Nanopore sequencing of cerebrospinal fluid of three patients with cryptococcal meningitis

Background Cryptococcal meningitis (CM) has a high morbidity and mortality due to the low detection of Cryptococcus in cerebrospinal fluid (CSF) during the early stage of the disease with traditional methods. Case presentation In addition to the traditional methods of India ink staining and cryptococcal antigen (CrAg), we used nanopore sequencing and next-generation sequencing (NGS) to detect pathogenic DNA in CSF samples of three patients with CM. The CSF samples of all three patients were positive by India ink staining and CrAg. NGS also detected Cryptococcus in all three CSF samples. Nanopore sequencing detected Cryptococcus in two CSF samples. Conclusion Nanopore sequencing may be useful in assisting with the clinical diagnosis of CM. Further research is needed to determine the sensitivity and specificity of nanopore sequencing of CSF.

Utility of nail enamel for inking of surgical margins in oral biopsy: a comparative study

Background: India Ink is the most commonly used ink in surgical pathology. The main disadvantages of India Ink are longer drying time, monochrome, toxicity and cost. Because of these disadvantages, alternative materials have been suggested to replace India ink. The aim of this study is to evaluate the effectiveness of nail enamel for inking of surgical margin and to compare it with India ink. Materials and methods: N = 20, which included 10 mucosal and 10 skin samples. Each selected margin is divided into 2 equal halves and one is inked with India ink and the other with nail enamel (Vernis A Ongles: Dark green). After routine processing and staining, the effectiveness of nail enamel and India ink were compared based on macroscopic and microscopic parameters. Results: Less drying time and visibility on paraffin wax block were excellent for nail enamel. Microscopic visibility of nail enamel was comparable with that of India ink. However, processing fluids contamination is the main drawback of nail enamel. Conclusion: Nail enamel can be used as an alternative to India ink because of its less drying time, ease of application, good visibility on wax blocks and microscopically, availability in multiple colours, cost effectiveness and non-toxicity.

Quantification of Cartilage Surface Degeneration by Curvature Analysis Using 3D Scanning in a Rabbit Model

Objective Accurate analysis to quantify cartilage morphology is critical for evaluating degenerative conditions in osteoarthritis (OA). Three-dimensional (3D) optical scanning provides 3D data for the entire cartilage surface; however, there is no consensus on how to quantify it. Our purpose was to validate a 3D method for evaluating spatiotemporal alterations in degenerative cartilages in a rabbit OA model by analyzing their curvatures at various stages of progression. Design Twelve rabbits underwent anterior cruciate ligament transection (ACLT) unilaterally and were divided into 4 groups: 4 weeks control, 4 weeks OA, 8 weeks control, and 8 weeks OA. 3D scanning, India ink staining, and histological assessments were performed in all groups. In 3D curvature visualization, the surfaces of the condyles were divided into 8 areas. The standard deviations (SD) of mean curvatures from all vertices of condylar surfaces and subareas were calculated. Results Regarding the site of OA change, curvature analysis was consistent with India ink scoring. The SD of mean curvature correlated strongly with the India ink Osteoarthritis Research Society International (OARSI) score. In curvature histograms, the curvature distribution in OA was more scattered than in control. Of the 8 areas, significant OA progression in the posterolateral part of the lateral condyle (L-PL) was observed at 4 weeks. The histology result was consistent with the 3D evaluation in terms of representative section. Conclusions This study demonstrated that 3D scanning with curvature analysis can quantify the severity of cartilage degeneration objectively. Furthermore, the L-PL was found to be the initial area where OA degeneration occurred in the rabbit ACLT model.

Comparison of clinical and biological characteristics of HIV-infected patients presenting Cryptococcus neoformans versus C. curvatus/C. laurentii meningitis

