Nonrespiratory Specimens Research Articles

Expect the unexpected: endocarditis caused by Legionella feeleii.

We report a fatal case of Legionella feeleii endocarditis in a post-lung transplant patient. The diagnosis was delayed, as routine microbiological testing of nonrespiratory specimens does not account for extrapulmonary Legionella, and urine antigen testing only reliably detects Legionella pneumophila serogroup 1. This case also illustrates the utility of molecular sequencing for blood culture-negative endocarditis.

Description, validation, and review of a decade of experience with a laboratory-developed PCR test for detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens

Rapid detection of Mycobacterium tuberculosis complex directly from clinical specimens is critical for patient care. Mycobacterial culture requires days to weeks for results and therefore many laboratories employ rapid molecular methods for the diagnosis of tuberculosis. There are two FDA-cleared molecular assays for the detection of M. tuberculosis complex in the United States and both are cleared for testing of respiratory specimens only. The detection of M. tuberculosis complex in extrapulmonary specimens is often done using laboratory-developed PCR methods. In this work, the verification and subsequent validation of test performance over a decade is detailed for a laboratory-developed PCR assay (MTBRP) that detects M. tuberculosis complex from respiratory and non-respiratory specimens. The assay also provides information about potential isoniazid resistance. The performance of the MTBRP PCR assay was compared to the Cepheid Xpert MTB/RIF assay in acid-fast smear positive and smear negative specimens and mycobacterial culture for acid-fast smear positive specimens. The MTBRP assay demonstrated 99% correlation with the Xpert MTB/RIF assay using 499 respiratory specimens. The performance of the MTBRP PCR assay compared with mycobacterial culture for 867 AFB smear positive respiratory and non-respiratory specimens demonstrated a sensitivity of 100% and a specificity of 99.1%. This work provides longitudinal evidence using real-world clinical laboratory conditions and specimens to demonstrate that laboratory-developed PCR assays such as the MTBRP can provide a rapid and sensitive method for detection of pulmonary and extra-pulmonary tuberculosis from a wide-variety of smear positive specimen sources.

Role of GeneXpert MTB/RIF assay for the diagnosis of cervical lymph node tuberculosis and rifampicin resistance.

Tuberculosis (TB) of lymph node (TB lymphadenitis) is one of the most common forms of extrapulmonary TB (EPTB) whose diagnosis is critically challenging. Although new diagnostic methods have been developed, especially in patients without a history of TB, the cervical tuberculous lymphadenitis diagnosis is still elusive. This study assessed the applicability of GeneXpert in early diagnosis of EPTB, especially cervical lymphadenopathy. The study was conducted in a tertiary care hospital from January 2018 to December 2020 at the department of microbiology. All the samples of cervical lymph node tissue and lymph node aspirate were followed as per the routine protocol for mycobacterial identification. The sample was divided into two parts: one was used for the new molecular-based GeneXpert MTB/RIF assay and the second one was tested by direct and concentrated acid-fast bacilli microscopy by Z-N staining and culture for the detection of MTB. Among the 145 samples tested, the GeneXpert detected the DNA of MTB in 89 samples (61.37%), whereas the culture test was positive in 42 (28.93%) specimens. GeneXpert also detected 7 rifampicin resistance cases. GeneXpert sensitivity and specificity results were assessed according to culture results. The sensitivity and specificity of the GeneXpert assay were 85.71% and 48.54%, respectively. GeneXpert MTB/RIF should be used in conjunction with clinical presentation and other molecular investigation in nonrespiratory specimens.

Antibiotic Resistance in COVID-19 with Bacterial Infection: Laboratory-Based Surveillance Study at Single Tertiary Hospital in Indonesia.

