Role of Genetic Testing in the Management of Indeterminate Thyroid Nodules in the Indian Setting.

The increased detection of thyroid nodules in the human population has led to an increase in the number of thyroid surgeries without an improvement in survival outcomes. Though the choice for surgery is straightforward in malignant thyroid nodules, the decision is far more complex in those nodules that get categorized into indeterminate thyroid nodules (ITN) by fine needle aspiration. Therefore, there is a pressing need to develop a tool that will aid in decision-making among the ITN. In this context, the development of various molecular testing (MT) panels has helped to confirm or rule out malignancy, reducing unnecessary surgeries and potentially guiding the extent of surgery as well. Currently, such tests are widely used among the Western population but these MT panels are not used by the South Asian population because of non-availability of validated panels and the high cost involved. There is a need to develop a suitable panel which is population-specific and validate the same. In this review, we would focus on current trends in the management of ITN among the South Asian population and how to develop a novel MT panel which is cost-effective, with high diagnostic accuracy obviating the need for expensive panels that already exist.

A case report of sepsis and death caused by Parvimonas micra, a rare anaerobe.

Parvimonas micra is a type of Gram-positive anaerobic cocci widely distributed in the oral cavity, gastrointestinal tract, respiratory tract, and female reproductive system mucosa. It is a conditional pathogen that can cause infections in the human oral cavity, wounds, and other areas as well as sepsis. In this case report, the patient's immune system was compromised by various underlying diseases and a pulmonary infection, which led to the entry of P. micra infection into the bloodstream. P. micra is a slow-growing organism (When a bloodstream infection occurs, flagging an anaerobic bottle of blood culture as positive will usually take >48 h), which makes it hard to secure timely blood culture results. Our patient's poor physical condition eventually led to sepsis, and she died after 5 days in the hospital.

Intraoperative Molecular Analysis of Total Tumor Load in Sentinel Lymph Node: A Predictor of Axillary Status in Early Breast Cancer.

BACKGROUND: Axillary lymph node dissection (ALND) remains the standard of care in breast cancer patients with positive sentinel lymph node (SLN). However, approximately 40-60% of patients with positive SLNs have not developed to non-SLN metastasis and ALND seems to be an overtreatment. The purpose of this study was to analyze predictors and define a specific cut-off of total tumor load (TTL) of CK19 that can be used as a predictive factor of non-SLN metastasis in early breast cancer patients. MATERIALS AND METHODS: The records of 238 patients with cT1-3N0 breast cancer who had an intraoperative SLN evaluation performed through One-Step nucleic acid (OSNA) assay at Songklanagarind Hospital between 1 January 2015 and 31 December 2019 were examined. Univariate and Multivariate analysis was used to identify clinicopathologic features in SLN-positive patients that predict metastasis to non-SLNs. Finally, receiver operative characteristics (ROC) curves were used to choose an optimal TTL cut-off value. RESULTS: Of a total of 110 patients who had a positive SLN, only 48 (43.64%) were found to have positive nodes in non-SLN. Multivariate analysis revealed that lymphovascular invasion, type of SLN metastasis and SLN TTL (copies/µL) were independent predictors of positive non-SLNs.  TTL cut-off value was 19,000 copies/µL, with an AUC of 0.838 with 72.7% sensitivity and 84.7% specificity to predict non-SLN metastasis. CONCLUSIONS: The likelihood of positive non-SLNs in patients who showed a positive SLN correlates with lymphovascular invasion, type of SLN metastasis and SLN TTL (copies/µL). Our result revealed that the patients with a SLN TTL ≥19,000 copies/µl continue to attract the recommendation to proceed with ALND. This cut-off value can then help clinicians to assess which patients would benefit from ALND.

