Fluid Polymerase Chain Reaction Research Articles

Paradoxical deterioration during treatment of tuberculous meningitis responded to infliximab but not to steroids

Paradoxical deterioration in patients with tuberculous meningitis is a significant diagnostic and treatment challenge. We present the case of a 55-year-old previously healthy male, human immunodeficiency virus (HIV)-negative, presenting with headache, fever, agitation and slurred speech, who was ultimately diagnosed with tuberculous meningoencephalitis confirmed by cerebrospinal fluid polymerase chain reaction positive for Mycobacterium tuberculosis complex. After initiation of a standard antitubercular therapy combined with glucocorticoids, the patient experienced an initial neurological improvement. However, after 3 months of therapy, he had a major stroke with neurological deterioration, clinically and radiologically attributable to a paradoxical deterioration or an immune reconstruction syndrome as described in HIV-positive patients. Due to the persistence of inflammatory symptoms and radiological evidence of worsening tuberculomas despite ongoing antituberculosis therapy, off-label use of the tumour necrosis factor (TNF) inhibitor infliximab was initiated. The patient received three doses of infliximab at 5 mg/kg at monthly intervals, which led to marked clinical and radiological improvement. Follow-up magnetic resonance imaging showed regression of lesions, and the patient was eventually discharged with a significant recovery of motor and speech functions. This case underscores the potential of TNF inhibitors in managing severe cases of paradoxical deterioration in patients with tuberculous meningitis, challenging existing treatment paradigms and suggesting a need for further research into the pathophysiology and treatment of this condition.

Challenges of Congenital HHV6 Infection Diagnosis and Treatment: Two Case Reports and Literature Review

Introduction: Congenital human herpesvirus 6 (HHV6) infection occurs in 1% of the general population and may result from the transmission of an inherited chromosomally integrated HHV6 (iciHHV6) or transplacental infection. It is mostly asymptomatic. Case reports: Case 1: a 29th-week-old female preterm newborn, admitted to the neonatal intensive care unit, became clinically unstable and irritable on the 20th day of hospitalization. Cranial ultrasound, revealed a significant posthemorrhagic tetraventricular dilation, with signs of ventriculitis. Investigations revealed HHV6 positivity on cerebrospinal fluid polymerase chain reaction multiplex panel testing and HHV6-DNA high viral loads in plasma samples. Case 2: a female late preterm newborn was admitted to the neonatal intensive care unit due to early-onset sepsis. Investigations revealed group B streptococcus positive blood cultures and cerebrospinal fluid HHV6 positivity on polymerase chain reaction multiplex panel testing, with negative bacterial culture. After 3 days of adequate antibiotic treatment, she maintained persistent moaning, which motivated a cranial ultrasound, revealing mild brain edema. Clinical improvement was observed only after beginning antiviral treatment in both newborns. Due to the persistency of high viral loads in both cases, despite antiviral treatment and clinical improvement, an iciHHV6 was suspected and posteriorly confirmed. Discussion/conclusion: Congenital iciHHV6 infection diagnosis is challenging because the presence of an iciHHV6 results in persistently high viral loads, even in the absence of active infection. Only a few diagnostic techniques can confirm active replication; unfortunately, these are not available in most countries. The decision to initiate antiviral treatment should be based on clinical judgment. Better ways for the diagnosis of active infection are needed.

Infantile Intraparenchymal Brain Abscess due to Streptococcus pyogenes

Introduction Group A ß-hemolytic streptococcus (GABHS) are the most common bacterial cause of tonsillitis, and can cause noninvasive diseases such as pharyngitis and impetigo, as well as more severe invasive diseases. The incidence of invasive disease is 1-3/100,000 per year, and the morbidity and mortality rate is high. GABHS is rarely lead to brain abscesses. Case Report Acute phase reactants were found to be high in a 40-day-old patient who presented with fever, irritability and focal seizures. Acute phase reactants were high. Transfontanel ultrasonography showed increased thickness, echo and blood supply in the meninges. Brain magnetic resonance imaging (MRI) and diffusion MRI revealed a thick-walled abscess in the right cerebral hemisphere and parietooccipital parenchyma with diffusion restriction on diffusion-weighted images. Cerebrospinal fluid (CSF) polymerase chain reaction (PCR) test revealed S. pyogenes,and S. pyogenes grown in CSF culture. Initially, meropenem, vancomycin, metronidazole antibiotherapy was applied. Treatment was revised to cefotaxime and vancomycin after culture antibiogram and was completed for 8 weeks. Seizures regressed. Control brain imaging showed improvement. Restriction in left lower extremity movements remained. Physical therapy and rehabilitation was recommended and he was discharged. Conclusion GABHS, which is the causative agent of acute tonsillitis, rarely causes invasive disease which has high mortality and morbidity.