PurposeCoronavirus disease 2019 (COVID-19) is a new pandemic affecting the respiratory system and caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to the increased use of antibiotics, the length of stay of hospitalized patients affects the risk of bacterial infections among the COVID-19 patients. However, this pandemic has interrupted antibiotic surveillance activity and led to an information gap about the prevalence and characteristics of bacterial infection. This study aims to describe the antibiotic resistance in COVID-19 patients with culture-proven bacterial infection using a laboratory-based surveillance approach.Patients and MethodsA retrospective study with a cross-sectional design was conducted on adult patients that confirmed positive for COVID-19 according to the International Classification of Diseases 10th Revision (ICD-10). From March 2020 to October 2021, data were obtained from the hospital information system and merged with the culture and antibiotic susceptibility test from laboratory information system at Hasan Sadikin General Hospital. The outcome is the prevalence percentage of resistance to selected antibiotics in patients with COVID-19. The resistance percentage is considered high when equal to or more than 20%.ResultsThere was 2786 adult patient confirmed for COVID-19 according to the ICD-10, and 26.3% (n = 733) of them submitted clinical specimen for culture. The prevalence of bacterial infection among COVID-19 patients was 16.4%, predominating Gram-negative bacteria (GNB). The respiratory specimen dominated the positive growth culture. The GNB were predominantly discovered among the respiratory and non-respiratory specimens. High range resistance to ampicillin-sulbactam (24–100%), ceftriaxone (22–81%), cefotaxime (22–73%) and ciprofloxacin (20–86%) are observed among the GNB.ConclusionThere is high resistance to fluoroquinolone and cephalosporins in identified isolate, commonly used as the first-line empirical treatment for respiratory and non-respiratory infection in Indonesia. The continuous antibiotic surveillance is mandatory and crucial to prevent the long-term effects of the COVID-19 pandemic, particularly bacterial infection.

CRISPR-based assay reveals SARS-CoV-2 RNA dynamic changes and redistribution patterns in non-human primate model

ABSTRACT Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT–PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT–PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT–PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT–PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT–PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT–PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT–PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.

SARS-CoV-2 Rapid Antigen Detection in Respiratory and Nonrespiratory Specimens in COVID-19 Patients.

Rapid antigen testing for coronavirus disease 2019 (COVID-19) available at present provides immediate results at low cost with less expertise and without any need of sophisticated infrastructure. Most of these test kits available are for nasopharyngeal samples. This is a novel study to detect the presence of COVID antigen in samples other than throat and oropharyngeal. Various samples received from patients admitted in the COVID-19 dedicated center were tested for the presence of antigen. Same procedure was followed as done for the nasopharyngeal sample. A total of 150 samples were tested, which included ascitic fluid, pleural fluid, drain fluid, bile, bronchoalveolar lavage, cerebrospinal fluid, endotracheal tube aspirate, sputum, tissue, and urine. Out of 150, 11 (7.33%) were positive and 138 (92.66%) were negative for the antigen test. The COVID-19 antigen test kit, though designed for nasopharyngeal samples, was able to detect the presence of antigen in other clinical samples.

Clinical characteristics and imaging features of patients with nontuberculous mycobacteria in a tertiary care center

BackgroundNon-tuberculous mycobacteria (NTM) are ubiquitous organisms that occasionally causes invasive diseases in humans, but they are under-reported in Saudi Arabia. We aimed to describe NTM infections and apply the American Thoracic Society/Infectious Diseases Society of America ATS/IDSA criteria. MethodPositive laboratory reports for NTM between January 2006 and December 2017 were retrospectively reviewed, and then classified into respiratory and non-respiratory specimens. ATS/IDSA criteria were applied to all respiratory specimens. Host status, clinical presentation, species identification, imaging, treatment, and outcome data were collected using a standardized form and analyzed. Cases with duplication or incomplete data were excluded. Results183 unique patients with positive NTM culture were included. Median age was 52 years and males represented 59%. Majority of cases were in the respiratory specimens group (n = 146), of which only 15 cases have met the ATS/IDSA criteria. Overall, cases were primarily known to have non-immunocompromising condition but 27% had either an active malignancy (n = 35), HIV (n = 13), or primary immunodeficiency (n = 8). 68.3% of cases presented with respiratory symptoms with or without fever. Among the identified NTM species (51.9%), slowly growing NTM were predominant. Anti-NTM therapy was provided in only 22.4% of this cohort. Death was documented only in 5 cases; all were in the respiratory group and were not treated. ConclusionThough uncommon to isolate, only one in every ten respiratory NTM isolates was found potentially true pathogen in a single center in Saudi Arabia. Future studies on NTM prevalence in Saudi Arabia are recommended.