Molecular characterization of nontuberculous Mycobacteria in a tuberculosis and HIV reference unit in the State of Amazonas, Brazil

ABSTRACT Background: In recent years, the prevalence of nontuberculous mycobacterial (NTM) infections has increased in different regions of the world. The American Thoracic Society (ATS) recommends standardized identification criteria, reinforcing the need for faster and less complicated clinical and laboratory techniques. Methods: In this retrospective study, NTM species isolated from pulmonary, extrapulmonary, and disseminated samples from patients treated at a TB/HIV reference unit in the State of Amazonas from 2011 to 2014 were identified through a combination of molecular techniques. Results: To identify the molecular technique, 50 cryopreserved NTM cultures were recovered and subcultivated in culture medium. The potentially pathogenic NTM species identified were M. avium, M. intracellulare, M. kansasii, M. chelonae, M. abscessus, M. fortuitum, and M. peregrinum. Results of GenoType® showed moderate agreement with those of genomic sequencing (kappa = 0.60), whereas the results obtained by the PRA-hsp65 technique disagreed with the results obtained by sequencing (kappa = 0.49). Conclusions: Our findings highlight that GenoType CM is a good method for the identification of NTM, as well as the need for the application of standardized criteria, such as those set forth by the ATS.

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India.

With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021-March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.

Highly Sensitive and Specific Molecular Test for Mutations in the Diagnosis of Thyroid Nodules: A Prospective Study of BRAF-Prevalent Population.

Molecular testing offers more objective information in the diagnosis and personalized decision making for thyroid nodules. In Korea, as the BRAF V600E mutation is detected in 70-80% of thyroid cancer specimens, its testing in fine-needle aspiration (FNA) cytology specimens alone has been used for the differential diagnosis of thyroid nodules until now. Thus, we aimed to develop a mutation panel to detect not only BRAF V600E, but also other common genetic alterations in thyroid cancer and to evaluate the diagnostic accuracy of the mutation panel for thyroid nodules in Korea. For this prospective study, FNA specimens of 430 nodules were obtained from patients who underwent thyroid surgery for thyroid nodules. A molecular test was devised using real-time PCR to detect common genetic alterations in thyroid cancer, including BRAF, N-, H-, and K-RAS mutations and rearrangements of RET/PTC and PAX8/PPARr. Positive results for the mutation panel were confirmed by sequencing. Among the 430 FNA specimens, genetic alterations were detected in 293 cases (68%). BRAF V600E (240 of 347 cases, 69%) was the most prevalent mutation in thyroid cancer. The RAS mutation was most prevalently detected for indeterminate cytology. Among the 293 mutation-positive cases, 287 (98%) were diagnosed as cancer. The combination of molecular testing and cytology improved sensitivity from 72% (cytology alone) to 89% (combination), with a specificity of 93%. We verified the excellent diagnostic performance of the mutation panel applicable for clinical practice in Korea. A plan has been devised to validate its performance using independent FNA specimens.

Use of a rapid recombinase-aided amplification assay for Mycoplasma pneumoniae detection.

BACKGROUND: Mycoplasma pneumoniae is one of the most common causative pathogens of community-acquired pneumonia (CAP), accounting for as many as 30-50% of CAP during peak years. An early and rapid diagnostic method is key for guiding clinicians in their choice of antibiotics. METHODS: The recombinase-aided amplification (RAA) assay is a recently developed, rapid detection method that has been used for the detection of several pathogens. The assays were performed in a one-step single tube reaction at 39° Celsius within 15-30 min. In this study, we established an RAA assay for M. pneumoniae using clinical specimens for validation and commercial real-time PCR as the reference method. RESULTS: The analytical sensitivity of the RAA assay was 2.23 copies per reaction, and no cross-reactions with any of the other 15 related respiratory bacterial pathogens were observed. Compared with the commercial real-time PCR assay used when testing 311 respiratory specimens, the RAA assay obtained 100% sensitivity and 100% specificity with a kappa value of 1. CONCLUSIONS: These results demonstrate that the proposed RAA assay will be of benefit as a faster, sensitive, and specific alternative tool for the detection of M. pneumoniae.

Molecular testing in metastatic colorectal adenocarcinoma cytology cell pellets.