Persistence Pays: Diagnosing Tuberculous Meningitis after 11 Negative Polymerase Chain Reaction Evaluations.

Managing tuberculous meningitis (TBM) is challenging because of its poor prognosis and the difficulty in making an early diagnosis due to the low sensitivity of cerebrospinal fluid (CSF) polymerase chain reaction (PCR) evaluations. A 75-year-old woman presented with fatigue and multiple enlarged lymph nodes and was initially suspected of having metastatic cancer of unknown primary origin. Differential diagnoses included carcinomatous meningitis, neurosarcoidosis, and TBM, as suggested by the presence of multiple enhancing cerebral nodules. Despite 11 negative PCR evaluations, including nested PCR of CSF and biopsied lymph nodes within the first 3 days of empirical anti-tubercular treatment, TBM was eventually confirmed by CSF cultures 32 days later. This case highlights the need for repeated sampling.

Enteroviral meningitis in children: Clinical and laboratory features with and without pleocytosis.

Enteroviruses (EV) are most common causes of the etiologically known aseptic meningitis in children. EV can be detected with polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) samples. We aimed to evaluate the clinical and laboratory characteristics of children diagnosed with PCR-confirmed EV meningitis in CSF samples. Patients aged 1 month to 17 years who underwent lumbar puncture (LP) with suspected meningitis and had CSF viral PCR and culture results between September 2012 and January 2021 at a tertiary care hospital in Turkey were included. Patients with no virus detected in CSF samples by PCR was comprised PCR-negative group. The EV PCR-positive patients were divided into two groups based on CSF pleocytosis as enteroviral meningitis (EVM) with CSF pleocytosis and EVM without CSF pleocytosis, and compared in terms of clinical and laboratory features. 78 (38.2%) were EV PCR-positive, and 126 (61.8%) were PCR-negative. Pleocytosis was detected in 55 (70.5%) EV PCR-positive patients and 94 (74.6%) of PCR-negative patients. Convulsion was significantly frequent (p=0.017) in EV PCR-positive patients with no pleocytosis. Protein and lactate concentrations in CSF were significantly higher in EV PCR-positive patients with pleocytosis (p=0.048, p=0.001, respectively). Median hospital stay was significantly longer in PCR-negative group (p<0.001). Diagnosing EVM with PCR prevents long-term hospitalization, unnecessary antibiotic use and healthcare-related complications.

A case of a cerebellar form of progressive multifocal leukoencephalopathy in a patient undergoing peritoneal dialysis.

Progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system, is caused by the reactivation of the polyomavirus JC virus (JCV). It favors the cerebrum and typically occurs in patients with immunodeficiencies, with a progressive course and fatal outcome in the majority of cases. However, the cerebellar form of PML, characterized by isolated posterior fossa lesions, such as those in the cerebellum or brainstem at disease onset, is rare, and reports of its occurrence in peritoneal dialysis (PD) patients are lacking. In this paper, we describe a rare case of a cerebellar form of PML in a PD patient. A 64-year-old man undergoing PD was referred to our hospital for anorexia, nausea, and vomiting in the past month. He had finger-to-nose test abnormalities, gaze-directed nystagmus, and scanning speech. He was diagnosed with the cerebellar form of PML based on his progressive cerebellar symptoms, the typical magnetic resonance imaging findings, and the presence of JCV-DNA in the cerebrospinal fluid polymerase chain reaction test. He developed nocturnal delirium, aggravated disquiet, and died of pneumonia on the 69th day. Clinicians should consider the cerebellar form of PML as a differential diagnosis if PD patients develop progressive cerebellar symptoms.