Detection of three pandemic causing coronaviruses from non-respiratory samples: systematic review and meta-analysis

SARS-CoV-2 has posed an unprecedented challenge to the world. Pandemics have been caused previously by viruses of this family like Middle East Respiratory Corona Virus (MERS CoV), Severe Acute Respiratory Syndrome Corona Virus (SARS CoV). Although these viruses are primarily respiratory viruses, but they have been isolated from non-respiratory samples as well. Presently, the detection rate of SARS‐CoV‐2 RNA from different clinical specimens using Real Time Reverse Transcriptase Polymerized Chain Reaction (qRT‐PCR) after onset of symptoms is not yet well established. Therefore, the aim of this systematic review was to establish the profile of detecting SARS‐CoV‐2, MERS CoV, SARS CoV from different types of clinical specimens other than the respiratory using a standard diagnostic test (qRT‐PCR). A total of 3429 non-respiratory specimens were recorded: SARS CoV (total sample—802), MERS CoV (total sample—155), SARS CoV-2 (total sample—2347). Out of all the samples studied high positive rate was seen for saliva with 96.7% (14/14; 95% CI 87.6–100.0%) for SARS CoV and 57.5% (58/250; 95% CI − 1.2 to 116.2%) for SARS CoV-2, while low detection rate in urine samples for SARS CoV-2 with 2.2% (8/318; 95% CI 0.6–3.7%) and 9.6% (12/61; 95% CI − 0.9 to 20.1%) for SARS CoV but there was relatively higher positivity in urine samples for MERS CoV with detection rate of 32.4% (2/38; 95% CI − 37.3 to 102.1%). In Stool sample positivity was 54.9% (396/779; 95% CI 41.0–68.8%), 45.2% (180/430; 95% CI 28.1–62.3%) and 34.7% (4/38; 95% CI − 29.5 to 98.9%) for SARS CoV-2, MERS CoV, and SARS CoV, respectively. In blood sample the positivity was 33.3% (7/21; 95% CI 13.2–53.5%), 23.7% (42/277; 95% CI 10.5–36.9%) and 2.5% (2/81; 95% CI 0.00–5.8%) for MERS CoV, SARS CoV-2 and SARS CoV respectively. SARS‐CoV‐2 along with previous two pandemic causing viruses from this family, were highly detected stool and saliva. A low positive rate was recorded in blood samples. Viruses were also detected in fluids along with unusual samples like semen and vaginal secretions thus highlighting unique pathogenic potential of SARS‐CoV‐2.

The diagnostic impact of implementing a molecular-based algorithm to standard mycobacterial screening at a reference laboratory with an intermediate prevalence for non-respiratory samples.

Rapid, reliable results can be given by molecular, direct detection and identification of the Mycobacterium tuberculosis (MTB/Mtb) complex from clinical samples. The Xpert MTB/RIF assay is an assay that has been availablefor more than a decade for identification of Mycobacterium tuberculosis and resistance to rifampicin. However, there is minimal evidence on its clinical usefulness in paucibacillary, non-respiratory samples. The Xpert MTB/RIF assay clinical utility index, its diagnostic characteristics and the number required to diagnose 2935 non-respiratory specimens submitted for routine mycobacterial work-up in a reference laboratory in an intermediate prevalence setting per specimen form were evaluated. The Xpert MTB/RIF assay showed a variable clinical utility index and number required to diagnose (NND) depending on the type of specimen, which was moderate in tissue biopsies (NND = 1.8) and excellent in pus and urine samples, compared to acid-fast microscopy and culture as a gold standard technique (NND = 1.1 and 1.2). Microscopy, on the other hand, consistently showed a weak to fair index of clinical usefulness in all specimen forms, with in NND of 2.3–12.5. The NND for detecting tuberculous infection in the cerebrospinal fluid by the Xpert MTB/RIF assay was noted to be 1.2, with a moderate clinical utility index of 0.8. The evidence presented indicates that the overall appropriate diagnostic utility of the Xpert MTB/RIF assay is clinically successful in most non-respiratory samples. To check the cost-effectiveness and prognostic effect of integrating this completely automated molecular-based assay into the routine testing algorithm for non-respiratory mycobacterial specimens, further data must be collected.

