difference and reduce the mortality rates. At first, we highlight the reasons behind the diagnostic delays and treatment failures and provide proposals to establish a quick diagnosis in both PAM and GAE. Secondly, we emphasize the use of a transcribrial device, and a prompt, but vigilant surgical reduction of the intracranial pressure in these patients which could be life-saving. We also debate that an exudate obtained from the olfactory region by irrigation via a modified transcribrial device or by conventional methods, instead of a cerebrospinal fluid sample, could serve as a source of obtaining amoeba in PAM for a real-time polymerase chain reaction-based definitive diagnosis of PAM. Also, introduced is the rationale that has the potential to deliver the drugs to the brain in patients with PAM and the GAE localized to the frontal lobe of the brain, by bypassing the blood brain barrier. We put forward these proposals for debate and deliberation to our fellow colleagues in order to spot the potential of their application to reduce the mortality rates caused by the rare but fatal encephalitis caused by these FLA.

Adapting Reactive Case Detection Strategies for falciparum Malaria in a Low-Transmission Area in Cambodia.

Reactive case detection around falciparum malaria cases in Cambodia presents a low output. We improved it by including individuals occupationally coexposed with index case patients and using polymerase chain reaction-based diagnosis. The positivity rate increased from 0.16% to 3.9%.

Development of nested polymerase chain reaction-based diagnosis of duck enteritis virus and detection of DNA polymerase gene from non-descriptive duck breeds of West Bengal, India.

Aim:The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP) infected eggs, and histopathological changes of chorioallantoic membrane (CAM) in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR).Materials and Methods:After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell) embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus.Results:Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076) showed homology with the sequences data available in GenBank.Conclusion:The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.

Clinical consequences of polymerase chain reaction-based diagnosis of intestinal parasitic infections.

The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa has led to higher sensitivity and (subtype) specificity, more convenient sampling, and the possibility for high-throughput screening. PCR for routine detection of human intestinal protozoa in fecal samples is used by an increasing number of clinical laboratories. This paper discusses the recent developments in the diagnosis of intestinal protozoa, with an emphasis on PCR-based diagnostics. Although many reviews have described the technical aspects of PCR-based diagnostics, this review focuses on the clinical consequences that result from the shift from microscopic toward PCR-based diagnostics. Despite its undisputed superiority, the use of PCR comes with challenges that clinicians should be aware of.

Comparison of Penile Skin Swab with Intra-Urethral Swab and First Void Urine for Polymerase Chain Reaction-Based Diagnosis of Chlamydia trachomatis Urethritis in Male Patients

Comparison of Penile Skin Swab with Intra-Urethral Swab and First Void Urine for Polymerase Chain Reaction-Based Diagnosis of Chlamydia trachomatis Urethritis in Male Patients

Serodiagnosis of Human Bocavirus Infection

Background. A new human-pathogenic parvovirus, human bocavirus (HBoV), has recently been discovered and associated with respiratory disease in small children. However, many patients have presented with low viral DNA loads, suggesting HBoV persistence and rendering polymerase chain reaction-based diagnosis problematic. Moreover, nothing is known of HBoV immunity. We examined HBoV-specific systemic B cell responses and assessed their diagnostic use in young children with respiratory disease. Patients and methods. Paired serum samples from 117 children with acute wheezing, previously studied for 16 respiratory viruses, were tested by immunoblot assays using 2 recombinant HBoV capsid antigens: the unique part of virus protein 1 and virus protein 2. Results. Virus protein 2 was superior to the unique part of virus protein 1 with respect to immunoreactivity. According to the virus protein 2 assay, 24 (49%) of 49 children who were positive for HBoV according to polymerase chain reaction had immunoglobulin (Ig) M antibodies, 36 (73%) had IgG antibodies, and 29 (59%) exhibited IgM antibodies and/or an increase in IgG antibody level. Of 22 patients with an increase in antibody levels, 20 (91%) had a high load of HBoV DNA in the nasopharynx, supporting the hypothesis that a high HBoV DNA load indicates acute primary infection, whereas a low load seems to be of less clinical significance. In a subgroup of patients who were previously determined to have acute HBoV infection (defined as a high virus load in the nasopharynx, viremia, and absence of other viral infections), 9 (100%) of 9 patients had serological evidence of primary infection. In the control group of 68 children with wheezing who had polymerase chain reaction results negative for HBoV in the nasopharynx, 9 (13%) had IgM antibodies, including 5 who displayed an increase in IgG antibody levels and were viremic. No cross-reactivity with human parvovirus B19 was detected. Conclusions. Respiratory infections due to HBoV are systemic, elicit B cell immune responses, and can be diagnosed serologically. Serological diagnoses correlate with high virus loads in the nasopharynx and with viremia. Serological testing is an accurate tool for disclosing the association of HBoV infection with disease.

Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples.