Changes in respiratory infection trends during the COVID-19 pandemic in patients with haematologic malignancy

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has changed respiratory infection patterns globally. However, its impact on community-acquired pneumonia (CAP) in high-risk patients with haematological malignancies (HM) is uncertain. We aimed to examine how community-acquired pneumonia aetiology in patients with haematological malignancies changed during the COVID-19 pandemic.MethodsThis was a retrospective study that included 524 patients with haematological malignancies hospitalised with community-acquired pneumonia between March 2018 and February 2022. Patients who underwent bronchoscopy within 24 h of admission to identify community-acquired pneumonia aetiology were included. Data on patient characteristics, laboratory findings, and results of bronchioalveolar lavage fluid cultures and polymerase chain reaction tests were analysed and compared to identify changes and in-hospital mortality risk factors.ResultsPatients were divided into the ‘pre-COVID-19 era’ (44.5%) and ‘COVID-19 era’ (55.5%) groups. The incidence of viral community-acquired pneumonia significantly decreased in the COVID-19 era, particularly for influenza A, parainfluenza, adenovirus, and rhinovirus (pre-COVID-19 era vs. COVID-19 era: 3.0% vs. 0.3%, P = 0.036; 6.5% vs. 0.7%, P = 0.001; 5.6% vs. 1.4%, P = 0.015; and 9.5% vs. 1.7%, P < 0.001, respectively), whereas that of bacterial, fungal, and unknown community-acquired pneumonia aetiologies remain unchanged. Higher Sequential Organ Failure Assessment scores and lower platelet counts correlated with in-hospital mortality after adjusting for potential confounding factors.ConclusionsIn the COVID-19 era, the incidence of community-acquired pneumonia with viral aetiologies markedly decreased among patients with haematological malignancies, with no changes in the incidence of bacterial and fungal pneumonia. Further studies are required to evaluate the impact of COVID-19 on the prognosis of patients with haematological malignancies and community-acquired pneumonia.

Listeria monocytogenes: a rare cause of rhomboencephalitis in an immunocompetent patient.

We present an unusual case of Listeria monocytogenes rhomboencephalitis in a young, healthy patient. Although L. monocytogenes meningitis is usually associated with immunodeficiency, rhomboencephalitis is more commonly seen in immunocompetent patients. The wide differential for rhomboencephalitis can create a diagnostic challenge. Without prompt pathogen identification and appropriate antibiotic regimen, L. monocytogenes central nervous system infections can be fatal. Cerebro-Spinal Fluid (CSF) Polymerase Chain Reaction (PCR) aided a prompt diagnosis and adjustment of therapy to achieve a good patient outcome.

Detection of Perkinsus sp. in Chionista fluctifraga cultivated in the southeast Gulf of California

Objective. To determine the presence of Perkinsus sp. in the sand clam Chionista fluctifraga cultivated in an intertidal zone of the southeastern Gulf of California, using Ray's thioglycollate fluid medium staining (MFTR) and polymerase chain reaction (PCR). Material and Methods. The prevalence and parasite load of Perkinsus sp. were monthly obtained and correlated with environmental water conditions and clam biometric indicators (n = 540), from May 2018 to September 2019. Results. Presumptive hypnospores of the protozoan were detected in eight months of culture. The prevalence fluctuated from 3.3% to 13.3%, while the average parasite load ranged from 2 to 1286 hypnospores/g of tissue. The intensity of infection varied from negative to light. The prevalence and parasite load were correlated with each other (r=0.61, P˂0.05) but not with the environmental parameters, nor with the biometric indicators of the clam. The PCR test was negative for the MFTR positive cases. Conclusions. Presumptive hypnospores of Perkinsus sp. were detected in C. fluctifraga without clear indications of infection that compromised the clam health in culture. The constant monitoring of Perkinsus sp. in C. fluctifraga is highly recommended to establish possible infections.

Clinical Spectrum and Epidemiology of Human Parechovirus Infections in Infants: A Retrospective Study in the Western Part of Sweden.