"A Rapid Review on the sensitivity of SARS-CoV-2 RT-PCR done on different clinical specimens "

Background: RT-PCR using respiratory tract specimens, most commonly nasopharyngeal swab (NPS), has been used to confirm the diagnosis of COVID-19. NPS is a relatively invasive procedure that causes patient discomfort and risks viral transmission. Other specimens are therefore being investigated for the detection of SARS-CoV-2 RNA. Objective: To determine the sensitivity of non-respiratory tract specimens in detecting SARS-CoV-2 RNA in patients with COVID-19. Methodology: This review summarized the results of eight studies obtained from a literature search done in May 2020 in PubMed MEDLINE, Cochrane Library and MedRxiv. Two independent investigators reviewed and appraised the studies that were included, and pooled estimates of sensitivity for each specimen were determined using Stata’s Metaprop function. Results: The sensitivity in detecting SARS-CoV-2 RNA in non-respiratory tract specimens of diagnosed COVID-19 patients are as follows: Saliva 77% (95% CI 71-83%), stool/rectal swab/anal swab 22% (95% CI 22-37%), blood/serum/plasma 2% (95% CI 1-3%), and urine 22% (95% CI 18-25%). Conclusion: SARS-CoV-2 RNA is detected in saliva, stool/rectal swab/anal swab, blood/serum/plasma and urine. Among these, saliva has the highest estimated sensitivity. However, more studies are needed to correct the heterogeneity brought about by factors such as timing of specimen collection, disease severity and treatment.

Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2.

The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary tuberculosis and rifampicin resistance in adults.

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary tuberculosis and rifampicin resistance in adults.

Performance Comparison of GeneXpert MTB/RIF and ProbeTec ET Tests for Rapid Molecular Diagnosis of Extrapulmonary Tuberculosis in a Low TB/MDR-TB Incidence Country

Objective: This study evaluated the performance of GeneXpert MTB/RIF (Xpert) and ProbeTec ET (PTec-ET) assays in diagnosing extrapulmonary tuberculosis (EPTB) in Kuwait. Materials and Methods: We tested nonrespiratory clinical specimens (n = 3,995) collected from 3,995 patients suspected to have EPTB. These included cavitary fluids (n = 2,054), fine-needle aspirate (FNA)/pus/tissue biopsy (n = 1,461), urine (n = 302), cerebrospinal fluid (CSF, n = 118), and others (n = 60). All specimens were processed for acid-fast bacilli (AFB), culture in mycobacteria growth indicator tube 960 system, and nucleic acid detection by Xpert and PTec-ET according to manufacturer’s instructions. Results: Of 3,995 specimens, 95 were AFB-positive, 403 were culture-positive, and an additional 86 samples had histopathology suggestive of TB. Using culture as reference, the sensitivity and specificity values were 88.33 and 97.3% for Xpert and 72.95 and 97.80% for PTec-ET, respectively. Although performance of both tests was comparable in AFB-positive samples, Xpert detected significantly more cases in culture-positive samples. Among culture-negative samples, Xpert detected 18 more cases including 16 with histopathological evidence of TB. Lowest positivity was detected for both tests in cavitary fluids. Xpert performed better than PTec-ET in culture-positive FNA/pus/tissue biopsy and CSF samples. Conclusions: Although performance of both tests was suboptimal for AFB-negative/culture-positive samples, Xpert performed better than PTec-ET and also detected more cases of AFB-negative/culture-negative/histopathology-positive samples. PTec-ET was positive in 3, while Xpert was positive in all 6 culture-positive CSF specimens for rapid diagnosis of TB meningitis. Xpert was thus superior to PTec-ET or smear microscopy in rapid diagnosis of EPTB.

New Insight into Laboratory Tests and Imaging Modalities for Fast and Accurate Diagnosis of COVID-19: Alternative Suggestions for Routine RT-PCR and CT-A Literature Review.