Background Cryptococcal meningitis is mainly caused by Cryptococcus neoformans/C. gattii complex. We compared the clinical, biological, and antifungal susceptibility profiles of isolates from HIV-Infected Patients (HIVIP) with C. neoformans (Cn) versus C. curvatus/C. laurentii (Cc/Cl) meningitis. Methods Comparative analytical study were conducted. Apart from patients’ clinical data, the following analysis were performed and the results were compared in both groups: biochemical examination, cryptococcal antigen test, India ink staining, and culture on Cerebral Spinal Fluid (CSF), strains identification by mass spectrometry, ITS sequencing, PCR serotyping and antifungal susceptibility. The main outcome variable was the “species of Cryptococcus identified”, which was compared to other variables of the same type using the Pearson Chi-square test or the Fisher exact test. Results A total of 23 (79.3%) Cn meningitis cases versus 6 (20.7%) Cc/Cl meningitis were retained.Cn meningitis was more frequently associated with headache (100% vs 50%, p = 0.005) than Cc/Cl meningitis and meningeal signs were more frequent in Cn infected patients. Biologically, hypoglycorrhachia and low CD4 count were more observed in Cn group (90% vs 20% of patients, p = 0.01; 45.6 vs 129.8 cells/µL, p = 0.02, respectively). A higher proportion of Cn strains (91.3%) showed a low Minimum Inhibitory Concentration (MIC) (< 8 mg/L) for fluconazole compared to Cc/Cl strains (66.7%). Also, Cc/Cl strains resistant to 5-flucytosine and amphotericin B were found in 16.7% of cases for each of the two antifungal agents. Cryptococcus detection by routine analysis (India ink, culture, and antigens) was better for Cn samples than Cc/Cl. Except ITS sequencing, which identified all strains of both groups, mass spectrometry and serotyping PCR identified Cn strains better than Cc/Cl (100% vs 80%, p = 0.1; 100% vs 0%, p < 0.0001, respectively). After treatment with amphotericin B, 5-flucytosine, and fluconazole in both groups, the outcome was similar. Conclusions Clinical presentation of Cn meningitis is certainly more severe than that of Cc/Cl meningitis, but Cc/Cl infection should be considered in the management of HIVIP with meningeal syndrome because of the diagnostic difficulty and the high MICs of antifungal agents required for the treatment of meningitis due to these cryptococcal species.

Cryptococcal Meningoencephalitis in an Immunosuppressed Patient with Chronic Lymphocytic Leukemia

Cryptococcal meningoencephalitis, an invasive fungal infection caused by an encapsulated fungus Cryptococcus neoformans should be suspected in immune compromised individuals with defective cell-mediated immunity and patients on immunosuppressive drugs with recent development of fever, confusion and loss of consciousness. A rapid diagnosis is fundamental for decreasing morbidity and mortality from cryptococcal disease. Cerebrospinal fluid (CSF) study and simple stain like India Ink Stain can be performed for diagnosis of cryptococcal meningoencephalitis. Here, we report a case of cryptococcal meningoencephalitis in chronic lymphocytic leukemia (CLL) patient on immunosuppressive drugs diagnosed by CSF study and India ink stain which responded dramatically with antifungal agents after diagnosis. JAFMC Bangladesh. Vol 16, No 2 (December) 2020: 87-89

Case Report: Streptococcus Suis Meningitis Diagnosed in a HIV-Infected Patient With Cryptococcal Meningitis Using Next-Generation Sequencing

Background: Streptococcus suis has been recognized as a zoonotic pathogen that may cause infections in humans. Although rarely described, it is not surprising that both cryptococcal and streptococcus suis meningitis infections can co-exist in a HIV-infected patient with a low CD4 count. However, a fast and accurate diagnose of meningitis of multipathogenic infections is challenging. In this report, we describe such a case of a HIV-infected patient with meningitis of multipathogenic infections.Case Presentation: The patient was a 34-year-old Chinese male who was diagnosed with cryptococcal meningitis and HIV at the same time about 1 year ago. During the same time period, he had received (with good compliance) fluconazole and tenofovir-lamivudine- dolutegravir based antiretroviral therapy (ART). However, symptom of progressively worsening occipital headache appeared after he was exposed to a truck which was used for transporting pigs. Initial workup indicated an increase of the cerebrospinal fluid (CSF) opening pressure (OP) and an increase in the number of lymphocytes and proteins in CSF. A magnetic resonance imaging (MRI) scan revealed that partial cerebellar surface enhancement. The cryptococcus capsular antigen test of CSF was positive. The results of the India Ink microscopy for cryptococcus, nucleic acid of CMV and EBV and mycobacterium tuberculosis (MTB) tests of CSF were negative. The results of the bacteria and fungi smear and culture of CSF were also negative. Eventually, streptococcus suis was detected using next-generation sequencing (NGS) in CSF. The diagnosis of Streptococcus suis meningitis was made based on the patient's contact history with carrier pigs and the clinical findings addressed above. The treatment of 2 weeks of intravenous ceftriaxone and 1 week of oral moxifloxacin resulted in improvement of the condition of CSF. The anti-fungal treatment using fluconazole continued until the CFS OP went down to a normal level and the cryptococcus capsular antigen test of CSF was negative 6 months later.Conclusion: This case highlights that NGS might be beneficial to HIV-infected patients who have meningitis with negative CSF culture results. Multiple etiologies for such condition in the immunocompromised patients must be taken into consideration and early stage NGS is recommended.