A nested polymerase chain reaction (PCR) assay has been developed and used in the diagnosis of fatal and benign cases of Mediterranean spotted fever (MSF). The test was based on specific primers derived from a Rickettsia conorii 17-kD protein gene. A positive signal was obtained from spotted fever group (SFG) and typhus group (TG) rickettsiae. Discrimination between SFG and TG rickettsiae was based on a restriction fragment length polymorphism test. Other gram-negative bacterial species tested did not generate a signal, attesting for the specificity of the assay. The SFG-specific DNA fragment was detected in four of 29 acute-phase sera from serologically confirmed patients with MSF, while acute-phase sera from 25 patients without MSF were PCR negative. Acute-phase sera samples (five of five) and tissue autopsies (six of seven) from fatal suspected cases of MSF were PCR positive. The results demonstrate that sera and tissue samples are suitable specimens for the nested PCR tests, especially in fatal cases.

Polymerase chain reaction-based diagnosis of bone marrow involvement in 170 cases of non-Hodgkin lymphoma.

Up to the current time, diagnosis of bone marrow (BM) involvement in non-Hodgkin lymphoma (NHL) has been based on morphologic findings. Polymerase chain reaction (PCR) for antigen receptor gene rearrangements has the potential to increase the detection sensitivity of minimal degrees of BM involvement. The authors therefore assessed PCR-based clonalities of BM concurrently with morphology from 170 cases with NHL and evaluated the usefulness of comparative analysis of clonalities between bilateral BMs and the lymph node and the clinical significance of PCR based clonalities of BM. Bilateral BM clot sections of 170 cases and 47 lymph nodes were tested for immunoglobulin heavy chain gene rearrangement or T-cell receptor gamma gene rearrangement according to the B- or T-lineage of the lymph node. When compared with morphology, the results of PCR showed an unexpectedly low positive concordance rate of 61.0% for B-cell NHL and 57.1% for T-cell NHL. When the clonality of BM was compared with that of lymph nodes in B-cell NHL, bilateral clonalities of BM showed high concordance with the clonality of the lymph nodes. PCR-based clonality did not show significant impact on survival. Morphology remains the gold standard in the evaluation of BM involvement by NHL. Although the comparative analysis of BM clonality and that of the lymph nodes is considered a valuable tool that increases the reliability of clonality, PCR-based clonality of BM does not significantly add to the sensitivity of diagnosing BM involvement by NHL.

New polymerase chain reaction-based diagnostic techniques for bacterial respiratory infection

Polymerase chain reaction protocols are now available for the diagnosis of all of the major bacterial respiratory tract pathogens. Molecular techniques are also being used to determine the susceptibility of Streptococcus pneumoniae and Mycobacterium tuberculosis. In addition, polymerase chain reaction-based diagnosis will find a place in the investigation of the epidemiology of some pathogens such as Bordetella pertussis and Chlamydia pneumoniae. The added sensitivity will allow the detection of mild or asymptomatic cases or carriers who may play a role in sustaining the pathogen in the community.

Long range polymerase chain reaction-based diagnosis of friedreich ataxia in the clinical molecular diagnostics laboratory

Long range polymerase chain reaction-based diagnosis of friedreich ataxia in the clinical molecular diagnostics laboratory

Polymerase Chain Reaction-Based Diagnosis of Del(5q) in Acute Myeloid Leukemia and Myelodysplastic Syndrome Identifies a Minimal Deletion Interval

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.

Polymerase chain reaction-based diagnosis of mollicture infection of commercial animal sera

A total of 72 commercial animal sera were assayed for mollicute infection by a polymerase chain reaction (PCR)-based detection method and by four classical detection methods. The methods included microbiological assay by inoculation onto agar and into broth, DNA staining of an indicator cell line with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) fluorochrome, enzyme-linked immnosorbent assay (ELISA) and adenosine phosphorylase (AdoP) screening. Mollicutes were detected in 2 of the 72 sera assayed. The species isolated was Acholeplasma laidlawii in both cases. When detection methods were compared, PCR, microbiological culture and DNA staining were perfectly concordant. ELISA and AdoP detection produced false-negative results for 1 of the 2 infected sera each. False positive results appeared in 1 of the 70 mollicute-free sera with ELISA and in 16 of the 70 with AdoP detection. It was concluded that the PCR-based detection method is a useful tool in addition to current methods of detection of mollicutes in animal sera, with regard to reliability, sensitivity, specificity and time.

Campylobacter jejuni: Specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis

Campylobacter jejuni: Specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis

Campylobacter jejuni: specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis.

A 1189 base-pair long DNA fragment, VS1, was isolated from a Campylobacter jejuni CIP 70.2 cosmid library and was found to contain regions specific for this bacterial species. For detection and identification of C. jejuni, two oligonucleotides derived from the VS1 sequence were used as primers in polymerase chain reaction tests on genomic DNAs from 38 campylobacter and from 10 non-Campylobacter strains. A specific, 358 base-pair long DNA fragment was amplified only when C. jejuni DNA was used as a target. The detection limit of the amplification reaction was as low as 1.86 fg DNA, which is the equivalent of one C. jejuni genome.