The globally inimitable and unremitting outbreak of COVID-19 infection confirmed the emergency need for critical detection of human coronavirus infections. Laboratory diagnostic tests and imaging modalities are two test groups used for the detection of COVID-19. Nowadays, real-time polymerase chain reaction (RT-PCR) and computed tomography (CT) have been frequently utilized in the clinic. Some limitations that confront with these tests are false-negative results, tests redone for follow-up procedure, high cost, and unable to do for all patients. To overcome these limitations, modified and alternative tests must be considered. Among these tests, RdRp/Hel RT-PCR assay had the lowest diagnostic limitation and highest sensitivity and specificity for the detection of SARS-CoV-2 RNA in both respiratory tract and nonrespiratory tract clinical specimens. On the other hand, lung ultrasound (LUS) and magnetic resonance imaging (MRI) are CT-alternative imaging modalities for the management, screening, and follow-up of COVID-19 patients.

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Preventive Guidelines for Disinfection and Microbiological Processes During Handling of COVID-19 Samples

Since the first detection and emergence of SARS-CoV-2 in Wuhan, China in December 2019, it has become a worldwide health concern. Human to human airborne transmission and persistence of SARS-CoV-2 on inanimate surfaces or contaminated hands have been documented. Droplets transmission has been recognized as a major source of virus spread. Most of the samples received in the pathology laboratories were for the diagnosis of the corona virus disease-2019 (COVID-19). The highest concentration of SARS-CoV-2 has been observed in the respiratory specimens compared to the non-respiratory specimens as the virus is present in lower concentration in urine, blood, and stool. The guidelines in this article present the procedures for disinfection and microbiological processes that should minimize the risk of infection to the laboratory personnel. Surface disinfection with sodium hypochlorite (0.1%), ethanol (62%-71%), or hydrogen peroxide (0.5%) with an exposure time of 1 min is likely to be efficient against SARS-CoV-2. However, the ultraviolet light at 254 nm, glutaraldehyde, formalin, heat treatment of 65 ◦C or above, and acidic (pH <3) may be effective for inactivating SARS-CoV-2. The SARS-CoV-2 can remain infectious on the inanimate surfaces for up to 9 days so laboratory personnel should be aware of the need for following the biosafety protocols while processing the specimens.

Comparing The Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 in Different Human Specimens

This meta-analysis study analyzed the data of 47 recent studies with data related SARS-COV-2 viral load detection in different human specimens. 1099 patients were tested for SARS-COV-2 viral load using up to 19 different respiratory and non-respiratory specimens using RT-PCR by targeting different types of viral genes of which ORF1ab is the most commonly used target gene. 9909 specimens were taken from the patients. The mean of viral load cycle threshold value is 17.8 (±11.7), with a median of 15.95 with minimum value of 0.2 and a maximum value of 36.5. Nasopharyngeal swab has the highest positivity rate (90.5%) for viral load detection followed by Bronchoalveolar lavage, nasal swab, nasopharyngeal aspirate, throat swab and sputum. For the non-respiratory specimen, stool and rectal swab are most appropriate specimens followed by blood. The urine is not appropriate specimen for viral load detection due to very low sensitivity. The sputum was positive up to 23 days in a daily manner since start of symptoms except for the days 19, 21, and 23 that were negative for the virus. Three specimens, the nasopharyngeal swab, throat swab, and rectal swab, showed positive RT-PCR results before the appearance of COVID-19 clinical features. Possible positive results can be present up to 43 days in throat swab, stool, and rectal swab. After negative conversion of respiratory specimens, the viral shedding can continue more than one month from stool and rectal swab. The 3rd day since onset of symptoms is the most day of testing (223/2935). The highest positivity of SARS-COV-2 viral load was recorded in day 16 since the onset of symptoms.

Comparison between TRCReady MTB and MTB ELITe MGB kit for the direct detection of Mycobacterium tuberculosis complex in respiratory and non-respiratory samples