Human parechovirus (HPeV) infections can cause sepsis and meningoencephalitis in infants. To improve our knowledge of the consequences of HPeV infections in young children, the incidence, clinical spectrum, and short-term outcome among infants infected with HPeV were investigated retrospectively. The presence of HPeV RNA was investigated by polymerase chain reaction in cerebrospinal fluid from 327 children aged 0 to 12 months sampled between 2014 and 2017. Eighty-one were infected with HPeV and included in the study. These infants were divided into 3 groups based on clinical assessment: HPeV was the presumed cause of disease (n = 35); HPeV could have contributed to or been considered the cause of disease (n = 24); and HPeV was not considered the cause of disease (n = 22). Infection with HPeV type 3 was common in all groups (n = 54), and most children were younger than 3 months (n = 63). The children in the first group (HPeV as presumed cause) had meningoencephalitis (n = 20), viral sepsis (n = 9), or non-severe viral infection (n = 6). The youngest were more prone to develop meningoencephalitis, while the slightly older children had symptoms of viral sepsis or nonsevere viral infection (P < .05). Eleven had symptom onset within 2 days after birth. Two infants diagnosed with sudden infant death syndrome were HPeV infected when tested postmortem. HPeV infections were identified in 25% of children with suspected central nervous system infection. The clinical presentation of those infected with HPeV varied with age. HPeV infections may be associated with sudden infant death syndrome, although this is not well studied. The results suggest that HPeV infections may be underdiagnosed in young infants.

Whole-Blood PCR Preferred for Timely Diagnosis of Neuroinvasive West Nile Virus Infections: Lessons From the 2021 Arizona Outbreak.

In 2021, the state of Arizona experienced the largest focal outbreak of West Nile virus (WNV) in US history. Timely and accurate diagnostic testing remains a challenge for WNV due to transient viremia and limited immunoassay specificity. Recent studies have identified whole blood (WB) and urine as more sensitive specimen types for the detection of WNV RNA. We evaluated ordering practices, test performance, and patient characteristics of probable and confirmed cases. In total, we identified 190 probable and proven cases, including 127 patients (66.8%) with neuroinvasive disease. Among all cases, only 29.5% had WNV polymerase chain reaction (PCR) testing ordered on WB, of which 80.3% resulted as positive, including 7 cases in which WNV serologic testing was negative and 5 cases for which serologic testing was not ordered. In comparison, only 23.7% of cases that had cerebrospinal fluid (CSF) PCR ordered had a positive result, including 3 cases that were negative by PCR on WB. In contrast, WNV PCR on WB detected 12 neuroinvasive cases that were CSF PCR negative. WNV PCR testing in urine was only ordered on 2 patients, both of whom were positive. Crossing cycle threshold (Ct) values were not significantly different between WB and CSF specimen types, nor was there a correlation between Ct value and days from symptom onset at the time of sample collection; all specimen types and time points had Ct values, with 98% above 30. WB was positive by WNV PCR in several patients for >7 days (range, 7-25 days) after symptom onset, as was the CSF PCR. Taken together, these findings indicate that WNV PCR testing on WB may be the best initial test for timely diagnosis of WNV infection, irrespective of clinical manifestation; however, if negative in patients with suspected neuroinvasive disease, WNV PCR testing on CSF should be ordered.

OUTCOMES OF INFECTIOUS PANUVEITIS ASSOCIATED WITH SIMULTANEOUS MULTI-POSITIVE OCULAR FLUID POLYMERASE CHAIN REACTION.

To evaluate features of infectious panuveitis associated with multiple pathogens detected by ocular fluid sampling. Single-center, retrospective, consecutive case series of patients with aqueous/vitreous polymerase chain reaction testing with >1 positive result in a single sample from 2001 to 2021. Of 1,588 polymerase chain reaction samples, 28 (1.76%) were positive for two pathogens. Most common pathogens were cytomegalovirus (n = 16, 57.1%) and Epstein-Barr virus (n = 13, 46.4%), followed by varicella zoster virus (n = 8, 28.6%), Toxoplasma gondii (n = 6, 21.4%), herpes simplex virus 2 (n = 6, 21.4%), herpes simplex virus 1 (n = 6, 21.4%), and Toxocara (n = 1, 3.6%). Mean initial and final visual acuity (logarithm of the minimum angle of resolution) were 1.3 ± 0.9 (Snellen ∼20/400) and 1.3 ± 1.1 (Snellen ∼20/400), respectively. Cytomegalovirus-positive eyes (n = 16, 61.5%) had a mean final visual acuity of 0.94 ± 1.1 (Snellen ∼20/175), whereas cytomegalovirus-negative eyes (n = 10, 38%) had a final visual acuity of 1.82 ± 1.0 (Snellen ∼20/1,320) ( P < 0.05). Main clinical features included intraocular inflammation (100%), retinal whitening (84.6%), immunosuppression (65.4%), retinal hemorrhage (38.5%), and retinal detachment (34.6%). Cytomegalovirus or Epstein-Barr virus were common unique pathogens identified in multi-PCR-positive samples. Most patients with co-infection were immunosuppressed with a high rate of retinal detachment and poor final visual acuity. Cytomegalovirus-positive eyes had better visual outcomes compared with cytomegalovirus-negative eyes.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing.