BACKGROUND: Mutational status for KRAS, NRAS, and BRAF genes should be performed on all colorectal carcinoma (CRC) specimens in order to guide targeted therapy selection for metastatic disease. Mutations are typically assessed via polymerase chain reaction and/or next generation sequencing (NGS) on formalin-fixed paraffin-embedded tissues. With minimally invasive diagnostic methodologies, the cytology cell pellet obtained by fine-needle aspiration (FNA) can serve as an alternative source of tumor deoxyribonucleic acid. METHODS: An electronic record review of the cytopathology files (CoPathPlus, Cerner Corp., North Kansas City, Missouri) from September 1, 2015 through December 31, 2018 was conducted. All cytology specimens obtained via FNA and diagnosed as metastatic CRC on which NGS was performed were included. NGS for KRAS, NRAS, and BRAF mutations using the AmpliSeq Cancer Hotspot Panel v2.0 kit (Thermo Fisher Scientific, Waltham, Massachusetts) was performed on cytology cell pellets. RESULTS: Forty-eight cases were identified. Forty-six of 48 specimens (96%) were adequate for molecular testing. Of those adequate specimens, proportion of malignant cells in the sample ranged from 5% to 95% (mean 46%). Twenty-seven of 48 cases (56%) were positive for clinically relevant mutations. Twenty-four of 27 cases (89%) were positive for KRAS mutations, with exon 2 most frequently involved (22/24 cases, 92%). Two of 27 cases (7%) were positive for NRAS mutations and one case (1/27, 4%) was positive for a BRAF mutation involving codon 594. CONCLUSION: Mutational analysis performed on cytology cell pellets serves as a useful means of gathering clinically actionable information on tumor mutation status in metastatic CRC.

Polymerase-chain reaction/electrospray ionization-mass spectrometry for the detection of bacteria and fungi in bronchoalveolar lavage fluids: a prospective observational study.

PLEX-ID uses polymerase chain reaction-electrospray ionization/mass spectrometry for rapid identification of infectious agents in clinical samples. We evaluated its concordance with our centre's standard methods (SM) for bacterial and fungal detection in bronchoalveolar lavage (BAL) fluid in a prospective observational cohort study. The primary outcome was concordance (%) between SM and PLEX-ID. Secondary outcomes included concordance when excluding commensal oral flora, detection of resistance genes, and PLEX-ID's potential impact on clinical management, as determined by two independent reviewers. Included were 101 specimens from 94 patients. BALs were performed primarily for suspected pneumonia (76/101, 75%) and lung transplant work-ups (12/101, 12%). Most specimens yielded at least one organism by either method (92/101, 91%). Among all microorganisms detected (n = 218), 83% and 17% were bacterial and fungal, respectively. Overall concordance between SM and PLEX-ID was 45% (45/101). Concordance increased to 66% (67/101) when discordance for commensal flora was excluded. PLEX-ID failed to detect 21% of all 183 SM-identified organisms, while SM did not identify 28% of the 191 PLEX-ID-identified organisms (p <0.001). There was low concordance for mecA detection. Two infectious-disease specialists' analyses concluded that in most of the 31 discordant, non-commensal cases, PLEX-ID results would have had little or no impact on patient management; in eight cases, however, PLEX-ID would have led to 'wrong decision-making'. The tested version of PLEX-ID concurred weakly with standard methods in the detection of bacteria and fungi in BAL specimens, and is not likely to be useful as a standalone tool for microbiological diagnosis in suspected respiratory infections.

An overview of mutation detection methods in genetic disorders.

GENETIC DISORDERS ARE TRADITIONALLY CATEGORIZED INTO THREE MAIN GROUPS: single-gene, chromosomal, and multifactorial disorders. Single gene or Mendelian disorders result from errors in DNA sequence of a gene and include autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders. Chromosomal disorders are due to chromosomal aberrations including numerical and structural damages. Molecular and cytogenetic techniques have been applied to identify genetic mutations leading to diseases. Accurate diagnosis of diseases is essential for appropriate treatment of patients, genetic counseling and prevention strategies. Characteristic features of patterns of inheritance are briefly reviewed and a short description of chromosomal disorders is also presented. In addition, applications of cytogenetic and molecular techniques and different types of mutations are discussed for genetic diagnosis of the pediatric genetic diseases. The purpose is to make pediatricians familiar with the applications of cytogenetic and molecular techniques and tools used for genetic diagnosis.