<em>Background</em>: A recent World Health Organization survey estimated about 10.4 million new tuberculosis (TB) cases per year with a high mortality rate. A rapid and accurate diagnosis of TB is very important for the optimal treatment and the prevention of spread. In the last few years rapid nucleic acid amplification tests for the detection of <em>Mycobacterium</em> <em>tuberculosis</em> <em>complex</em> (MTB) were developed. <br /><em>Materials and methods</em>: In this study we have compared TRCReady MTB, based on the Transcription Reverse Transcription Concerted Reaction technology (TRC) and MTB ELITe MGB Kit, a qualitative nucleic acid amplification assay, for the direct detection of MTB in 56 respiratory and 24 non-respiratory specimens, collected from September to November 2015. <br /><em>Results</em>: Both methods did not detect any MTB in 45 respiratory samples and in 22 non-respiratory specimens with negative cultures. MTB ELITe MGB Kit identified MTB in 8 respiratory samples with MTB positive cultures, 7 of which were detected by TRCReady MTB as well. Two non-respiratory MTB positive cultures were correctly identified by both methods. In two respiratory samples with <em>Mycobacterium Other Than Tuberculosis</em> positive cultures both methods provided negative results. <br /><em>Conclusions</em>: In conclusion, TRCReady MTB performance proved comparable to that of MTB ELITe MGB Kit in the diagnosis of pulmonary and extra-pulmonary TB, with a shorter analytical time (50 vs 110 min).

Clinical evaluation of the Abbott RealTime MTB Assay for direct detection of Mycobacterium tuberculosis-complex from respiratory and non-respiratory samples

Rapid and reliable diagnosis is crucial for correct management of tuberculosis. The Abbott RealTime MTB Assay represents a novel qualitative real-time PCR assay for direct detection of M. tuberculosis-complex (MTB) DNA from respiratory samples. The test targets two highly conserved sequences, the multi-copy insertion element IS6110 and the protein antigen B (PAB) gene of MTB, allowing even the detection of IS6610-deficient strains. We evaluated this commercial diagnostic test by analyzing 200 respiratory and, for the first time, 87 non-respiratory clinical specimens from our tertiary care institution and compared its results to our IS6110-based in-house real-time PCR for MTB as well as MTB culture. Overall sensitivity for Abbott RealTime MTB was 100% (19/19) in smear positive and 87.5% (7/8) in smear negative specimens, while the specificity of the assay was 100% (260/260). For both non-respiratory smear positive and smear negative specimens Abbott RealTime MTB tests showed 100% (8/8) sensitivity and 100% (8/8) specificity. Cycle threshold (Ct) value analysis of 16 MTB positive samples showed a slightly higher Ct value of the Abbott RealTime MTB test compared to our in-house MTB assay (mean delta Ct = 2.55). In conclusion, the performance of the new Abbott RealTime MTB Assay was highly similar to culture and in-house MTB PCR. We document successful analysis of 87 non-respiratory samples with the highly automated Abbott RealTime MTB test with no inhibition observed.

Assessment of Sputum Quality and Its Importance in the Rapid Diagnosis of Pulmonary Tuberculosis

Background: Globally, Tuberculosis (TB) still remains a major public health problem. In India, Pulmonary Tuberculosis (PTB) is the most common form of the disease; however, Extrapulmonary Tuberculosis (EPTB) comprises 10 to 20% of all TB cases. The diagnosis of EPTB cases is difficult because of paucibacillary nature and consequently is associated with low sensitivity of Zhiel- Neelson (ZN) smear and culture on Lowenstein-Jensen (LJ) media as gold standard. The present study comparatively evaluates the utility of real time quantitative PCR over nested PCR and other conventional techniques for the detection of M. tuberculosis Complex (Mtc) in clinical isolates of PTB and EPTB samples at a tertiary care centre, Shri Ram Murti Smarak Institute of Medical Sciences (SRMSIMS), Bareilly. Methods and Findings: In total, 205 both pulmonary (24) and extrapulmonary (181) specimens were processed for ZN smear, culture on LJ media, nested PCR and real time quantitative PCR using IS6110 as a gene target. Out of 205 samples, none of the sample was found to be smearpositive and only 28 (14%) samples were found to be culture positive. The nested PCR and real time quantitative PCR positivity was observed 100% in culture positive specimens. However in majority of culture negative specimens the sensitivity was 100% in both nested PCR and real time quantitative PCR assays for EPTB and PTB specimens. The specificity was better in case of real time quatitative PCR as compared to nested PCR, 45.8% and 20% in case of nested PCR for nonrespiratory and respiratory specimens respectively. The specificity was increased upto 86.1 and 100% with real time quantitative PCR for EPTB and PTB cases respectively. Conclusions: The combined analysis of nested PCR, real time quantitative PCR and other lab investigations can be very useful in the rapid diagnosis of M. tuberculosis in paucibacillary extrapulmonary tuberculosis samples in Indian scenario.