The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.

Psychiatric Manifestations Caused by Mycoplasma pneumoniae Encephalitis Mimicking Autoimmune Encephalitis

AbstractA significant etiological factor for upper respiratory tract infections and community-acquired pneumonia is Mycoplasma pneumoniae. The incidence of extrapulmonary neurological problems in infected patients has been shown to range from 0.1 to 7%, often manifesting within a timeframe of 2 to 14 days following the onset of respiratory symptoms. Acute disseminated encephalomyelitis, Guillain–Barré syndrome, and transverse myelitis are among the immune-mediated illnesses encompassed under the syndrome. A 3-year-old male child exhibited symptoms of acute encephalopathy and behavioral disruption subsequent to an infection caused by M. pneumoniae. He presented with irritability, sleep disturbance, slurred speech, increased appetite, episodes of unresponsiveness, moving in circles, staring, and laughing episodes lasting for up to 15 to 30 minutes over a week. He lost his previous toilet training. Abnormal jerks were noted while awake and asleep. Symptoms were preceded by exposure to vague febrile illness 3 weeks prior to presentation. The patient's brain magnetic resonance imaging was normal. Electroencephalography showed a slow background with no epileptiform discharges. Cerebrospinal fluid analysis and polymerase chain reaction for viruses were negative. The workup for autoimmune encephalitis was negative. Mycoplasma serology IgM was detected. Marked improvement was noted after methylprednisolone pulse therapy, intravenous immunoglobulin, valproic acid, and azithromycin. In conclusion, our report serves as a reminder that M. pneumoniae infection is a possible cause of encephalopathy and behavioral disturbance in children. Early recognition and promotion of immunomodulatory and antimicrobial treatment can prevent the affected child from experiencing different levels of long-lasting impairments in cognitive, physical, or visual abilities.

Sepsis-like HPeV encephalitis in young infants – case series

Human parechovirus is the most common underrecognised aetiological agent of encephalitis in children younger than 90 days. The aim of this study is to describe the clinical manifestation, laboratory features, and potential risk factors for severe parechovirus-related sepsis-like encephalitis. The case series included five infants aged below three months diagnosed with sepsis-like parechovirus encephalitis. The diagnosis was based on the patients’ neurological symptoms, the criteria of sepsis defined by the National Institute for Health and Care Excellence, and parechovirus-positive cerebrospinal fluid polymerase chain reaction (PCR). The majority of patients presented with irritability, fever, abnormal muscle tone, mottling, decreased activity, tachycardia, abdominal distension, and absence of neonatal developmental reflexes. No abnormalities in the cerebrospinal fluid were identified. Abnormal blood laboratory measurements included lymphopaenia, leukopaenia, and anaemia. The case series highlights the necessity to perform cerebrospinal fluid PCR for parechovirus in infants with sepsis-like illness and neurological signs but without cerebrospinal fluid signs of inflammation.

1324. Impact of Implementation of CSF PCR Panel in Patients with Meningitis

Abstract Background Cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) panel is routinely used for etiologic diagnosis of meningitis. While its role in this setting has been proven, there is no data on its benefits on duration of antibiotic use and hospital length of stay. Methods Retrospective cohort study was performed to compare meningitis patients who received the CSF PCR panel versus those who did not in a large database of HCA hospitals across the United States. Data was collected from January 2014 to May 2022 on all patients diagnosed with meningitis with a CSF WBC greater than 10. Using logistic regression and negative binomial regression, the study compared utility of CSF PCR panel use versus non-use with regards to length of stay, antibiotic duration, resolution of fever and leukocytosis, and hospital readmission rate. Demographics by Panel Use A total of 4895 patients were included in the study, of which 379 received the CSF PCR Meningitis Panel. Results A total of 4895 distinct patient encounters were included, of whom 379 patients had the CSF PCR panel. CSF PCR use was associated with a significant decrease in readmission rates within 30 days (x2 5.64, p&amp;lt; 0.05) and had decreased log count days of antibiotic duration (x2 17.56, p&amp;lt; 0.0001) with a mean duration of antibiotic use of 73.91 hours in CSF PCR panel patients versus 82.05 hours. Panel use was not significantly associated with length of stay (x2 0.96, p 0.328). The likelihood of fever resolution and leukocytosis resolution was the same for those who received the CSF PCR Panel versus those who did not. Antimicrobial Medications Received by Panel Use Antimicrobial Duration and Clinical Presentation by Panel Use There was a decrease in antibiotic use of 73.91 hours in CSF PCR panel patients versus 82.05 hours. There was also a significant decrease in hospital readmission rates within 30 days Conclusion CSF PCR panel benefits included reduced antibiotic duration of use and reduced 30-day readmission rates. Fever resolution and leukocytosis or leukopenia resolution was not impacted by CSF PCR panel use. Disclosures All Authors: No reported disclosures