The external oblique intercostal block: anatomical evaluation and case series

Study Objective We report a modified block technique aimed at obtaining upper midline and lateral abdominal wall analgesia: the external oblique intercostal (EOI) block. Design A cadaveric study and retrospective cohort study assessing the potential analgesic effect of the EOI block. Setting Cadaver lab and operating room. Patients Two unembalmed cadavers and 22 patients. Interventions Bilateral ultrasound-guided EOI blocks on cadavers with 29 ml of bupivacaine 0.25% with 1 ml of India ink; single-injection or continuous EOI blocks in patients. Measurements Dye spread in cadavers and loss of cutaneous sensation in patients. Main Results In the cadaveric specimens, we identified consistent staining of both lateral and anterior branches of intercostal nerves from T7–T10. We also found consistent dermatomal sensory blockade of T6–T10 at the anterior axillary line and T6–T9 at the midline in patients receiving the EOI block. Conclusions We demonstrate the potential mechanism of this technique with a cadaveric study that shows consistent staining of both lateral and anterior branches of intercostal nerves T7–T10. Patients who received this block exhibited consistent dermatomal sensory blockade of T6–T10 at the anterior axillary line and T6–T9 at the midline. This block can be used in multiple clinical settings for upper abdominal wall analgesia.

Frequency of number of roots and root canals of maxillary first premolar teeth.

Objective: To determine the frequency of the number of roots and root canals in a sample of 250 extracted maxillary first premolar teeth of patients visiting Peshawar Dental College and Hospital Khyber Pakhtunkhwa. Study Design: Cross Sectional. Setting: Department of Oral Biology, Peshawar Dental College and Hospital Khyber Pakhtunkhwa. Period: April 2016 to December 2016. Material & Methods: A total of 250 extracted human maxillary first premolars were collected from the Department of Oral & Maxillofacial Surgery, Peshawar Dental College, and Hospital Khyber Pakhtunkhwa. All teeth were visually inspected to count the number of roots. Subsequently, the access cavity was prepared, and pulp extirpated from each tooth. Endodontic explorer was used to locating the canal orifice(s) at the pulp chamber floor. Later the root canal orifices were injected with India ink to stain the canals. After that roots of teeth were sectioned at different levels to note down the number of canals. Data were analyzed using SPSS version 19. The statistical significance of the variations from mean values was considered significant if the p-value was less than 0.05. Results: Out of 250 maxillary first premolar teeth, 44.8% had one root, 40.4% had two separate and 12.8% had two fused roots. Three roots were seen in 2.0% teeth. Two root canals were present in the vast majority (70.4%), whereas one and three root canals were seen in 27.6% and 2.0% teeth, respectively. The correlation between the number of roots and root canals of maxillary first premolar teeth was highly significant. Conclusions: There was a high frequency of maxillary first premolars with two roots and two root canals.

Disseminated Glioneuronal Tumor: A rare presentation in children

Gliomas, though most common pediatric central nervous system tumor, can manifest as disseminated glio-neuronal tumor, a rare variant in children. Clinical presentation depends on its location, type and age of child. We are presenting 8 years old male child with fever, projectile vomiting and severe headache which woke him up from deep sleep for 1 month. He had positive meningeal signs and raised intracranial tension with cerebrospinal fluid picture suggestive of partially treated meningitis. There was no improvement even on adequate duration of intravenous antibiotics and had appearance of new onset false localizing signs, MRI brain showed features of cryptococcal meningitis for which India ink staining was negative. As clinical picture was unlike of meningitis, repeat 3 tesla MRI brain was done. Expert neuro-radiologist’s opinion was in favor of disseminated glio-neuronal tumor which was confirmed on histopathological examination. Child underwent laminectomy in TATA memorial hospital and advised palliative care. Child succumbed at home within 6 months of illness.