Ocular manifestations and diagnosis of tuberculosis involving the uvea: a case series

BackgroundOcular tuberculosis (TB) affects 1–2% of patients with TB, with TB uveitis being the most common. This series aims to look at different manifestations of tuberculosis associated uveitis and the different tests used to make a presumptive or definitive diagnosis.MethodsPatients diagnosed with TB related uveitis in Hong Kong SAR between 2017 and 2020 were reviewed. Demographics, clinical features, investigations and treatments of patients were collected.ResultsFifteen eyes in 10 patients with a mean age 57.30 ± 10.17 years were included. The ocular manifestations on presentation included anterior uveitis (50%), posterior uveitis (40%) and panuveitis (10%), where 70% of them were unilateral and 30% were bilaterally infected; on subsequent visits the manifestations further developed into posterior uveitis (40%), panuveitis (40%) and anterior uveitis (20%), where 50% of them were unilateral and 50% bilateral infected. Tuberculosis tests were positive in 5 out of 7 Mantoux tests, 4 out of 4 T-SPOT TB tests, 3 out of 4 QuantiFERON-TB gold tests, 1 out of 1 lymph node biopsy, 0 out of 9 chest x-rays, and no aqueous fluid polymerase chain reaction (PCR) was tested. Vision impairing complications were seen in 6 patients where retinal vasculitis was most commonly seen. With anti-TB treatment prescribed in 9 patients, side effects occurred in 5 patients, including ocular hypertension, disc swelling, and hepatitis.ConclusionsOcular TB infections may manifest in various forms, and can involve different parts of the eye. Bilateral involvement of TB is commonly presented, and both eyes should be evaluated at every follow up. When TB is suspected in a patient, diagnostic confirmation requires multimodal investigations where a negative chest x-ray is not useful in ruling out ocular TB infections, especially in an endemic region like Hong Kong. In these patients, it is crucial to have a high index of suspicion for TB, even when they do not demonstrate classical respiratory signs and symptoms of TB.

A-308 The Molecular Diagnosis of Varicella Zoster Virus (VZV): A Validation of a Real-Time PCR (qPCR) Assay for the Qualitative Detection

Abstract Background Varicella zoster virus (VZV) is the etiological agent of chicken pox and herpes zoster. After the primary infection, which usually occurs in childhood, VZV remains latent in sensory nerve roots for life. When reactivation occurs, especially in immunocompromised patients, the virus migrates to the skin and causes painful rashes that can evolute to a dermatome. Even after the end of the viral acute infection, some individuals may experience continued pain. Furthermore, VZV reactivation can lead to a wide spectrum of clinical manifestations such as self-limited radicular pain and central nervous system involvement. During acute illness, 90% of patients experience pain and 12% have symptoms similar to influenza infection (influenza-like). Immunocompromised patients may have an unfavorable course with disseminated infection, pneumonitis, hepatitis, and encephalitis. The cerebrospinal fluid polymerase chain reaction (PCR) test is indicated for suspected involvement of the central nervous system and complications of ocular or periocular lesions. Compared to other diagnostic methods, real-time PCR has great advantages as its high throughput, efficiency, and sensitivity. Therefore, the detection of VZV by real-time PCR presents great importance for the diagnosis and monitoring of the disease due to its precision and agility. Thus, we intended to validate a qualitative method for VZV detection by real-time PCR. Methods We evaluated the performance of the inventoried TaqMan assay (Vi06439647_s1) (Thermo Fisher Scientific). For this validation, we used 64 samples - whole blood (n = 31), cerebrospinal fluid (n = 32), vaginal lesion scraping (n = 1) - from the Genetics sector of the Pardini Group, previously tested by in-house PCR for the qualitative detection of VZV. Samples were extracted using the MagNA Pure 24 System platform (Roche Diagnostics) with the MagNA Pure 24 Total NA Isolation Kit, according to the manufacturer's instructions. All assays were performed using Taqman Universal Master Mix (Thermo Fisher Scientific). The inventoried B-actin Assay (assay ID: Hs01060665_g1) (Thermo Fisher Scientific) was used as endogenous control. Assay verification tests, evaluation of reaction efficiency, and determination of the detection limit (LOD) were performed using a synthetic control of known concentrations and a pool of negative samples (healthy volunteers). At the end of the tests, were made a comparison of the previous results with the results obtained in the ThermoFisher Real-Time PCR Assay. Results The Inventoriad Taqman Assay (VZV) had a limit of detection of 10 copies/µL and the Cq &amp;gt; 35 was defined as a cut-off to consider a sample as positive. Whole blood samples had higher Cts than cerebrospinal fluid samples, indicating lower viremia. Whitin all evaluated samples, 11 samples had positive results detected by in-house PCR (9 from whole blood and 2 from cerebrospinal fluid). Of these, 10 had a result detected in Real-time PCR and the agreement with the positive results between compared methodologies was 91.0%. The divergent sample was whole blood and can be related to DNA degradation. The 47 negative samples evaluated were concordant in both methods. Conclusion The inventoried TaqMan Assay with Taqman Universal Master Mix protocol evaluated was sensitive, fast, and applicable as a tool for the qualitative detection of VZV.

Polymicrobial community-acquired pneumonia: Coinfection with tuberculosis, Klebsiella and Staphylococcus

Abstract A 71-year-old male a chronic smoker, who also had diabetes mellitus and hypertension presented with complaints of fever, cough with foul-smelling sputum and dyspnoea for 25 days. Coarse crepitations were evident in the interscapular area, infrascapular, axillary, infra-axillary and mammary areas on the left side. Contrast-enhanced computed tomography of the chest showed necrotic mediastinal lymph nodes and areas of consolidation in the left upper lobe. Bronchoalveolar lavage fluid polymerase chain reaction identified Staphylococcus, New Delhi metallo-β-lactamase (NDM) producing Klebsiella; GeneXpert detected Mycobacterium tuberculosis complex. Pleural fluid analysis was suggestive of exudative effusion with elevated adenosine deaminase. The patient was managed with anti-tuberculosis treatment, ceftazidime-avibactam, aztreonam and linezolid.

Role of fungal burden in risk stratification of HIV-negative patients with Pneumocystis pneumonia: A 12-year, retrospective, observational, multicenter cohort

ObjectivesThis study aimed to explore the role of fungal burden in risk stratification of patients without HIV-negative patients with Pneumocystis pneumonia (PCP). MethodsThis was a retrospective analysis of the characteristics associated with 30-day mortality in patients who were positive for P. jirovecii using polymerase chain reaction in bronchoalveolar lavage fluid between 2006 and 2017 in a multicenter cohort from Central Norway. The fungal burden was indicated by the cycle threshold (CT) values from semiquantitative real-time polymerase chain reaction targeting the β-tubulin gene. ResultsWe included 170 patients with proven or probable PCP. The all-cause 30-day mortality was 18.2%. After adjusting for host characteristics and premorbid corticosteroid use, a higher fungal burden was associated with a higher risk of dying: adjusted odds ratio 1.42 (95% confidence interval 0.48-4.25) for a CT value 31-36, increasing to odds ratio 5.43 (95% confidence interval 1.48-19.9) for a CT value ≤30 compared with patients with a CT value ≥37. The Charlson comorbidity index (CCI) improved the risk stratification: patients with a CT value ≥37 and CCI ≤2 had a 9% mortality risk compared with 70% among those with a CT value ≤30 and CCI ≥6. Comorbid cardiovascular disease, solid tumors, immunological disorders, premorbid corticosteroids, hypoxemia, abnormal leukocyte counts, low serum albumin, and C-reactive protein ≥100 were also independently associated with 30-day mortality. The sensitivity analyses did not suggest selection bias. ConclusionFungal burden may improve the risk stratification of patients without HIV-negative patients with PCP.